[Federal Register Volume 59, Number 28 (Thursday, February 10, 1994)]
[Unknown Section]
[Page 0]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 94-2587]


[[Page Unknown]]

[Federal Register: February 10, 1994]


_______________________________________________________________________

Part II





Environmental Protection Agency





_______________________________________________________________________



40 CFR Part 141



Monitoring Requirements for Public Drinking Water Supplies; Proposed 
Rule
ENVIRONMENTAL PROTECTION AGENCY

40 CFR Part 141

[WH-FRL-4818-8]

 

National Primary Drinking Water Regulations: Monitoring 
Requirements for Public Drinking Water Supplies: Cryptosporidium, 
Giardia, Viruses, Disinfection Byproducts, Water Treatment Plant Data 
and Other Information Requirements

AGENCY: Environmental Protection Agency (EPA).

ACTION: Proposed rule.

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SUMMARY: EPA is proposing to require public water systems which serve 
10,000 people or greater to generate and provide the Agency with 
specific monitoring data and other information characterizing their 
water systems. Systems which use surface water, or ground water under 
the influence of surface water, and serve between 10,000-100,000 people 
would be required to (a) monitor their source water at the intake of 
each plant for two disease-causing protozoa, Giardia and 
Cryptosporidium; fecal coliforms or Escherichia coli; and total 
coliforms; and (b) provide specific engineering data as it pertains to 
removal of disease-causing microorganisms. Systems which use surface 
water, or ground water under the influence of surface water, and serve 
more than 100,000 people would be required to monitor their source 
water at the intake of each plant for the microorganisms indicated 
above, plus viruses, and, when pathogen levels exceed one pathogen/
liter in the source water, finished water for these microorganisms; 
monitor for certain disinfection byproducts (DBPs) as well as other 
water quality indicators; and provide specific engineering data as they 
pertain to removal of disease causing organisms and control of DBPs. 
All ground water systems that serve more than 100,000 people would be 
required to monitor for certain DBP, other water quality indicators, 
and to provide specific physical and engineering data. Systems which 
use surface water and serve more than 100,000 people and systems which 
use ground water and serve more than 50,000 people would be required to 
conduct bench or pilot scale studies to evaluate treatment performance 
for the removal of precursors to DBPs unless they have met certain 
source water or treated water quality criteria. This information will 
be used to consider possible changes to the current Surface Water 
Treatment Rule (SWTR) and to develop drinking water regulations for 
disinfectants and DBPs. If the SWTR is amended, information collected 
under this monitoring rule would assist utilities in complying with 
such amendments.

DATES: Comments should be postmarked or delivered by hand on or before 
March 14, 1994. Comments received after this date may not be considered 
because of time constraints.

ADDRESSES: Send written comments to ESWTR/DBPR Monitoring Docket Clerk, 
Water Docket (MC-4101); U.S. Environmental Protection Agency; 401 M 
Street, SW; Washington, DC 20460. Please submit any references cited in 
your comments. EPA would appreciate an original and three copies of 
your comments and enclosures (including references). Commenters who 
want EPA to acknowledge receipt of their comments should include a 
self-addressed, stamped envelope. No facsimiles (faxes) will be 
accepted because EPA cannot ensure that they will be submitted to the 
Water Docket.
    The proposed rule with supporting documents and all comments 
received are available for review at the Water Docket at the address 
above. For access to Docket materials, call (202) 260-3027 between 9 
a.m. and 3:30 p.m. for an appointment.

FOR FURTHER INFORMATION CONTACT: The Safe Drinking Water Hotline, 
Telephone (800) 426-4791. The Safe Drinking Water Hotline is open 
Monday through Friday, excluding Federal holidays, from 9 a.m. to 5:30 
p.m. Eastern Time. For technical inquiries, contact Stig Regli or Paul 
S. Berger, Ph.D., Office of Ground Water and Drinking Water (WH-550D), 
U.S. Environmental Protection Agency, 401 M Street SW., Washington DC 
20460, telephone (202) 260-7379 (Regli) or (202) 260-3039 (Berger).

SUPPLEMENTARY INFORMATION:

Table of Contents

I. Statutory Authority
II. Regulatory Background
III. Discussion of Proposed Rule
    A. Enhanced Surface Water Treatment Requirements (ESWTR)
    1. Need for Enhanced SWTR
    2. Monitoring and reporting requirements and rationale
    3. Reasons for monitoring listed pathogens and indicators
    4. Rationale for frequency of microbial monitoring
    5. Rationale for reporting physical data and engineering 
information
    6. Analytical methods
    7. Laboratory approval
    8. Quality assurance
    B. Disinfection Byproducts Rule (Stage 2)
    1. Need for additional data
    2. Monitoring and reporting requirements and rationale
    3. Treatment process information collection
    4. Database development
    5. Analytical methods
    6. Quality assurance
    7. Bench/pilot scale testing
    C. Dates
    D. Reporting Requirements
    E. List of Systems Required to Submit Data
IV. State Implementation
V. Cost of Rule
VI. Other Statutory Requirements
    A. Executive Order 12866
    B. Regulatory Flexibility Act
    C. Paperwork Reduction Act
    D. Science Advisory Board, National Drinking Water Advisory 
Council, and Secretary of Health and Human Services
VII. Request for Public Comments
VIII. References

I. Statutory Authority

    The Safe Drinking Water Act (SDWA or the Act), as amended in 1986, 
requires EPA to promulgate National Primary Drinking Water Regulations 
(NPDWRs) which specify maximum contaminant levels (MCLs) or treatment 
techniques for drinking water contaminants (42 U.S.C. 300g-1). NPDWRs 
apply to public water systems (42 U.S.C. 300f(1)(A). Section 1412(b)(3) 
of the Act requires EPA to publish regulations for at least 25 
contaminants at three year intervals. Section 1412(b)(9) of the Act 
requires EPA to review existing national primary drinking water 
regulations at least once every 3 years.
    According to section 1445(a)(1) of the Act, every public water 
system ``shall establish and maintain such records, make such reports, 
conduct such monitoring, and provide such information as the 
Administrator may reasonably require by regulation to assist him in 
establishing regulations, [or] * * * in evaluating the health risks of 
unregulated contaminants''. This section authorizes EPA to require 
systems to monitor and provide the Agency with these data as well as 
other data characterizing the system, including source and treated 
water quality.
    In addition, section 1401(1)(d) of the Act defines NPDWRs to 
include ``criteria and procedures to assure a supply of drinking water 
which dependably complies with such maximum contaminant levels; 
including quality control and testing procedures * * * ''. This section 
authorizes EPA to require systems and laboratories to use Agency-
approved methods and quality assurance criteria for collecting and 
analyzing water samples.

II. Regulatory Background

    Two regulations attempt to control disease-causing microorganisms 
(pathogens) in public water supplies--the Total Coliform Rule (54 FR 
27544; June 29, 1989) and the Surface Water Treatment Requirements 
(SWTR) (54 FR 27486; June 29, 1989). A third regulation, the 
Groundwater Disinfection Rule, which is currently under development, 
will add further protection for systems using ground water. The Agency 
is considering revising the SWTR in conjunction with the development of 
other new regulations.
    Another rule EPA is currently developing will address chemical 
byproducts that form when disinfectants used for microbial control in 
drinking water react with various organic chemicals in the source 
water. Some of these disinfection byproducts are toxic or are probable 
human carcinogens. As such, they were included on the 1991 Drinking 
Water Priority List (56 FR 1470; January 14, 1991) as candidates for 
future regulations. They are among the candidate contaminants for which 
EPA must meet a Court-ordered deadline that is currently being 
negotiated.
    To develop the Disinfectant/Disinfection Byproducts (D/DBP) Rule, 
EPA instituted a formal regulation negotiation process in 1992 (57 FR 
53866; Nov 13, 1992) including representatives from water utilities, 
State and local agencies, environmental groups, consumer groups, and 
EPA. The Negotiating Committee agreed to propose three rules: a) an 
information collection rule (ICR), which is proposed herein, b) an 
``interim'' enhanced surface water treatment rule (ESWTR), to be 
proposed within the next few months, and c) D/DBP regulations, to be 
proposed concurrently with the interim ESWTR.
    During the development of the D/DBP Rule, a number of members of 
the Negotiating Committee did not believe that there were adequate data 
available to address some of the DBPs on EPA's priority list (56 FR 
1473; January 14, 1991). They believed that insufficient data were 
available on many aspects of DBPs necessary to make appropriate 
regulatory decisions including health effects and health effect related 
issues, occurrence of and exposure to contaminants, and the 
capabilities of treatment technologies. Also of concern were the 
limited data on microbial contaminants for making regulatory decisions.
    The Negotiating Committee's development of the three proposed rules 
mentioned above was based on the premise of (1) taking prudent 
immediate steps by proposing a two staged D/DBP rule and an interim 
ESWTR, and (2) developing additional data through monitoring and 
research for future regulatory decisions that would support refinements 
to the proposed interim ESWTR and the Stage 2 D/DBP rule. For example, 
decisions on the direction of an ESWTR will be limited without more 
data on the occurrence of microorganisms, the effectiveness of current 
and advanced treatment schemes, potential consumer exposure, dose 
response relationships for certain pathogens, pathogen strain 
differences, and cyst/oocyst viability measures. Likewise, important 
decisions on the Stage 2 D/DBP rule would benefit from additional data 
on occurrence of DBPs, effects of current and advanced treatment 
approaches on DBP formation, potential consumer exposures, acute short-
term health effects, chronic health effects, and the use of surrogates 
as tools for defining adequacy of treatment for specific contaminants 
and reduced monitoring.
    The ICR was developed to obtain both microbial and DBP occurrence, 
exposure, and treatment data for input to the ESWTR and Stage 2, as 
outlined below, and would require the expenditure of an estimated $130 
million over three and a half years by a segment of public water 
suppliers. The commitment by the public water supply community to 
support the collection of additional data was linked to EPA's 
commitment to provide (1) adequate quality control procedures for 
collecting and managing the information obtained under the ICR and (2) 
additional funding, especially on health effects, for properly 
interpreting the data collected under the ICR. As evidence of this 
linkage, non-EPA members of the Negotiating Committee sought to assist 
the Agency in obtaining funding for the health effects and other 
research equally critical to the future decisions. On May 20, 1993, 
these committee members sent letters to the Administration and members 
of Congress requesting support for a federal commitment of $4 million 
per year for five years to support the needed research. The letters 
noted that the American Water Works Association Research Foundation 
had, independent of the negotiations, presented a public-private 
partnership research plan under which they committed to provide up to 
$2 million per year for the research under a one for two match.
    On a related effort, non-EPA Negotiating Committee members 
requested on July 14, 1993, in a letter to EPA's Administrator, 
consideration of reallocation of Agency research funds to support the 
research needs described above. The July 14, 1993 letter also spoke of 
the need for the Agency to commit funds necessary to adequately 
collect, manage, and analyze data collected under the ICR. A number of 
Negotiating Committee members believed that, without additional federal 
research and data management funding, the ICR data generated by systems 
would not be particularly useful in developing the ESWTR or Stage 2 D/
DBP Rule.
    The Negotiating Committee agreed that more data, especially 
monitoring data, should be collected under the ICR to assess possible 
shortcomings of the SWTR and develop appropriate remedies, if needed, 
to prevent increased risk from microbial disease when systems began 
complying with the new D/DBP Rule. It was also agreed that EPA would 
propose an interim ESWTR for systems serving greater than 10,000 people 
that included a wide range of regulatory alternatives. Data gathered 
under the ICR would form the basis for developing the most appropriate 
criteria among the options presented in the proposed interim ESWTR, and 
eventually for a long-term ESWTR that would include possible 
refinements to the interim ESWTR and be applicable to all system sizes. 
Both of these ESWTR rules would become effective concurrently with the 
requirements of the Stage 1 D/DBP rule for the respective different 
system sizes.
    The Negotiating Committee also agreed that additional data on the 
occurrence of disinfectants, DBPs, potential surrogates for DBPs, 
source water and within-treatment conditions affecting the formation of 
DBPs, and bench-pilot scale information on the treatability for removal 
of DBP precursors would be useful for developing Stage 2 D/DBP 
regulatory criteria beyond those currently being considered for 
proposal in Stage 1. To this end, today's proposed ICR rule, which 
would require this additional information, was accepted as necessary 
and reasonable by the Negotiating Committee.

III. Discussion of Proposed Rule

A. Enhanced Surface Water Treatment Requirements

1. Need for Enhanced SWTR
    The SWTR, which became effective on December 31, 1990, requires all 
systems using surface water, or ground water under the direct influence 
of surface water, to disinfect. It also requires all such systems to 
filter their water unless they can demonstrate that they have an 
effective watershed protection program and can meet other EPA-specified 
requirements. The SWTR also specifies that systems using surface water 
must treat water to remove/inactivate at least 99.9% (3 logs10) of 
the Giardia lamblia cysts (a protozoan) and at least 99.99% (4 
logs10) of the viruses. The SWTR does not require a system to 
monitor its source water or drinking water for these pathogens.
    During the development of the SWTR, the United States experienced 
its first large recognized waterborne disease outbreak of 
cryptosporidiosis, caused by the protozoan, Cryptosporidium (Hayes et 
al., 1989). Other outbreaks caused by this pathogen have since been 
reported both in the United States and other countries. Because of the 
lack of data before 1989 on Cryptosporidium oocyst occurrence and 
susceptibility to treatment, EPA decided to regulate this pathogen in a 
future rulemaking, rather than to delay publication of the SWTR until 
these data were available. EPA and others are now performing research 
to understand the health risks posed by Cryptosporidium. Although some 
occurrence and treatment data are now available, EPA believes that much 
more is needed before EPA can promulgate a suitable regulation for 
Cryptosporidium. EPA is planning to propose an MCLG and treatment 
technique requirement for Cryptosporidium in the ESWTR, and use the 
data from this rule to determine the need for, and specifics of, that 
regulation.
    Another shortcoming of the SWTR is that a 3-log removal/
inactivation of Giardia and a 4-log removal/inactivation of enteric 
viruses may be inadequate when a system is supplied by a poor quality 
source water. In developing the SWTR, EPA assumed on the basis of data 
available at that time, that this level of treatment was adequate for 
most systems. The Agency published associated guidance recommending 
greater treatment for systems supplied by poor quality source waters 
(EPA, 1991).
    Subsequent data on Giardia densities in source water and drinking 
water (LeChevallier et al., 1991a,b), however, bring into question the 
assumption that the treatment specified in the SWTR was adequate for 
most systems. These new data suggest that Giardia cyst concentrations 
in the source waters of many systems may be too great for the specified 
minimum level of treatment to adequately control waterborne giardiasis 
(to be discussed in the preamble of the forthcoming proposed interim 
ESWTR).
    As a result of this uncertainty, EPA needs much more data on the 
concentration of Giardia cysts and viruses for various qualities of 
source waters, with variation over time and seasonal influences, to 
determine the need for additional treatment to provide adequate Giardia 
and virus control. In addition, EPA needs more field data on the 
effectiveness of different types of water treatment for controlling 
these pathogens.
    If these new data indicate that EPA's original assumption was 
correct, i.e., that only a small percentage of systems have source 
water Giardia and virus concentrations that are too great for adequate 
control under the SWTR, then guidance (EPA, 1991) may suffice and no 
revision of the SWTR would be needed. In contrast, if a high percentage 
of systems have elevated concentrations of Giardia, then EPA believes 
that the SWTR may need to be revised to require additional treatment 
for such systems.
    If the data indicate that a revision of the SWTR is needed, then 
one regulatory option would be to tailor required treatment levels to 
Giardia concentrations in the source water. For example, the Agency 
might require a system to achieve at least a 99.9 percent (3-log) 
reduction if the source water(s) contained less than 1 cyst/100 liters, 
a 99.99 percent (4-log) reduction if the source water(s) contained 1 to 
9 cysts/100 liters, a 99.999 percent (5-log) reduction if the source 
water(s) contained 10 to 99 cysts/100 liters, and a 99.9999 percent (6-
log) reduction if the source water(s) contained more than 99 cysts/100 
liters. These suggested level of treatment requirements are consistent 
with existing EPA Guidance (USEPA 1991). Based on the dose response 
curve developed by Rose et al (1991) these levels of treatment have 
been predicted to ensure a risk of less than 1 infection per 10,000 
people per year. The concept of utilities providing higher levels of 
treatment to meet a desired acceptable risk level will be one of the 
options discussed in the preamble of the forthcoming proposed ESWTR. 
The data collected under today's monitoring rule, if promulgated, could 
be used as the basis for the treatment level prescribed.
    If EPA decides to revise the SWTR according to the above or similar 
approach, then the monitoring data would assist the Agency in 
determining the most appropriate manner for calculating source water 
pathogen densities. For example, options include the arithmetic means, 
geometric means, highest value, or a 90th percentile value (e.g., for 
ten data points, the system would select the second highest, or for 18 
data points, the system would select the third highest). These options 
will be discussed in greater detail in the forthcoming proposed interim 
ESWTR. These proposed revisions would be modified or withdrawn based on 
monitoring data collected under the present rule.
    In summary, today's proposed rule, if promulgated, would provide 
the Agency with much needed field data to determine the need for 
amending the SWTR to control microorganisms in an appropriate manner. 
Data collected under this proposed rule could also form the basis by 
which systems could establish levels of treatment, perhaps beyond those 
minimally required under the SWTR, that are appropriate for controlling 
microbial risk while complying with new D/DBP regulations. EPA 
understands that the water industry may voluntarily provide additional 
useful data for these purposes. The data collected under today's 
proposed rule, if promulgated, would also support the long-term ESWTR 
rule.
2. Monitoring and Reporting Requirements and Rationale
    The rule would require systems using surface water that serve a 
population greater than 100,000 (about 233 systems nationally) to 
monitor their influent to each plant for Giardia cysts, Cryptosporidium 
oocysts, ``total culturable viruses'' (hereafter referred to as 
``viruses'', unless otherwise indicated), fecal coliforms or 
Escherichia coli, and total coliforms. Monitoring would be monthly for 
18 months. If a plant has several sources of water, the system must 
sample the blended water from all sources or, if this is not possible, 
sample the source with the expected highest pathogen concentration. If, 
during the first twelve months of monitoring, any pathogen were to 
exceed a density of one/liter, or if the detection limit for any 
pathogen exceeds one/liter, the system would be required to monitor 
their finished water for the entire set of pathogens and indicators at 
the same frequency as source water sampling for the remaining months.
    Under this rule, systems would not be required to continue 
monitoring for viruses if: (1) viruses are not detected in the source 
water at the intake (for each plant) during the first twelve months of 
monitoring, or (2) the system has tested the source water at the intake 
(for each plant) for either total coliforms or fecal coliforms at least 
five times per week between [insert first day of month, 4 months prior 
to the promulgation date of this rule] and [insert first day of month, 
2 months after the promulgation date of this rule], and the density of 
total coliforms or fecal coliforms is less than 100 colonies/100 ml or 
20 colonies/100 ml, respectively, for at least 90% of the samples.
    For surface water systems that serve between 10,000 and 100,000 
people, the rule would require source water monitoring at the intake of 
each plant for the organisms listed above, except that they would not 
have to monitor for viruses. Monitoring for this category of systems 
would be every two months for 12 months. The rule would require all 
systems serving more than 10,000 people to provide the above monitoring 
data and other, system-specific information to EPA. The rule would not 
apply to systems that purchase all of their water from other systems.
    The rationale for requiring this information is to provide EPA with 
much needed data on the concentrations and variations with time of 
viral and protozoan pathogens in various types of source waters. It 
would also help EPA evaluate whether current assumptions on water 
treatment removal efficiencies for pathogenic protozoa and viruses are 
appropriate. Together, these data and the data on source water 
concentrations would provide EPA and the system a better understanding 
of pathogen concentrations following treatment, which would allow for a 
more accurate assessment of the pathogen levels and the associated 
health risk to which the public may be exposed. These data, along with 
possible additional data on dose-response patterns, pathogen strain 
differences, and cyst/oocyst viability measures, would allow EPA to 
determine the circumstances under which the SWTR is not adequate and to 
revise this rule accordingly to overcome any shortcomings.
    The data would also help EPA characterize occurrence relationships 
among Giardia cysts, Cryptosporidium oocysts, and viruses. For example, 
these data would help the Agency evaluate the merits of using Giardia 
as the primary target to define treatment requirements, as it did in 
the SWTR. In addition, the data may help EPA identify and prevent 
treatment changes that systems might inappropriately consider to meet 
the forthcoming D/DBP rule.
    The source water data collected under this rule might also be used 
for determining appropriate levels of treatment for particular systems 
serving more than 10,000 people, if minimum treatment requirements were 
specified as a function of source water quality conditions under the 
interim ESWTR.
    EPA would not require systems serving between 10,000 and 100,000 
people to monitor treated water because the Agency believes that 
sufficient data for microorganisms would be provided by the larger 
systems, which are generally better able to fund the collection of the 
needed data. EPA would also not require these sized systems to monitor 
viruses in source waters because the Agency believes that the larger 
systems would provide sufficient data to establish any relationship 
between the viruses and the two protozoan pathogens being monitored, 
regarding source water densities and treatment effectiveness. The 
Agency, in the absence of data suggesting otherwise, would continue to 
use Giardia, possibly including Cryptosporidium, as the primary target 
organism(s) for regulation, given their greater disinfection resistance 
compared to most other organisms, and consequently less data would be 
needed for the viruses.
    The data from these larger systems would also be useful for 
estimating pathogen concentrations in many source waters serving 
systems with fewer than 10,000 people, which EPA believes typically do 
not have the financial resources or technical expertise to collect and 
process the samples as part of the above monitoring requirements. The 
Agency would use the large system data to define the relationship 
between the pathogen concentrations in the source water and the 
concentrations of potential/existing microbial indicators of water 
quality. If such a relationship were found, then small systems could 
use one or more of these easily-measured indicators to estimate 
pathogen concentrations in their source waters.
    In addition, small systems that use the same source water and are 
in the same vicinity as a large system may be able to use the same 
pathogen concentrations measured by the large system as a basis for 
determining the minimum level of treatment required. Finally, EPA may 
be able to use these data to develop national occurrence patterns that 
would allow the Agency to establish more appropriate treatment criteria 
for small systems. By characterizing source water quality using any one 
or a combination of these three approaches, a small system could 
evaluate the effectiveness of treatment in place for pathogen control 
and determine the need for additional treatment steps.
    The Agency requests suggestions for assessing pathogen exposure in 
small systems in addition to the three approaches provided above. 
Following the full compilation of data under the ICR and other research 
developments, EPA is considering proposing a long-term ESWTR that would 
include criteria by which systems serving less than 10,000 people could 
determine appropriate levels of treatment for different source water 
qualities.
    As stated above, under this proposed ICR, systems using surface 
water and serving more than 100,000 people would be required to monitor 
their finished water for the entire set of pathogens and indicators if 
any pathogen density in the source water were to exceed one/liter. 
Since pathogen occurrence in a particular source water can vary by 
several orders of magnitude, a pathogen density of slightly greater 
than one/liter during one month might be followed by considerably 
greater densities in subsequent months. Requiring a system to monitor 
its raw and filtered water concurrently in the months following a 
source water pathogen concentration of greater than one/liter would be 
more likely to result in pathogen detection in the filtered water 
compared to a situation where source water pathogen densities are less 
than one/liter. EPA believes that, at Giardia occurrence levels above 
one/liter or virus occurrence levels above 10/liter, a 3-log Giardia 
reduction or 4-log virus reduction, depending upon the efficacy of 
treatment, should still be countable in the treated water. At a density 
less than one/liter in source water, the sample volume needed to detect 
pathogens in treated waters would be unreasonably high and technically 
difficult to achieve.
    To avoid virus monitoring that is likely to be uninformative 
because of exceptionally good source water quality, EPA would allow two 
circumstances under which a system that serves more than 100,000 people 
could forgo all or part of the virus monitoring requirement. In one 
case, a system that does not detect any viruses during the first twelve 
months of monitoring would not be required to monitor viruses during 
the last six months of monitoring. In the other case, if a system has 
monitored for total coliforms or fecal coliforms in the source water 
for at least five days/week every week for six months before the 
effective date of this rule, and 90 percent of all samples are no 
greater than 100 total coliform/100 ml or 20 fecal coliforms/100 ml, 
the system may forgo the virus monitoring requirement, per approval by 
EPA upon submission of this data. EPA believes that systems that do not 
detect viruses during a full year of monitoring, or where the densities 
of total coliforms or fecal coliforms do not exceed the values 
specified in the SWTR above which a system is required to filter, could 
assume that treatment that removes/inactivates Giardia satisfactorily 
would also reduce viruses to a safe level.
    One issue raised during rule development is whether a system could 
submit previously collected monitoring data in lieu of part or all of 
the data required by this rulemaking. EPA believes such data would be 
useful only if (1) the laboratory used the same analytical methods 
approved under this rulemaking, (2) the Agency has some assurance that 
the laboratory used adequate quality assurance procedures in analyzing 
the samples, (3) the system provides all data, rather than selected 
data, and that these data include seasonal information, and (4) the 
laboratory analyzed the full set of pathogens and bacterial indicators 
required by this rule so that microbial interrelationships can be 
evaluated. The Agency solicits comment on whether to allow systems to 
submit previously collected data in lieu of the requirements of this 
rulemaking and, if so, the appropriateness of the criteria outlined 
above regarding the admissibility of such data.
    Another issue is whether EPA should require systems to submit some 
percentage of their processed microbiological samples to the Agency or 
some other repository for archiving. Such a repository would allow EPA, 
States, systems, and research centers to study the samples in the 
future for any newly identified pathogens or any additional 
relationships. Also, a repository could allow for very efficient 
research since particular samples of interest could be selected from 
the same sites based on previous ICR monitoring results. The previous 
data could, in part, be validated using new analytical methods that 
become available in the future. An examination of archived data may 
allow EPA to require monitoring of an easily measured indicator rather 
than pathogens in any future rulemaking.
    If the Agency determines that archiving is appropriate, based on 
public comments received, EPA would facilitate its implementation by 
making any requirement as simple as possible for systems and 
laboratories. For this purpose, EPA intends to serve as the repository 
for all archived samples under this rule. For Giardia/Cryptosporidium 
samples, systems/laboratories would collect a total volume of at least 
140L and 1400L for raw and treated waters, respectively, and send 
approximately one-fourth of the sample concentrate (\1/4\ of the 
pellet), i.e., about 5 ml of sediment in 5 ml of formalin, to EPA for 
archiving under refrigeration. For viruses, systems/laboratories would 
collect a total volume of at least 200L and 1400L for raw and treated 
waters, respectively, and ship a 100-ml filter eluant (pH neutralized) 
on dry ice to EPA for each sample.
    EPA solicits comment on the feasibility and utility of archiving 
samples.
    EPA also requests comment on the option for requiring systems to 
collect particle size count data within the treatment plant in lieu of, 
or in addition to, finished water monitoring for Giardia and 
Cryptosporidium. The intent of the finished water monitoring is to 
provide data on removal efficiencies throughout the treatment process, 
and applicability of pathogen removal credits for various treatment 
processes. However, because suspended solids in some source waters may 
clog the filters and thus limit the sample volume collected, systems 
may only be able to determine an upper limit for pathogen 
concentration, i.e., less than the detection limit, rather than an 
actual concentration. This problem would preclude a system from 
calculating pathogen reduction efficiencies by treatment. Additionally, 
the analytical method currently specified does not clearly 
differentiate between live or dead cysts/oocysts of Giardia and 
Cryptosporidium. Potential public misunderstandings of cysts/oocysts 
detected in plant effluent is another reason to allow particle count 
data.
    Removal efficiencies indicated by particle count data may 
approximate removal efficiencies of Giardia cysts and Cryptosporidium 
oocysts. Particle size counting may be used as a tool for evaluating 
removal efficiencies of physical removal processes. Ongoing research 
may provide enough information to establish a quantitative relationship 
between reductions by treatment of particle counts of specific size and 
reductions of Giardia cysts and Cryptosporidium oocysts. Due to 
recovery problems of Giardia and Cryptosporidium by the methodology and 
the inability to quantitate removal efficiencies in many waters, the 
use of particle counts in the same or smaller size range as Giardia and 
Cryptosporidium may be a better method for the evaluation of removal 
efficiencies by treatment.
    The intent of the option for allowing particle size measurements in 
lieu of finished water monitoring for Giardia and Cryptosporidium is to 
obtain data on the use of particle count data as a surrogate for 
Giardia and Cryptosporidium removal. Under this option particle counts 
would be taken on the plant influent, settled water, filter effluent, 
and plant effluent. The particle count data would be taken on the same 
day as the plant influent data for Giardia and Cryptosporidium.
    The particle count data would be recorded on a form similar to that 
shown in Appendix A of this preamble. The data would be recorded as 
particle size counts for each treatment step between the plant influent 
and effluent. By requiring particle size counts in increments of 
``greater than'' values for some specified volume of flow, removal 
efficiency for a specified particle size range (e.g., 5-10 m), 
could be calculated for a particular treatment process. This would be 
done by subtracting the count in the higher size range (e.g., >10 
m) from the count in the lower size range (e.g., >5 
m) for the effluent of one treatment process (or the raw 
water) and comparing this value, ``a'', to a similarly calculated 
value, ``b'', for a subsequent treatment process (i.e., [``a'' - 
``b'']/``a''  x  100). Removal efficiencies calculated based upon 
particle size counts in the ranges of 2-5 m and 5-10 
m, as indicated in Appendix A of this preamble, may be 
conservative indicators for estimating the removal efficiency of 
Giardia or Cryptosporidium which are generally in the respective size 
ranges of 3-7 microns and 8-12 microns, respectively.
    EPA solicits comment on the following issues pertaining to 
monitoring of particle size counts: Under what circumstances, if any, 
should monitoring of particle size counts be allowed in lieu of 
monitoring finished water for Giardia and Cryptosporidium? What 
particle size ranges and sample volumes should be monitored? What 
analytical method(s), including instrumentation, should be used for 
such monitoring? What criteria should be specified to ensure that 
particle size data collected from different systems could be 
appropriately compared? What criteria should be specified to ensure 
that the particle size measurements would be most representative of 
removal of Giardia and Cryptosporidium? Should methods in addition to, 
or in lieu of, particle size counting, such as Microscopic Particulate 
Analysis (MPA), be included as a condition for avoiding finished water 
monitoring of Giardia and Cryptosporidium?
3. Reasons for Monitoring Listed Pathogens and Indicators
    EPA would require monitoring of Giardia concentrations because this 
pathogen causes more reported waterborne disease outbreaks than any 
other single known pathogen and is more resistant to environmental 
stresses and disinfection than almost all other known waterborne 
pathogens. The Agency would require monitoring of Cryptosporidium 
because this pathogen has caused major waterborne disease outbreaks in 
the United States, England, and elsewhere, and is even more resistant 
to disinfection than is Giardia. Cryptosporidium may also not be as 
readily removed by filtration as Giardia, given its smaller size.
    A number of enteric viruses have caused waterborne disease and they 
may be responsible for many, if not most, of the outbreaks where a 
causative agent was not specifically identified (about half of all 
reported outbreaks). EPA believes, however, that it would be 
prohibitively expensive to monitor for all of them, using current 
technology. Moreover, adequate analytical methodology is not yet 
available for routine analysis for many of them. For this reason, the 
Agency would require systems to monitor total culturable viruses (as 
determined by BGM (Buffalo Green Monkey) tissue cultures), a group of 
enteric viruses that are commonly found in fecally polluted waters and 
which EPA believes are at least somewhat representative of other 
pathogenic enteric viruses. Total culturable viruses contain some 
strains that are capable of causing waterborne disease, have been 
widely studied for many years, and analytical methods are far better 
defined for them than is the case for many specific enteric viruses. 
EPA believes that monitoring for total culturable viruses is useful 
both because this group of viruses contains pathogens and is a 
potential indicator for other viral pathogens.
    Some individuals believe that systems which satisfactorily control 
for Giardia cysts will adequately control for pathogenic viruses, since 
viruses generally are much less resistant to disinfection than are 
Giardia cysts, and thus virus monitoring is not warranted under this 
rulemaking. They point out that, based on the Guidance Manual to the 
Surface Water Treatment Requirements (EPA, 1991), the disinfection CT 
values (disinfection concentration in mg/l x disinfection contact time 
in minutes) for achieving the SWTR compliance level inactivation of 
viruses, which is based on hepatitis A inactivation data, is about one 
to two orders of magnitude below that for achieving the SWTR compliance 
level of inactivation of Giardia.
    EPA, however, does not believe that sufficient data are yet 
available to conclude that the Giardia density in source waters is an 
adequate gauge to define the necessary treatment for viruses in all 
types of source waters. The Agency is not aware of data on relative 
densities between Giardia and viruses in source water. If the virus 
concentration in some source waters greatly exceeds that of Giardia, 
and some pathogenic viruses are significantly more resistant to 
disinfection than is hepatitis A, an adequate treatment for Giardia may 
not result in adequate control of viruses. Moreover, the Agency notes 
that viruses have often been detected in fully treated waters (i.e., 
coagulation, sedimentation, filtration, and disinfection) (Gerba and 
Rose, 1990; Payment et al., 1985; Hurst, 1991), and it is not aware of 
any data demonstrating that viruses in raw water or treated water are 
usually or always accompanied by Giardia cysts. The Agency also notes 
that the CT values for viruses in the Guidance Manual to the SWTR (EPA, 
1991) were based upon laboratory studies on free (i.e., non-aggregated) 
viruses; in environmental waters, viruses are usually aggregated or 
associated with cell debris, some of which may not be removed entirely 
by filtration processes. Such cell-associated aggregates are 
considerably more resistant to disinfection than free viruses 
(Williams, 1985; Sobsey et al., 1991). Moreover, some pathogenic 
enteric viruses may be substantially more resistant to disinfection 
than others (Keswick et al., 1985).
    Because of these uncertainties, it may not be appropriate to assume 
that by controlling Giardia densities, systems will adequately control 
viral pathogens. EPA needs monitoring data from many systems nationwide 
to determine the level of treatment needed to control viruses. 
Specifically, the Agency needs to determine the extent to which Giardia 
are present in source waters when viruses are present. The Agency also 
needs to determine what minimum level of disinfection inactivation is 
necessary for surface water supplies to ensure adequate virus control, 
regardless of Giardia densities. These data will allow the Agency to 
determine whether a system that consistently provides an overall 
Giardia reduction of 3-logs (of which at least 0.5-log is due to 
disinfection alone) or any greater reduction level for Giardia, will 
also consistently provide an adequate control for viruses, especially 
in cases where virus densities in source waters are much higher than 
those for Giardia. Information collected under this rule would provide 
part of these data. The Agency believes that these data, along with a 
more intensive voluntary monitoring effort among a small number of 
systems, should clarify this situation sufficiently to allow it to 
develop suitable revisions to the SWTR.
    With regard to bacterial pathogens, EPA believes that pathogenic 
protozoa and many waterborne viruses are more resistant to 
environmental stress and disinfection than most enteric bacteria that 
cause waterborne disease. Thus a system that protects the public from 
pathogenic protozoa and viruses will concurrently protect them from 
most pathogenic bacteria (except possibly for those bacteria that can 
proliferate within the distribution system or which have special 
protective factors). For this reason, EPA would not require these 
systems to monitor pathogenic bacteria in the source water or in 
treated water.
    While EPA would not require systems to monitor pathogenic bacteria, 
the Agency would require them to monitor potential bacterial indicators 
for waterborne pathogens in source water and treated water. Under this 
rule, EPA is proposing to require systems to monitor for total 
coliforms and either fecal coliforms or E. coli. Total coliforms and 
fecal coliforms have been used widely for decades to assess source 
water quality, testing for these two groups of bacteria is very simple 
and inexpensive, and systems are familiar with these tests. Total 
coliforms are usually much more numerous in water than fecal coliforms, 
and therefore enumeration in source waters and treated water is more 
sensitive than with fecal coliforms. However, fecal coliforms are a 
better indicator of fresh fecal contamination than are total coliforms. 
Because the bacterium E. coli is more closely related to fresh fecal 
pollution and to gastrointestinal illness among bathers than are fecal 
coliforms, EPA would allow a system to analyze for E. coli in lieu of 
fecal coliforms.
    EPA solicits comment on the requirement to monitor the specific 
pathogens and bacterial indicators mentioned above. The Agency 
specifically seeks comment on whether to require systems to monitor 
both fecal coliforms and E. coli, rather than one or the other. In 
addition, the Agency may include a requirement to monitor for two other 
potential indicators--Clostridium perfringens (C. perfringens) and 
coliphage which are discussed below.
    Clostridium perfringens. C. perfringens is a bacterium that is 
common in the intestinal tract of warm-blooded animals. This organism 
forms an endospore in the environment that is extremely resistant to 
environmental stresses and disinfection. Of the more than 60 species of 
Clostridium, C. perfringens is the one most consistently associated 
with human fecal wastes (Cabelli, 1977). It is consistently present in 
human feces at a relatively high density (Bisson and Cabelli, 1980) and 
appears to be excreted in greater numbers than are fecal pathogens 
(NATO, 1984). There is controversy over whether other important animal 
hosts exist, since C. perfringens spores are widely found in 
terrestrial and aquatic environments (Cabelli, 1977). The survivability 
of C. perfringens spores in water and their resistance to treatment 
compared to the pathogens is much greater than other indicators (Bonde, 
1977), except possibly for Giardia and Cryptosporidium. Analysis is 
relatively easy and inexpensive. The European Community has a 
supplementary standard for the endospores of sulfite-reducing 
Clostridium for drinking waters.
    Recently, Payment and Franco (1993) published a paper that showed 
that C. perfringens may be a suitable indicator for viral and protozoan 
pathogens in both raw water and filtered water. In this study, the 
investigators collected large-volume samples from three water treatment 
plants and analyzed them for Giardia cysts, Cryptosporidium oocysts, 
cultivable human enteric viruses, Clostridium, and somatic and male-
specific coliphage. They found that Clostridium densities were 
significantly correlated with the densities of viruses, cysts, and 
oocysts in river water and with viruses and oocysts (but not Giardia 
cysts) in filtered water.
    For the above reasons, EPA is considering a requirement that 
systems monitor their source and filtered water for C. perfringens at 
the same frequency as is being proposed for the other organisms. C. 
perfringens may be appropriate as a low cost monitoring indicator for 
estimating pathogen densities in the source water and/or for defining 
treatment effectiveness. If feasible, such an indicator could greatly 
reduce monitoring costs for determining appropriate levels of treatment 
to address microbial concerns. This would be of special benefit for 
smaller systems under the long-term ESWTR. EPA solicits comment on this 
issue.
    Coliphage. The Agency also seeks comment on the utility of 
coliphage as an indicator of pathogen presence. Coliphages, which are 
viruses that infect the bacterium E. coli, are far simpler to analyze 
than other viruses and are, like E. coli, generally associated with 
fecal contamination. They have often been discussed as a possible 
indicator of treatment effectiveness for enteric viruses. Coliphages 
are commonly categorized into two groups: the somatic phage and the 
male-specific (or F-specific) phage. The somatic phage gain entry into 
E. coli cells via the cell wall, while the male-specific phage gain 
entry only through the sex-pili of those E. coli cells that have them 
(referred to as male cells).
    Because coliphages are so much simpler to analyze than human 
viruses, EPA wants to determine whether systems can use coliphages to 
indicate the presence of the human viruses in source waters and 
filtered water. Data on relative densities in natural waters are 
sparse. Somatic phages are common in the feces of humans and other 
animals but, unlike human viruses, some of them apparently can multiply 
in natural water, probably in species other than E. coli. Male-specific 
phages are not common in humans and other animals, but are common in 
sewage, suggesting they can multiply in the sewerage system (IAWPRC, 
1991). Data on the relative resistance and removal of coliphages and 
human viruses during the water treatment process is also scarce, and 
the data which exist are inconsistent, especially for the somatic 
phages (IAWPRC, 1991). Some of the male-specific phages (e.g., MS2), 
however, appear to be more resistant to chemical disinfection than most 
waterborne pathogens (Sobsey, 1989).
    One recent study suggests that coliphages are suitable as an 
indicator for viruses, at least in filtered water. In the Payment and 
Franco (1993) study indicated above, the densities of somatic 
coliphages (E. coli CN13 host) were statistically correlated with human 
enteric viruses and Cryptosporidium oocysts (but not Giardia cysts) in 
filtered water, and not in river water. Male-specific coliphages 
(Salmonella typhimurium WG49 host) were correlated with human enteric 
viruses in filtered water, but not in river water. The male-specific 
coliphages were also correlated with Giardia cysts, but not 
Cryptosporidium oocysts, in river water.
    In another study, Havelaar et al. (1993) compared the 
concentrations of culturable viruses (BGM cell line) with those of 
thermotolerant coliforms, fecal streptococci, and male-specific RNA 
phages (Salmonella typhimurium WG49 host) for a variety of water types. 
The investigators found that the male- specific phages were 
significantly correlated (significant at P <1%) with culturable virus 
concentrations in river water, coagulated river water, and lake water, 
but not for raw and biologically treated sewage. They conclude that 
male-specific phages may be a suitable indicator for enteric viruses in 
fresh waters.
    If data suggest that one or both groups of coliphages are adequate 
as an indicator of pathogen presence for source waters and/or treatment 
effectiveness, EPA may, in the long-term ESWTR, require systems, 
especially those serving populations fewer than 10,000, to monitor 
these organisms as one basis for determining what level of treatment is 
needed to safeguard the drinking water. The Agency solicits comment on 
this issue.
4. Rationale for Frequency of Microbial Monitoring
    The rule would require systems serving more than 100,000 people to 
monitor monthly for a consecutive period of 18 months, and for systems 
serving between 10,000-100,000 people to monitor every two months for a 
consecutive 12 month period, between [insert month beginning three 
months following promulgation date] and March 1997. Moreover, unlike 
larger systems, systems serving between 10,000-100,000 people would not 
be required to monitor treated water.
    The extended interval of time within which the monitoring can occur 
is to allow adequate lab capacity to be developed and approved by EPA. 
EPA encourages that monitoring begin as soon as the system identifies 
an EPA approved lab for conducting the analysis. Criteria that EPA will 
use to approve laboratories for conducting ICR analysis are discussed 
later. Any D/DBP monitoring required under this rule should not 
commence until the microbial monitoring can begin to allow EPA to 
characterize how treatment concurrently affects microbial and DBP 
occurrence.
    The microbial monitoring under this rule would provide EPA with 
over 15,000 data points for each monitored organism in source water 
(about 8,000 data points for viruses) and probably up to 4,000 data 
points for each monitored organism in treated water. EPA believes that 
this amount of data, complemented with additional research, will be 
sufficient for allowing the Agency to accurately assess the pathogen 
exposure and decipher the relationships in source water densities among 
pathogens and between pathogens and their potential indicators. 
Importantly, the data provided by this monitoring schedule would allow 
the Agency to establish a database on pathogen and indicator densities 
and their variations with time, including seasonal variations, and thus 
allow the Agency to revise the SWTR, if appropriate, in a reasonable 
manner.
    Under this rule, all monitoring for microbiological related 
parameters would end no later than March 31, 1997, with a substantial 
portion of this monitoring completed much sooner. EPA expects 
monitoring completed during this period will allow the Agency to a) 
develop the most suitable revisions to the SWTR, if required, and 
promulgate such a rule by December 1996, and b) for individual systems, 
provide sufficient data to establish an appropriate level of treatment 
by June 1998, the effective date of the interim ESWTR that was agreed 
to by the Negotiating Committee (should such a rule become necessary). 
The schedule for such rule development is further described in section 
III.C of this preamble.
5. Rationale for Reporting Physical Data and Engineering Information
    In addition to requiring systems to monitor for specific 
microorganisms, the rule would also require each system to provide 
certain information to EPA about the nature of the source water and 
treatment processes. Systems serving greater than 100,000 or more 
people would be required to submit the data indicated in Table III.6 
(see section III.B.3) using data entry software developed by EPA. This 
information, in conjunction with the microbial occurrence data 
indicated in Appendix A of the rule and DBP occurrence data indicated 
in Tables III.1-III.5 (see section III.B.2), would be used by EPA to 
analyze relationships between source water quality, treatment 
characteristics, and finished water quality as it pertains to both 
pathogens and DBPs. EPA would use the information collected in Table 
III.6 and from other research to predict the ability of systems to 
comply with different ESWTR regulatory options, i.e., achieve different 
levels of pathogen removal and inactivation, either within existing 
design and operation capacity, or with system upgrades.
    The information cited above would assist EPA in evaluating the 
monitoring data and treatment removal efficiencies, thus clarifying 
pathogen exposure levels in finished water entering the distribution 
system under real world conditions. This would allow EPA to develop 
more refined regulations or guidance to limit pathogen exposure. The 
information would also help systems comply with the forthcoming D/DBP 
Rule without undermining pathogen control.
    With regard to treatment processes, EPA would require information 
on the type of disinfectant used and its dosage, contact time, and pH; 
and the type of filter process used and the media size, depth, and 
hydraulic loading rate. This information, along with information on 
pathogen densities in the source water and treated water (including 
particle size count data if this monitoring option is adopted), would 
help the Agency determine the validity of existing treatment efficiency 
assumptions and models for pathogens.
    EPA would also require systems that do not detect Giardia, 
Cryptosporidium, or viruses in a sample to report the sample volume 
used and the organism detection limit. This information would allow EPA 
to determine the maximum theoretical pathogen density in that sample.
    EPA solicits comment on the need to report the listed physical data 
and engineering information, and whether additional reporting 
requirements are warranted.
    Systems serving between 10,000 and 100,000 people would not have 
the extensive DBP occurrence data or finished water microbial data 
required of large systems and, therefore, would only be required to 
submit part of the information in Appendix A of the rule (i.e., raw 
water occurrence information for Giardia, Cryptosporidium, total 
coliforms, and fecal coliforms or E.coli) and treatment data as it 
pertains to microbial concerns (Appendix B of the rule). The purpose of 
the treatment plant information is to enable EPA to predict the 
national impact on systems in this size category for meeting different 
ESWTR regulatory options.
    The Negotiating Committee agreed that all systems of the pertinent 
size categories be required to submit physical and engineering data 
even though this might provide more data than was needed to develop 
national cost estimates. Nevertheless, the Negotiating Committee 
believed the requirement to be appropriate because of the large number 
of systems with diverse characteristics and of the difficulties in 
otherwise equitably funding the collection of a smaller but still large 
and representative data set.
    EPA solicits comment on whether alternative more efficient means 
for obtaining treatment plant information are available for systems 
serving between 10,000 and 100,000 people. For example, is it 
appropriate to only require the treatment plant data from a random 
subset of systems in this size category (e.g., from 200 systems), and 
to extrapolate such data to all the other systems in this size 
category? Would it be appropriate to assume that systems in the size 
category 10,000 to 100,000 have, in general, the same design and 
operating conditions as those in the size category 100,000 and above, 
and therefore could avoid submitting the required treatment plant 
information?
6. Analytical Methods
    General. EPA must approve all analytical methods used in this rule. 
In the present rulemaking, the Agency would require all systems to use 
the same methods for the analysis of Giardia, Cryptosporidium, and 
viruses to facilitate comparisons among the systems.
    Total coliforms, fecal coliforms, and E. coli. Analytical methods 
for monitoring total coliforms and fecal coliforms in source water are 
already approved by the SWTR under Sec. 141.74(a), and would be used 
for monitoring under the present rulemaking. For monitoring E. coli in 
source waters, EPA would approve the following methods, all of which 
have been approved for detecting E. coli in drinking water under the 
Total Coliform Rule (Sec. 141.21(f)):
    (1) EC medium supplemented with 50 g/ml of 4-
methylumbelliferyl-beta-D-glucuronide (MUG), as specified in 
Sec. 141.21(f)(6)(i). In this method, each total coliform-positive 
broth culture from the Multiple Tube Fermentation (MTF) Technique 
(Sec. 141.74(a)(2)) or each total coliform-positive colony from the 
Membrane Filter Technique (Sec. 141.74(a)(2)) is transferred to 10 ml 
of EC + MUG. After incubation, the inoculated medium is examined with 
an ultraviolet light. If fluorescence is observed, the medium contains 
E. coli.
    (2) Nutrient agar supplemented with 100 g/ml of MUG, as 
specified in Sec. 141.21(f)(6)(ii), with the additional requirement 
that E. coli colonies be counted.
    (3) Minimal Medium ONPG-MUG Test, often referred to as the Colilert 
Test, as specified in Sec. 141.74(a)(2), with the additional 
requirement that total coliform-positive tubes be examined with an 
ultraviolet light. If fluorescence is observed, the medium contains E. 
coli.
    Giardia, cryptosporidium, and total culturable viruses. In August 
1993, EPA sponsored a workshop of invited experts in Giardia, 
Cryptosporidium, and virus analysis and quality assurance procedures to 
help the Agency develop standardized methods for these organisms for 
use with the ICR. Workshop participants included representatives from 
academia; water industry; commercial laboratories; and federal, State 
and local governments. As the basis for the discussion, the workshop 
used the Giardia/Cryptosporidium method published by ASTM (1992) and 
the method to be published shortly in the 18th edition Supplement to 
Standard Methods for the Examination of Water and Wastewater. Two virus 
methods in the 18th edition of Standard Methods (Method 9510C for virus 
collection and elution; Method 9510G for virus assay) (APHA, 1992) were 
used. The methods in ASTM (1992) and Standard Methods were used as the 
basis for this discussion because these texts are highly respected and 
widely used references that have been peer-reviewed throughout the 
scientific community. The workshop generally recommended use of the 
methods above, but, because these methods allow many sub-options, 
decided to refine and standardize them to achieve more precise 
comparisons among systems under the ICR (USEPA, 1993a).
    The method for Giardia/Cryptosporidium, as revised, is in Appendix 
C of the proposed rule. This method includes sample collection, 
purification, and microscopic assay, and allows the density of Giardia 
and Cryptosporidium to be determined simultaneously on the same sample. 
The microscopic assay includes the use of epifluorescence along with 
differential-interference- (or Hoffman Modulation) contrast optics to 
identify morphological characteristics.
    One issue with regard to the Giardia/Cryptosporidium method 
concerns how to express the results. The total number of cysts and 
oocysts are counted, based on immunofluorescence, size, shape, and 
presence of internal structures. Then the total number of cysts with 
internal structures is tallied. The issue is what terminology to use 
for these two steps. One procedure is to categorize the first step as a 
``presumptive'' test and the second step as the ``confirmed'' test. The 
terminology ``confirmed'' could be used if at least two internal 
structures are identified as being Giardia/Cryptosporidium cysts/
oocysts. The second procedure is to categorize the first step as the 
``total number of cysts and/or oocysts per 100L'' (which would be 
equivalent to ``presumptive'') and the second step as the ``total 
number of cysts and/or oocysts with internal structures.'' The 
terminology ``with internal structures'' could be used if at least one 
internal structure is identified as being Giardia/Cryptosporidium 
cysts/oocysts.
    The rationale for considering the two steps as presumptive and 
confirmed is: (1) Some algal and yeast cells recovered with this 
procedure cross-react with the protozoan monoclonal antibodies used, 
(2) many algae and other particles autofluoresce and thereby confuse 
the analyst, and (3) depending upon the criteria that will be used for 
defining level of treatment requirements in the interim ESWTR, use of 
the terminology ``confirmed'' may reduce the number of false positives 
and thereby not lead to excessive levels of treatment to achieve the 
desired health risk goal. However, the use of these terms is somewhat 
inaccurate in that it diminishes the importance of the total count 
(i.e., the presumptive test). The confirmed test only reflects those 
particles where internal structures can be specifically observed, which 
may represent only a small fraction of the cysts/oocysts on the slide.
    EPA requests comment on which terminology is most suitable for 
referring to the two steps.
    Other methods for the assay of Giardia and Cryptosporidium are 
currently being developed. One of these assays (the electrotation 
assay) is based on the observation that particles in a rotating 
electric field also rotate if the frequency is right. In addition to 
this assay, other potential assays for the protozoa include polymerase 
chain reaction and flow cytometry. The Agency requests comment about 
the most appropriate means for incorporating new and easier analytical 
methods for Giardia and Cryptosporidium into the ICR.
    The method for viruses, as revised, is in Appendix D of the 
proposed rule. This method relies on a most probable number technique 
using BGM tissue culture monolayers, with cytopathic effect (CPE) as 
the sole enumeration endpoint. Attendees at the workshop considered 
plaque-forming units (PFU) as an endpoint, but rejected it. Although 
the PFU endpoint can be determined without the use of a microscope, 
unlike the CPE endpoint, it may not be as sensitive as CPE, i.e., use 
of CPE should result in greater virus densities. The workshop members 
determined that sensitivity was more important than precision in 
quantitation for comparing virus and protozoan data to determine the 
appropriateness of using Giardia and possibly Cryptosporidium as the 
primary target organism(s) for defining adequacy of treatment.
    Clostridium perfringens. If EPA decides to require systems to 
monitor Clostridium perfringens, as was discussed in Section IIIA3 
above, the Agency would also specify a method for this bacterium. The 
Agency believes that the most appropriate method is a membrane filter 
procedure using M-CP medium (Bisson and Cabelli, 1979), possibly as 
modified by Armon and Payment (1988). The Agency solicits comment on 
whether this method is most suitable for monitoring Clostridium 
perfringens. The Agency notes that this organism must be grown under 
strict anaerobic conditions (i.e., without oxygen).
    Coliphage. If EPA decides to require the monitoring of somatic 
coliphages and/or male-specific coliphage, as was discussed in Section 
IIIA3, the Agency believes that the most appropriate method is a simple 
agar overlay procedure. For somatic phage testing, the Agency believes 
that the most suitable host is E. coli C. The Agency solicits comment 
on whether this procedure and host are most suitable for monitoring the 
somatic coliphage. The Agency also seeks comment, with data, on what 
bacterial host is most suitable for monitoring male-specific 
coliphages. The method for sample collection, sample processing, and 
assay for somatic and male-specific coliphage is presented in Appendix 
D of the proposed rule.
    EPA requests comment on the appropriateness of the above methods.
7. Laboratory Approval
    General. EPA is developing a program for approving laboratories to 
analyze the pathogens that would be monitored under this rule. This 
program would ensure that these laboratories are competent to perform 
the analyses. Analytical skill is especially important for the 
difficult and sophisticated processing and analyses specified for the 
total culturable viruses and Giardia and Cryptosporidium. Another 
prominent reason for approving laboratories is to ensure that 
laboratory procedures are as standardized as possible for uniform data 
comparison among systems.
    Currently, EPA has a laboratory certification program for drinking 
water analyses. All laboratories that analyze drinking water samples to 
determine compliance with MCLs must be certified by EPA or the State, 
as specified by 40 CFR 142.10(b)(4) and 141.28. Under this program, EPA 
certifies the principal State laboratory and, with certain exceptions 
(see 40 CFR 142.10), each State certifies all drinking water 
laboratories within the State. Laboratories certified to perform 
analysis for coliforms under the Total Coliform Rule would be approved 
to analyze for total coliforms, fecal coliforms, and E. coli under the 
ICR without further action. The current program does not address 
pathogens.
    Rather than broaden the present laboratory certification program to 
include Giardia, Cryptosporidium, and the viruses, EPA believes that it 
would be more appropriate to develop a separate program and to 
differentiate the two programs by using the term laboratory 
``approval'' instead of ``certification'' to refer to laboratories 
performing pathogen analyses required by the ICR. The rationale for 
this approach is that (1) EPA expects that only a small number of 
laboratories will be qualified to perform analyses for the protozoa and 
viruses because of the complexity of the methods, (2) few States and 
EPA Regions are currently able to certify laboratories for the 
pathogens of interest, and (3) the short time constraints for 
implementing this rule and the short-term nature of the sampling (up to 
18 months) do not provide time for a full certification program.
    Nevertheless, EPA is proposing to use several major elements of the 
current certification program in its program to ``approve'' 
laboratories for pathogen analysis, including performance evaluation 
(PE) samples, training, and on-site evaluations. If an interim or long-
term ESWTR were to require some systems to monitor the same pathogens 
as those specified by the ICR, then the laboratory approval criteria 
would probably be incorporated into the drinking water laboratory 
certification program.
    Performance evaluation samples. Under the laboratory approval 
program proposed herein, a laboratory would need to analyze 
satisfactorily a set of PE samples to become approved and subsequent 
sets of PE samples (e.g., 6, 12, 18 months) to maintain approval. 
Workshop participants recommended that a set of PE samples for Giardia/
Cryptosporidium consist of (1) a mixture of Giardia cysts and 
Cryptosporidium oocysts, (2) a mixture of Giardia cysts and 
Cryptosporidium oocysts plus algal cells, and (3) algal cells only 
(negative control). According to workshop recommendations, a set of PE 
samples for viruses should include virus samples of varying titers 
(concentrations) that the laboratory would process as if they were 
filter eluates. Currently, EPA is developing a PE sample program 
intended to satisfy these recommendations.
    Training. In addition to PE samples, at least one principal analyst 
in each laboratory would need to complete an EPA-specified training 
course or meet the requirements of equivalent training, as defined by 
the Agency. Although EPA has not yet defined ``equivalent training'', 
the Agency is considering an approach involving a training video or an 
apprenticeship with an expert. EPA is developing two training courses--
one in Giardia/Cryptosporidium analysis, and the other in environmental 
virus analysis. Each of these courses would also include training in 
sample collection.
    On-site evaluation. EPA is also proposing to require a laboratory 
to pass an on-site evaluation before receiving approval. The EPA 
Regional Administrator would be the ultimate approval authority. The 
Agency would develop criteria for determining whether an individual has 
the necessary expertise to conduct the intended tests.
    The Agency has drafted a laboratory approval manual that lists the 
specific criteria that an on-site evaluator would examine. These 
criteria are based on workshop recommendations. This manual, which is 
available in the Water Docket, includes a number of certification 
criteria from Chapters III and V of EPA's laboratory certification 
manual (USEPA, 1990). For example, as part of the on-site evaluation, 
the certification officer would ensure that the laboratory has prepared 
and is using a written laboratory Quality Assurance Plan. This plan is 
described in EPA's laboratory certification manual (Chapter III). Some 
draft criteria pertaining to the qualifications of laboratory personnel 
are indicated below.
    For Giardia and Cryptosporidium analysis:
      Technician: This person performs at the bench level and 
is actively involved in collecting samples, extracting filters, and/or 
processing the filter eluent for Giardia/Cryptosporidium analysis. The 
technician must have two years of college (full time) in life sciences 
or a related field.
      Analyst: This person must have two years of college (full 
time) in the life sciences or a related field and have at least three 
months experience in examining indirect fluorescent antibody stains 
under the microscope.
      Principal Analyst/Supervisor: This person is a qualified, 
experienced microbiologist with a minimum of a B.A./B.S. degree in 
microbiology or a closely related field. The principal analyst must 
have completed the ICR protozoan training course (mentioned above) or 
have equivalent experience, as approved by EPA.
    For virus analysis:
      Technician: This person extracts the filter and processes 
the sample, but does not perform tissue culture work. The technician 
must have at least three months experience in filter extraction of 
virus samples and sample processing.
      Analyst: This person performs at the bench level and is 
involved in all aspects of the analysis, including sample collection, 
filter extraction, sample processing, and assay. The analyst must have 
two years of college (full time) in the life sciences or at least six 
months of bench experience in cell culturing and animal virus analyses.
      Principal Analyst/Supervisor: This person is a qualified, 
experienced microbiologist who oversees the entire analysis. The 
individual must have a B.A./B.S. degree in the life sciences with three 
years experience in cell culture and animal virus analyses. This 
individual must have completed the ICR environmental virology training 
course or have equivalent experience, as approved by EPA.
    Because of the tight time constraints and the limited number of 
national experts capable of participating in on-site evaluations, EPA 
proposes to give highest priority in evaluating those laboratories 
(e.g., commercial, academic, utility, State) that (1) have been 
analyzing Giardia and Cryptosporidium or virus samples for at least one 
year, (2) have nationally recognized experts in protozoan or virus 
analyses, or (3) have the technical capability, capacity, and 
willingness to analyze at least four samples/month under the ICR 
requirements for Giardia and Cryptosporidium or viruses.
    Laboratory capacity. If, following the beginning effective date of 
this rule, a system cannot locate an approved laboratory to analyze its 
water samples for the indicated pathogens, the system would be required 
to notify EPA in writing (see Section III.C). EPA will inform the 
system which laboratories are available for performing the requisite 
analysis, or when new approved laboratories become available to do such 
analysis.
    EPA solicits comment on the approach above for approving 
laboratories and, more broadly, on the most appropriate means for 
ensuring that laboratories performing the pathogen analyses are 
competent. Laboratories wishing to become approved for doing these 
analyses should contact ICR Laboratory Coordinator, USEPA, Office of 
Ground Water and Drinking Water, Technical Support Division, 26 West 
Martin Luther King Drive, Cincinnati, Ohio 45268, for an application 
form to initiate the approval process.
8. Quality Assurance
    Sample collection. For the collection of samples for pathogens, the 
laboratory would document that each sample collector, either from the 
laboratory or the system, is properly trained. Without such 
documentation, the laboratory would not proceed with analyzing the 
system's samples. EPA encourages approved laboratories to provide 
adequate training, if needed, not only to laboratory sample collectors, 
but to individuals at client water systems who collect their own 
samples for pathogens. Other criteria for sampling are included in the 
draft laboratory approval manual mentioned in Section 7, above.
    Data reporting. EPA proposes to require a laboratory to submit data 
results to both the Agency and the client system for the pathogens. The 
water system would also be required to submit the same data results to 
the Agency. By receiving and comparing both data submissions, EPA can 
reduce reporting errors. EPA would require that systems report data in 
a computer-readable form; in addition, systems serving at least 100,000 
people would be required to report data in an EPA-specified electronic 
format (see Section III.B6 for more discussion). EPA encourages systems 
serving 10-100,000 people to also submit data using the electronic 
format.
    EPA also proposes to require a laboratory, when the laboratory 
submits pathogen data to the Agency, to include its results on the most 
recent set of PE samples for that pathogen. This quality assurance 
criterion would allow EPA to assess the quality of that data, 
especially if the data appear to be atypical or equivocal.

B. Stage 2 Disinfection By-Products Rule

1. Need for Additional Data
    When drinking water is disinfected, the organic material and 
bromide that are naturally present in the water react with the 
disinfectant to form hundreds of DBPs. Only a small subset of these 
chemicals have been identified due to the complexities of measuring 
them. Many of them are not stable, so they decompose during the 
sampling or analytical process. Others are polar and so are not easily 
extracted from the water for further analysis.
    Most of the DBPs that can be measured in drinking water (i.e., 
there are analytical techniques available to detect them) are 
byproducts from the use of chlorine. However, there is limited 
occurrence information on even these DBPs, so the extent of exposure 
cannot be estimated. Only a subset of them have been studied to 
determine whether exposure to them presents a risk to health.
    Several DBPs were included on the 1991 Drinking Water Priority List 
(56 FR 1470; January 14, 1991), as candidates for future regulations. 
During development of the proposed Stage 1 D/DBP Rule, the Negotiating 
Committee did not believe there were adequate data available to address 
most of the DBPs on the Priority List, so MCLs were recommended for a 
subset of the Priority List DBPs (trihalomethanes [THMs], haloacetic 
acids [HAAs], chlorite and bromate). The Stage 1 D/DBP Rule would 
address the ``other'' DBPs in two ways: 1) EPA would assume that 
control of other Priority List DBPs would occur if systems could meet 
the MCLs for THMs and HAAs; and 2) EPA would require some surface water 
systems using conventional treatment to implement optimized coagulation 
to remove as much organic material as possible before disinfection, 
thereby minimizing the formation of all DBPs. Total organic carbon 
(TOC) was designated as the surrogate for the organic precursor 
material removed during optimized coagulation.
    Many members of the Negotiating Committee expressed concern on the 
adequacy of data to support the use of surrogate limits such as TOC for 
inclusion in the Stage 1 regulatory criteria. The lack of field data 
led the Negotiating Committee to base its decisions on the Stage 1 D/
DBP Rule using a water treatment plant model to predict DBP 
concentrations resulting from various changes in treatment practices.
    The THM and HAA compliance monitoring requirements being considered 
for proposal in the Stage 1 D/DBP Rule were modeled after the 
requirements of the current Total Trihalomethane (TTHM) Rule (44 FR 
68624, November 1979). Some members of the Negotiating Committee were 
concerned that quarterly monitoring for THMs and HAAs would not 
accurately reflect consumer exposure to DBPs. An under-prediction of 
consumer exposure would be especially serious if research indicated 
there were short-term adverse health effects from exposure to DBPs. 
Field data were not available to assess the spatial and seasonal 
variability of DBP concentrations within distribution systems. Data 
were also lacking concerning the usefulness of surrogates, such as 
total organic halide (TOX), as tools for reducing compliance monitoring 
costs.
    As a result of the above uncertainties, the Negotiating Committee 
strongly recommended that additional information be collected and 
analyzed to assist in the development of a Stage 2 D/DBP Rule. Field 
data are needed to: (1) Characterize source water parameters that 
influence DBP formation, (2) determine the concentrations of DBPs in 
drinking water, (3) refine models for predicting DBP formation based on 
treatment and water quality parameters, and (4) establish cost-
effective monitoring requirements that are protective of the public 
health. Today's proposed rule would provide EPA with the data necessary 
to accomplish the above tasks.
2. Monitoring and Reporting Requirements and Rationale
    The rule would require all community and nontransient, noncommunity 
systems serving at least 100,000 persons to: (1) Perform the monitoring 
summarized in Table III.1-.2 and (2) report treatment plant operational 
data specified in Table III.6. Treatment plants that use alternate 
disinfectants (chloramines, ozone, or chlorine dioxide) or hypochlorite 
solutions would also be required to perform monitoring for DBPs that 
are of particular concern for the disinfectant being used. Community 
and nontransient, noncommunity systems that use groundwater not under 
the direct influence of surface water and serve between 50,000 and 
99,999 persons would be required to conduct monthly monitoring for 
total organic carbon (TOC) in water entering the distribution system. 

             Table III.1.--Sampling Points for All Systems              
------------------------------------------------------------------------
      Sampling point                Analyses\1\             Frequency   
------------------------------------------------------------------------
Treatment plant influent..  pH, alkalinity, turbidity,  Monthly.        
                             temperature, calcium and                   
                             total hardness, TOC,                       
                             UV254, bromide, and                        
                             ammonia.                                   
Treatment plant influent    Optional oxidant demand     Monthly.        
 (optional for waters with   test.                                      
 high oxidant demand due                                                
 to the presence of                                                     
 inorganics).                                                           
Treatment plant influent..  TOX.......................  Quarterly.      
After air stripping.......  Ammonia...................  Monthly.        
Before and after            pH, alkalinity, turbidity,  Monthly.        
 filtration.                 temperature, calcium and                   
                             total hardness, TOC, and                   
                             UV254.                                     
At each point of            pH, alkalinity, turbidity,  Monthly.        
 disinfection\2\.            temperature, calcium and                   
                             total hardness, TOC, and                   
                             UV254.                                     
At end of each process in   Disinfectant residual\3\..  Monthly.        
 which chlorine is applied.                                             
After filtration (if        THMs, HAAs(6), HANs, CP,    Quarterly.      
 chlorine is applied prior   HK, CH, and TOX.                           
 to filtration).                                                        
Entry point to              pH, alkalinity, turbidity,  Monthly.        
 distribution system.        temperature, calcium and                   
                             total hardness, TOC,                       
                             UV254, and disinfectant                    
                             residual\3\.                               
Entry point to              THMs, HAAs(6), HANs, CP,    Quarterly.      
 distribution system.        HK, CH, TOX, and SDS\4\.                   
4 THM Compliance            THMs, HAAs (6), HANs, CP,   Quarterly.      
 Monitoring Points in        HK, CH, TOX, pH,                           
 Distribution System (1      Temperature, Alkalinity,                   
 sample point will be        Total Hardness and                         
 chosen to correspond to     Disinfectant Residual\3\.                  
 the SDS sample,\4\ 1 will                                              
 be chosen at a maximum                                                 
 detention time, and the                                                
 remaining 2 will be                                                    
 representative of the                                                  
 distribution system).                                                  
------------------------------------------------------------------------
\1\TOC is total organic carbon. UV254 is absorbance of ultraviolet light
  at 254 nanometers. THMs are chloroform, bromodichloromethane,         
  dibromochloromethane, and bromoform. HAAs(6) is mono-, di-, and       
  trichloroacetic acid; mono- and di- bromoacetic acid; and             
  bromochloroacetic acid. HANs are dichloro-, trichloro-, bromochloro-, 
  and dibromo- acetonitrile. CP is chloropicrin. HK is 1,1-             
  dichloropropanone and 1,1,1- trichloropropanone. CH is chloral        
  hydrate. TOX is total organic halide. SDS is the simulated            
  distribution system test.                                             
\2\For utilities using ozone or chlorine dioxide, Tables III.4 and      
  III.5, respectively, show additional monitoring requirements at this  
  sampling point.                                                       
\3\Free chlorine residual will be measured in systems using free        
  chlorine as the residual disinfectant; total chlorine residual will be
  measured in systems using chloramines as the residual disinfectant.   
\4\The SDS (simulated distribution system test) sample will be stored in
  such a manner that it can be compared to the results from one of the  
  distribution system sampling points. This distribution system sampling
  point will be selected using the following criteria: 1) No additional 
  disinfectant added between the treatment plant and this point, 2)     
  Approximate detention time of water is available, and 3) No blending  
  with water from other sources. The SDS sample will be analyzed for    
  THMs, HAAs(6), HANs, CP, HK, CH, TOX, pH and disinfectant residual.   
\5\Five THM samples.                                                    



    Monitoring of source water quality. EPA would require all community 
and nontransient noncommunity water systems serving at least 100,000 
persons to conduct monthly monitoring of the raw water entering each 
treatment plant for pH, alkalinity, turbidity, temperature, calcium and 
total hardness, total organic carbon (TOC), ultraviolet absorbance at 
254 nm (UV254), bromide ion, and ammonia. If the raw water were to 
contain a sufficiently high concentration of inorganic chemicals (i.e., 
hydrogen sulfide, iron, manganese) to cause a high oxidant demand, then 
the system would be encouraged to monitor for this inorganic oxidant 
demand at the same frequency. Systems would collect samples from the 
plant influent after water from multiple sources is blended. The 
sampling point would be before the first treatment step to characterize 
the chemical quality of the water being treated. A system that uses 
ground water not under the direct influence of surface water and with 
multiple wells in the same aquifer would only be required to collect 
raw water samples from representative wells in the two aquifers serving 
the largest portion of the system's population.
    The above parameters were selected because they influence the 
quantity and chemical character of the DBPs formed when the 
disinfectant is added to the water. High oxidant demand water should be 
characterized because the availability of the disinfectant for reaction 
with organic material to form DBPs will depend on the amount of 
disinfectant that is consumed by inorganic chemicals. EPA solicits 
comments on the definition of high oxidant demand water and the type(s) 
of measurements necessary to characterize it.
    Monthly sampling at the treatment plant influent would provide an 
estimate of the variability in raw water quality. EPA would use data 
from this portion of the rule to characterize source water parameters 
that influence DBP formation.
    Monitoring within the treatment plant. EPA would require systems 
serving at least 100,000 people to monitor for most of the same 
parameters at several points within the treatment plant. These 
requirements are summarized in Table III.1. Samples from representative 
points before and after the filters collected on a monthly basis would 
be measured for pH, alkalinity, turbidity, temperature, calcium and 
total hardness, TOC, and UV254. These measurements would provide 
data on changes in water quality between the plant influent and the 
last filtration step. Of particular importance are data on how the 
organic precursor material (as represented by TOC and UV254) is 
removed prior to and through filtration.
    Monthly monitoring of the same parameters (pH, alkalinity, 
turbidity, temperature, calcium and total hardness, TOC, and 
UV254) would be required at each point of disinfection. These data 
are critical, because most data now available for comparing these 
variables with DBP concentrations are based on source water data. Most 
utilities do some treatment of the water prior to the addition of 
disinfectant, so source water measurements do not accurately reflect 
the quality of the water when the disinfectant is added. These data 
would provide a more accurate determination of how these parameters 
influence DBP formation.
    Disinfectant residuals would be measured monthly at the end of each 
treatment process in which chlorine is applied. Free and total chlorine 
residual would be reported if free chlorine is used as the 
disinfectant; total chlorine residual would be reported if ammonia is 
added in combination with chlorine or when sufficient ammonia is 
present in the source water that breakpoint chlorination is not 
achieved. These data combined with information on the applied 
disinfectant dosages and contact times (from the plant operational data 
discussed in the next section) would give a more accurate picture on 
DBP formation, because the chlorine or chloramine demand of the water 
can be estimated. Part of this demand is reflected in the formation of 
DBPs.
    If a water plant practices air stripping to remove volatile organic 
compounds (VOCs) from the raw water prior to the addition of a 
disinfectant and the raw water contains ammonia, then a monthly sample 
collected immediately following the air stripper and analyzed for 
ammonia would be required. Air stripping might change the concentration 
of ammonia, and an accurate concentration of ammonia is necessary to 
determine DBP formation.
    EPA would also require systems serving at least 100,000 people to 
analyze samples from the entry point to the distribution system 
monthly. The monitoring would consist of pH, alkalinity, turbidity, 
temperature, calcium and total hardness, TOC, UV254, and 
disinfectant residual.
    Systems are already monitoring for many of the parameters listed 
above, either to comply with other drinking water regulations or for 
operational considerations. Therefore, the additional costs of 
providing monthly data would not be excessive for these parameters.
    The monthly data from the treatment plants would provide EPA with 
the necessary information to conduct two analyses essential for the 
development of the Stage 2 D/DBP Rule: (1) The variability in source 
water quality and treatment operation and its impacts on the parameters 
that influence the formation of DBPs, and (2) when the data are 
combined with the DBP data described below, EPA will have a better 
understanding of how water quality and treatment practices influence 
DBP formation. This understanding would allow EPA to refine models for 
predicting DBP formation based on treatment and water quality 
parameters and thus to further clarify the interrelationships between 
disinfectant concentrations and DBPs under field conditions.
    EPA would require community and nontransient, noncommunity water 
systems that use only ground water not under the direct influence of 
surface water and serve between 50,000 and 99,999 people to analyze TOC 
samples monthly from the entry points to the distribution system.
    Additional monitoring for chlorination by-products. EPA would 
require monitoring for specific chlorination by-products quarterly to 
fulfill three objectives: (1) To relate water quality and treatment 
practices to DBP formation, (2) to determine the concentration of DBPs 
in drinking water, and (3) to establish cost effective monitoring 
requirements that are protective of public health. The Agency would 
require analysis for the following chlorination by-products: 
chloroform, bromodichloromethane, dibromochloromethane, bromoform, 
monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, 
monobromoacetic acid, dibromoacetic acid, bromochloroacetic acid, 
trichloroacetonitrile, dichloroacetonitrile, bromochloroacetonitrile, 
dibromoacetonitrile, 1,1- dichloropropanone, 1,1,1-trichloropropanone, 
chloropicrin, and chloral hydrate. Each time a DBP sample is collected, 
the system would also be required to measure and report pH, 
temperature, alkalinity, and disinfectant residual. Free chlorine 
residual would be measured in systems using free chlorine as the 
disinfectant. Total chlorine residual would be measured at sampling 
points after the addition of ammonia, because the residual disinfectant 
would be chloramines.
    To relate DBP formation to water quality and treatment practices, 
EPA would require systems to monitor the above DBPs at the following 
locations: (1) At a representative point immediately after the last 
filtration step (if chlorine is applied prior to the filters), (2) at 
the entry point to the distribution system, and (3) at a TTHM 
compliance monitoring sampling point in the distribution system which 
can be related to a simulated distribution system (SDS) sample. This 
distribution system sampling point would be selected using the 
following criteria: (1) No additional disinfectant is added to the 
water between entry to the distribution system and the sampling point, 
(2) the approximate detention time of the water is available, and (3) 
there is no blending with water from other treatment plants. A sample 
would also be collected at the entry point to the distribution system 
and incubated at a time and temperature corresponding to the 
distribution system sample. This SDS sample would be analyzed for the 
same DBPs as the distribution system sample and it would provide a 
measure of DBP formation under controlled conditions. Data from SDS 
samples would also be evaluated as a cost-effective alternative to 
distribution system compliance monitoring.
    The concentration of chlorination by-products would be determined 
by requiring the utilities to conduct quarterly monitoring at four 
points in the distribution system using the same criteria for sampling 
point selection as specified in the THM Rule. One sample would be taken 
from a point representing a maximum detention time in the system. The 
sample point with the highest THM concentrations would meet this 
criterion. The second sample would correspond to the SDS sampling point 
described above. The remaining two points would be representative of 
the distribution system. All four sampling points would be routine 
sampling points for TTHM compliance monitoring. This regimen minimizes 
the sampling costs, since additional sampling points are not required. 
It also provides a link between the measurements made for this rule and 
the historical TTHM compliance monitoring data for each system. Systems 
that have two or more treatment plants serving the same distribution 
system would only be required to collect four DBP samples in the 
distribution system.
    Six quarters of DBP monitoring would provide EPA with information 
concerning the spatial and seasonal variability of DBPs within 
distribution systems. In an effort to evaluate lower cost monitoring 
options, EPA would also require systems to monitor total organic halide 
(TOX) concentrations at the same sampling points and at the same time 
DBP concentrations are measured. Total organic halide (TOX) is an 
indicator of the total quantity of dissolved halogenated organic 
material present in water. Essentially all of the TOX present in 
chlorinated drinking water in the United States is the result of 
reactions between chlorine and the organic material and bromide ion 
present in the source water. The eighteen chlorination by-products 
listed above typically account for less than 50% of the TOX that is 
measured in chlorinated drinking water. Since TOX also includes the 
halogenated by-products not routinely measured, it might be a better 
surrogate of chlorination by-product concentrations than are TTHMs and 
THAAs. The TOX analysis of treatment plant influent would also be 
required quarterly, because the source water could contain background 
concentrations of halogenated organic compounds as a result of chemical 
contamination or upstream discharges of chlorinated water. The DBP, 
TOX, and surrogate precursor (i.e., TOC and UV254) data will be 
evaluated to determine the most cost-effective monitoring requirements 
that are protective of public health.
    All the samples for the above-named parameters would be collected 
as close together in time as feasible (during the same working day if 
possible). Samples would be collected during normal plant operating 
conditions, when there were no obvious changes in source water quality 
due to storm events, chemical spills, etc. The quarterly sampling for 
DBPs would be conducted at the same time as the sampling from the 
treatment plant(s). The quarterly samples would be collected at a time 
when the source water quality and plant operations had been stable for 
several days, so that the distribution system sample can be related to 
the SDS sample that is collected at the same time.
    Additional monitoring required for systems using chloramines. EPA 
would require systems serving at least 100,000 people and using 
chloramines to analyze for one additional DBP, cyanogen chloride. This 
by-product is formed when chlorine reacts with organic material in the 
presence of the ammonium ion (Ohya and Kanno, 1985). There are little 
data available to assess the occurrence of this compound and the 
factors influencing its formation are poorly understood. Therefore, 
these data are necessary to determine how the distribution of by-
products would change if utilities switched from free chlorine to 
chloramines as the residual disinfectant to meet the MCLs for TTHM and 
THAA.
    Monitoring for cyanogen chloride would be required quarterly, as 
summarized in Table III.2. Only one sample would be required from the 
distribution system, because of the analytical complexities of 
measuring the compound. By sampling at the entry point to the 
distribution system and at a point of maximum detention time, EPA would 
be able to assess the concentration range at which this compound 
occurs. Cyanogen chloride is very reactive, and would be expected both 
to decompose and be produced within the distribution system. 

Table III.2.--Additional Sampling Required of Systems Using Chloramines 
------------------------------------------------------------------------
      Sampling point                 Analyses               Frequency   
------------------------------------------------------------------------
Entry point to              Cyanogen chloride.........  Quarterly.      
 distribution system.                                                   
One THM compliance          Cyanogen chloride.........  Quarterly.      
 monitoring sample point                                                
 representing a maximum                                                 
 detention time in                                                      
 distribution system.                                                   
------------------------------------------------------------------------

    Additional monitoring required of systems using hypochlorite 
solutions. EPA would require systems serving at least 100,000 people 
and using hypochlorite solutions for chlorination to perform the 
additional monitoring presented in Table III.3. The monitoring would 
include quarterly measurements for chlorate in the treatment plant 
influent, hypochlorite feedstock solution, and water at the entry point 
to the distribution system. Chlorate is a decomposition product found 
in hypochlorite feedstock (Lister, 1956; Bolyard, et al., 1992; and 
Gordon et al., 1993). Its concentration in the drinking water would not 
be expected to change in the distribution system unless additional 
hypochlorite solution was added, because it is not a DBP from chlorine 
reactions under drinking water conditions. Quarterly monitoring of the 
hypochlorite stock solution to assess the factors that influence 
chlorate formation (pH, storage temperature, and hypochlorite ion 
concentration) would also be required. These data would allow EPA to 
assess the significance of chlorate ion resulting from the use of 
hypochlorite solutions. EPA anticipates chlorate would be regulated as 
part of the Stage 2 DBP Rule. 

      Table III. 3.--Additional Sampling Required of Systems Using      
                         Hypochlorite Solutions                         
------------------------------------------------------------------------
      Sampling point                 Analyses               Frequency   
------------------------------------------------------------------------
Treatment plant influent..  Chlorate..................  Quarterly.      
Hypochlorite stock          pH, temperature, free       Quarterly.      
 solution.                   residual chlorine, and                     
                             chlorate.                                  
Entry point to              Chlorate..................  Quarterly.      
 distribution system.                                                   
------------------------------------------------------------------------

    Additional monitoring required of systems using ozone. EPA would 
require systems serving at least 100,000 people and using ozone in 
their treatment process to perform the additional monitoring listed in 
Table III.4. The ozone contactor influent would be monitored monthly 
for parameters that influence formation of by-products: pH, alkalinity, 
turbidity, temperature, calcium and total hardness, TOC, UV254, 
bromide, and ammonia. The ozone residual would be measured in the 
contactor effluent and immediately prior to filtration. These data 
would be combined with the operational data and the DBP data to better 
understand and predict DBP formation. 

   Table III.4.--Additional Sampling Required of Systems Using Ozone    
------------------------------------------------------------------------
      Sampling point                 Analyses               Frequency   
------------------------------------------------------------------------
Ozone contactor influent..  pH, alkalinity, turbidity,  Monthly.        
                             temperature, calcium and                   
                             total hardness, TOC,                       
                             UV254, bromide, and                        
                             ammonia.                                   
Ozone contactor influent..  Aldehydes\1\ and AOC/       Quarterly.      
                             BDOC\2\.                                   
Ozone contactor effluent..  Ozone residual............  Monthly.        
Ozone contactor effluent..  Aldehydes\1\ and AOC/       Quarterly.      
                             BDOC\2\.                                   
Before filtration.........  Ozone residual............  Monthly.        
Entry point to              Bromate...................  Monthly.        
 distribution system.                                                   
Entry point to              Aldehydes\1\ and AOC/       Quarterly.      
 distribution system.        BDOC\2\.                                   
------------------------------------------------------------------------
\1\The aldehydes to be included in this analysis are: formaldehyde,     
  acetaldehyde, butanal, propanal, pentanal, glyoxal, and methyl        
  glyoxal. Measurement of other aldehydes is optional.                  
\2\Submission of data for assimilable organic carbon (AOC) or           
  biodegradeable organic carbon (BDOC) is optional.                     

    Water systems using ozone would also be required to monitor for 
specific DBPs that are known to be formed as the result of oxidation 
reactions. The contactor influent, contactor effluent and water from 
the entry point to the distribution system would be monitored on a 
quarterly basis for aldehydes. Utilities would also be encouraged to 
voluntarily measure assimilable organic carbon (AOC) or biodegradeable 
dissolved organic carbon (BDOC) at the same sampling points and at the 
same frequency and voluntarily submit the data. The concentration of 
bromate would be monitored on a monthly basis at the entry point to the 
distribution system. The concentration of bromate is not expected to 
increase in the water after it leaves the treatment plant.
    Additional monitoring required of systems using chlorine dioxide. 
EPA would require systems serving 100,000 people and using chlorine 
dioxide in their treatment process to conduct the additional monitoring 
listed in Table III.5. Parameters that influence the formation of by-
products would be measured on a monthly basis at sampling point(s) 
prior to each application of chlorine dioxide. The analyses would 
include: pH, alkalinity, turbidity, temperature, calcium and total 
hardness, TOC, UV254, and bromide.

   Table III.5--Additional Sampling Required of Systems Using Chlorine  
                                 Dioxide                                
------------------------------------------------------------------------
      Sampling point                 Analyses               Frequency   
------------------------------------------------------------------------
Treatment plant influent..  Chlorate..................  Quarterly.      
Before each chlorine        pH, alkalinity, turbidity,  Monthly.        
 dioxide application.        temperature, calcium and                   
                             total hardness, TOC,                       
                             UV254, and bromide.                        
Before first chlorine       Aldehydes\1\ and AOC/       Quarterly.      
 dioxide application.        BDOC\2\.                                   
Before application of       pH, chlorine dioxide        Monthly.        
 ferrous salts, sulfur       residual, chlorite,                        
 reducing agents, or GAC.    chlorate.                                  
Before downstream chlorine/ Aldehydes\1\ and AOC/       Quarterly.      
 chloramine application.     BDOC\2\.                                   
Entry point to              Chlorite, chlorate,         Monthly.        
 distribution system.        chlorine dioxide                           
                             residual, bromate.                         
Entry point to              Aldehydes\1\ and AOC/       Quarterly.      
 distribution system.        BDOC\2\.                                   
3 distribution system       chlorite, chlorate,         Monthly.        
 sampling points (1 near     chlorine dioxide                           
 first customer, 1 in        residual, pH, and                          
 middle of distribution      temperature.                               
 system, and 1 at a                                                     
 maximum detention time in                                              
 the system).                                                           
------------------------------------------------------------------------
\1\The aldehydes to be included in this analysis are: formaldehyde,     
  acetaldehyde, butanal, propanal, pentanal, glyoxal, and methyl        
  glyoxal. Measurement of other aldehydes is optional.                  
\2\Submission of data for AOC or BDOC is optional.                      

    The by-products of particular concern from the use of chlorine 
dioxide are chlorite and chlorate. Since the application of ferrous 
salts or sulfur reducing agents changes the concentrations of these by-
products, utilities would be required to monitor for chlorite and 
chlorate prior to and following each of these treatment processes. 
Monitoring would also be required before and after granular activated 
carbon (GAC) filtration. These data would provide a better 
understanding of the formation and control of these two by-products and 
would allow the development of predictive models for use in development 
of the Stage 2 D/DBP Rule.
    Very little data are available concerning the chlorite and chlorate 
concentrations generally present in drinking water as a result of 
chlorine dioxide use. Therefore, utilities would be required to monitor 
for these by-products at the entry point to the distribution system and 
at three sites within the distribution system. The concentrations of 
chlorite and chlorate are expected to change as the water is 
distributed through the system, so distribution system samples are 
needed to assess the magnitude of the changes. One sample would be 
collected near the first customer; another sample would be collected at 
a point representing the maximum detention time in the distribution 
system and the last sample would be collected at a point representative 
of the average consumer.
    These water systems would also be required to monitor the chlorine 
dioxide residual concentrations, pH and temperature at the above 
sampling points. Of particular concern is the possible re-formation of 
chlorine dioxide in the distribution system as a result of reactions 
between chlorite and chlorine. Since chlorine dioxide and its by-
products may pose acute health risks, monitoring for them would be 
required on a monthly basis. The proposed Stage 1 D/DBP Rule may 
require daily monitoring for chlorine dioxide at the point of entry 
into the distribution system and monthly monitoring for chlorite at 
three points in the distribution system.
    Because low levels of chlorate have been reported in source water 
(Bolyard, et al., 1993; and Gordon, et al., 1993), EPA would also 
require systems using chlorine dioxide to monitor the treatment plant 
influent monthly for chlorate. This monitoring would provide data to 
assess the relative amounts of chlorate from source water versus the 
amount produced as the result of chlorine dioxide use.
    EPA would also require systems using chlorine dioxide to perform 
quarterly monitoring for several oxidation by-products, because there 
is a small amount of data indicating their presence as the result of 
chlorine dioxide use. Quarterly monitoring for aldehydes would be 
required: (1) Before the first chlorine dioxide application in order to 
determine background levels from the source waters; (2) before 
application of the secondary disinfectant to determine what was 
produced by chlorine dioxide; and (3) at the entry point to the 
distribution system to evaluate the total level delivered to the 
consumers based upon all the treatment processes and disinfectants. EPA 
would also encourage systems to voluntarily measure AOC or BDOC at the 
same sampling points and at the same frequency and voluntarily submit 
the data. The Agency would require systems to report the bromate 
concentration present in the sample analyzed for chlorite and chlorate 
from the entry point to the distribution system, because there are 
limited data indicating that bromate may be formed as a result of 
sunlight catalyzed reactions between chlorine dioxide and bromide ion 
present in the source water (Zika et al., 1985). This would be an 
additional sample, because the measurement of low levels of bromate 
(<10 g/L) in the presence of much higher levels of chlorite 
(100-1000 g/L) would require special treatment of the sample.
3. Treatment Process Information Collection
    Background/justification. EPA proposes collecting treatment process 
information as part of this rule to characterize the various forms of 
treatment currently being used by treatment plants serving more than 
100,000 persons. The treatment process information will be used to 
evaluate options available to large water utilities to monitor and 
reduce DBP formation. The Water Treatment Plant (WTP) Model 
(Harrington, et al., 1992) was used to predict THM and HAA levels in 
the development of the Stage 1 D/DBP Rule. The model is available from 
the Safe Drinking Water Act Hotline (1-800-426-4791). It uses raw water 
quality and treatment process data to predict THM and HAA formation. 
The WTP model is calibrated on fewer than 100 bench-, pilot-, and full-
scale studies. This rule would provide a sufficiently large database to 
upgrade the model to include additional processes, predict other DBPs, 
and better calibrate the model based on hundreds of plant experiences.
    The process data would be coupled with the water quality data 
described in Tables III.1 through III.5 to assess how treatment impacts 
precursor removal; how treatment affects the formation of THMs, HAAs 
and other DBPs; and how parameters like TOX and SDS compare to 
distribution system compliance parameters. Relationships between the 
process data and water quality data collected under this rule would be 
evaluated to help define Stage 2 requirements of the D/DBP Rule and to 
better evaluate and refine prediction models that will be used for the 
Stage 2 D/DBP Rule development.
    Specific Process Information. The treatment plant information and 
unit processes listed in Table III.6 and the water quality data 
described in previous sections will provide the information necessary 
to develop predictions between raw water quality, treatment conditions, 
precursor removal, and DBP formation. EPA selected the parameters 
listed to characterize the unit process for use in developing the 
predictions and Stage 2 D/DBP Rule development. For example, 
coagulation parameters are needed for evaluation of efficiencies to 
better define the impact of enhanced coagulation for precursor (TOC) 
control. The depth of the filter is needed to evaluate the feasibility 
of adding GAC to the filter for precursor removal. The complete process 
train details are needed to evaluate the feasibility and costs of 
treatment changes being considered for DBP control. The list does not 
include every possible water treatment process parameter, but does 
include the parameters that would be used to characterize the treatment 
practices for the purpose of this monitoring rule. 

               Table III.6.--Treatment Plant Information                
                                                                        
                                                                        
Utility information:                                                    
  Utility Name                                                          
  Mailing Address                                                       
  Contact Person & Phone Number                                         
  Public Water Supply Identification Number FRDS (PWSID)                
  Population Served                                                     
Plant information:                                                      
  Name of plant                                                         
  Design flow (MGD)                                                     
  Annual minimum water temperature (C)                                  
  Annual maximum water temperature (C)                                  
  Hours of operation (hours per day)                                    
Source water information:                                               
  Name of source                                                        
  Type of source (One of the following)                                 
      1River                                                            
      2Stream                                                           
      3Reservoir                                                        
      4Lake                                                             
      5Ground water under the direct influence of surface water         
      6Ground water                                                     
      7Spring                                                           
      8Purchased from Utility Name, FRDS PWSID                          
      9Other                                                            
  Surface water as defined by SWTR (TRUE/FALSE)                         
  Monthly Average Flow of this Source (MGD)                             
  Upstream sources of microbiological contamination                     
      Wastewater plant discharge in watershed (yes/no)                  
          Distance from intake (miles)                                  
          Monthly average flow of plant discharge (MGD)                 
          Point source feedlots in watershed (yes/no)                   
          Distance of nearest feedlot discharge to intake (miles)       
          Non-point sources in watershed                                
          Grazing of animals (yes/no)                                   
          Nearest distance of grazing to intake (miles)                 
Plant influent (ICR influent sampling point):                           
  Monthly average flow (MGD)                                            
  Monthly peak hourly flow (MGD)                                        
  Flow at time of sampling (MGD)                                        
Plant effluent (ICR effluent sampling point):                           
  Monthly average flow (MGD)                                            
  Monthly peak hourly flow (MGD)                                        
  Flow at time of sampling (MGD)                                        
Sludge treatment:                                                       
  Monthly average solids production (lb/day)                            
  Installed design sludge handling capacity (lb/day)                    
General process parameters:                                             
The following data will be required for all unit processes:             
  Number of identical parallel units installed                          
  Number of identical parallel units in service at time of sampling     
The following parameters will be required for all unit processes except 
 chemical feeders:                                                      
  Design Flow per unit (MGD)                                            
  Liquid volume per unit (gallons)                                      
  Tracer study flow (MGD)                                               
  T50 (minutes)                                                         
  T10 (minutes)                                                         
Presedimentation basin:                                                 
  Surface loading at design flow (gpm/ft2)                              
Chemical feeder:                                                        
  Type of feeder (one of the following)                                 
      1Liquid                                                           
      2Gas                                                              
      3Dry                                                              
  Capacity of each unit (lb/day)                                        
  Purpose (one or more of the following)                                
      1Coagulation                                                      
      2Coagulation aid                                                  
      3Corrosion control                                                
      4Dechlorination                                                   
      5Disinfection                                                     
      6Filter aid                                                       
      7Fluoridation                                                     
      8Oxidation                                                        
      9pH adjustment                                                    
      10Sequestration                                                   
      11Softening                                                       
      12Stabilization                                                   
      13Taste and odor control                                          
      14Other                                                           
Chemical feeder chemicals (one of the following):                       
  Alum                                                                  
  Anhydrous ammonia                                                     
  Ammonium hydroxide                                                    
  Ammonium sulfate                                                      
  Calcium hydroxide                                                     
  Calcium hypochlorite                                                  
  Calcium oxide                                                         
  Carbon dioxide                                                        
  Chlorine dioxide--acid chlorite                                       
  Chlorine dioxide--chlorine/chlorite                                   
  Chlorine gas                                                          
  Ferric chloride                                                       
  Ferric sulfate                                                        
  Ferrous sulfate                                                       
  Ozone                                                                 
  Polyaluminum chloride                                                 
  Sodium carbonate                                                      
  Sodium chloride                                                       
  Sodium fluoride                                                       
  Sodium hydroxide                                                      
  Sodium hypochlorite                                                   
  Sodium hexametaphosphate                                              
  Sodium silicate                                                       
  Sulfuric acid                                                         
  Zinc orthophosphate                                                   
  Other                                                                 
Notes:                                                                  
  1. The above list is intended to be a comprehensive list of chemicals 
   used at water treatment plants. If the name of a chemical does not   
   appear in the list then ``Other Chemical'' information will be       
   requested.                                                           
  2. Formulas and feed rate units will be included in data reporting    
   software.                                                            
  Monthly average feed rate based on inventory (mg/L) Feed rate at time 
   of sampling (mg/L)                                                   
Other chemical:                                                         
Note:                                                                   
  In addition to Chemical Feeder information the following will be      
   required for any chemical not included in the Chemical Feeder list of
   chemicals.                                                           
      Trade name of chemical                                            
      Formula                                                           
      Manufacturer                                                      
Rapid mix:                                                              
  Type of mixer (one of the following)                                  
      1Mechanical                                                       
      2Hydraulic jump                                                   
      3Static                                                           
      4Other                                                            
  If mechanical: horsepower of motor                                    
  If hydraulic: head loss (ft)                                          
  If static: head loss (ft)                                             
Flocculation basin:                                                     
  Type of mixer (one of the following)                                  
      1Mechanical                                                       
      2Hydraulic                                                        
      3Other                                                            
  If mechanical: Mixing power (HP)                                      
  If hydraulic: head loss (ft)                                          
Sedimentation basin:                                                    
  Loading at Design Flow (gpm/ft2)                                      
  Depth (ft)                                                            
Filtration:                                                             
  Loading at Design Flow (gpm/ft2)                                      
  Media Type (one or more of the following)                             
      1Anthracite                                                       
      2GAC                                                              
      3Garnet                                                           
      4Sand                                                             
      5Other                                                            
  Depth of top media (in)                                               
  If more than 1 media: Depth of second media (in)                      
  If more than 2 media: Depth of third media (in)                       
  If more than 3 media: Depth of fourth media (in)                      
  If GAC media: Carbon replacement frequency (months):                  
  Water depth to top of media (ft)                                      
  Depth from top of media to bottom of backwash trough (ft)             
  Backwash Frequency (hours)                                            
  Backwash volume (gallons)                                             
Contact basin (Stable liquid level):                                    
  Baffling Type (one of the following as defined in SWTR guidance       
   manual)                                                              
      1Unbaffled (mixed tank)                                           
      2Poor (inlet/outlet only)                                         
      3Average (Inlet/Outlet and intermediate)                          
      4Superior (Serpentine)                                            
      5Perfect (Plug flow)                                              
Clearwell (Variable liquid level):                                      
  Baffling Type (one of the following as defined in SWTR guidance       
   manual)                                                              
      1Unbaffled (mixed tank)                                           
      2Poor (inlet/outlet only)                                         
      3Average (Inlet/Outlet and intermediate)                          
      4Superior (Serpentine)                                            
      5Perfect (Plug flow)                                              
  Minimum liquid volume (gallons)                                       
  Liquid volume at time of tracer study (gallons)                       
Ozone contact basin:                                                    
  Basin Type                                                            
      1Over/Under (Diffused O3)                                         
      2Mixed (Turbine O3)                                               
  Number of Stages                                                      
  CT (min mg/L)                                                         
EPA requests comments on the design and operating parameters to be      
 reported for ozone contact basins.                                     
Tube settler:                                                           
  Surface loading at design flow (gpm/ft2)                              
  Tube angle from horizontal (degrees)                                  
Upflow clarifier:                                                       
  Design horse power of turbine mixer (HP)                              
  Surface loading at design flow (gpm/ft2)                              
  Special Equipment (none, one, or more of the following)               
      1Lamella plates                                                   
      2Tubes                                                            
Plate settler:                                                          
  Surface loading at design flow (gpm/ft2)                              
DE filter:                                                              
  Surface loading at design flow (gpm/ft2)                              
  Precoat (lb/ft3)                                                      
  Bodyfeed (mg/L)                                                       
  Run length (hours)                                                    
Granular activated carbon:                                              
  Empty bed contact time at design flow (minutes)                       
  Design regeneration frequency (days)                                  
  Actual regeneration frequency (days)                                  
Membranes:                                                              
  Type (one of the following)                                           
      1Reverse osmosis                                                  
      2Nanofiltration                                                   
      3Ultrafiltration                                                  
      4Microfiltration                                                  
      5Electrodialysis                                                  
      6Other                                                            
  Name of Other type                                                    
  Membrane type (one of the following)                                  
      1Cellulose acetate and derivatives                                
      2Polyamides                                                       
      3Thin-film composite                                              
      4Other                                                            
  Name of other membrane type                                           
  Molecular weight cutoff (gm/mole)                                     
  Configuration (one of the following)                                  
      1Spiral wound                                                     
      2Hollow fiber                                                     
      3Tube                                                             
      4Plate and frame                                                  
      5Other                                                            
  Name of other configuration                                           
  Design flux (gpd/ft2)                                                 
  Design pressure (psi)                                                 
  Purpose of membrane unit (one or more of the following)               
      1Softening                                                        
      2Desalination                                                     
      3Organic removal                                                  
      4Other                                                            
      5Contaminant removal--name of contaminant                         
  Percent recovery (%)                                                  
  Operating pressure (psi)                                              
Air stripping:                                                          
  Packing height (ft)                                                   
  Design liquid loading (gpm/ft2)                                       
  Design air to water ratio                                             
  Type of packing (Name)                                                
  Nominal size of packing (inch)                                        
  Operating air flow (SCFM)                                             
Adsorption clarifier:                                                   
  Surface loading at design flow (gpm/ft2)                              
Dissolved air flotation:                                                
  Surface loading at design flow (gpm/ft2)                              
Slow sand filtration:                                                   
  Surface loading at design flow (gpd/ft2)                              
Ion exchange:                                                           
  Purpose (one or more of the following)                                
      1Softening                                                        
      2Contaminant removal                                              
  Contaminant name                                                      
  Media type (Name)                                                     
  Design exchange capacity (equ/ft3)                                    
  Surface loading at design flow (gpm/ft2)                              
  Bed depth (ft)                                                        
  Regenerant Name (one of the following)                                
      1Sodium Chloride (NaCl)                                           
      2Sulfuric Acid (H2SO4)                                            
      3Sodium Hydroxide (NaOH)                                          
      4Other                                                            
  If other: Name and formula                                            
  Operating regeneration frequency (hr)                                 
  Regenerant concentration (%)                                          
  Regenerant Used (lb/day)                                              
Other treatment:                                                        
  Name                                                                  
  Purpose                                                               
  Design Parameters                                                     


    EPA will be working with the industry to develop the software to 
collect this process information as described in the following section. 
Utilities would use the data collection software to input the process 
data once at the beginning of the monitoring period with monthly 
updates of the operating data and any treatment modifications.
    EPA requests comments on the completeness of Table III.6 to 
describe treatment plant configurations and the specific design 
parameters for the unit processes that would be relevant to Stage 2 D/
DBP rule development and future model development for predicting DBPs. 
Is all the requested information essential? Are more efficient 
mechanisms available than those proposed herein for obtaining the 
desired information? Will the treatment plant information requested be 
adequate for developing models by which to predict the ability of 
utilities to achieve various potential regulatory criteria under Stage 
2 (e.g., DBP and TOX occurrence levels in the distribution system)? 
Will the treatment plant information required for systems serving 
100,000 or more people be adequate for developing predictive models of 
DBP formation for systems serving less than 100,000 people? What 
additional information, if any, would be important to obtain to predict 
the formation of DBPs in systems serving less than 10,000 people? If 
additional information is needed, what mechanisms should be used for 
obtaining it? For example, would any survey techniques of 
representative systems be useful for obtaining this information?
    Data collection software design. Since the collection of DBP 
occurrence data and source water quality data must be combined with 
information about the treatment processes, EPA proposes using data 
collection software as a mechanism for obtaining the monitoring data 
and treatment plant process information necessary for developing the 
Stage 2 D/DBP Rule. The software would capture information about source 
water quality, treatment plant design, unit processes, chemical 
dosages, and the monitoring results listed in Tables III.1-III.6. EPA 
would provide technical assistance for use of the data collection 
software.
    To capture both water quality data and process information from 
each plant, the data collection software and database would be designed 
to handle various treatment configurations including split flow, 
process parameters relevant to each configuration, and water quality 
monitoring data described in earlier sections.
    EPA would provide each utility a diskette containing the data 
collection software. The software would generate screen driven data 
entry forms that are customized for the water utility depending on the 
treatment process configuration entered by the utility. The water 
quality parameters listed in Tables III.1 through III.5 and the results 
of the microbiological monitoring would also be entered by the utility. 
The water utility would only enter monitoring results pertinent to its 
system. Table III.6 lists the unit process choices that would be used 
to develop the process train for a given water treatment plant. The 
computer program would be designed to prompt the user for the process 
parameters based on the process choices selected. For example, a plant 
using only chlorine for disinfection would not see prompts for chlorine 
dioxide residual, bromate, or chlorite on its data entry screen.
    The software will determine such details as where sampling points 
should generally be located and which water quality parameters should 
be measured. The user would have the option of printing a series of 
data forms to be used as a guide in identifying sample point locations, 
requesting laboratory analysis, and gathering design and operation 
parameters. The software will be designed in data segments and will 
save data to a monthly data file on a hard drive or diskette. The 
utility will send data to EPA as described in the following section.
4. Database Development
    The proposed procedure would entail each PWS collecting the data on 
a computer diskette provided by EPA using the data collection software, 
sending the data via modem or by diskette to a database coordinator, 
having the data reviewed for correctness by an engineer or scientist 
familiar with water treatment, loading the data into a master database, 
having the data analyzed periodically throughout the monitoring period, 
generating interim reports, and having the database in final usable 
form for Stage 2 D/DBP Rule development shortly after the conclusion of 
the sixth quarter monitoring period. Any interested party would have 
access to the data at various points in time during the collection 
period. EPA would provide technical assistance throughout the data 
collection and reporting process.
    EPA proposes that a personal computer with an MS-DOS operating 
system be used for data entry. EPA would provide the ICR data 
collection software to the utilities for data collection. The utilities 
would provide the personal computer. The software will have many built 
in features to guide the user through the process train configuration 
and data input. In addition, EPA intends to make technical assistance 
available, if needed, to help assure the quality of information 
provided.
    The output from the data collection software would be monthly data 
files in ASCII format. Data files on diskette would be mailed to EPA 
and transferred to the master data base. Data files transferred via 
modem would be sent using telecommunication software supplied by the 
utility. EPA requests comment on the use of diskettes, modem or other 
means for data reporting.
    Design of the database, its input/output mechanisms, and its output 
formats would be considered before start-up of the monitoring effort. 
The output would target the requirements being considered for the Stage 
2 D/DBP Rule and the Enhanced SWTR. Examples of the many questions the 
output would address are: (1) What is the national distribution of 
bromide, TOC, etc., i.e., the factors that affect DBP formation? (2) 
What is the distribution of HAAs, chloral hydrate, etc. in distribution 
system waters? (3) What treatment processes and operating conditions 
are associated with minimum DBP levels? (4) What levels of bromate form 
in ozonation plants under different conditions?
    Testing data collection and transfer. Before monitoring begins, EPA 
would need to beta test the ICR data collection software for 
transferring data from the utility to a master database to identify 
unforeseen problems with the data collection procedure. Therefore, the 
Agency's schedule for beta testing must have enough lead time to modify 
the process, if needed, before monitoring begins. EPA intends to 
conduct the data collection software beta testing with the cooperation 
of a small number of utilities with diverse characteristics. The master 
database and its data manipulation and output procedures would also be 
beta tested to identify unforeseen problems with the data handling 
procedures after the data are reported to EPA.
    Frequency of reporting. EPA would require systems to submit data to 
the Agency two months after monitoring begins and thereafter monthly. 
Periodic reporting would allow EPA to review the data and resolve 
problems associated with data collection and submission, and also to 
quicken the pace of regulatory development of the interim and long-term 
ESWTRs.
    Data availability. EPA would make raw (unanalyzed) data available 
to interested organizations and individuals periodically throughout the 
monitoring period via electronic transfer. EPA proposes that the data 
be made available after the first two quarters' raw data have been 
verified, and for every 6 months of data thereafter following the 
verification of that data until the conclusion of the monitoring 
period. This access would be a ``read only'' mode.
    EPA would make analyzed data available in summary form. The 
analyzed data would be grouped by source water type, utility size, type 
of treatment, distribution of DBPs, distribution of TOC, treatment 
effectiveness, etc. These data would be used in developing the interim 
and long-term ESWTR and the Stage 2 D/DBP rule.
5. Analytical Methods
    Approved methods. Analytical methods that are currently approved 
for monitoring purposes under other drinking water regulations would be 
approved for use under this rule. These include the parameters: (1) pH; 
(2) alkalinity; (3) turbidity; (4) temperature; (5) calcium hardness; 
(6) free residual chlorine; (7) total residual chlorine; (8) chlorine 
dioxide residual; (9) ozone residual; (10) chloroform; (11) 
bromodichloromethane; (12) dibromochloromethane; and (13) bromoform.
    Analytical methods for several of the above named parameters have 
also been updated in the 18th edition of Standard Methods for the 
Examination of Water and Wastewater for the Examination of Water and 
Wastewater. These include: (1) pH; (2) alkalinity; (3) turbidity; (4) 
temperature; (5) calcium hardness; (6) free residual chlorine; (7) 
total residual chlorine; (8) chlorine dioxide residual; and (9) ozone 
residual. The updated versions of these methods would also be approved 
for compliance monitoring under this rule.
    In addition to the methods currently approved for monitoring 
purposes under other drinking water regulations and their most recent 
versions, approved methods for the remainder of the parameters that 
must be measured for this rule are listed in Table III.7. The methods 
are published and contain descriptions of the methodology and 
information on the precision and accuracy of the methods.
    EPA is proposing one new method (EPA Method 551) for trihalomethane 
(chloroform, bromodichloromethane, dibromochloromethane, and bromoform) 
monitoring under this rule. EPA is also soliciting comment on whether 
use of this method should also be approved for compliance with the 
monitoring requirements under the Trihalomethane rule [44 FR 68264, 
November 29, 1979].
    Monitoring for the six haloacetic acids (HAAs) would be done using 
EPA Method 552.1 or an expanded version of Method 6233 B which is 
published in the 18th edition of Standard Methods. Bromochloroacetic 
acid is not listed as an analyte in the published version of Method 
6233 B, because an analytical standard was not commercially available 
when the method was first developed. The feasibility of including it in 
Method 6233 B has been demonstrated (Barth and Fair, 1992), and it will 
be added to the method during the next revision.
    Method 6233 B is undergoing revision for the 19th edition of 
Standard Methods, so EPA proposes that a draft version be made 
available to laboratories performing HAA analyses for this monitoring 
rule.
    EPA would require laboratories to use EPA Method 551 for measuring 
trichloroacetonitrile, dichloroacetonitrile, bromochloroacetonitrile, 
dibromoacetonitrile, 1,1-dichloropropanone, 1,1,1-trichloropropanone, 
and chloropicrin. The use of pentane instead of methyl-tertiary-butyl 
ether (MTBE), the solvent described in the method, would be permissible 
when analyzing for these analytes and for the THMs.
    Chloral hydrate (CH) would also be measured using EPA Method 551, 
but its concentration would be determined by analyzing a separate 
sample from the one collected for the other 551 analytes. CH requires a 
different dechlorinating agent than the other DBPs included in the 
method. The THMs can also be measured in the chloral hydrate sample. 
MTBE must be used as the extracting solvent when measuring CH.
    EPA Method 551 specifies that the pH of the sample be adjusted to 
between 4.5 and 5.0 when the sample is collected, to prevent base-
catalyzed hydrolysis of several of the analytes. Sample stability has 
been demonstrated for 14 days when this technique is used in the 
laboratory. However, field application of this preservation technique 
(i.e., titration) has not been tested and may not be practical. EPA 
proposes that the samples be collected without adjusting the pH and 
that the laboratories be required to extract the samples within 24-48 
hours of sample collection. This requirement would result in a negative 
bias in the data for several of the analytes, with the bias increasing 
as the pH of the samples increases. EPA solicits comments on this 
approach or suggestions on alternative approaches.
    Chlorate, chlorite, bromide, and bromate would be measured using 
EPA Method 300.0. Laboratories would be permitted to use alternate 
eluents (e.g., borate eluent) or sample cleanup or concentration 
techniques in order to lower the detection limit for bromate, as long 
as the quality assurance criteria specified in the method are met.
    EPA is aware that the above method may not be sensitive enough to 
provide quantitative data for bromate at concentrations <10 g/
L. Some laboratories may be able to detect bromate in samples at 
concentrations as low as 5 g/L, but the data will not be 
precise enough to be used for making decisions on how treatment 
practices and source water characteristics influence bromate formation. 
Since the Stage 1 D/DBP Rule may propose a maximum contaminant level 
goal (MCLG) of zero for bromate, it is important to extend the 
quantitation for bromate to as low a concentration as possible during 
this information collection process.
    One of EPA's laboratories has the capability to measure bromate at 
concentrations of <1 g/L using a selective anion concentration 
technique prior to ion chromatography analysis (Hautman, D.P., Nov. 
1992). EPA does not think this new technique could be readily 
transferred to laboratories doing routine analyses, because the 
required instrumentation is not commercially available and the 
technique is complex and time consuming. Therefore, in order to obtain 
low level bromate measurements, EPA is proposing that utilities be 
required to collect duplicate samples and to send one sample from each 
duplicate set to EPA. EPA could then obtain more sensitive quantitation 
to better characterize bromate formation as a function of water quality 
treatment characteristics. EPA would use the data generated by 
utilities to evaluate the ability of laboratories to accurately and 
precisely measure bromate near the anticipated MCL of 10 g/l 
in the Stage 1 D/DBP rule that was agreed to by the Negotiating 
Committee. EPA would be responsible for obtaining the required analyses 
using the new technique. EPA solicits comments on this approach for 
obtaining low level bromate measurements.
    Cyanogen chloride (CNCl) concentrations would be monitored using a 
modified version of EPA Method 524.2. This compound is not listed in 
the method, but feasibility has been demonstrated (Flesch and Fair, 
1988). Cyanogen chloride is unstable, so laboratories would be required 
to perform the analysis within 24-48 hours of sample collection. 
Samples for CNCl analysis must be dechlorinated using ascorbic acid.
    EPA is aware of one other technique for measuring CNCl. A headspace 
analytical technique using gas chromatography with electron capture 
detection was recently described in the literature (Xie and Reckhow, 
1993). It can also be used to measure cyanogen bromide which may be 
preferentially formed when the source water contains bromide ion. EPA 
solicits comments on whether this technique should be used to generate 
data for this monitoring rule. Use of the technique would be contingent 
upon preparation of a written protocol for performing the analysis 
including specific quality control requirements. The protocol would be 
published in the ICR DBP Analytical Methods Guidance Manual.
    A method for the analysis of aldehydes in source water and drinking 
water is being written for the 19th edition of Standard Methods. The 
methodology involves the use of O-(2,3,4,5,6-pentafluorobenzyl)-
hydroxylamine (PFBHA) as a derivatizing agent. PFBHA reacts with low 
molecular weight carbonyl compounds, including aldehydes, in aqueous 
solutions to form the corresponding oximes. These derivatives are 
extractable with organic solvents and can be measured using gas 
chromatography with either electron capture (ECD) or selective ion 
monitoring-mass spectrometry (SIM-MS) detection (Glaze et al., 1989; 
Cancilla et al., 1992). EPA proposes that the draft version of the 
method be used by laboratories performing aldehyde analyses for this 
monitoring rule.
    Analyses for aldehydes are usually begun immediately or within 24 
to 48 hours after sample collection, because a preservation technique 
has not been demonstrated. EPA proposes that all aldehyde analyses for 
this rule be initiated within 48 hours of sample collection. EPA 
solicits comments on alternative approaches.
    Total organic halide (TOX) would be monitored using Standard Method 
5320 B. All samples for this monitoring rule would be dechlorinated and 
acidified at the time of collection.
    Total organic carbon (TOC) would be monitored using Standard Method 
5310 C (persulfate-ultraviolet oxidation) or 5310 D (wet-oxidation). 
The samples must not be filtered prior to analysis. Turbid samples 
would be diluted using organic free water in order to remove 
interferences from high concentrations of particulate matter.
    EPA is aware of recent advances in the measurement of TOC using 
high temperature catalytic oxidation (Benner and Hedges, 1993; Kaplan, 
1992). The instrumentation is commercially available and is being used 
in some drinking water laboratories. Published data suggest the new 
technique may be slightly more effective than the proposed methods in 
oxidizing refractory organic material. If this is true, then results 
produced using the new technique would indicate higher TOC levels than 
would be measured using the proposed methods, when samples contained 
refractory organic material. The methodology has not been evaluated by 
EPA and it is not published in a reference text such as Standard 
Methods or an EPA Methods Manual. EPA solicits comments on whether (or 
under what conditions) the use of this new oxidation technique should 
be permitted for monitoring under this rule.
    No written method exists for measuring ultraviolet absorbance at 
254 nm (UV254). EPA proposes that a protocol be developed by a 
workgroup composed of persons familiar with techniques currently being 
used to study precursor removal. The protocol would be distributed to 
all laboratories that generate UV254 data for this rule and its 
use would be required. The protocol would also be published in the ICR 
DBP Analytical Methods Guidance Manual. The protocol will specify 
sample filtration and pH adjustment procedures.
    Simulated distribution system (SDS) samples would be incubated at 
the same temperature and pH as the distribution system for a reaction 
time comparable to the estimated detention time of the distribution 
system sampling point selected for comparison purposes. The general 
protocol is described in the 18th edition of Standard Methods under 
Method 5710 E. Exact details of how the SDS samples would be handled 
will be specified in the ICR DBP Analytical Methods Guidance Manual. 
Since the temperature and incubation time of the SDS samples will be 
utility specific, EPA will recommend that the utility incubate the 
sample for the specified time period. The pH and disinfectant residual 
would be measured at the end of the incubation period. The sample would 
then be poured into sample bottles containing the appropriate 
dechlorinating agents and preservatives and sent to the laboratory for 
analysis. This procedure would alleviate concern over laboratory 
logistics in dealing with many SDS samples requiring different 
incubation temperatures and times. The SDS sample would be analyzed for 
chloroform, bromodichloromethane, dibromochloromethane, bromoform, 
monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, 
monobromoacetic acid, dibromoacetic acid, bromochloroacetic acid, 
chloral hydrate, trichloroacetonitrile, dichloroacetonitrile, 
bromochloroacetonitrile, dibromoacetonitrile, 1,1- dichloropropanone, 
1,1,1-trichloropropanone, chloropicrin, total organic halide, pH, and 
disinfectant residual.

                          Table III.7--Analytical Methods Approved for Monitoring Rule                          
----------------------------------------------------------------------------------------------------------------
                                                            Methodology                                         
                  ----------------------------------------------------------------------------------------------
     Analyte             40 CFR                                                                     Standard    
                      reference\1\                           EPA method                            method\2\    
----------------------------------------------------------------------------------------------------------------
pH...............  141.74(a)(7),                                                                4500-H+         
                    141.89(a).                                                                                  
Alkalinity.......  141.89(a)........                                                            2320 B          
Turbidity........  141.22(a),                                                         180.1\3\  2130 B          
                    141.74(a)(4).                                                                               
Temperature......  141.74(a)(6),                                                                2550 B          
                    141.89(a).                                                                                  
Calcium hardness.  141.89(a)........                                                  200.7\4\  3111 B, 3120 B, 
                                                                                                 3500-Ca D      
Free residual      141.74(a)(5).....                                                            4500-Cl D, 4500-
 chlorine.                                                                                       Cl F, 4500-Cl  
                                                                                                 G, 4500-Cl H   
Total residual     141.74(a)(5).....                                                            4500-Cl D, 4500-
 chlorine.                                                                                       Cl E, 4500-Cl  
                                                                                                 F, 4500-Cl G,  
                                                                                                 4500-Cl I      
Chlorine dioxide   141.74(a)(5).....                                                            4500-ClO2 C,    
 residual.                                                                                       4500-ClO2 D,   
                                                                                                 4500-ClO2 E    
Ozone residual...  141.74(a)(5).....                                                            4500-O3 B       
Chloroform.......  141 Subpt C, App.                                502.2\5\, 524.25,6, 5517,8  ................
                    C.                                                                                          
Bromodichlorometh  141 Subpt C, App.                                502.2\5\, 524.25,6, 5517,8  ................
 ane.               C.                                                                                          
Dibromochlorometh  141 Subpt C, App.                                502.2\5\, 524.25,6, 5517,8  ................
 ane.               3.                                                                                          
Bromoform........  141 Subpt C, App.                               502.2\5\, 524.25,6, 5517,8,  ................
                    C.                                                                                          
Monochloroacetic   .................                                                  552.1\6\  6233 B          
 acid.                                                                                                          
Dichloroacetic     .................                                                  552.1\6\  6233 B          
 acid.                                                                                                          
Trichloroacetic    .................                                                  552.1\6\  6233 B          
 acid.                                                                                                          
Monobromoacetic    .................                                                  552.1\6\  6233 B          
 acid.                                                                                                          
Dibromoacetic      .................                                                  552.1\6\  6233 B          
 acid.                                                                                                          
Bromochloroacetic  .................                                                  552.1\6\  6233 B\9\       
 acid.                                                                                                          
Chloral Hydrate..  .................                                                    551\7\  ................
Trichloroacetonit  .................                                                    5517,8  ................
 rile.                                                                                                          
Dichloroacetonitr  .................                                                    5517,8  ................
 ile.                                                                                                           
Bromochloroaceton  .................                                                    5517,8  ................
 itrile.                                                                                                        
Dibromoacetonitri  .................                                                    5517,8  ................
 le.                                                                                                            
1,1-               .................                                                    5517,8  ................
 Dichloropropanon                                                                                               
 e.                                                                                                             
1,1,1-             .................                                                    5517,8  ................
 Trichloropropano                                                                                               
 ne.                                                                                                            
Chloropicrin.....  .................                                                    5517,8  ................
Chlorite.........  .................                                                 300.0\10\  ................
Chlorate.........  .................                                                 300.0\10\  ................
Bromide..........  .................                                                 300.0\10\  ................
Bromate..........  .................                                                 300.0\10\  ................
Cyanogen Chloride  .................                                                  524.2\6\  ................
Aldehydes........  .................  ........................................................  draft method    
                                                                                                 submitted to   
                                                                                                 19th Edition   
Total Organic      .................  ........................................................  5320 B          
 Halide (TOX).                                                                                                  
Total Organic      .................  ........................................................  5310 C, 5310 D  
 Carbon.                                                                                                        
UV absorbance at   .................  ........................................................  ................
 254 nm (method                                                                                                 
 described in                                                                                                   
 preamble--protoc                                                                                               
 ol will be                                                                                                     
 developed).                                                                                                    
Simulated          .................  ........................................................  5710 E          
 Distribution                                                                                                   
 System Test                                                                                                    
 (SDS).                                                                                                         
Total Hardness...  .................  ........................................................  2340 B, 2340 C  
Ammonia..........  .................  ........................................................  4500-NH3 D, 4500-
                                                                                                 NH3 F          
Oxidant Demand/    .................  ........................................................  2350 B, 2350 C, 
 Requirement                                                                                     2350 D         
 (optional).                                                                                                    
AOC/BDOC           .................  ........................................................  9217 B/         
 (optional).                                                                                                    
----------------------------------------------------------------------------------------------------------------
\1\Currently approved methodology for drinking water compliance monitoring is listed in Title 40 of the Code of 
  Federal Regulations in the sections referenced in this column.                                                
\2\Standard Methods for the Examination of Water and Wastewater, 18th ed., American Public Health Association,  
  American Water Works Association, Water Pollution Control Federation, 1992.                                   
\3\``Methods of Chemical Analysis of Water and Wastes,'' EPA Environmental Monitoring Systems Laboratory,       
  Cincinnati, OH EPA-600/4-79-020, Revised March 1983.                                                          
\4\Methods for the Determination of Metals in Environmental Samples. Available from National Technical          
  Information Service (NTIS), U.S. Department of Commerce, Springfield, Virginia, PB91-231498, June 1991.       
\5\USEPA, ``Methods for the Determination of Organic Compounds in Drinking Water,'' EPA/600/4-88/039, PB91-     
  231480, National Technical Information Service (NTIS), December 1988 (revised July 1991).                     
\6\USEPA, ``Methods for the Determination of Organic Compounds in Drinking Water--Supplement II,'' EPA/600/R-92/
  129, PB92-207703, NTIS, August 1992.                                                                          
\7\USEPA, ``Methods for the Determination of Organic Compounds in Drinking Water--Supplement I,'' EPA/600/4-90- 
  020, PB91-146027, NTIS, July 1990.                                                                            
\8\Pentane may be used as the extraction solvent for this analyte, if the quality control criteria of the method
  are met.                                                                                                      
\9\This analyte is not currently included in the method. However, Barth and Fair (1992) present data            
  demonstrating it can be added to the method. The method is being revised for the 19th edition of Standard     
  Methods and it will include this analyte.                                                                     
\10\USEPA, ``Methods for the Determination of Inorganic Substances in Environmental Samples,'' EPA/600/R/93/100-
  , August 1993.                                                                                                


    Laboratory approval. EPA recognizes that the usefulness of the data 
generated as the result of this rule depends on the ability of 
laboratories to reliably analyze the disinfectants, disinfection by-
products and other parameters. EPA has a laboratory certification 
program for drinking water analyses. All laboratories that analyze 
drinking water samples to determine compliance with drinking water 
regulations must be certified by EPA or the State, as specified by 40 
CFR 142.10(b)(4) and 141.28. Under this program, EPA certifies the 
principal State Laboratory and, with certain exceptions (see 40 CFR 
142.10), each State certifies drinking water laboratories within the 
State.
    Laboratories currently certified to perform analyses using EPA 
Methods 501.1, 501.2, 502.2 or 524.2 for TTHMs or volatile organic 
compound (VOC) would be approved to analyze for chloroform, 
bromodichloromethane, dibromochloromethane, and bromoform using the 
same analytical method under the ICR without further action. In 
addition, all persons or laboratories already approved by EPA or the 
State for analyzing alkalinity, pH, temperature, turbidity, 
disinfectant residual, and calcium hardness analyses would be approved 
to perform these measurements under the ICR without further action. 
Parties approved by a State for calcium hardness analyses using 
Standard Methods 3500-Ca D would also be approved for total hardness 
measurements using Standard Method 2340 C under this rule. Parties 
approved by a State for calcium hardness analyses using Standard 
Methods 3111 B or 3120 B would also be approved for total hardness 
measurements using Standard Methods 2340 B under this rule. Parties 
approved by a State for pH measurements using Standard Methods 4500-
H+ would also be approved for ammonia measurements using Standard 
Method 4500-NH3 F under this rule.
    For other parameters to be monitored under this rule, EPA proposes 
to develop a separate laboratory evaluation process apart from the 
drinking water laboratory certification program. A new process is being 
proposed for several reasons: 1) few States and EPA Regions are 
currently able to certify laboratories for the new analytes of interest 
in this rule and it is unlikely that they could develop the capacity in 
the time frame to implement this rule; 2) the short-term nature of the 
monitoring period may not warrant a full certification program, since 
monitoring would not be required for many of the analytes after the 18 
month monitoring period; and 3) large numbers of laboratories are not 
needed to perform the DBP-related monitoring, because the monitoring 
requirements only affect approximately 300 systems.
    Under the new process, EPA would require laboratories to meet 
specific criteria (described below) before approving them to perform 
monitoring of the new analytes covered in the DBP portion of the ICR. 
Laboratories would be approved on a method-by-method basis.
    Laboratory approval criteria would consist of the following 
elements:
    (1) The laboratory would be required to contact ICR Laboratory 
Coordinator, USEPA, Office of Ground Water and Drinking Water, 
Technical Support Division, 26 West Martin Luther King Drive 
Cincinnati, Ohio, 45268, for an application form to initiate the 
approval process. The form would request information on the laboratory 
personnel, facilities, analytical methods/protocols in use for ICR 
analyses, current State certification status, and laboratory capacity 
to process DBP/ICR samples. The laboratory could submit a copy of the 
most recent application form it had filed with the State and the most 
recent copy of the State's on-site visit report, in lieu of completing 
portions of the EPA form. The laboratory could also provide EPA with 
copies of its PE data for ICR analytes in the three most recent PE 
studies. The PE data must have been generated using the methods for 
which the laboratory is seeking approval.
    (2) EPA would require the laboratory to use the analytical methods 
or protocols specified in this rule and contained in the ICR DBP 
Analytical Methods Guidance Manual. A laboratory that desires to use 
EPA Method 551 for trihalomethane analyses under this rule would have 
to apply for approval under this process, even though it may be 
certified for THM compliance monitoring using a different method.
    (3) EPA would require the laboratory to have a Quality Assurance 
(QA) Manual specific to this rule. Guidance for preparing this manual 
will be provided in the ICR DBP Analytical Methods Guidance Manual. 
(Examples of the types of information that should be included in the QA 
Manual are: (1) Laboratory organization; (2) sampling handling 
procedures; (3) analytical method references and quality control; and 
(4) data handling and reporting procedures. The QA manual would also 
include or reference the standard operating procedure (SOP) for each 
analytical method/protocol in use for ICR analyses.) The QA manual must 
be available for review, if requested.
    (4) EPA would require the laboratory to conduct an initial 
demonstration of capability (IDC) and method detection limit (MDL) 
determinations for each analysis for which it requests approval for 
this monitoring rule, and submit these data to the Agency. EPA would 
require laboratories to determine the MDL according to the procedure 
outlined in 40 CFR part 136 Appendix B, with additional guidance being 
given in the ICR DBP Analytical Methods Guidance Manual. The manual 
will also outline minimum requirements for performing the IDC 
determinations. Minimum performance criteria for each method IDC and 
MDL would also be specified in the ICR DBP Analytical Methods Guidance 
Manual based on what is feasible to achieve and what is necessary to 
obtain the data quality objectives of this rule. (EPA is proposing that 
the minimum performance criteria for IDCs and MDLs be based on IDC and 
MDL data obtained from a minimum of three laboratories that are 
experienced in conducting each specific analysis.)
    (5) If the laboratory does not have a history of successfully 
analyzing PE samples for the ICR analytes using the methods specified 
in this rule, then EPA would require the laboratory to satisfactorily 
analyze two PE samples, if available, for each of the methods it uses 
to generate data for this monitoring rule. Historical performance in PE 
studies could be applied toward meeting this requirement if the 
laboratory had satisfactory performance on at least two of three PE 
samples analyzed by the method in question and the last PE sample was 
satisfactorily analyzed. EPA proposes that satisfactory performance on 
PE samples be defined as achieving within 40% of the study 
mean concentration for this rule. EPA considers this criteria as 
reasonable relative to what laboratories should be able to achieve in 
order to meet the objectives of the rule.
    PE samples are currently available for THMs, six HAAs, chloral 
hydrate, bromate, chlorite, and chlorate. EPA plans to conduct special 
PE studies for the ICR which will also include trichloroacetonitrile, 
dichloroacetonitrile, bromochloroacetonitrile, dibromoacetonitrile, 
1,1- dichloropropanone, and 1,1,1-trichloropropanone, bromide, TOC, TOX 
and UV254 PE samples. A PE sample for chloropicrin will not be 
required because laboratory performance using EPA Method 551 can be 
assessed using the data from the other method analytes.)
    EPA is considering using a third party (independent organization) 
to review the application form, IDC, MDL, and PE study data and conduct 
an on-site inspection, if necessary. Based upon the third party's 
assessment of the laboratory, EPA would approve laboratories. EPA 
solicits comment on this process or other options such as laboratories 
paying for the review by a third party.
    Implementation of the laboratory approval process would begin upon 
promulgation of the ICR and it would extend until the end of the first 
quarter period of monitoring, following the beginning effective date of 
this rule, but possibly later, if EPA determines that insufficient 
laboratories through that date had been approved. No additional 
laboratories would be evaluated after this period unless there was not 
adequate laboratory capacity to handle the monitoring required by the 
DBP ICR. If additional capacity was required, then new laboratories 
would be evaluated until the necessary capacity was reached.
    EPA proposes that a list of ``approved'' laboratories be made 
available to all the utilities required to monitor for DBPs, their 
precursors and surrogates. The list would be distributed directly to 
the utilities, as well as to each EPA Regional Office and State Primacy 
Agency. The list would also be available for public distribution from 
EPA.
    EPA would monitor the performance of ``approved'' laboratories 
throughout the ICR monitoring period by requiring the laboratories to: 
(1) periodically (either quarterly or semiannually, depending on 
feasibility) analyze PE samples; and (2) report specific quality 
control (QC) data with the analytical results from the monitoring 
samples. Maintaining laboratory ``approval'' throughout the ICR 
monitoring period would be contingent upon successfully meeting the 
acceptance criteria for the PE samples and the quality control data. 
The required QC data and performance criteria would be included in the 
ICR DBP Analytical Methods Guidance Manual. (An overview is presented 
in Section 6 of this preamble under Analytical Data.) Laboratories that 
do not pass a PE sample would receive another PE sample before the next 
regularly scheduled EPA PE study, to demonstrate successful completion 
of corrective action. EPA, either directly or by third party, would 
provide technical assistance to laboratories that had initially been 
``approved'' and then develop problems, if the operation of such 
laboratories is necessary to maintain the lab capacity to fulfill the 
requirements of this rule.
    Laboratory capacity. EPA recognizes that obtaining the necessary 
laboratory capacity to complete the DBP monitoring required by this 
rule may be difficult. For this reason, as for pathogen monitoring, EPA 
is proposing a period within which monitoring could be initiated and 
completed. Systems would be required to conduct microbial and DBP 
monitoring simultaneously, beginning as soon as EPA approved 
laboratories could be identified for conducting both analysis. However, 
TOC monitoring would not be delayed because these data are required to 
assess which systems would need to do bench or pilot scale testing of 
precursor removal technologies. Therefore, all TOC monitoring must 
begin by [insert date 3 months following the promulgation of this 
rule]. EPA also proposes to delay or omit the monitoring of certain 
analytes, if their inclusion would cause undue delay in the start of 
monitoring for the remainder of the analytes. Monitoring would not be 
omitted for the following parameters: (1) Trihalomethanes; (2) 
haloacetic acids; (3) bromate; (4) chlorite; (5) chlorate; (6) total 
organic halide; (7) total organic carbon; and (8) bromide. EPA requests 
comments on this issue.
    EPA is concerned about the feasibility of developing laboratory 
capacity for measuring cyanogen chloride (CNCL) and aldehydes. In 
addition, EPA is concerned about its ability to evaluate laboratories 
that may develop capabilities for measuring these analytes, because PE 
samples will not be available. These issues are described below.
    EPA has several concerns about the measurement of CNCl. The first 
issue is one of safety. Analytical standards must be prepared from pure 
CNCl, because pure CNCl is the only commercially available material. 
The worker who prepares the stock liquid CNCl standards must be 
experienced in the preparation of liquid standards from gases. Due to 
the toxicity of the compound, special precautions must be taken to 
ensure the safety of the worker. Few laboratories that specialize in 
analyses of drinking water are equipped to prepare CNCl standards from 
pure gas.
    One solution to the safety issue would be for EPA to provide liquid 
CNCl standards to laboratories that perform this analysis for the ICR. 
EPA is not certain that development of liquid CNCl standards is 
feasible within the time frame of this rule. In addition, EPA is 
concerned about the ability to evaluate the performance of laboratories 
that conduct this analysis.
    EPA does not have the resources to develop performance evaluation 
(PE) samples for CNCl or aldehydes in time to meet the requirements of 
this regulation. An alternative approach to compare laboratory 
performance would be to conduct round robin interlaboratory studies 
using whole volume samples. Due to issues concerning the stability of 
CNCl and aldehydes and limited data on the intralaboratory performance 
of the methods, the results from round robin interlaboratory studies 
would be very difficult to interpret.
    One of EPA's laboratories has the capability to measure CNCl in 
water using EPA Method 524.2 and to measure aldehydes using the PFBHA 
methodology. Utilities could be required to send all samples for CNCl 
and aldehyde analyses to EPA. Having one laboratory perform all these 
analyses for the ICR would eliminate the data variability that results 
from multiple laboratory analyses, thus producing more precise data. 
Greater precision would make it easier to determine how treatment 
practices and source water characteristics influence CNCl and aldehyde 
formation. EPA solicits comment on this approach for obtaining CNCl and 
aldehyde measurements.
6. Quality Assurance
    The integrity of the DBP monitoring database is contingent upon 
accurate and precise analytical data from the samples, accurate plant 
process information from each utility, and correct input of the data 
into the database. EPA proposes that each utility prepare a Quality 
Assurance Project Plan (QAPP) specific for the ICR monitoring. The QAPP 
would cover the entire project starting with the objectives of the 
project, through the sampling strategy and procedures, laboratory 
procedures and analytical methods and finally, the data handling and 
reporting processes. Guidance for preparing it would be provided in an 
ICR Guidance Manual.
    Sampling. The sampling for this rule would primarily be done by the 
system. Each system has its own sampling regime and protocol for the 
currently regulated contaminants. Sampling for the unregulated DBPs is 
more complex, and will require greater coordination with the analytical 
laboratory. As a result, EPA intends to develop a sampling guidance 
manual to describe the proper sampling techniques for use in complying 
with this rule. The manual would describe: (1) Sample containers; (2) 
sampling techniques; (3) required preservatives and dechlorinating 
agents; (4) sample shipping conditions; and (5) sample holding times 
and conditions. Samplers would be required to follow the specifications 
outlined in the manual. EPA solicits comments concerning alternative 
mechanisms for ensuring consistency in the sampling aspects of the 
study.
    Analytical data. The analytical data for this rule may be generated 
by many laboratories. As a result, the data will have variable 
characteristics such as: (1) Detection level; (2) precision; and (3) 
bias. As a first step to ensuring data comparability, EPA would require 
laboratories to use the specific analytical methods or protocols 
outlined in the ICR and described in the ICR DBP Analytical Methods 
Guidance Manual. An additional technique that may be employed to assist 
in data comparability is to require all laboratories to obtain their 
primary standards (i.e., standards which laboratories use to calibrate 
their instruments) from the same source. EPA is evaluating the cost of 
providing primary standards for the major ICR analytes to laboratories 
``approved'' for performing analyses for the ICR.
    In addition, EPA proposes that minimum quality control acceptance 
criteria be established for all data that are entered into the DBP 
database. A workgroup will establish acceptance criteria for each 
parameter being measured based on the data quality objectives necessary 
for successfully completing the monitoring study objectives. These 
criteria will be included in the ICR DBP Analytical Methods Guidance 
Manual. The performance of the method as it is routinely used in 
laboratories currently doing the same analysis will be used as a guide 
for determining feasibility in meeting the data quality objectives. 
Laboratories will be required to: (1) Demonstrate the absence of 
interferences from background contamination by analyzing method and/or 
shipping blanks, depending upon the method at a specified frequency; 
(2) achieve quantitative recovery of surrogate standards that are 
spiked into samples for some analytical methods; (3) achieve 
quantitative recovery of the internal standard when its use is 
specified in the method/protocol; (4) perform a specified minimum 
number of duplicate analyses and analyses of fortified samples (or 
reagent water, depending upon the analysis) with each batch of samples 
processed through the analytical procedure; (5) achieve a specified 
level of precision and accuracy for each batch of samples. Where 
appropriate, calibration will require a specified number of procedural 
standards, as well as periodic verification of quantitation at the 
minimum reporting level. The ICR Analytical Methods Guidance Manual 
will contain specific criteria for: (1) The quality control (QC) 
procedures that must be followed with each analytical method or 
protocol; (2) the minimum reporting level for each method/protocol and 
a method for demonstrating it (The minimum reporting level, which is 
the level at which laboratories will be able to accurately and 
precisely measure the analyte, will be higher than the method detection 
limit [MDL]); and (3) data quality acceptance criteria for each method/
protocol. The QC procedures and acceptance criteria may be more 
stringent than the specifications in the current versions of the 
methods based on ICR data quality objectives. Concentrations below the 
minimum reporting level specified for each method/protocol will be 
reported as ``zero'' in the database. EPA requests comments on the use 
of zero in the database to indicate concentrations below the reporting 
level, or whether data should be reported as low as the MDL level.
    EPA would require laboratories to include the above mentioned QC 
data with the analytical results for the samples in the reports they 
send to the systems. The Agency would provide systems guidance on how 
to evaluate the QC data. Monitoring data that meet the minimum QC 
acceptance criteria (as specified in the ICR DBP Analytical Methods 
Manual) would be reported to EPA along with a subset of the associated 
QC data. The utility would send the QC information and identification 
of the laboratories to EPA using the same mechanism as it uses to 
report plant process and monitoring data. In some cases, the QC data 
for a batch of samples will be shared by two or more utilities (e.g., 
analyses of laboratory fortified blanks). EPA would require both the 
laboratory and utility to report to EPA the extraction and analysis 
dates for each batch of samples.
    The QC data would be entered into the DBP database along with the 
analytical data. Computer algorithms will be used to determine if the 
data meet the specified QC criteria and the data will be classified as 
acceptable or marginally acceptable. Systems would not submit to EPA 
data that do not meet the minimum QC criteria. Instead, the utility 
will notify EPA of the reason for losing the sample (i.e, breakage, 
sample holding time exceeded, laboratory QC out of control, etc.). When 
the laboratory fails to consistently meet performance criteria, EPA 
would assist the system in finding an alternate laboratory for future 
monitoring. EPA would also provide technical assistance, upon request, 
either directly or through a contractor, to laboratories who develop 
technical difficulties in measuring critical ICR analytes, to maintain 
the necessary laboratory capacity and capability to complete the ICR 
monitoring. EPA requests comments on the QA/QC criteria for data entry 
into the database.
    Treatment plant process data. To maintain quality and integrity of 
data input, EPA would undertake some level of review of system data. 
The Agency would screen the data for proper use of the input software, 
proper electronic transfer of data, submission of all required data and 
plant operating information, reasonableness and completeness of the 
data, consistency with previous reports, etc. EPA requests comment on 
how the data review should be conducted.
7. Bench/Pilot Scale Testing
    During the negotiation of the D/DBP rule, the Negotiating Committee 
agreed to require surface water systems serving greater than 100,000 
people and ground water systems serving greater than 50,000 people to 
conduct bench or pilot studies on DBP precursor removal, using either 
GAC or membrane filtration, unless these systems met certain water 
quality conditions or already had such full scale treatment in place. 
The purpose of this requirement was twofold: (a) To obtain more 
information on the cost effectiveness of GAC and membrane technology 
for removing DBP precursors and reducing DBP levels, and (b) to 
accelerate the time that systems would need to install such full scale 
technology if they were required to do so under the Stage 2   D/DBP 
rule. The proposed rule would require each system to complete the 
study, including a report describing the results and conclusion of the 
study, by September 1997.
    The Negotiating Committee also considered whether these objectives 
could be met without all systems conducting the studies, and if so, how 
resources that would otherwise be devoted to bench/pilot scale testing 
could be used to fill other possible data gaps. EPA is exploring 
alternatives to the proposed regulations if it is determined that not 
all systems need to undertake the studies in order to fulfill the 
objectives of these requirements. One possibility is for the final rule 
to provide that some systems that would otherwise conduct the studies 
could instead pool their resources (in an amount equivalent to the cost 
of such studies) to contribute to funding key research identified 
during the negotiated rule-making process. EPA is exploring an 
arrangement with a third party organization to use those pooled 
resources to undertake such efforts. Such a project would be conducted 
under the guidance of an advisory group representing the participants 
in the negotiated rule-making. EPA solicits comments on the approach 
and which criteria could be used in the final rule for determining 
which systems could participate in this alternative. EPA also solicits 
comments on other means for accomplishing the objective of maximizing 
data collection resources.
    The Negotiating Committee agreed that systems using surface water 
would not have to conduct the bench pilot scale studies if they met 
either of the following conditions: (1) System uses chlorine as the 
primary and residual disinfectant and had an annual average of less 
than 40 g/l for total trihalomethanes and less than 30 
g/l for total haloacetic acids (HAAS), or (2) the TOC level in 
the raw water before disinfection is less than 4.0 mg/l, based on an 
average of monthly measurements for one year beginning [insert 3 months 
following the promulgation date of this rule]. Systems using ground 
water would not have to conduct a study if the TOC in the finished 
water is less than 2.0 mg/l, based on an average of monthly 
measurements for one year beginning [insert 3 months following the 
promulgation date of this rule].
    EPA is proposing that the treatment studies be designed to yield 
representative performance data and allow the development of treatment 
cost estimates for different levels of organic disinfection byproduct 
control. The treatment study would be conducted with the effluent from 
treatment processes already in place that remove disinfection byproduct 
precursors and TOC. Depending upon the type of treatment study that is 
made, the study would be conducted in accordance with the following 
criteria.
    Bench scale testing. Bench-scale testing would be defined as 
continuous flow tests: (1) Rapid small scale column test (RSSCT) for 
GAC (Crittenden et al. 1991; Sontheimer et al. 1988; Summers et al. 
1992; Cummings et al., 1992); and (2) reactors with a configuration 
that yield representative flux loss assessment for membranes. Both the 
RSSCT and membrane system test can be adversely affected by the 
presence of particles. Therefore, both tests would be preceded by 
particle removal processes, such as microfiltration.
    GAC bench-scale testing would include the following information on 
each RSSCT: Pretreatment conditions, GAC type, GAC particle diameter, 
height and dry weight (mass) of GAC in the RSSCT column, RSSCT column 
inner diameter, volumetric flow rate, and operation time at which each 
sample is taken. EPA would require the testing of at least two empty 
bed contact times (EBCTs) using the RSSCT. The Agency would require 
these RSSCT EBCTs to be designed to represent a full-scale EBCT of 10 
min and a full-scale EBCT of 20 min. Additional EBCTs could be tested. 
The RSSCT testing would include the water quality parameters and 
sampling frequency listed in Table III.8. The RSSCT would be run until 
the effluent TOC concentration is 75% of the average influent TOC 
concentration or a RSSCT operation time that represents the equivalent 
of one year of full-scale operation, whichever is shortest. The average 
influent TOC would be defined as the running average of the influent 
TOC at the time of effluent sampling. RSSCTs would be conducted 
quarterly over one year to obtain the seasonal variation. Thus a total 
of four RSSCTs at each EBCT is required. If, after completion of the 
first quarter RSSCTs, the system finds that the effluent TOC reaches 
75% of the average influent TOC within 20 full-scale equivalent days on 
the EBCT=10 min test and within 30 full-scale equivalent days on the 
EBCT=20 min test, then the last three quarterly tests would be 
conducted using membrane bench-scale testing with only one membrane, as 
described in Section 141.142 (b)(1)(B). (Crittenden et al. 1991; 
Sontheimer et al. 1988; Summers et al. 1992; Cummings et al. 1992)

           Table III.8.--Sampling of GAC Bench-Scale Systems            
------------------------------------------------------------------------
 Sampling point        Analyses                Sample frequency         
------------------------------------------------------------------------
GAC influent...  Alkalinity, total &   Two samples per batch of influent
                  calcium hardness,     evenly spaced over the RSSCT    
                  ammonia and bromide.  run.                            
GAC influent...  pH, turbidity,        Three samples per batch of       
                  temperature, TOC      influent evenly spaced over the 
                  and UV254. SDS1 for   RSSCT run.                      
                  THMs, HAA6, TOX,                                      
                  and chlorine demand.                                  
GAC effluent @   pH, temperature, TOC  A minimum of 12 samples. One     
 EBCT=10 min      and UV254. SDS1 for   after one hour, and thereafter  
 (scaled).        THMs, HAA6, TOX,      at 5% to 8% increments of the   
                  and chlorine demand.  average influent TOC.           
GAC effluent @   pH, temperature, TOC  A minimum of 12 samples. One     
 EBCT=20 min      and UV254. SDS1 for   after one hour, and thereafter  
 (scaled).        THMs, HAA6, TOX,      at 5% to 8% increments of the   
                  and chlorine demand.  average influent TOC.           
------------------------------------------------------------------------
\1\SDS conditions are defined in Section 141.142 (b)(4).                


    (B) EPA would require the membrane bench-scale testing to include 
the following information: pretreatment conditions, membrane type, 
membrane area, configuration, inlet pressure and volumetric flow rate, 
outlet (reject) pressure and volumetric flow rate, permeate pressure 
and volumetric flow rate, recovery, and operation time at which each 
sample is taken. A minimum of two different membrane types with nominal 
molecular weight cutoffs of less than 1000 would be investigated. The 
membrane test system would need to be designed and operated to yield a 
representative flux loss assessment. The system would conduct membrane 
tests quarterly over one year to obtain the seasonal variation. Thus, 
the system would run a total of four membrane tests with each membrane. 
The membrane bench-scale testing would include the water quality 
parameters and sampling frequency, as listed in Table III.9.

        Table III. 9.--Sampling of Bench-Scale Membrane Systems         
------------------------------------------------------------------------
Sampling point         Analyses                Sample frequency2        
------------------------------------------------------------------------
Membrane         Alkalinity, total     Two samples per batch of influent
 influent.        dissolved solids,     evenly spaced over the membrane 
                  total & calcium       run. If a continuous flow (non- 
                  hardness and          batch) influent is used then    
                  bromide.              samples are taken at the same   
                                        time as the membrane effluent   
                                        samples.                        
Membrane         pH, turbidity,        Three samples per batch of       
 influent.        temperature, HPC,     influent evenly spaced over the 
                  TOC and UV254. SDS1   membrane run. If a continuous   
                  for THMs, HAA6,       flow (non-batch) influent is    
                  TOX, and chlorine     used then samples are taken at  
                  demand.               the same time as the membrane   
                                        effluent samples.               
Membrane         pH, alkalinity,       A minimum of 8 samples evenly    
 permeate for     total dissolved       spaced over the membrane run.   
 each membrane    solids, turbidity,                                    
 tested.          temperature, total                                    
                  & calcium hardness,                                   
                  bromide, HPC, TOC                                     
                  and UV254. SDS1 for                                   
                  THMs, HAA6, TOX,                                      
                  and chlorine demand.                                  
------------------------------------------------------------------------
1SDS conditions are defined in Section 141.142(b)(4).                   
2More frequent monitoring of flow rate and pressure would be required to
  accurately assess flux loss.                                          


    Pilot-scale testing. Under the proposal, EPA defines pilot-scale 
testing as continuous flow tests: (1) Using GAC of particle size 
representative of that used in full-scale practice, a pilot GAC column 
with a minimum inner diameter of 2.0 inches, and hydraulic loading rate 
(volumetric flow rate/column cross-sectional area) representative of 
that used in full-scale practice, and (2) using membrane modules with a 
minimum of a 4.0 inch diameter for spiral wound membranes or equivalent 
membrane area if other configurations are used.
    GAC pilot-scale testing would include the following information on 
the pilot plant: Pretreatment conditions, GAC type, GAC particle 
diameter, height and dry weight (mass) of GAC in the pilot column, 
pilot column inner diameter, volumetric flow rate, and operation time 
at which each sample is taken. If pilot scale testing were conducted, 
at least two EBCTs would be required to be tested, EBCT=10 min and 
EBCT=20 min, using the pilot-scale plant. Additional EBCTs could be 
tested. The pilot testing would include the water quality parameters 
listed in Table III.10. The pilot tests would be run until the effluent 
TOC concentration is 75% of the average influent TOC concentration, 
with a maximum run length of one year. The average influent TOC would 
be defined as the running average of the influent TOC at the time of 
sampling. The pilot-scale testing should be sufficiently long to 
determine the seasonal variation.

           Table III.10.--Sampling of GAC Pilot-scale Systems           
------------------------------------------------------------------------
Sampling point         Analyses                 Sample frequency        
------------------------------------------------------------------------
GAC influent...  pH, alkalinity,       A minimum of 15 samples taken at 
                  turbidity,            the same time as the samples for
                  temperature, total    GAC effluent at EBCT=20 min.    
                  & calcium hardness,                                   
                  ammonia, bromide,                                     
                  TOC and UV254. SDS1                                   
                  for THMs, HAA6,                                       
                  TOX, and chlorine                                     
                  demand.                                               
GAC effluent @   pH, turbidity,        A minimum of 15 samples. One     
 EBCT=10 min.     temperature,          after one day, and thereafter at
                  ammonia,2 TOC and     3% to 7% increments of the      
                  UV254. SDS1 for       average influent TOC.           
                  THMs, HAA6, TOX,                                      
                  and chlorine demand.                                  
GAC effluent @   pH, turbidity,        A minimum of 15 samples. One     
 EBCT=20 min.     temperature,          after one day, and thereafter at
                  ammonia,2 TOC and     3% to 7% increments of the      
                  UV254. SDS1 for       average influent TOC.           
                  THMs, HAA6, TOX,                                      
                  and chlorine demand.                                  
------------------------------------------------------------------------
1SDS conditions are defined in Section 141.142 (b.4).                   
2If present in the influent.                                            
                                                                        
 Note: More frequent effluent monitoring may be necessary in order to   
  predict the 3% to 7% increments of average influent TOC.              


    If membrane pilot-scale testing were conducted it would include the 
following information on the pilot plant: pretreatment conditions, 
membrane type, configuration, staging, inlet pressure and volumetric 
flow rate, outlet (reject) pressure and volumetric flow rate, permeate 
pressure and volumetric flow rate, recovery, operation time at which 
each sample is taken, recovery, cross flow velocity, recycle flow rate, 
backwashing and cleaning conditions, and characterization and ultimate 
disposal of the reject stream. The membrane test system would be 
designed to yield a representative flux loss assessment. The pilot-
scale testing shall be sufficient in length, and conducted throughout 
the year in order to capture the seasonal variation, with a maximum run 
length of one year. The pilot testing would include the water quality 
parameters as listed in Table III.11. 

        Table III.11.--Sampling of Pilot-scale Membrane Systems         
------------------------------------------------------------------------
Sampling point         Analyses                Sample frequency3        
------------------------------------------------------------------------
Membrane         pH, alkalinity,       A minimum of 15 samples to be    
 influent.        total dissolved       taken at the same time as the   
                  solids, turbidity,    membrane effluent samples.      
                  temperature, total                                    
                  & calcium hardness,                                   
                  ammonia, bromide,                                     
                  HPC, TOC and UV254.                                   
                  SDS1 for THMs,                                        
                  HAA6, TOX, and                                        
                  chlorine demand.                                      
Membrane         pH, alkalinity,       A minimum of 15 samples evenly   
 permeate.        total dissolved       spaced over the membrane run.   
                  solids, turbidity                                     
                  temperature, total                                    
                  & calcium hardness,                                   
                  ammonia2, bromide,                                    
                  HPC, TOC and UV254.                                   
                  SDS1 for THMs,                                        
                  HAA6, TOX, and                                        
                  chlorine demand.                                      
------------------------------------------------------------------------
1SDS conditions are defined in Section 141.142(b)(4).                   
2If present in the influent.                                            
3More frequent monitoring of flow rate and pressure will be required to 
  accurately assess flux loss.                                          


    Pretreatment analysis. EPA would require that influent water to 
either bench- or pilot-scale tests be taken at a point before the 
addition of any oxidant or disinfectant that forms chlorinated 
disinfection byproducts. If the oxidant or disinfectant addition 
precedes any full-scale treatment process that removes disinfection 
byproduct precursors, then bench- or pilot-scale treatment processes 
that simulate this full-scale treatment process would be required prior 
to the GAC or membrane process.
    Simulated distribution system analysis. EPA would require the use 
of simulated distribution system (SDS) conditions with chlorine before 
the measurement of THMs, HAA6, TOX and chlorine demand. These 
conditions would be based on the site-specific SDS sample, as defined 
in Section 141.141(c) (Table 1) with regard to holding time, 
temperature, and chlorine residual. If chlorine is not used as the 
final disinfectant in practice, then a chlorine dose should be set to 
yield a free chlorine residual of 0.2 mg/l after a holding time equal 
to the longest period of time the water is expected to remain in the 
distribution system or seven days, whichever is shortest. The holding 
time prior to analysis of THMs, HAA6, TOX and chlorine demand would be 
required to remain as that of the SDS sample as defined in 
Sec. 141.141(c) (Table 1).
    Systems with multiple source waters. For systems with multiple 
source waters, bench-or pilot scale testing would be required for each 
treatment plant that serves a population greater than 100,000 (surface 
water supplies) or 50,000 (ground water supplies) and uses a 
significantly different source water. EPA would provide guidance for 
making such determinations.
    EPA would require a groundwater system with multiple wells from the 
same aquifer to monitor TOC from one sampling point to determine if a 
bench or pilot scale study is required. A ground water system with 
multiple wells from different aquifers must sample TOC from at least 
two wells from each of the aquifers with the highest TOC 
concentrations, as determined from at least one sample from each 
aquifer.
    Reporting. Under this rule, EPA would require all systems 
conducting bench or pilot scale studies to report to the Agency the 
additional information in Table 6 of Sec. 141.141, as appropriate, for 
source water and treatment processes that precede the bench/pilot 
systems. This information is to be reported for full-scale pretreatment 
processes and for pilot- or bench-scale pretreatment processes where 
appropriate.
    Selection of bench versus pilot scale and membrane versus GAC 
studies. Bench-scale GAC studies (RSSCTs) are less expensive than pilot 
studies and produce information based on the ability of GAC to adsorb 
TOC. Pilot-scale studies of GAC produce information more representative 
of TOC removal at full-scale.
    Removal of TOC by GAC in full-scale water treatment plants is a 
function of two processes that occur simultaneously: adsorption on the 
surface of GAC and biological degradation. Pilot scale studies are the 
most economical way to demonstrate both processes on a continuous flow 
basis.
    By their nature, RSSCT studies are of short duration and designed 
to measure adsorption of organic compounds. Biological activity is 
discouraged through various means and if biological degradation does 
occur, the RSSCT results are invalid.
    Pilot-scale GAC studies produce a time-averaged result of the 
influent TOC, whereas RSSCT studies are run on batches of water (50-100 
gallons) collected at discrete time periods. Pilot-scale GAC effluent 
data will reflect large spikes of influent TOC concentrations which can 
degrade the process performance. The RSSCT procedure cannot duplicate 
this process, and can only reflect higher than normal influent TOC 
concentrations if the batch sample collects the TOC spike.
    Bench-scale membrane studies would only generate limited data on 
DBP removal, primarily TOC removal. Moreover, what data is generated 
would be constrained by limited membrane flux information that is 
critical for generating membrane cost data. Consequently, EPA 
recommends that membrane performance and cost data for DBP control be 
generated by pilot-scale studies rather than bench studies.
    Most large systems may choose GAC for DBP removal studies, rather 
than membrane technology, due to the economies of scale associated with 
full-scale GAC treatment. However, systems with very poor source waters 
may more easily achieve low TOC levels in the treated water with 
membrane technology. A goal of this portion of the ICR is to obtain 
data from a number of pilot-scale studies for both membrane and GAC 
technologies as input to Stage 2 rule development. Without EPA 
specifically requiring that these pilot-scale studies be conducted, it 
remains unclear whether an adequate number of such studies will be 
done. A major issue is how to equitably encourage utilities to produce 
these studies.
    Table III.12 is a summary of the type and number of pilot studies 
expected to be needed for Stage 2 Rule development as discussed by the 
Negotiating Committee during the rule negotiation process. 

 Table III.12.--Number of Pilot Studies Needed for Stage 2 Organized by 
                              TOC Category                              
------------------------------------------------------------------------
                                       TOC concentrations, mg/L         
                              ------------------------------------------
      Pilot study type                   >8 to    \12 to                
                               >4 to 8     12       16          \16     
------------------------------------------------------------------------
GAC..........................       10       10       10      XXXXXXXXXX
Membrane.....................        2        2        2               2
------------------------------------------------------------------------



    EPA does not recommend GAC studies at very high TOC concentrations, 
due to the rapid breakthrough of TOC at empty bed contact times (EBCTs) 
of 10 and 20 minutes. The Agency believes that to ensure that the 
categories in Table III.12 are properly covered, the Agency would need 
to tell individual systems which concentration category to use. The 
water system representatives on the Negotiating Committee agreed to 
conduct a survey of systems serving more than 100,000 people, in 
conjunction with EPA, to identify which systems have a pilot plant 
suitable for running GAC studies in the post-filter adsorber mode or 
intend to build one in the near future. These systems will also be 
asked if they are willing to conduct pilot-scale membrane studies.
    EPA would also request systems to provide limited water quality 
data to enable EPA to assess a TOC concentration range and, if 
possible, a TOC ``type'' to the water to be tested. If the nature of 
the TOC cannot be classified, EPA would select waters from different 
sections of the country to cover the matrix in Table III.12.
    Based on the results of the survey, EPA may request systems with 
pilot plants to perform GAC or membrane pilot studies instead of an 
RSSCT. Systems with pilot plants in place should be able to perform GAC 
pilot studies at a fraction of the cost of having to build one from 
scratch. The cost should not be much greater than running an RSSCT.
    EPA developed the above described survey approach with follow up 
voluntary pilot plant studies among candidate utilities to encourage a 
wide range of studies for different types of waters and DBP precursors 
needed to be studied. The Negotiating Committee also discussed the 
advisability of requiring Subpart H systems to perform a pilot-scale 
study if (1) the systems have a raw water TOC concentration greater 
than 4.0 mg/L and serve more than 500,000 people, or (2) the systems 
have a raw water TOC concentration above a specified concentration and 
serve more than 100,000 people.
    The Negotiating Committee developed all of the above options 
because of the uncertainty of the distribution of TOC concentrations in 
the source waters for large systems and the desire to produce useful 
data for developing the Stage 2 D/DBP Rule. EPA solicits comment on how 
to ensure an adequate number of pilot scale studies for both membranes 
and GAC technology. If EPA finds that an insufficient number of systems 
are willing to conduct pilot-scale testing as a follow-up to the 
survey, what should the Agency require to ensure that the desired 
number of studies indicated in Table III.12 are done? Should EPA select 
the sites for GAC and membrane pilot studies, according to system size, 
TOC concentration, or both? Also, how can the site selection process 
ensure that membranes are used in some of the pilot studies?

C. Dates

    EPA is proposing to require systems serving 100,000 or more people 
to begin to monitor microbial (for Subpart H systems only), chemical, 
and treatment process parameters no earlier than [insert date three 
months following promulgation date of this rule] and no later than 
October 1995. The exception to this is for TOC monitoring which must 
begin [insert first day of month three months following promulgation 
date]. Once monitoring has begun, these systems would be required to 
monitor for 18 consecutive months and would have to be finished no 
later than March 31, 1997.
    Systems required to monitor both microbiological (under 
Sec. 141.140) and chemical parameters would have to conduct both types 
of monitoring concurrently for 18 consecutive months. This monitoring 
regimen would allow for evaluation of both treatment efficacy and DBP 
formation.
    Systems serving between 10,000 and 99,999 people would begin to 
monitor microbial and treatment process parameters no earlier [insert 
month three months following promulgation date] and no later than April 
1996. Once monitoring has begun, these systems would be required to 
monitor every other month for 12 consecutive months and would have to 
be finished no later than March 31, 1997.
    Subpart H systems serving 100,000 or more people and ground water 
systems serving 50,000 or more people would begin bench- or pilot-scale 
studies no later than [insert month 18 months after promulgation of 
rule] and be required to complete the studies by September 1997, unless 
the system met one of the criteria to avoid studies.
    Prior to the start of monitoring, systems must arrange to have 
samples analyzed by an EPA approved laboratory. If systems serving 
greater than 100,000 people are not able to arrange to have samples 
analyzed by such a laboratory by [insert date six months after 
publication of the final rule in the Federal Register], they are 
required to notify EPA. If systems serving between 10,000 and 100,000 
people are not able to arrange to have samples analyzed by such a 
laboratory by [insert date nine months after publication of the final 
rule in the Federal Register], they are required to notify EPA. EPA 
will then provide a list of approved labs or other necessary guidance.
    In summary of what has been stated previously in parts, the purpose 
of the monitoring under this rule is to (a) determine if an ESWTR is 
necessary, and if so, to support the development of appropriate 
criteria in both the interim and long-term ESWTR, (b) assist utilities 
in the implementation of the interim ESWTR if such a rule becomes 
necessary, and (c) support the development of the Stage 2 D/DBP Rule.
    The above monitoring schedules, albeit tight, were agreed to by the 
Negotiating Committee as part of the regulation negotiation process. 
The schedules for compiling monitoring data are tight because the 
Negotiating Committee placed a time limit of December 1996 for 
promulgating an interim ESWTR and a Stage 1 D/DBP Rule. For this 
schedule to be realized a large number of utilities will need to 
initiate monitoring beginning shortly after October 1994 so that EPA 
can analyze the data and consider them in promulgating the interim 
ESWTR. EPA is making every possible effort to ensure that enough 
laboratories can be approved to generate the necessary data within the 
desired time frame. Systems are encouraged to generate data as quickly 
as possible so that their data will be considered in the interim ESWTR. 
Data generated after the time EPA believes it has sufficient data to 
promulgate the interim ESWTR will be used to develop the long-term 
ESWTR, and assist utilities in the implementation of the interim ESWTR.
    Before promulgating the interim ESWTR, EPA intends to issue a 
Notice of Availability to: (a) Discuss the pertinent data collected 
under the ICR rule, (b) discuss additional research that would 
influence determination of appropriate regulatory criteria, (c) discuss 
criteria EPA considered appropriate to promulgate in the interim ESWTR 
(which would be among the regulatory options of the proposed interim 
ESWTR) and (d) solicit public comment on the intended criteria to be 
promulgated. Following consideration of public comments received, EPA 
would promulgate the interim ESWTR and the Stage 1 D/DBP rule at the 
same time to reduce the possibility that a system might unduly 
compromise its control of pathogens while complying with the Stage 1 D/
DBP rule. Table III.13 indicates the anticipated schedule by which the 
various rules would be proposed, promulgated and become effective. Even 
though the December 1993 date has not been met, EPA is hopeful that 
other dates will not slip commensurately.

Table III.13.--Proposed Time Frame of D/DBP, ESWTR, ICR Rule Development
                                                                        
------------------------------------------------------------------------
                    Stage 1 D/DBP      Stage 2 D/DBP                    
   Time line            rule               rule              ESWTR      
------------------------------------------------------------------------
12/93...........  .................  Propose            Propose         
                                      information        information    
                                      collection         collection     
                                      requirements for   requirements   
                                      systems >100k.     for systems    
                                                         >10k.          
3/94............  Propose enhanced   Propose Stage 2.   Propose interim 
                   coagulation        MCLs for TTHMs =   ESWTR for      
                   requirement for    40 g/l,   systems >10k.  
                   systems with       THAAs = 30                        
                   conventional       g/l,                     
                   treatment; MCLs    BAT as precursor                  
                   for TTHMs = 80     removal with                      
                   g/l ,     chlorination.                     
                   HAAs = 60 g/l. MCLs for                                      
                   bromate,                                             
                   chlorite, limits                                     
                   for                                                  
                   disinfectants                                        
                   for all                                              
                   systems.except                                       
                   TNCWSs.                                              
6/94............  .................  Promulgate ICR...  Promulgate ICR. 
8/94............  Close of public    .................  Close of public 
                   comment period.                       comment period 
                                                         to proposed    
                                                         ESWTR.         
10/94...........  .................  Systems >100,000   Systems begin   
                                      begin ICR          ICR monitoring.
                                      monitoring.                       
10/95...........  .................  SW systems >100k                   
                                      and GW systems                    
                                      >50k begin bench/                 
                                      pilot studies                     
                                      unless source                     
                                      water quality                     
                                      criteria met..                    
11/95...........  .................  .................  Notice of       
                                                         availability on
                                                         monitoring data
                                                         and direction  
                                                         of interim     
                                                         ESWTR.         
1/96............  .................  .................  Close of public 
                                                         comment period 
                                                         to NOA.        
12/96...........  Promulgate Stage   .................  Promulgate      
                   1.                                    interim ESWTR  
                                                         systems >10k.  
3/97............  .................  Systems complete   Systems complete
                                      ICR monitoring.    ICR monitoring.
6/97............  .................  Notice of          Propose long-   
                                      availability for   term ESWTR for 
                                      Stage 2            systems <10k,  
                                      reproposal.        possible       
                                                         changes for    
                                                         systems >10k.  
10/97...........  .................  Complete and       ................
                                      submit results                    
                                      of bench/pilot                    
                                      studies.                          
12/97...........  .................  Initiate           ................
                                      reproposal--begi                  
                                      n with 3/94                       
                                      proposal.                         
6/98............  Effective.         Close of public    Interim ESWTR   
                   Effective for SW   comment period.    effective for  
                   systems serving                       systems >10k   
                   greater >10k,                         1994, 1995,    
                   extended                              1996 monitoring
                   compliance date                       data used for  
                   for GAC or                            level of       
                   membrane                              treatment      
                   technology.                           determination. 
12/98...........  .................  Propose for all    Promulgate long-
                                      CWSs, NTNCWSs.     term ESWTR.    
6/00............  Stage 1 limits     Promulgate Stage   Long-term ESWTR 
                   effective for      2 for all CWSs,    effective for  
                   surface water      NTNCWSs.           all system     
                   systems <10k,                         sizes.         
                   and ground water                                     
                   systems >10k.                                        
1/02............  Stage 1 limits     Effective lower    ................
                   effective for GW   MCLs or other                     
                   systems <10k       criteria,                         
                   unless Stage 2     extended                          
                   criteria           compliance to                     
                   supersede.         2004 for GAC or                   
                                      membranes.                        
------------------------------------------------------------------------

    EPA believes it will need about one year of microbial monitoring 
data from a large number of utilities to determine candidate regulatory 
criteria for discussion in the Notice of Availability concerning the 
interim ESWTR. EPA also believes it will need about one year, following 
the issuance of the NOA, to promulgate the interim ESWTR. Microbial and 
DBP monitoring are required at the same time to facilitate data 
management and to allow comparisons to be made concerning simultaneous 
control of both pathogens and DBPs.
    EPA requests comment on the feasibility of the schedule for the 
monitoring requirements proposed under this ICR. EPA also solicits 
comments on alternative microbial monitoring schemes, that would need 
less laboratory capacity and would still provide the requisite data for 
developing the interim ESWTR, as well as providing adequate data by 
which systems could implement such a rule.
    EPA requests comment on a proposed alternative to require those 
systems serving 100,000 or more persons to initiate all microbial, 
chemical, and treatment process monitoring requirements (not including 
TOC monitoring which would begin [insert date three months following 
promulgation date of this rule]) within the first 3 months of the 
proposed 30 month monitoring period, and that those systems serving 
between 10,000 and 100,000 people complete all monitoring requirements 
during the last 12 months of the 30 month monitoring period. Systems 
serving between 10,000 and 100,000 people that desire and are able to 
initiate monitoring through an EPA approved laboratory before their 
required start date would be given credit toward meeting the 
requirements of this rule. EPA believes that this proposed alternative 
monitoring schedule may facilitate the generation of more microbial 
data within a shorter time, thereby increasing the likelihood of 
meeting the schedule for promulgating the interim ESWTR. This 
alternative schedule would also increase efficiencies of available EPA 
resources to manage and track data, and to provide technical assistance 
to utilities as they attempt to comply with this rule.
    EPA also requests comments on the appropriateness of separating the 
final ICR rule into two separate rules: one for data collection to 
support the development and implementation of the interim ESWTR, and 
another for data collection to support the development of the Stage 2 
D/DBP and ESWTR rules. The purpose of such a strategy would be to 
promulgate the microbial data collection requirements sooner than 
otherwise might be possible to avoid undue delay in developing and 
promulgating the interim ESWTR, as well as the Stage 1 D/DBP rule.

D. Reporting Requirements

    Under this rule, systems would provide the monitoring data and 
other indicated information directly to EPA. States, as well as the 
public, would have access to all the reported data via a national 
electronic data base. The Agency is using this approach to avoid 
increasing the implementation burden to the States and to obtain and 
analyze the data more quickly to meet the accelerated schedule of 
future rulemakings agreed to by the Negotiating Committee negotiating 
the DBP Rule.
    Under this ICR rule, systems serving more than 100,000 people would 
be required to provide the requisite data beginning [insert date 6 
months following the promulgation date of this rule], and every three 
months thereafter until completion of the required monitoring. Systems 
serving between 10,000 and 100,000 people would be required to provide 
the requisite data beginning four months after starting monitoring and 
every 2 months thereafter, until completion of the required monitoring. 
With this approach, a substantial amount of the data should become 
available in time for consideration in evaluating different regulatory 
options for the interim ESWTR. The initial data submissions will allow 
EPA to screen the data for problems and begin entering it into a 
national data base which would be accessible by the public. Systems 
would need to report the required physical and engineering information 
on the initial submission only, unless this information changes. To 
assist EPA in processing quickly the large amount of data anticipated, 
the Agency requests that systems serving more than 100,000 people 
submit data either electronically or on computer diskettes, and that 
systems serving between 10,000 and 100,000 people do so if possible.
    To assist the systems and facilitate EPA's effort to screen the 
data and enter it into a computer, the Agency has developed specific 
forms for data and information entry as previously described. These 
forms include the EPA address where the system should send data and the 
other required information.
    EPA requests comment on the feasibility of the above reporting 
schedule. The Agency also requests comment on alternative approaches 
that might be as, or more, efficient than the one above.

E. List of Systems Required To Submit Data

    Between now and the time of promulgation EPA will attempt to 
determine which systems would appropriately be required to meet the 
different requirements of the ICR. Appendix B of this preamble includes 
a preliminary list of candidate systems in the three main size 
categories that would be required to submit data to EPA to fulfill the 
requirements of this rule. However, systems which exclusively purchase 
water from other systems, and do not further disinfect, are not 
required to do any monitoring and are not intended to be included in 
these lists. Some systems are both wholesalers and retailers and are 
included in the lists. The intent of the ICR is for the requirements to 
pertain to systems which treat water for populations equivalent to more 
than 100,000 people or between 10,000 and 100,000 people.
    The intent of the first list (Appendix B-1 of this preamble) is to 
provide a tabulation of all systems using ground water or surface water 
and which produce treated drinking water for populations equivalent to 
serving 100,000 or greater. Systems using ground water in this size 
category would be required to monitor for DBPs and other water quality 
indicators, provide specific physical and engineering data, and conduct 
bench or pilot scale studies depending upon their water quality (see 
section III.B.7). Systems using surface water in this size category 
would also be required to submit this data, as well as microbial 
occurrence data.
    Data in Appendix B-1 of this preamble includes classification of 
populations serving retail and wholesale populations under two 
different data bases: The Federal Reporting Data System (FRDS) and the 
Water Industry Data Base (WIDB). Since there may be errors or 
incomplete data in either data base, data from both data bases are 
listed. Also included are data on the average daily production of water 
in millions of gallons per day (MGD). Based on data included in the 
WIDB, 95% of the time the average daily flow production associated with 
a population of 100,000 or greater is > 9 MGD. Therefore, systems with 
average daily flows (assuming the flows reported are correct) 
significantly greater than 9 MGD, although not necessarily listed with 
populations above 100,000, are included on the list should be 
considered candidates for regulation.
    The intent of the second list (Appendix B-2 of this preamble), 
generated from FRDS, is to provide a tabulation of all systems using 
surface water and which produce treated drinking water equivalent to 
serving populations between 10,000 and 100,000 people. These systems, 
if appropriately classified, would only be required to submit data on 
microbial occurrence in the source water and provide treatment plant 
data regarding microbial treatment.
    The intent of the third list (Appendix B-3 of this preamble), 
generated from FRDs, is to provide a tabulation of all systems using 
ground water and serving between 50,000 and 100,000 people. A portion 
of these systems would be required to monitor for TOC, and depending 
upon the TOC level in their ground water (see Section III. B.7), could 
be required to conduct bench or pilot scale studies for DBP precursor 
removal using GAC or membrane technology. No other data collection 
requirements pertain to these systems under this rule.
    EPA solicits comment on whether the three lists of systems included 
in Appendix B of this preamble accurately reflect the appropriate 
systems which would be required to comply with the requirements of this 
rule. Which systems should be added or deleted from the list and on 
what basis?

IV. State Implementation

    The Agency would not set requirements for States to obtain primary 
enforcement responsibility or require the States to enforce this rule. 
Rather, EPA would enforce the provisions of this rule, which is an 
information collection requirement only. EPA requests comment on this 
approach.

V. Cost of Rule

    The Information Collection Rule will result in total costs of 
between $118 and $149 million dollars to be expended over a three-and-
a-half year period. Since this cost does not exceed 100 million dollars 
per year, it does not qualify as a ``major rule'' for purposes of 
Executive Order 12866. EPA has prepared an economic impact analysis 
which establishes that this action would not be a major rule within the 
meaning of the Executive Order. This analysis has been submitted to the 
Office of Management and Budget for review. The following is a summary 
of cost estimates for implementation of this rule.
    The estimated cost is indicated in the third column of Table V.1. 
There are five elements contributing to the total cost estimates. The 
first cost element is start-up activities, estimated to cost a total of 
$515,000. These activities consist of reading and understanding the 
requirements of the rule. Start-up costs will be spread across 1,560 
non-purchased community water systems, resulting in an average cost of 
$330 per system.
    EPA would specify two types of monitoring requirements in the rule: 
microbial monitoring and DBP monitoring. The microbial monitoring 
applies to 1,725 plants in 1395 community surface water systems serving 
more than 10,000 persons. Microbial monitoring is estimated to cost a 
total of $11.76 million nationally, $9.21 million in systems serving 
more than 100,000 persons and $2.55 million in systems serving between 
10,000 and 100,000 persons. The average cost per plant will be $21,000 
in systems serving more than 100,000 persons and $2,000 in systems 
serving between 10,000 and 100,000 persons.
    The DBP monitoring applies to 292 non-purchased surface and ground 
water community systems serving more than 100,000 persons. The DBP 
monitoring is estimated to cost $56.53 million, averaging $26,500 to 
$50,000 per treatment site. The associated labor burden is estimated to 
be 421,227 hours nationally, averaging 199 to 373 hours per treatment 
site. Detailed calculations are presented in Tables V.2 through V.7.
    The fourth cost element of the Information Collection Rule is a 
requirement for reporting of various process parameters of surface 
water treatment processes related to microbial treatment (1,725 plants 
in 1395 non-purchased systems serving more than 10,000 persons) and 
related to DBP formation (440 plants in 233 non-purchased systems 
serving more than 100,000 persons). The total cost is estimated to be 
$3.88 million nationally, averaging $2,250 per plant.
    The fifth cost element is a requirement for pilot and bench scale 
testing. With some exceptions, this requirement applies to all surface 
water treatment plants in systems serving more than 100,000 persons 
that have an influent TOC concentration greater than 4 mg/l. It also 
applies to all groundwater systems serving more than 50,000 persons 
that have a treated effluent TOC concentration greater than 2 mg/l. The 
total national cost of this testing requirement is estimated to be 
between $45 and $76 million. The cost per facility is estimated to be 
between $150,000 per bench-scale test and $750,000 per pilot test. The 
low end cost estimate assumes that 200 bench scale studies (at $150,000 
per study assumed to be GAC) and 20 pilot scale studies (at $750,000 
per study) will be conducted for surface supplies and that 33 bench 
scale studies (at $250,000 per study--assumed to be membrane 
filtration) will be conducted for ground water supplies. The high end 
cost estimate assumes that 162 bench scale studies (at $150,000 per 
study) and 58 pilot scale studies (at $750,000 per study) will be 
conducted for surface supplies and that 27 bench scale studies (at 
$150,000 per study) and 6 pilot scale studies (at $750,000 per study) 
will be conducted for ground water supplies. At this time EPA cannot 
predict with any certainty the numbers of the different types of 
studies that will be conducted.

                                      Table V-1.--Total Cost and Burden Estimates for Information Collection Rule*                                      
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                           Cost (K$)                           Burden (hrs.)            
                                                        Respondents affected ---------------------------------------------------------------------------
                                                                               All respondents    Per respondent     All respondents     Per respondent 
--------------------------------------------------------------------------------------------------------------------------------------------------------
Compliance Activities:                                                                                                                                  
Start-Up Activities:                                                                                                                                    
    1395 Surface Water Systems > 10K..................  1,725 plants........                468               0.27             14,579                8.4
    165 Ground Water Systems > 50k....................  165 systems.........                 47               0.29              1,485                9.0
                                                       -----------------------                                                                          
        Subtotal......................................  ....................                515                                16,064                   
                                                       =======================                                                                          
Microbial Monitoring:                                                                                                                                   
    1395 Surface Water Systems > 10K..................  1,725 plants........             11,761                  7            200,205                116
DBP Monitoring:                                                                                                                                         
    233 Surface Water Systems > 100K..................  440 plants..........             22,126                 50            163,967                373
    59 Ground Water Systems > 100K....................  1,295 treat.sites...             34,402                 27            257,260                199
                                                       -----------------------                                                                          
        Subtotal......................................  ....................             56,529                               421,227                   
                                                       =======================                                                                          
Process Data Reporting:                                                                                                                                 
    1395 Surface Water Systems > 10K..................  1,725 plants........              3,881                  2            124,200                 72
Pilot Studies                                                                                                                                           
    233 Surface Water Systems > 100K..................  178 plants**........             48,300                271            322,000              1,809
    165 Ground Water Systems > 50K....................  33 systems**........              8,550                259             57,000              1,727
                                                       -----------------------                                                                          
        Subtotal......................................  ....................             56,850                               379,000                   
                                                       -----------------------                                                                          
        Total.........................................  ....................            129,536                             1,140,696                   
--------------------------------------------------------------------------------------------------------------------------------------------------------
*Total costs and burden over 18 months, except for pilot studies which extend over two and one-half years.                                              
**Surface water treatment plants with influent TOC >4 mg/l; ground water treatment plants with effluent TOC >2 mg/l.                                    


                                                                   Table V-2.--Summary                                                                  
                                  [Cost and burden estimates for DBP monitoring under the information collection rule]                                  
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                   Tot.              Unit     Unit                                                                      
                                          Tot.    ground   Total     cost    burden                                                                     
                                        surface   number   number    per      per      Surface    Ground cost   Surface   Ground    Total cost    Total 
                Analyte                  number     of       of     sample   sample    cost in     in dollars   burden    burden    in dollars   burden 
                                           of    samples  samples     in       in      dollars                 in hours  in hours               in hours
                                        samples                    dollars  minutes                                                                     
--------------------------------------------------------------------------------------------------------------------------------------------------------
Aldehydes.............................      756        0      756     $250      120     $189,000            0     1,512         0     $189,000     1,512
Alkalinity............................   38,886   54,504   93,390       21        6      816,606    1,144,584     3,889     5,450    1,961,190     9,339
Ammonia...............................    8,676   25,058   33,734       25       15      216,900      626,456     2,169     6,265      843,356     8,434
AOC/BDOC..............................      756        0      756      175      220      132,300            0     2,772         0      132,300     2,772
Bromate...............................      756        0      756      100       20       75,600            0       252         0       75,600       252
Bromide...............................    8,676   23,310   31,986       40       15      347,040      932,400     2,169     5,828    1,279,440     7,997
Ca. Hardness..........................   31,284   54,504   85,788       16       14      500,544      872,064     7,300    12,718    1,372,608    20,017
Chloral Hydrate.......................   12,288   15,540   27,828      275       50    3,379,200    4,273,500    10,240    12,950    7,652,700    23,190
Chlorate..............................    2,358    3,096    5,454      100       20      235,800      309,600       786     1,032      545,400     1,818
Chlorine..............................   23,130   47,652   70,782       20       10      462,600      953,040     3,855     7,942    1,415,640    11,797
Chlorine Dioxide......................    1,188        0    1,188       20       10       23,760            0       198         0       23,760       198
Chlorite..............................    1,512        0    1,512      125       20      189,000            0       504         0      189,000       504
Chloropicrin..........................   12,288   15,540   27,828       66       57      804,864    1,017,870    11,674    14,763    1,822,734    26,437
Chloropropanones......................   12,288   15,540   27,828       30       60      368,640      466,200    12,288    15,540      834,840    27,828
CNCI..................................    1,182      852    2,034      250       60      295,500      213,000     1,182       852      508,500     2,034
H2S, Fe, Mn, etc......................        ?        ?        ?        ?        ?            ?            ?         ?         ?            ?         ?
HAA...................................   12,288   15,540   27,828      200       50    2,457,600    3,108,000    10,240    12,950    5,565,600    23,190
HAN...................................   12,288   15,540   27,828      150       60    1,843,200    2,331,000    12,288    15,540    4,174,200    27,828
Ozone.................................      324        0      324       20       30        6,480            0       162         0        6,480       162
pH....................................   39,924   55,536   95,460       11       10      439,164      610,896     6,654     9,256    1,050,060    15,910
SDS...................................    2,640    7,770   10,410      957      387    2,025,160    7,432,005    17,028    50,117    9,957,165    67,145
Temperature...........................   39,330   55,536   94,866        4        4      157,320      222,144     2,622     3,702      379,464     6,324
THM...................................   12,288   15,540   27,828      100       30    1,228,800    1,554,000     6,144     7,770    2,782,800    13,914
TOC...................................   32,040   54,504   86,544       55       30    1,762,200    2,997,720    16,020    27,252    4,759,920    43,272
Tot. Hardness.........................   38,292   54,504   92,796       32       10    1,225,344    1,744,128     6,382     9,084    2,969,472    15,466
TOX...................................   12,288   15,540   27,828      105       60    1,290,240    1,631,700    12,288    15,540    2,921,940    27,828
Turbidity.............................   32,040   54,504   86,544       11       10      352,440      599,544     5,340     9,084      951,984    14,424
UV 254................................   32,040   54,504   86,544       25       15      801,000    1,362,600     8,010    13,626    2,163,600    21,636
                                       -----------------------------------------------------------------------------------------------------------------
    Total.............................  .......  .......  .......  .......  .......  $22,126,302  $34,402,451   163,967   257,260  $56,528,753   421,227
--------------------------------------------------------------------------------------------------------------------------------------------------------
Total number of Surface Plants: 440                                                                                                                     
Total number of Ground Trt. Sites: 1,295                                                                                                                


                                                Table V-3.--Requirements for All Systems Serving >100,000                                               
                                  [Cost and Burden Estimates for DBP Monitoring under the Information Collection Rule]                                  
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                      Sampling requirements for treatment sites          Surface     Ground         Sampling         Surface     Ground 
                              -------------------------------------------------------------------------------   requirements for   ---------------------
                               SurfaceSystems=233Treatement  GroundSystems=59Treatment                        distribution systems                      
                                           sites                       sites              Total      Total   ----------------------                     
           Analyte            --------------------------------------------------------- number of  number of               Total     Combined   Combined
                                                                                         samples    samples   Number of  number of    total      total  
                                                                               W/O         for        for      samples    samples   number of  number of
                                 W/Filt.429    W/O Filt.11    W/Filt.219    Filt.1076   treatment  treatment     per     for dist.   samples    samples 
                                                                                          sites      sites      system    systems                       
--------------------------------------------------------------------------------------------------------------------------------------------------------
No. of Samples/month/trt.                                                                                                                               
 site:                                                                                                                                                  
    pH.......................            4              2              4            2      31,284     54,504          4      7,008     38,292     54,504
    Alkalinity...............            4              2              4            2      31,284     54,504          4      7,008     38,292     54,504
    Turbidity................            4              2              4            2      31,284     54,504  .........          0     31,284     54,504
    Temperature..............            4              2              4            2      31,284     54,504          4      7,008     38,292     54,504
    Ca. Hardness.............            4              2              4            2      31,284     54,504  .........          0     31,284     54,504
    Tot. Hardness............            4              2              4            2      31,284     54,504          4      7,008     38,292     54,504
    TOC......................            4              2              4            2      31,284     54,504  .........          0     31,284     54,504
    UV 254...................            4              2              4            2      31,284     54,504  .........          0     31,284     54,504
    Bromide..................            1              1              1            1       7,920     23,310  .........          0      7,920     23,310
    Ammonia*.................          1.1            1.1            1.1          1.1       8,514     25,058  .........          0      8,514     25,058
    Dis. Resid...............            2              2              2            2      15,840     46,620          4      7,008     22,848     46,620
    H2S, Fe, Mn, etc.........            1              1              1            1           ?  .........  .........          ?          ?          ?
    Occurrence to be                                                                                                                                    
     determined).............                                                                                                                           
No. of Samples/quarter/trt.                                                                                                                             
 site:                                                                                                                                                  
    THM......................            2              2              2            2       5,280     15,540          4      7,008     12,288     15,540
    HAA......................            2              2              2            2       5,280     15,540          4      7,008     12,288     15,540
    HAN......................            2              2              2            2       5,280     15,540          4      7,008     12,288     15,540
    Chloropicrin.............            2              2              2            2       5,280     15,540          4      7,008     12,288     15,540
    Chloropropanones.........            2              2              2            2       5,280     15,540          4      7,008     12,288     15,540
    Chloral Hydrate..........            2              2              2            2       5,280     15,540          4      7,008     12,288     15,540
    TOX......................            2              2              2            2       5,280     15,540          4      7,008     12,288     15,540
    SDS......................            1              1              1            1       2,640      7,770  .........          0      2,640     7,770 
--------------------------------------------------------------------------------------------------------------------------------------------------------
*Number of samples is a weighted average to take into account the number of systems using air stripping for VOC removal.                                


                                   Table V-4.--Additional Requirements for Systems Using Chloramines Serving >100,000                                   
                                  [Cost and Burden Estimates for DBP Monitoring under the Information Collection Rule]                                  
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                Sampling requirements for treatment sites                           Sampling         Surface     Ground 
                            ---------------------------------------------------------------------------------   requirements for   ---------------------
                                                                                                              distribution systems                      
                                                                                   Surfacetotal  Groundtotal ----------------------                     
          Analyte                                                                    number of    number of                Total     Combined   Combined
                             SurfaceSystems=66Sites=125  GroundSystems=6Sites=142   samples for  samples for  Number of  number of    total      total  
                                                                                     treatment    treatment    samples    samples   number of  number of
                                                                                       sites        sites        per     for dist.   samples    samples 
                                                                                                                system    systems                       
--------------------------------------------------------------------------------------------------------------------------------------------------------
Number of samples/quarter/                                                                                                                              
 site:                                                                                                                                                  
    CNCl...................                    1                          1                750          852           1        432      1,182        852
--------------------------------------------------------------------------------------------------------------------------------------------------------


                                   Table V-5.--Additional Requirements for Systems Using Hypochlorite Serving >100,000                                  
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                 Sampling requirements for treatment sites                          Sampling         Surface     Ground 
                             --------------------------------------------------------------------------------   requirements for   ---------------------
                                                                                                              distribution systems                      
                                                                                   Surfacetotal  Groundtotal ----------------------                     
           Analyte                                                                   number of    number of                Total     Combined   Combined
                              SurfaceSystems=25Sites=47  GroundSystems=8Sites=172   samples for  samples for  Number of  number of    total      total  
                                                                                     treatment    treatment    samples    samples   number of  number of
                                                                                       sites        sites        per     for dist.   samples    samples 
                                                                                                                system    systems                       
--------------------------------------------------------------------------------------------------------------------------------------------------------
Number of samples/quarter/                                                                                                                              
 site:                                                                                                                                                  
    Chlorate................                   3                          3                846        3,096           0          0        846      3,096
    pH......................                   1                          1                282        1,032           0          0        282      1,032
    Temperature.............                   1                          1                282        1,032           0          0        282      1,032
    Free Cl.................                   1                          1                282        1,032           0          0        282      1,032
--------------------------------------------------------------------------------------------------------------------------------------------------------


            Table V-6.--Additional Requirements for Systems Using Chlorine Dioxide Serving > 100,000            
              [Cost and Burden Estimates for DBP Monitoring under the Information Collection Rule]              
----------------------------------------------------------------------------------------------------------------
                         Sampling requirements for treatment sites          Sampling         Surface    Ground  
                     ------------------------------------------------   requirements for   ---------------------
                                                                      distribution systems                      
                                                 Surface     Ground                                             
                                                  total      total   ----------------------  Combined   Combined
       Analyte          Surface       Ground    number of  number of               Total      total      total  
                       systems=18   systems=0    samples    samples   Number of  number of  number of  number of
                        sites=33     sites=0       for        for      samples    samples    samples    samples 
                                                treatment  treatment     per     for dist.                      
                                                  sites      sites     system     systems                       
----------------------------------------------------------------------------------------------------------------
Number of samples/                                                                                              
 month/site:                                                                                                    
    pH..............            2            2      1,188          0  .........          0      1,188          0
    Alkalinity......            1            1        594          0  .........          0        594          0
    Turbidity.......            1            1        594          0  .........          0        594          0
    Temperature.....            1            1        594          0  .........          0        594          0
    TOC.............            1            1        594          0  .........          0        594          0
    UV 254..........            1            1        594          0  .........          0        594          0
    Bromide.........            1            1        594          0  .........          0        594          0
    C1O2............            2            2      1,188          0  .........          0      1,188          0
    Chloride........            2            2      1,188          0          3        324      1,512          0
    Chlorate........            2            2      1,188          0          3        324      1,512          0
    Bromate.........            1            1        594          0  .........          0        594          0
Number of samples/                                                                                              
 quarter/site:                                                                                                  
    Aldehydes.......            3            3        594          0  .........          0        594          0
    AOC/BDOC........            3            3        594          0  .........          0        594         0 
----------------------------------------------------------------------------------------------------------------


                                      Table V-7.--Additional Requirements for Systems Using Ozone Serving > 100,000                                     
                                  [Cost and Burden Estimates for DBP Monitoring under the Information Collection Rule]                                  
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                            Sampling requirements for treatment sites               Sampling         Surface     Ground 
                                                   ----------------------------------------------------------   requirements for   ---------------------
                                                                                                              distribution systems                      
                                                                                   Surfacetotal  Groundtotal ----------------------                     
                      Analyte                                                        number of    number of                Total     Combined   Combined
                                                    Surfacesites=9  Groundsites=0   samples for  samples for  Number of  number of    total      total  
                                                                                     treatment    treatment    samples    samples   number of  number of
                                                                                       sites        sites        per     for dist.   samples    samples 
                                                                                                                system    systems                       
--------------------------------------------------------------------------------------------------------------------------------------------------------
Number of samples/month/site:                                                                                                                           
    pH............................................              1              1           162            0   .........          0        162          0
    Alkalinity....................................              1              1           162            0   .........          0        162          0
    Turbidity.....................................              1              1           162            0   .........          0        162          0
    Temperature...................................              1              1           162            0   .........          0        162          0
    TOC...........................................              1              1           162            0   .........          0        162          0
    UV 254........................................              1              1           162            0   .........          0        162          0
    Bromide.......................................              1              1           162            0   .........          0        162          0
    Ammonia.......................................              1              1           162            0   .........          0        162          0
    Ozone.........................................              2              2           342            0   .........          0        342          0
    Bromate.......................................              1              1           162            0   .........          0        162          0
Number of samples/quarter/site:                                                                                                                         
    Aldehydes.....................................              3              3           162            0   .........          0        162          0
    AOC/BDOC......................................              3              3           162            0   .........          0        162          0
--------------------------------------------------------------------------------------------------------------------------------------------------------

VI. Other Statutory Comments

A. Executive Order 12866

    Under Executive Order 12866, (58 FR 51735 (October 4, 1993)) the 
Agency must determine the regulatory action is ``significant'' and 
therefore subject to OMB review and the requirements of the Executive 
Order. The Order defines ``significant regulatory action'' as one that 
is likely to result in a rule that may:
    (1) Have an annual effect on the economy of $100 million or more or 
adversely affect in a material way the economy, a sector of the 
economy, productivity, competition, jobs, the environment, public 
health or safety, or State, local, or tribal governments or 
communities;
    (2) Create a serious inconsistency or otherwise interfere with an 
action taken or planned by another agency;
    (3) Materially alter the budgetary impact or entitlements, grants, 
user fees, or loan programs or the rights and obligations of the 
recipients thereof; or
    (4) Raise novel legal or policy issues arising out of legal 
mandates, the President's priorities, or the principles set forth in 
the Executive Order.
    This rule was reviewed by OMB under Executive Order 12866.

B. Regulatory Flexibility Act

    The Regulatory Flexibility Act requires EPA to explicitly consider 
the effect of proposed regulations on small entities. The Act requires 
EPA to consider regulatory alternatives if there is any economic impact 
on any number of small entities. The Small Business Administration 
defines a small water utility as one which serves fewer than 3,300 
people.
    The proposed rule is consistent with the objectives of the 
Regulatory Flexibility Act because it will not have any economic impact 
on any small entities. The proposed rule would only apply to systems 
serving more than 10,000 people; thus, systems serving fewer than 
10,000 people would not be affected. Therefore, pursuant to section 
605(b) of the Regulatory Flexibility Act, 5 U.S.C. 605(b), the 
Administrator certifies that this rule will not have an economic impact 
on a number of small entities.

C. Paperwork Reduction Act

    The information collection requirements in this proposed rule have 
been submitted for approval to the Office of Management and Budget 
(OMB) under the Paperwork Reduction Act, 44 U.S.C 3501 et seq. An 
Information Collection Request document has been prepared by EPA (ICR 
No. 270.31) and a copy may be obtained from Sandy Farmer, Information 
Policy Branch; EPA; 401 M St., SW. (PM-223); Washington, DC 20460 or by 
calling (202) 260-2740.
    Public reporting burden for this collection of information, 
including time for reviewing instructions, searching existing data 
sources, gathering and maintaining the data needed, and completing and 
reviewing the collection of information is estimated to total 1.1 
million hours over the three year clearance period. As shown in Table 
V.1., there are five elements contributing to the total burden 
estimate. The total burden associated with start-up activities is 
estimated to be 16,064 hours, an average of 10 hours per system. The 
total burden estimated for the microbial monitoring is 200,205 hours, 
averaging 295 hours per plant in systems serving more than 100,000 
persons, and 55 hours per plant in systems serving between 10,000 and 
100,000 persons. Total burden for DBP monitoring is 421,000 hours, 
averaging 370 hours per plant for surface water systems serving more 
than 100,000 persons, and 200 hours per plant in ground water systems 
serving more than 100,000 persons. The total burden for data reporting 
is estimated to be 124,200 hours, an average of 72 hours per plant. The 
per plant impact of this requirement on systems serving between 10,000 
and 100,000 persons will be significantly less than these estimates due 
to less extensive data processing requirements relating to DBPs in this 
system size range. The total burden estimate for bench and pilot scale 
testing is estimated to be approximately 379,000 hours. The labor 
burden per facility is estimated to be between 1,000 hours for bench-
scale tests and 5,000 hours for pilot tests.
    Send comments regarding the burden estimate or any other aspect of 
this collection of information, including suggestions for reducing this 
burden, to Chief, Information Policy Branch, PM-223, U.S. Environmental 
Protection Agency, 401 M St., SW., Washington, DC 20460; and to the 
office of Information and Regulatory Affairs, Office of Management and 
Budget, Washington, DC 20503, marked ``Attention: Desk Officer for 
EPA.'' The final rule will respond to any OMB or public comments on the 
information collection requirements contained in this proposal.

D. Science Advisory Board, National Drinking Water Advisory Council, 
and Secretary of Health and Human Services

    In accordance with section 1412(d) and (e) of the Safe Drinking 
Water Act, the Agency has submitted this proposed rule to the Science 
Advisory Board, National Drinking Water Advisory Council, and the 
Secretary of Health and Human Services for their review. The Agency 
will take their comments into account in developing the final rule.

VII. Request for Public Comments

    To ensure that EPA can read, understand and therefore properly 
respond to comments, the Agency would prefer for commenters to type or 
print comments in ink, and to cite where possible, the paragraph(s) in 
this proposed regulation (e.g., 141.140(a)) to which each comment 
refers. Commenters should use a separate paragraph for each issue 
discussed.
    EPA solicited public comments and requested suggestions on specific 
issues earlier in the ICR preamble and welcomes comments on other 
specific issues. For convenience the comment topics and requested 
suggestions are listed below.
     (III.A.2)  Collection of data for EPA evaluation of water 
treatment efficiencies

--Assessment of microbial concentrations in small systems (other than 
the three approaches given)
--Whether to allow systems to submit previously collected data
--Criteria for admissibility of previously collected data
--Feasibility and utility of archiving samples to develop data 
evaluations

     (III.A.2)  Particle size count data

--Under what circumstances should particle size count data within 
treatment plant be allowed in lieu of finished water monitoring for 
Giardia and Cryptosporidium
--What particle size ranges and sample volumes should be monitored
--What criteria should be specified to ensure particle size 
measurements collected from different systems could be appropriately 
compared and would be most representative of removal of Giardia and 
Cryptosporidium
--Should other monitoring by other methods, such as Microscopic 
Particulate Analysis (MPA) be included as condition for avoiding 
finished water monitoring of Giardia and Cryptosporidium

     (III.A.3)  Monitoring pathogens and indicators
--Requirements for monitoring Giardia and Cryptosporidium
--Requirements for monitoring total culturable viruses
--Requirements for monitoring bacterial pathogens
--Requirements for monitoring total coliforms, fecal coliforms or E. 
coli.
--Requirements for monitoring Clostridium perfringens
--Requirements for monitoring coliphage

     (III.A.5)  Need to Report physical data and engineering 
information

--Nature of source water (surface-ground, combination)
--Treatment processes (type of disinfectant, dosage, pH, contact time, 
type of filter process, media size, depth hydraulic loading rate)
--Whether additional reporting requirements are warranted
--Require fewer systems to submit data in size category 10,000-100,000
     (III.A.6)  Appropriateness of analytical methods

--EC medium supplemented with 50 g/ml of 4-methylumbelliferyl-
beta-D-glucuronide (MUG), as specified in 141.21 (f)(6)(i) for total 
coliforms, fecal coliforms and E. coli
--Nutrient agar supplemented with 100 g/ml of MUG, as 
specified in 141.21(f)(6)(ii). E. coli colonies to be counted
--Minimal Medium ONPG-MUG test (Colilert test), as specified in 141.74 
(a)(2) (coliform-positive tubes to be examined with UV light
--Method for Giardia/Cryptosporidium as described in Appendix C of the 
rule.
--Feasibility of other methods for analysis of protozoa
--Method for viruses as described in Appendix D of the rule
--Method for Clostridium perfringens.
--Method for coliphage as described in Appendix D of the rule

     (III.B.2)  Monitoring of Source Water Quality

--Definition of high oxidant demand water
--Types of measurements necessary to characterize high oxidant demand 
water

     (III.B.3)  Specific Process Information

--Design to be reported for ozone contact basins
--Operating parameters to be reported for ozone contact basins
--Completeness of Table III.6 (Treatment Plant Information) in 
describing treatment plant configurations and specific design 
parameters for the unit processes relevant to ESWTR and DBP Stage 2 
development
--Completeness of Table III.6 in describing treatment plant 
configurations and specific design parameters relevant to future model 
development for predicting DBPs

     (III.B.4)  Database development

--Use of diskettes and/or modem for data reporting, use of Windows 
based software

     (III.B.5)  Analytical methods

--Sample collection without adjusting pH and laboratories required to 
extract samples within 24-48 hours of sample collection
--Suggestions on alternative approaches to collecting sample without 
adjusting pH and laboratories extracting sample within 24-48 hours
--Alternative approaches to all aldehyde analyses being initiated 
within 48 hours of sample collection
--Proposal to drop or delay monitoring of certain analytes, if 
including them causes undue delay in other monitoring
--Proposal that any monitoring delay would not be cancelled or 
postponed for: (1) trihalomethanes; (2) haloacetic acids; (3) bromate; 
(4) chlorite; (5) chlorate; (6) total organic halide; (7) total organic 
carbon; and (8) bromide

     (III.B.6)  Quality Assurance

--Alternative mechanisms (other than following specifications outlined 
in manual to be developed) for ensuring consistency in sampling
--The use of zero in the database to indicate concentrations below the 
reporting level
--The QA/QC criteria for data entry into the database as presented in 
the text

     (III.B.7)  Selection of bench versus pilot scale and 
membrane versus GAC studies

--How to ensure an adequate number of pilot scale studies for both 
membranes and GAC technology to ensure quality results
--What specific requirements could be made to ensure that the necessary 
number of studies (as indicated in Table III.12) are done, if an 
insufficient number of volunteers are identified as willing to do pilot 
scale testing
--Should selection of sites for GAC and membrane pilot studies be 
required according to system size, TOC concentration, or both
--How the site selection process can ensure that some of the pilot 
studies use membranes

     (III.C)  Dates for completing data development monitoring 
requirements

--Feasibility of schedule for monitoring requirements

     (III.E) List of systems required to submit data

--Whether the list of systems accurately represents the systems 
required to comply with the ICR, should other systems be included, 
others deleted

    In addition to the specific comments solicited previously in this 
preamble, EPA solicits comments on the following: Are other mechanisms 
or procedures available than those proposed herein by which the desired 
information could be obtained more efficiently? What mechanisms might 
be available for transferring some of the resource commitments that 
large utilities have made during the D/DBP negotiated rulemaking, to 
fund other research in support of the development of the ESWTR or stage 
2 D/DBP rule?

VII. References

APHA. 1992. American Public Health Association. Standard methods for 
the examination of water and wastewater (18th ed.). Washington, DC.
ASTM. 1992. D-19 Proposal P 229, Proposed test method for Giardia 
cysts and Cryptosporidium oocysts in low-turbidity water by a 
fluorescent antibody procedure. 1992 Annual Book of ASTM Standards, 
Vol. 11.02 Water (II), pp. 925-935. ASTM, Philadelphia, PA.
Armon, R., and P. Payment. 1988. A modified M-CP medium for the 
enumeration of Clostridium perfringens from water samples. Can. J. 
Microbiol. 34:78-79.
Barth, R.C. and P.S. Fair. 1992. Comparison of the microextraction 
procedure and Method 552 for the analysis of HAAs and Chlorophenols. 
J. Amer. Water Works Assoc. 84(11):94-98.
Bisson, J.W., and V.J. Cabelli. 1979. Membrane filter enumeration 
method for Clostridium perfringens. Appl. Environ. Microbiol. 37:55-
66.
Bisson, J.W., and V.J. Cabelli. 1980. Clostridium perfringens as a 
water pollution indicator. J. Water Poll. Control Fed. 52:241-248.
Bolyard, M., P.S. Fair, and D.P. Hautman. 1992. Occurrence of 
chlorate in hypochlorite solutions used for drinking water 
disinfection. Environ. Sci. Technol. 26(8):1663-1665.
Bolyard, M., P.S. Fair, and D.P. Hautman. 1993. Sources of chlorate 
ion in US drinking water. J. Amer. Water Works Assoc. 85(9):81-88.
Bonde, G.J. 1977. Bacterial indication of water pollution. Pages 
273-364. In: M.R. Droop and H.W. Jannasch (eds.), Advances in 
aquatic microbiology, Vol. 1. Academic Press, NY.
Brenner, R., and J.I. Hedges. 1993. A test of the accuracy of 
freshwater DOC measurements by high-temperature catalytic oxidation 
and UV-promoted persulfate oxidation. Marine Chem. 41:161-165.
Cabelli, V.J. 1977. Clostridium perfringens as a water quality 
indicator. Pages 65-69. In: A.W. Hoadley and B.J. Dutka (eds.), 
Bacterial indicators/health associated with water. American Society 
for Testing and Materials. Philadelphia, PA.
Cancilla, D.A., C.-C. Chou, R. Barthel, and S.S. Que Hee. 1992. 
Characterization of the O-(2,3,4,5,6-pentafluorobenzyl)- 
hydroxylaminehydrochloride (PFBOA) derivatives of some aliphatic 
mono- and dialdehydes and quantitative water analysis of these 
aldehydes. J. AOAC Int. 75(5):842-854.
Carney, M. 1991. European Drinking Water Standards. J. Amer. Water 
Works Assoc. 83(7):48-55.
Crittenden et al., 1991. Predicting GAC performance with Rapid 
Small-Scale Column Tests. Journ. AWWA, 83(1), 77-87.
Cummings, Summers and Howe, 1992. Proc, 1992 AWWA Water Quality 
Tech. Conf., Toronto, Canada, AWWA, Denver, CO.
EPA. U.S. Environmental Protection Agency. 1990. Manual for the 
certification of laboratories analyzing drinking water (third ed.). 
EPA 570/9-90-008A), USEPA, Washington, DC. (Insure that Change 1 to 
Manual is included).
EPA. U.S. Environmental Protection Agency. 1991. Guidance manual for 
compliance with the filtration and disinfection requirements for 
public water systems using surface water sources. U.S. Environmental 
Protection Agency, Office of Ground Water and Drinking Water, 
Washington, DC.
EPA. U.S. 1993a. Summary Report: Protozoa, virus and coliphage 
monitoring workshop. August 10-12, 1993.
Flesch, J.J., and P.S. Fair. 1988. The analysis of cyanogen chloride 
in drinking water. Proceedings of Amer. Water Works Assoc. Water 
Qual. Technol. Conf. pp. 465-474.
Gerba, C., and J. Rose. 1990. Viruses in source and drinking water. 
Chapter 18, pp. 380-396. In: G. McFeters (ed.), Drinking Water 
Microbiology. Springer-Verlag New York, Inc.
Glaze, W.H., M. Koga, and D. Cancilla. 1989. Ozonation by-products. 
2. Improvement of an aqueous-phase derivatization method for the 
detection of formaldehyde and other carbonyl compounds formed by the 
ozonation of drinking water. Environ. Sci. Technol. 23(7):838-847.
Gordon, G. et al. 1993. Controlling the formation of chlorate ion in 
liquid hypochlorite feedstocks. J. Amer. Water Works Assoc. 
85(9):89-97.
Harrington, G., Z. Chowdhury, D. and D. Owen. 1992. Developing a 
computer model to simulate DBP formation during water treatment. J. 
Amer. Water Works Assoc. 84:78-87.
Hautman, D.P. 1992. Analysis of trace bromate in drinking water 
using selective anion concentration and ion chromatography. 
Proceedings of Amer. Water Works Assoc. Water Qual. Technol. Conf. 
pp. 993-1007.
Hayes EB, Matte, TD, O'Brien TR, et al. 1989. Large community 
outbreak of cryptosporidiosis due to contamination of a public water 
supply. N Engl J Med 320:1372-6.
Havelaar, A., M. van Olphen, and Y. Drost. 1993. F-specific RNA 
bacteriophages are adequate model organisms for enteric viruses in 
fresh water. Appl. Environ. Microbiol. 59:2956-2962.
Hurst, C. 1991. Presence of enteric viruses in freshwater and their 
removal by the conventional drinking water treatment process. Bull. 
World Health Org. 69(1):113-119.
IAWPRC. 1991. IAWPRC Study Group on Health Related Water 
Microbiology. Bacteriophages as model viruses in water quality 
control. Water Res. 25:529-545.
Kaplan, L.A. 1992. Comparison of high-temperature and persulfate 
oxidation methods for determination of dissolved organic carbon in 
freshwaters. Limnol. Oceanogr. 37(5):1119-1125.
Keswick, B.H. et al. 1985. Inactivation of Norwalk virus in drinking 
water by chlorine. Appl. Environ. Microbiol. 50:261-264.
LeChevallier, M., W. Norton, and R. Lee. 1991a. Occurrence of 
Giardia and Cryptosporidium spp. in surface water supplies. Appl. 
Environ. Microbiol. 57:2610-2616.
LeChevallier, M., W. Norton, and R. Lee. 1991b. Giardia and 
Cryptosporidium spp. in filtered drinking water supplies. Appl. 
Environ. Microbiol. 57:2617-2621.
Lister, M.W. 1956. Decomposition of sodium hypochlorite: The 
uncatalyzed decomposition. Can. J. Chem. 34:465.
NATO. 1984. North Atlantic Treaty Organization. Drinking water 
microbiology. Committee on the Challenge of Modern Society, EPA 570/
9-84-006, Washington, DC.
Ohya, T. and S. Kanno. 1985. Formation of cyanide ion or cyanogen 
chloride through the cleavage of aromatic rings by nitrous acid or 
chlorine. VIII. On the reaction of humic acid with hypochlorous acid 
in the presence of ammonium ion. Chemosphere. 14(11/12):1717-1722.
Payment, P., M. Trudel, and R. Plante. 1985. Elimination of viruses 
and indicator bacteria at each step of treatment during preparation 
of drinking water at seven water treatment plants. Appl. Environ. 
Microbiol. 1418-1428.
Payment, P. and E. Franco. 1993. Clostridium perfringens and somatic 
coliphages as indicators of the efficiency of drinking water 
treatment for viruses and protozoan cysts. Appl. Environ. Microbiol. 
59:2418-2424.
Sobsey, M., T. Fuji, and R. Hall. 1991. Inactivation of cell-
associated and dispersed Hepatitis A virus in water. J. Amer. Water 
Works Assoc. 83:64-67.
Sobsey, M.D. 1989. Inactivation of health-related microorganisms in 
water by disinfection processes. Water Sci. Technol. 21:179-195.
Sontheimer, Crittenden and Summers. 1988. Activated Carbon for Water 
Treatment, distributed by AWWA, Denver, CO.
Summers et al., 1992. Standardized Protocol for the Evaluation of 
GAC, AWWA, Denver, CO.
Williams, F. 1985. Membrane-associated viral complexes observed in 
stools and cell culture. Appl. Environ. Microbiol. 50:523-526.
Xie, Y. and D.A. Reckhow. 1993. A rapid and simple analytical method 
for cyanogen chloride and cyanogen bromide in drinking water. Wat. 
Res. 27(3):507-511.
Zika, R.G. et al. 1985. Sunlight-induced photodecomposition of 
chlorine dioxide. In: Water Chlorination Chemistry: Environmental 
Impact and Health Effects Vol. 5. Lewis Publ., Chelsea, Mich.

APPENDICES TO THE PREAMBLE

Appendix A--Sample Reporting Sheet for Particle Size Count Data

Name of Utility--------------------------------------------------------
Address----------------------------------------------------------------
----------------------------------------------------------------------

Name of Person Completing Form-----------------------------------------
Phone Number-----------------------------------------------------------

Source Water Type (example: river, lake)-------------------------------
    Microorganism count:
    Giardia ____ Cryptosporidium____ Virus ______ Coliform ______
Presedimentation process-----------------------------------------------
    Presedimentation effluent particle size distribution:
    >2 um____ >5 um____ >10 um____
    Microorganism count (optional):
    Giardia ____ Cryptosporidium ____ Virus ______ Coliform ______
Clarification/sedimentation process------------------------------------
    Clarification/sedimentation effluent particle size distribution:
    >2 um____ >5 um____ >10 um____
    Microorganism count (optional):
    Giardia ____ Cryptosporidium ____ Virus ______ Coliform ______
Roughing filter process------------------------------------------------
    Roughing filter effluent particle size distribution:
    >2 um____ >5 um____ >10 um____
    Microorganism count (optional):
    Giardia ____ Cryptosporidium ____ Virus ______ Coliform ______
Filtration process-----------------------------------------------------
    Filter effluent particle size distribution:
    >2 um____ >5 um____ >10 um____
    Microorganism count (optional):
    Giardia ____ Cryptosporidium ____ Virus ______ Coliform ______
    Clearwell effluent
    Clearwell effluent particle size distribution:
    >2 um____ >5 um____ >10 um____
    Microorganism count (optional):
    Giardia ____ Cryptosporidium ____ Virus ______ Coliform ______

           Appendix B-1.--Classification of Candidate Systems Using Ground or Surface Water Which May Be Subject to Requirements Pertaining to Systems Serving 100,000 or More People           
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                                                                        WIDB                   FRDS              WIDB           
                                                                                                                        ------------------------------------------------------------------------
                WIDB                                                                                            FRDS              Population served            Avg.       Avg. day flow (MGD)   
   PWS-ID       I.D.     Region    State                City                            Utility                retail   ------------------------------------   day   ---------------------------
                                                                                                                pop.                                          prod.                             
                                                                                                                           Retail     Wholesale     Total     (MGD)     Prod.    Purch.   Total 
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                          EPA Region--1                                                                                         
                                                                                                                                                                                                
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
CT0150011...   90*1620        1   CT      Bridgeport......................  Bridgeport Hydraulic Co........     367,577     382,300      10,000     392,300     66.2      57.6      1.2     58.8
CT0640011...   90*1624        1   CT      Hartford........................  The Metropolitan District......     391,250     400,000       8,000     408,000     53.1      63.0      0.0     63.0
CT0890011...   90*1626        1   CT      New Britain.....................  City of New Britain Water Dept.      90,677      80,000      20,000     100,000     11.9      11.0      0.0     11.0
CT0930011...   90*1627        1   CT      New Haven.......................  So Central Conn Reg Water Auth.     380,000     397,500      34,200     431,700     62.0      58.9      0.0     58.9
CT1350011...   90*1628        1   CT      Stamford........................  Stamford Water Company.........      85,000      85,500      19,500     105,000     14.6      16.0      0.8     16.8
CT1510011...   90*1629        1   CT      Waterbury.......................  City of Waterbury Bur of Water.     103,800     107,000      17,000     124,000     #N/A      18.7      0.0     18.7
               90*1144        1   MA      Boston..........................  MA Water Resources Authority...        #N/A           0   2,170,000   2,170,000     #N/A     323.4      0.0    323.4
MA4044000...  ........        1   MA      Brockton........................  Brockton Water Dept............     135,000  ..........  ..........  ..........     10.6  ........  .......  .......
MA1281000...   90*1163        1   MA      Springfield.....................  Springfield Water Dept.........     240,000     170,000     250,000     420,000     39.5      45.6      0.0     45.6
MA2348000...   90*1166        1   MA      Worcester.......................  City of Worcester..............     200,000     165,000       5,000     170,000     26.8      27.0      0.0     27.0
ME0091300...   90*1175        1   ME      Portland........................  Portland Water District........     132,000     160,000         200     162,000     22.0      24.0      0.0     24.0
NH1471010...   90*1270        1   NH      Manchester......................  Manchester Water Works.........     104,750     103,000      13,000     116,000     14.0      15.5      0.0     15.5
RI1592021...  ........        1   RI      Cumberland......................  Pawtucket, City Of.............     108,000  ..........  ..........  ..........     14.5  ........  .......  .......
RI1592024...  ........        1   RI      Scituate........................  Providence, City Of............     286,923  ..........  ..........  ..........     64.4  ........  .......  .......
                                                                                                                                                                                                
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                          EPA Region--2                                                                                         
                                                                                                                                                                                                
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
NJ1605002...   90*1280        2   NJ      Clifton.........................  Passaic Valley Water Comm......     270,000     600,000     400,000   1,000,000  g/ml of 4-
methylumbelliferyl-beta-D-glucuronide (MUG), as specified in 
Sec. 141.21(f)(6)(i) (In this method, a total coliform-positive broth 
culture from the Multiple Tube Fermentation (MTF) Technique 
(Sec. 141.74(a)(2)) or each total coliform-positive colony from the 
Membrane Filter Technique (Sec. 141.74(a)(2)) is transferred to at 
least 10 ml of EC + MUG); or Nutrient agar supplemented with 100 
g/ml of MUG, as specified in Sec. 141.21(f)(6)(ii), except 
that E. coli colonies are counted; or Minimal Medium ONPG-MUG Test, 
often referred to as the Colilert Test, as specified in 
Sec. 141.21(f)(6)(iii), using a five or ten tube Most Probable Number 
test.
* * * * *
    4. A new Subpart M is added to read as follows:

Subpart M--Information Collection Requirements (ICR) for Public 
Water Systems


Sec. 141.140  Microbiological ICR monitoring and reporting requirements 
for Subpart H systems serving 10,000 or more persons.

    (a) Applicability. (1) The requirements of this section apply to 
subpart H systems that serve 10,000 or more persons.
    (2) Consecutive systems. If a system supplies water to other 
systems, only the supplier, which uses raw water as a source, must 
comply with this section. In determining population served, the 
supplier must include the population of its system and those for all 
consecutive systems that do not further disinfect the water.
    (b) Schedule. Systems required to monitor under the provisions of 
Sec. 141.141 (Disinfection Byproduct ICR Monitoring) must begin 
monitoring for this section and Sec. 141.141 in the same month.
    (1) Subpart H systems serving 100,000 or more people must begin 
monitoring no earlier than three months after publication of the final 
rule in the Federal Register and no later than October 1995. Prior to 
the start of monitoring, systems must arrange to have samples analyzed 
by a laboratory which meets the standards specified in paragraph (d) of 
this section. If systems are not able to arrange to have samples 
analyzed by a laboratory which meets the standards specified in 
paragraph (c) of this section by six months after publication of the 
final rule in the Federal Register, they are required to notify 
Technical Support Division, ATTN: ICR Laboratory Coordinator (Micro), 
OGWDW, USEPA, 26 West Martin Luther King Drive, Cincinnati, OH 45268. 
EPA will then provide a list of approved labs or other necessary 
guidance. Once a system has begun monitoring, it must continue to 
monitor for 18 consecutive months. All monitoring must be completed no 
later than March 31, 1997.
    (2) Subpart H systems serving at least 10,000, but less than 
100,000 people, must begin monitoring no earlier than three months 
after publication of the final rule in the Federal Register and no 
later than April 1996. Prior to the start of monitoring, systems must 
arrange to have samples analyzed by a laboratory which meets the 
standards specified in paragraph (c) of this section. If systems are 
not able to arrange to have samples analyzed by a laboratory which 
meets the standards specified in paragraph (d) of this section by nine 
months after publication of the final rule in the Federal Register, 
they are required to notify Technical Support Division, ATTN: ICR 
Laboratory Coordinator (Micro), OGWDW, USEPA, 26 West Martin Luther 
King Drive, Cincinnati, OH 45268. EPA will then provide a list of 
approved labs or other necessary guidance. Once a system has begun 
monitoring, it must continue to monitor for 12 consecutive months. All 
monitoring must be completed no later than March 31, 1997.
    (c) Monitoring Requirements--(1) Parameters. Except as allowed 
below, systems must sample for the following parameters for the period 
specified in paragraph (b) of this section and at the frequency and 
location specified in this paragraph, using the analytical methods 
specified in this paragraph. For each sample, systems must determine 
the concentration of total coliforms, fecal coliforms or Escherichia 
coli, Giardia, and Cryptosporidium. In addition, subpart H systems 
serving 100,000 or more people must determine the concentration of 
total culturable viruses.
    (2) Frequency and sample location. (i) Subpart H systems serving 
100,000 or more people must collect one sample per month of the source 
water at the intake of each plant within that system. Subpart H systems 
serving at least 10,000 but less than 100,000 people must collect one 
sample every other month of the source water at the intake of each 
plant within that system. The ``intake'' is defined as a point 
subsequent to surface water runoff, as determined by the system, but 
before the first treatment step used to comply with the Giardia/virus 
removals required by the Surface Water Treatment Rule (40 CFR 141, 
subpart H). If a plant has several sources or intakes of water, the 
system must sample the blended water from all sources; if the system 
determines that this is not possible because of the plant 
configuration, the system must sample the source with the expected 
highest pathogen concentrations.
    (ii) Systems serving 100,000 or more people that (A) detect one or 
more Giardia cyst, Cryptosporidium oocyst, or total culturable virus in 
one liter of water during the first twelve months of monitoring, or (B) 
calculate a numerical value of the pathogen concentration equal to or 
greater than 1.00 per liter, must also collect one sample per month of 
the finished water, beginning in the first calendar month after the 
system learns of such a result. (E.g., if the numerical value is <1.00, 
the system does not have to monitor finished water; if the value is 
1.00, the system must monitor finished water.) For each 
finished water sample, systems must determine the density of total 
coliforms, fecal coliforms or E. coli, Giardia, Cryptosporidium, and 
total culturable viruses. Systems must continue finished water 
monitoring monthly until 18 months of source water monitoring has been 
completed.
    (iii) Systems required to monitor total culturable viruses under 
this section that do not detect total culturable viruses during the 
first 12 months of monitoring are not required to monitor for total 
culturable viruses during the last six months of monitoring.
    (iv) Systems required to monitor total culturable viruses under 
this section that have tested the source water at each plant for either 
total coliforms or fecal coliforms at least five times per week between 
four months before publication of this final rule in the Federal 
Register and two months after publication need not monitor for total 
culturable viruses if: (A) The density of total coliforms is less than 
100 colonies/100 ml for at least 90 percent of the samples, or (B) the 
density of fecal coliforms is less than 20 colonies/100 ml for at least 
90 percent of the samples. Coliform monitoring data must be reported as 
required in paragraph (d) of this section. Systems may use monitoring 
conducted under the provisions of Sec. 141.71(a)(1) to meet this 
requirement. Systems that elect to use such monitoring must submit 
separate monitoring reports to meet the requirements under both subpart 
H and this section.
    (3) Analytical methods. Methods for total coliforms, fecal 
coliforms, Giardia and Cryptosporidium, total culturable viruses, and 
E. coli are specified in Sec. 141.74(a) (1), (2), (8), (9) and (10), 
respectively. Analysis under this section for microbiological 
contaminants shall be conducted by laboratories that have received 
approval from EPA to perform sample analysis for compliance with this 
rule.
    (d) Reporting. (1) In addition to reporting specified in 
Sec. 141.141, systems serving 100,000 or more people must report data 
and information in the format described in appendix A using an EPA-
specified computer readable format beginning four months after starting 
monitoring and monthly thereafter. Systems serving between at least 
10,000 but fewer than 100,000 people must report raw water data and 
information (except for viruses) in the format described in appendices 
A and B beginning four months after starting monitoring and every two 
months thereafter.
    (2) Systems that wish to avoid monitoring for total culturable 
viruses under the provisions of Sec. 141.140(c)(2)(iv) must report the 
dates and results of all total coliform and/or fecal coliform 
monitoring not later than three months after ICR promulgation.
    (3) All reports required by this paragraph will be submitted to 
____________________. Coordination for electronic reports will be made 
through ____________________.


Sec. 141.141  Disinfection Byproduct ICR Monitoring.

    (a) Applicability. (1) All community and nontransient noncommunity 
water systems that serve a population of 100,000 or more people must 
comply with the requirements in this section. Community and 
nontransient noncommunity water systems that use only ground water not 
under the direct influence of surface water and serve a population 
between 50,000 and 99,999 people, must only comply with the total 
organic carbon (TOC) monitoring requirements at the entry point to the 
distribution system as indicated in Table 1; no other monitoring in 
this section is required for these systems.
    (2) Consecutive systems. (i) Systems that receive only some of 
their water from a supplier must comply with all requirements of this 
section.
    (ii) Systems that receive all their water from a supplier and 
further disinfect this water must comply with the monitoring 
requirements in this section associated with sampling locations at and 
subsequent to the entry point to the distribution system.
    (iii) Systems that receive all their water from a supplier and do 
not further disinfect this water need not comply with the requirements 
in this section.
    (3) In determining population served, systems must include their 
own population and populations for all consecutive systems.
    (b) Schedule. Systems required to monitor under the provisions of 
Sec. 141.140 (Microbiological ICR Monitoring) must begin monitoring for 
this section and Sec. 141.140 in the same month, except as noted in 
paragraph (b)(2) of this section.
    (1) Except as required by paragraph (b)(2), systems must begin 
monitoring no earlier than [three months after publication of the final 
rule in the Federal Register] and no later than October 1995. Prior to 
the start of monitoring, systems must arrange to have samples analyzed 
by a laboratory which meets the standards specified in paragraph (c) of 
this section. If systems are not able to arrange to have samples 
analyzed by a laboratory which meets the standards specified in 
paragraph (c) of this section by [six months after publication of the 
final rule in the Federal Register], they are required to notify 
Technical Support Division, ATTN: ICR Laboratory Coordinator (Chem), 
OGWDW, USEPA, 26 West Martin Luther King Drive, Cincinnati, OH 45268. 
EPA will then provide a list of approved labs or other necessary 
guidance. Once a system has begun monitoring, it must continue to 
monitor for 18 consecutive months. All monitoring must be completed no 
later than March 31, 1997.
    (2) Subpart H systems must begin monitoring for source water TOC 
[three months after publication of the final rule in the Federal 
Register] and continue this monitoring until all other monitoring 
required by this section is complete. Community and nontransient 
noncommunity water systems that use only ground water not under the 
direct influence of surface water and serve 100,000 or more people must 
begin monitoring for finished water TOC [three months after publication 
of the final rule in the Federal Register] and continue this monitoring 
until all other monitoring required by this section is complete. 
Community and nontransient noncommunity water systems that use only 
ground water not under the direct influence of surface water and serve 
at least 50,000 but fewer than 100,000 people must begin monitoring for 
finished water TOC [three months after publication of the final rule in 
the Federal Register] and continue this monitoring for 12 months.
    (c) Monitoring requirements. All systems must obtain representative 
samples at the frequency and location noted in Table 1 of this section.
    (1) Additional requirements for systems using chloramines. Systems 
that use chloramines for treatment must also conduct the additional 
sampling identified in Table 2 of this section.
    (2) Additional requirements for systems using hypochlorite 
solutions. Systems that use hypochlorite solutions for treatment must 
also conduct the additional sampling identified in Table 3 of this 
section.
    (3) Additional requirements for systems using ozone. Systems that 
use ozone for treatment must also conduct the additional sampling 
identified in Table 4 of this section.
    (4) Additional sampling requirements for systems using chlorine 
dioxide. Systems that use chlorine dioxide for treatment must also 
conduct the additional sampling identified in Table 5 of this section.
    (5) Additional information reporting requirements for all systems 
serving at least 100,000 people. Such systems must also report the 
applicable information in Table 6 of this section.
    (6) Analytical methods. Systems must use the methods identified in 
Table 7 of this section for conducting analyses required by this 
section. Analysis under this section for disinfection byproducts shall 
be conducted by laboratories that have received approval from EPA to 
perform sample analysis for compliance with this rule.
    (d) Reporting. (1) Systems serving 100,000 or more people must 
report the required data and information in Tables 1-6 to EPA, using an 
EPA-specified computer readable format, beginning two months after 
starting monitoring, and every month thereafter. At the time of the 
first report, subpart H systems must submit the results of monthly 
source water TOC monitoring to date and subsequent monthly results as 
part of subsequent monthly reports. At the time of the first report, 
systems that use only ground water not under the direct influence of 
surface water and serve at least 100,000 people must submit the results 
of monthly finished water TOC monitoring to date and subsequent monthly 
results as part of subsequent monthly reports. Systems that use only 
ground water not under the direct influence of surface water and serve 
between 50,000 and 99,999 people must submit the results of 12 months 
of finished water TOC monitoring not later than [date 17 months after 
ICR promulgation].
    (2) All reports required by this paragraph will be submitted to 
________________________. Coordination for electronic reports will be 
made through ________________________.


Sec. 141.142  Disinfection Byproduct Precursor Removal ICR.

    (a)(1) Applicability. Except for systems meeting one or more 
criteria in paragraphs (a) (2) through (4) of this section, the 
following community and nontransient noncommunity water systems must 
conduct a disinfection byproduct precursor removal study (treatment 
study):
    (i) Subpart H systems that serve a population of 100,000 or more; 
and
    (ii) Systems that serve a population of 50,000 or more that use 
only ground water not under the direct influence of surface water and 
add a disinfectant to the water at any point in the treatment process.
    (2) Systems that use chlorine as the primary and residual 
disinfectant and have, as an annual average of four quarterly averages 
(quarterly averages are the arithmetic average of the four distribution 
system samples collected under the requirements of Sec. 141.141(c)), 
levels of less than 40 g/l for total THMs and less than 30 
/l of HAA5, are not required to conduct a treatment study.
    (3) Subpart H systems that do not exceed a TOC level of 4.0 mg/l in 
the treatment plant influent, measured in accordance with 
Sec. 141.141(c) and calculated by averaging the initial 12 monthly TOC 
samples, are not required to conduct a treatment study.
    (4) Groundwater systems that do not exceed a TOC level of 2.0 mg/l 
in the treated water at the entry point to the distribution system, 
measured in accordance with Sec. 141.141(c) and calculated by averaging 
the initial 12 monthly TOC samples, are not required to conduct a 
treatment study.
    (5) For systems that already use full scale GAC or membrane 
technology, full scale plant data must be submitted along with copies 
of any prior bench/pilot studies. Systems meeting criteria for avoiding 
treatment studies must continue to monitor as prescribed in 
Sec. 141.141.
    (b) The treatment study shall consist of bench- and/or pilot-scale 
systems for at least one of the two appropriate candidate technologies 
(GAC or membrane processes) for the reduction of organic DBP 
precursors. The treatment studies shall be designed to yield 
representative performance data and allow the development of treatment 
cost estimates for different levels of organic disinfection byproduct 
control. The treatment study shall be conducted with the effluent from 
treatment processes already in place that remove disinfection byproduct 
precursors and TOC. Depending upon the type of treatment study, the 
study shall be conducted in accordance with the following criteria.
    (1) Bench-scale testing shall be defined as continuous flow tests 
using: (i) Rapid small scale column test (RSSCT) for GAC; and (ii) 
Reactors with a configuration that yield representative flux loss 
assessment for membranes. Tests shall be preceded by particle removal 
processes, such as microfiltration.
    (A) GAC bench-scale testing shall include the following information 
on each RSSCT: pretreatment conditions, GAC type, GAC particle 
diameter, height and dry weight (mass) of GAC in the RSSCT column, 
RSSCT column inner diameter, volumetric flow rate, and operation time 
at which each sample is taken. At least two empty bed contact times 
(EBCTs) shall be tested using the RSSCT. These RSSCT EBCTs must be 
designed to represent a full-scale EBCT of 10 min and a full-scale EBCT 
of 20 min. Additional EBCTs may be tested. The RSSCT testing shall 
include the water quality parameters and sampling frequency listed in 
Table 8. The RSSCT shall be run until the effluent TOC concentration is 
75% of the average influent TOC concentration or a RSSCT operation time 
that represents the equivalent of one year of full-scale operation, 
whichever is shortest. The average influent TOC is defined as the 
running average of the influent TOC at the time of effluent sampling. 
RSSCTs shall be conducted quarterly over one year in order to determine 
the seasonal variation. Thus, a total of four RSSCTs at each EBCT is 
required. If, after completion of the first quarter RSSCTs, the system 
finds that the effluent TOC reaches 75% of the average influent TOC 
within 20 full-scale equivalent days on the EBCT=10 min test and within 
30 full-scale equivalent days on the EBCT=20 min test, then the last 
three quarterly tests shall be conducted using membrane bench-scale 
testing with only one membrane, as described in Sec. 141.142 (b)(1)(B).
    (B) Membrane bench-scale testing shall include the following 
information: Pretreatment conditions, membrane type, membrane area, 
configuration, inlet pressure and volumetric flow rate, outlet (reject) 
pressure and volumetric flow rate, permeate pressure and volumetric 
flow rate, recovery, and operation time at which each sample is taken. 
A minimum of two different membrane types with nominal molecular weight 
cutoffs of less than 1000 must be investigated. The membrane test 
system must be designed and run to yield a representative flux loss 
assessment. Membrane tests must be conducted quarterly over one year to 
determine the seasonal variation. Thus, a total of four membrane tests 
with each membrane must be run. The membrane bench-scale testing shall 
include the water quality parameters and sampling frequency listed in 
Table 9 of this section.
    (2) Pilot-scale testing shall be defined as continuous flow tests: 
(i) Using GAC of particle size representative of that used in full-
scale practice, a pilot GAC column with a minimum inner diameter of 2.0 
inches, and hydraulic loading rate (volumetric flow rate/column cross-
sectional area) representative of that used in full-scale practice; and 
(ii) using membrane modules with a minimum of a 4.0 inch diameter for 
spiral wound membranes or equivalent membrane area if other 
configurations are used.
    (A) GAC pilot-scale testing shall include the following information 
on the pilot plant: Pretreatment conditions, GAC type, GAC particle 
diameter, height and dry weight (mass) of GAC in the pilot column, 
pilot column inner diameter, volumetric flow rate, and operation time 
at which each sample is taken. At least two EBCTs shall be tested, 
EBCT=10 min and EBCT=20 min, using the pilot-scale plant. Additional 
EBCTs may be tested. The pilot testing shall include the water quality 
parameters listed in Table 10 of this Section. The pilot tests shall be 
run until the effluent TOC concentration is 75% of the average influent 
TOC concentration, with a maximum run length of one year. The average 
influent TOC is defined as the running average of the influent TOC at 
the time of sampling. The pilot-scale testing shall be sufficiently 
long to capture the seasonal variation.
    (B) Membrane pilot-scale testing shall include the following 
information on the pilot plant: Pretreatment conditions, membrane type, 
configuration, staging, inlet pressure and volumetric flow rate, outlet 
(reject) pressure and volumetric flow rate, permeate pressure and 
volumetric flow rate, recovery, operation time at which each sample is 
taken, recovery, cross flow velocity, recycle flow rate, backwashing 
and cleaning conditions, and characterization and ultimate disposal of 
the reject stream. The membrane test system must be designed to yield a 
representative flux loss assessment. The pilot-scale testing shall be 
sufficient in length and conducted throughout the year in order to 
capture the seasonal variation, with a maximum run length of one year. 
The pilot testing shall include the water quality parameters listed in 
Table 11.
    (3) For either the bench- or pilot-scale tests, systems must 
collect influent water samples at a location before the first point at 
which oxidants or disinfectants that form chlorinated disinfection 
byproducts are added. If the use of these oxidants or disinfectants 
precedes any full-scale treatment process that removes disinfection 
byproduct precursors, then bench- and pilot-scale treatment processes 
that represent these full-scale treatment processes are required prior 
to the GAC or membrane process.
    (4) Simulated distribution system (SDS) conditions with chlorine 
will be used prior to the measurement of THMs, haloacetic acids (six) 
(HAA6), TOX, and chlorine demand. These conditions should be based on 
the site specific SDS sample as defined in Sec. 141.141(c) (Table 1) 
with regards to holding time, temperature, and chlorine residual. If 
chlorine is not used as the final disinfectant in practice, then a 
chlorine dose should be set to yield a free chlorine residual of at 
least 0.2 mg/l after a holding time equal to the longest period of time 
the water is expected to remain in the distribution system or 7 days, 
whichever is shortest. The holding time prior to analysis of THMs, 
HAA6, TOX, and chlorine demand shall remain as that of the SDS sample 
as defined in Sec. 141.141(c) (Table 1).
    (5) For systems with multiple source waters, bench- or pilot scale 
testing shall be required for each treatment plant that serves a 
population greater than that set forth in Sec. 141.142(a) and use other 
source waters that exceed the TOC criteria set forth in 
Sec. 141.142(a)(1) unless the source waters are of similar water 
quality.

    (Note: Guidance Manual will specify)

    (6) All systems conducting bench or pilot scale studies must report 
the additional information in Table 6 of Sec. 141.141 as appropriate 
for source water and treatment processes that precede the bench/pilot 
systems. This information is to be reported for full-scale pretreatment 
processes and for pilot- or bench-scale pretreatment processes where 
appropriate.
    (c) Schedule. Systems must begin the disinfection byproduct 
precursor removal study not later than [date 18 months following 
promulgation] and submit the report(s) of the completed study to EPA 
not later than September 30, 1997.

                Table 1.--Sampling Points for All Systems               
------------------------------------------------------------------------
     Sampling point               Analyses1               Frequency     
------------------------------------------------------------------------
Treatment Plant Influent5  pH, Alkalinity,           Monthly.           
                            Turbidity, Temperature,                     
                            Calcium and Total                           
                            Hardness, TOC, UV254,                       
                            Bromide, and Ammonia.                       
Treatment Plant Influent   Optional oxidant demand   Monthly.           
 (optional for waters       test.                                       
 with high oxidant demand                                               
 due to the presence of                                                 
 inorganics).                                                           
Treatment Plant Influent.  TOX.....................  Quarterly.         
After Air Stripping......  Ammonia.................  Monthly.           
Before and After           pH, Alkalinity,           Monthly.           
 Filtration.                Turbidity, Temperature,                     
                            Calcium and Total                           
                            Hardness, TOC, and                          
                            UV254.                                      
At each Point of           pH, Alkalinity,           Monthly.           
 Disinfection\2\.           Turbidity, Temperature,                     
                            Calcium and Total                           
                            Hardness, TOC, and                          
                            UV254.                                      
At End of Each Process in  Disinfectant Residual\3\  Monthly.           
 which Chlorine is                                                      
 Applied.                                                               
After Filtration (If       THMs, HAAs(6), HANs, CP,  Quarterly.         
 Chlorine is Applied        HK, CH, and TOX.                            
 Prior to Filtration).                                                  
Entry Point to             pH, Alkalinity,           Monthly.           
 Distribution System.       Turbidity, Temperature,                     
                            Calcium and Total                           
                            Hardness, TOC, UV254,                       
                            and Disinfectant                            
                            Residual\3\.                                
Entry Point to             THMs, HAAs(6), HANs, CP,  Quarterly.         
 Distribution System.       HK, CH, TOX, and SDS\4\.                    
4 THM Compliance           THMs, HAAs (6), HANs,     Quarterly.         
 Monitoring Points in       CP, HK, CH, TOX, pH,                        
 Distribution System (1     Temperature,                                
 sample point will be       Alkalinity, Total                           
 chosen to correspond to    Hardness and                                
 the SDS sample\4\, 1       Disinfectant                                
 will be chosen at a        Residual\3\.                                
 maximum detention time,                                                
 and the remaining 2 will                                               
 be representative of the                                               
 distribution system).                                                  
------------------------------------------------------------------------
1TOC: total organic carbon. UV254: absorbance of ultraviolet light at   
  254 nanometers. THMs: chloroform, bromodichloromethane,               
  dibromochloromethane, and bromoform. HAAs(6): mono-, di-, and         
  trichloroacetic acid; mono-, and di- bromoacetic acid; and            
  bromochloroacetic acid. HANs: dichloro-, trichloro-, bromochloro-, and
  dibromo- acetonitrile. CP: chloropicrin. HK: 1,1-dichloropropanone and
  1,1,1- trichloropropanone. CH: chloral hydrate. TOX: total organic    
  halide. SDS: simulated distribution system test.                      
2For utilities using ozone or chlorine dioxide, Tables 4 and 5,         
  respectively, show additional monitoring requirements at this sampling
  point.                                                                
3Free chlorine residual will be measured in systems using free chlorine 
  as the residual disinfectant; total chlorine residual will be measured
  in systems using chloramines as the residual disinfectant.            
4The simulated distribution system test sample will be stored in such a 
  manner that it can be compared to the results from one of the         
  distribution system sampling points. This distribution system sampling
  point will be selected using the following criteria: 1) No additional 
  disinfectant added between it and the treatment plant; 2) Approximate 
  detention time of water is available; and 3) No blending with water   
  from other sources. The SDS sample will be analyzed for THMs, HAAs(6),
  HANs, CP, HK, CH, TOX, pH and disinfectant residual.                  
5A ground water system with multiple wells from the same aquifer is only
  required to monitor TOC from one sampling point. A ground water system
  with multiple wells from different aquifers must collect at least one 
  sample from each aquifer and determine which two aquifers have the    
  highest TOC concentrations; thereafter, the system must sample TOC    
  from these two aquifers.                                              


  Table 2.--Additional Sampling Required of Systems Using Chloramines   
------------------------------------------------------------------------
      Sampling point              Analyses               Frequency      
------------------------------------------------------------------------
Entry Point to              Cyanogen Chloride....  Quarterly.           
 Distribution System.                                                   
One THM Compliance          Cyanogen Chloride....  Quarterly.           
 Monitoring Sample Point                                                
 Representing a Maximum                                                 
 Detention Time in                                                      
 Distribution System.                                                   
------------------------------------------------------------------------


  Table 3.--Additional Sampling Required of Systems Using Hypochlorite  
                               Solutions                                
------------------------------------------------------------------------
      Sampling point              Analyses               Frequency      
------------------------------------------------------------------------
Treatment Plant Influent..  Chlorate.............  Quarterly.           
Hypochlorite Stock          pH, Temperature, Free  Quarterly.           
 Solution.                   Residual Chlorine,                         
                             and Chlorate.                              
Entry Point to              Chlorate.............  Quarterly.           
 Distribution System.                                                   
------------------------------------------------------------------------


     Table 4.--Additional Sampling Required of Systems Using Ozone      
------------------------------------------------------------------------
     Sampling point                Analyses               Frequency     
------------------------------------------------------------------------
Ozone Contactor Influent.  pH, Alkalinity,           Monthly.           
                            Turbidity, Temperature,                     
                            Calcium and Total                           
                            Hardness, TOC, UV254,                       
                            Bromide, and Ammonia.                       
Ozone Contactor Influent.  Aldehydes1 and AOC/BDOC2  Quarterly.         
Ozone Contactor Effluent.  Ozone Residual..........  Monthly.           
Ozone Contactor Effluent.  Aldehydes1 and AOC/BDOC2  Quarterly.         
Before Filtration........  Ozone Residual..........  Monthly.           
Entry Point to             Bromate.................  Monthly.           
 Distribution System.                                                   
Entry Point to             Aldehydes1 and AOC/BDOC2  Quarterly.         
 Distribution System.                                                   
------------------------------------------------------------------------
1The aldehydes to be included in this analysis are: formaldehyde,       
  acetaldehyde, butanal, propanal, pentanal, glyoxal, and methyl        
  glyoxal. Measurement of other aldehydes is optional.                  
2Analysis or submission of data for assimilable organic carbon (AOC) or 
  biodegradeable organic carbon (BDOC) is optional.                     


Table 5.--Additional Sampling Required of Systems Using Chlorine Dioxide
                                                                        
------------------------------------------------------------------------
     Sampling point                Analyses               Frequency     
------------------------------------------------------------------------
Treatment Plant Influent.  Chlorate................  Quarterly.         
Before each Chlorine       pH, Alkalinity,           Monthly.           
 Dioxide Application.       Turbidity, Temperature,                     
                            Calcium and Total                           
                            Hardness, TOC, UV254,                       
                            and Bromide.                                
Before First Chlorine      Aldehydes\1\ and AOC/     Quarterly.         
 Dioxide Application.       BDOC\2\.                                    
Before Application of      pH, Chlorine Dioxide      Monthly.           
 Ferrous Salts, Sulfur      Residual, Chlorite,                         
 Reducing Agents, or GAC.   Chlorate.                                   
Before Downstream          Aldehydes\1\ and AOC/     Quarterly.         
 Chlorine/Chloramine        BDOC\2\.                                    
 Application.                                                           
Entry Point to             Chlorite, Chlorate,       Monthly.           
 Distribution System.       Chlorine Dioxide                            
                            Residual, Bromate.                          
Entry Point to             Aldehydes\1\ and AOC/     Quarterly.         
 Distribution System.       BDOC\2\.                                    
3 Distribution System      Chlorite, Chlorate,       Monthly.           
 Sampling Points (1 near    Chlorine Dioxide                            
 first customer, 1 in       Residual, pH, and                           
 middle of distribution     Temperature.                                
 system, and 1 at a                                                     
 maximum detention time                                                 
 in the system).                                                        
------------------------------------------------------------------------
\1\The aldehydes to be included in this analysis are: formaldehyde,     
  acetaldehyde, butanal, propanal, pentanal, glyoxal, and methyl        
  glyoxal. Measurement of other aldehydes is optional.                  
\2\Analysis or submission of data for AOC or BDOC is optional.          


                  Table 6.--Treatment Plant Information                 
                                                                        
                                                                        
Utility                                                                 
 Information:                                                           
  Utility Name                                                          
  Mailing Address                                                       
  Contact Person &                                                      
   Phone Number                                                         
  Public Water                                                          
   Supply                                                               
   Identification                                                       
   Number FRDS                                                          
   (PWSID)                                                              
  Population Served                                                     
                                                                        
--------------------                                                    
Plant Information:                                                      
  Name of plant                                                         
  Design flow (MGD)                                                     
  Annual minimum                                                        
   water                                                                
   temperature (C)                                                      
  Annual maximum                                                        
   water                                                                
   temperature (C)                                                      
  Hours of                                                              
   operation (hours                                                     
   per day)                                                             
                                                                        
--------------------                                                    
Source Water                                                            
 Information:                                                           
  Name of source                                                        
  Type of source                                                        
   (One of the                                                          
   following)                                                           
    1 River                                                             
    2 Stream                                                            
    3 Reservoir                                                         
    4 Lake                                                              
    5 Ground water                                                      
     under the                                                          
     direct                                                             
     influence of                                                       
     surface water                                                      
    6 Ground water                                                      
    7 Spring                                                            
    8 Purchased                                                         
     from Utility                                                       
     Name, FRDS                                                         
     PWSID                                                              
    9 Other                                                             
  Surface water as                                                      
   defined by SWTR                                                      
   (YES/NO)                                                             
  Monthly Average                                                       
   Flow of this                                                         
   Source (MGD)                                                         
  Upstream sources                                                      
   of                                                                   
   microbiological                                                      
   contamination                                                        
    Wastewater                                                          
     plant                                                              
     discharge in                                                       
     watershed (yes/                                                    
     no)                                                                
    Distance from                                                       
     intake (miles)                                                     
    Monthly average                                                     
     flow of plant                                                      
     discharge                                                          
     (MGD)                                                              
  Point source                                                          
   feedlots in                                                          
   watershed (yes/                                                      
   no)                                                                  
    Distance of                                                         
     nearest                                                            
     feedlot                                                            
     discharge to                                                       
     intake (miles)                                                     
  Non-point sources                                                     
   in watershed                                                         
    Grazing of                                                          
     animals (yes/                                                      
     no)                                                                
  Nearest distance                                                      
   of grazing to                                                        
   intake (miles)                                                       
                                                                        
--------------------                                                    
Plant Influent:                                                         
 (ICR influent                                                          
 sampling point)                                                        
  Monthly average                                                       
   flow (MGD)                                                           
  Monthly peak                                                          
   hourly flow                                                          
   (MGD)                                                                
  Flow at time of                                                       
   sampling (MGD)                                                       
                                                                        
--------------------                                                    
Plant Effluent:                                                         
 (ICR effluent                                                          
 sampling point)                                                        
  Monthly average                                                       
   flow (MGD)                                                           
  Monthly peak                                                          
   hourly flow                                                          
   (MGD)                                                                
  Flow at time of                                                       
   sampling (MGD)                                                       
                                                                        
--------------------                                                    
Sludge Treatment:                                                       
  Monthly average                                                       
   solids                                                               
   production (lb/                                                      
   day)                                                                 
  Installed design                                                      
   sludge handling                                                      
   capacity (lb/                                                        
   day)                                                                 
                                                                        
--------------------                                                    
General Process                                                         
 Parameters:                                                            
  The following                                                         
   will be                                                              
   requested for                                                        
   all unit                                                             
   processes.                                                           
      Number of                                                         
       identical                                                        
       parallel                                                         
       units                                                            
       installed.                                                       
      Number of                                                         
       identical                                                        
       parallel                                                         
       units in                                                         
       service at                                                       
       time of                                                          
       sampling.                                                        
  The following                                                         
   parameters will                                                      
   be requested for                                                     
   all unit                                                             
   processes except                                                     
   chemical                                                             
   feeders.                                                             
      Design flow                                                       
       per unit                                                         
       (MGD)                                                            
      Liquid volume                                                     
       per unit                                                         
       (gallons)                                                        
      Tracer study                                                      
       flow (MGD)                                                       
      T50 (minutes)                                                     
      T10 (minutes)                                                     
                                                                        
--------------------                                                    
Presedimentation                                                        
 Basin:                                                                 
  Surface loading                                                       
   at design flow                                                       
   (gpm/ft\2\)                                                          
                                                                        
--------------------                                                    
Chemical Feeder:                                                        
  Type of feeder                                                        
   (one of the                                                          
   following)                                                           
    1 Liquid                                                            
    2 Gas                                                               
    3 Dry                                                               
  Capacity of each                                                      
   unit (lb/day)                                                        
  Purpose (one or                                                       
   more of the                                                          
   following)                                                           
    1 Coagulation                                                       
    2 Coagulation                                                       
     aid                                                                
    3 Corrosion                                                         
     control                                                            
    4                                                                   
     Dechlorination                                                     
    5 Disinfection                                                      
    6 Filter aid                                                        
    7 Fluoridation                                                      
    8 Oxidation                                                         
    9 pH adjustment                                                     
    10                                                                  
     Sequestration                                                      
    11 Softening                                                        
    12                                                                  
     Stabilization                                                      
    13 Taste and                                                        
     odor control                                                       
    14 Other                                                            
                                                                        
--------------------                                                    
Chemical Feeder                                                         
 Chemicals: (one of                                                     
 the following)                                                         
    Alum                                                        
    Anhydro                                                     
     us ammonia                                                         
    Ammoniu                                                     
     m hydroxide                                                        
    Ammoniu                                                     
     m sulfate                                                          
    Calcium                                                     
     hydroxide                                                          
    Calcium                                                     
     hypochlorite                                                       
    Calcium                                                     
     oxide                                                              
    Carbon                                                      
     dioxide                                                            
    Chlorin                                                     
     e dioxide--                                                        
     acid chlorite                                                      
    Chlorin                                                     
     e dioxide--                                                        
     chlorine/chlor                                                     
     ite                                                                
    Chlorin                                                     
     e gas                                                              
    Ferric                                                      
     chloride                                                           
    Ferric                                                      
     sulfate                                                            
    Ferrous                                                     
     sulfate                                                            
    Ozone                                                       
    Polyalu                                                     
     minum chloride                                                     
    Sodium                                                      
     carbonate                                                          
    Sodium                                                      
     chloride                                                           
    Sodium                                                      
     fluoride                                                           
    Sodium                                                      
     hydroxide                                                          
    Sodium                                                      
     hypochlorite                                                       
    Sodium                                                      
     hexametaphosph                                                     
     ate                                                                
    Sodium                                                      
     silicate                                                           
    Sulfuri                                                     
     c acid                                                             
    Zinc                                                        
     orthophosphate                                                     
    Other                                                       
Notes:                                                                  
  1. The above list                                                     
   is intended to                                                       
   be a                                                                 
   comprehensive                                                        
   list of                                                              
   chemicals used                                                       
   at water                                                             
   treatment                                                            
   plants. If the                                                       
   name of a                                                            
   chemical does                                                        
   not appear in                                                        
   the list then                                                        
   ``Other                                                              
   Chemical''                                                           
   information will                                                     
   be requested.                                                        
  2. Formulas and                                                       
   feed rate units                                                      
   will be included                                                     
   in data                                                              
   reporting                                                            
   software.                                                            
  Monthly average                                                       
   feed rate based                                                      
   on inventory (mg/                                                    
   L)                                                                   
  Feed rate at time                                                     
   of sampling (mg/                                                     
   L)                                                                   
                                                                        
--------------------                                                    
Other Chemical:                                                         
  Note: In addition                                                     
   to Chemical                                                          
   Feeder                                                               
   information the                                                      
   following will                                                       
   be requested for                                                     
   any chemical not                                                     
   included in the                                                      
   Chemical Feeder                                                      
   list of                                                              
   chemicals.                                                           
      Trade name of                                                     
       chemical                                                         
      Formula                                                           
      Manufacturer                                                      
                                                                        
--------------------                                                    
Rapid Mix:                                                              
  Type of mixer                                                         
   (one of the                                                          
   following)                                                           
    1 Mechanical                                                        
    2 Hydraulic                                                         
     jump                                                               
    3 Static                                                            
    4 Other                                                             
  If mechanical:                                                        
   horsepower of                                                        
   motor                                                                
  If hydraulic:                                                         
   head loss (ft)                                                       
  If static: head                                                       
   loss (ft)                                                            
                                                                        
--------------------                                                    
Flocculation Basin:                                                     
  Type of mixer                                                         
   (one of the                                                          
   following)                                                           
    1 Mechanical                                                        
    2 Hydraulic                                                         
    3 Other                                                             
  If mechanical:                                                        
   Mixing power                                                         
   (HP)                                                                 
  If hydraulic:                                                         
   head loss (ft)                                                       
                                                                        
--------------------                                                    
Sedimentation                                                           
 Basin:                                                                 
  Loading at Design                                                     
   Flow (gpm/ft\2\)                                                     
  Dept (ft)                                                             
                                                                        
--------------------                                                    
Filtration:                                                             
  Loading at Design                                                     
   Flow (gpm/ft\2\)                                                     
  Media Type (one                                                       
   or more of the                                                       
   following)                                                           
    1 Anthracite                                                        
    2 GAC                                                               
    3 Garnet                                                            
    4 Sand                                                              
    5 Other                                                             
  Depth of top                                                          
   media (in)                                                           
  If more than 1                                                        
   media: Depth of                                                      
   second media                                                         
   (in)                                                                 
  If more than 2                                                        
   media: Depth of                                                      
   third media (in)                                                     
  If more than 3                                                        
   media: Depth of                                                      
   fourth media                                                         
   (in)                                                                 
  If GAC media:                                                         
   Carbon                                                               
   replacement                                                          
   frequency                                                            
   (months):                                                            
  Water depth to                                                        
   top of media                                                         
   (ft)                                                                 
  Depth from top of                                                     
   media to bottom                                                      
   of backwash                                                          
   trough (ft)                                                          
  Backwash                                                              
   Frequency                                                            
   (hours)                                                              
  Backwash volume                                                       
   (gallons)                                                            
                                                                        
--------------------                                                    
Contact Basin:                                                          
 (Stable liquid                                                         
 level)                                                                 
  Baffling Type                                                         
   (one of the                                                          
   following as                                                         
   defined in SWTR                                                      
   guidance manual)                                                     
    1 Unbaffled                                                         
     (mixed tank)                                                       
    2 Poor (inlet/                                                      
     outlet only)                                                       
    3 Average                                                           
     (Inlet/Outlet                                                      
     and                                                                
     intermediate)                                                      
    4 Superior                                                          
     (Serpentine)                                                       
    5 Perfect (Plug                                                     
     flow)                                                              
                                                                        
--------------------                                                    
Clearwell:                                                              
 (Variable liquid                                                       
 level)                                                                 
  Baffling Type                                                         
   (one of the                                                          
   following as                                                         
   defined in SWTR                                                      
   guidance manual)                                                     
    1 Unbaffled                                                         
     (mixed tank)                                                       
    2 Poor (inlet/                                                      
     outlet only)                                                       
    3 Average                                                           
     (Inlet/Outlet                                                      
     and                                                                
     intermediate)                                                      
    4 Superior                                                          
     (Serpentine)                                                       
    5 Perfect (Plug                                                     
     flow)                                                              
  Minimum liquid                                                        
   volume (gallons)                                                     
  Liquid volume at                                                      
   time of tracer                                                       
   study (gallons)                                                      
                                                                        
--------------------                                                    
 Ozone Contact                                                          
 Basin:                                                                 
  Basin Type                                                            
  1 Over/Under                                                          
   (Diffused O3)                                                        
    2 Mixed                                                             
     (Turbine O3)                                                       
  Number of Stages                                                      
  CT (min mg/L)                                                         
  EPA requests                                                          
   comments on the                                                      
   design and                                                           
   operating                                                            
   paramenters to                                                       
   be reported for                                                      
   ozone contact                                                        
   basins.                                                              
                                                                        
--------------------                                                    
Tube Settler:                                                           
  Surface loading                                                       
   at design flow                                                       
   (gpm/ft2)                                                            
  Tube angle from                                                       
   horizontal                                                           
   (degrees)                                                            
                                                                        
--------------------                                                    
Upflow Clarifier:                                                       
  Design horse                                                          
   power of turbine                                                     
   mixer (HP)                                                           
  Surface loading                                                       
   at design flow                                                       
   (gpm/ft2)                                                            
  Special Equipment                                                     
   (none, one, or                                                       
   more of the                                                          
   following)                                                           
    1 Lamella                                                           
     plates                                                             
    2 Tubes                                                             
                                                                        
--------------------                                                    
Plate Settler:                                                          
  Surface loading                                                       
   at design flow                                                       
   (gpm/ft2)                                                            
                                                                        
--------------------                                                    
DE Filter:                                                              
  Surface loading                                                       
   at design flow                                                       
   (gpm/ft2)                                                            
  Precoat (1b/ft3)                                                      
  Bodyfeed (mg/L)                                                       
  Run length                                                            
   (hours)                                                              
                                                                        
--------------------                                                    
Granular Activated                                                      
 Carbon:                                                                
  Empty bed contact                                                     
   time at design                                                       
   flow (minutes)                                                       
  Design                                                                
   regeneration                                                         
   frequency (days)                                                     
  Actual                                                                
   regeneration                                                         
   frequency (days)                                                     
                                                                        
--------------------                                                    
Membranes:                                                              
  Type (one of the                                                      
   following)                                                           
    1 Reverse                                                           
     osmosis                                                            
    2                                                                   
     Nanofiltration                                                     
    3                                                                   
     Ultrafiltratio                                                     
     n                                                                  
    4                                                                   
     Microfiltratio                                                     
     n                                                                  
    5                                                                   
     Electrodialysi                                                     
     s                                                                  
    6 Other                                                             
  Name of other                                                         
   type                                                                 
  Membrane type                                                         
   (one of the                                                          
   following)                                                           
    1 Cellulose                                                         
     acetate and                                                        
     derivatives                                                        
    2 Polyamides                                                        
    3 Thin-film                                                         
     composite                                                          
    4 Other                                                             
  Name of other                                                         
   membrane type                                                        
  Molecular weight                                                      
   cutoff (gm/mole)                                                     
  Configuration                                                         
   (one of the                                                          
   following)                                                           
    1 Spiral wound                                                      
    2 Hollow fiber                                                      
    3 Tube                                                              
    4 Plate and                                                         
     frame                                                              
    5 Other                                                             
  Name of other                                                         
   configuration                                                        
  Design flux (gpd/                                                     
   ft\2\)                                                               
  Design pressure                                                       
   (psi)                                                                
  Purpose of                                                            
   membrane unit                                                        
   (one or more of                                                      
   the following)                                                       
    1 Softening                                                         
    2 Desalination                                                      
    3 Organic                                                           
     removal                                                            
    4 Other                                                             
    5 Contaminant                                                       
     removal--name                                                      
     of contaminant                                                     
  Percent recovery                                                      
   (%)                                                                  
  Operating                                                             
   pressure (psi)                                                       
                                                                        
--------------------                                                    
 Air Stripping:                                                         
  Packing height                                                        
   (ft)                                                                 
  Design liquid                                                         
   loading (gpm/                                                        
   ft\2\)                                                               
  Design air to                                                         
   water ratio                                                          
  Type of packing                                                       
   (name)                                                               
  Nominal size of                                                       
   packing (inch)                                                       
  Operating air                                                         
   flow (SCFM)                                                          
                                                                        
--------------------                                                    
 Adsorption                                                             
 Clarifier:                                                             
  Surface loading                                                       
   at design flow                                                       
   (gpm/ft\2\)                                                          
                                                                        
--------------------                                                    
 Dissolved Air                                                          
 Flotation:                                                             
  Surface loading                                                       
   at design flow                                                       
   (gmp/ft\2\)                                                          
                                                                        
--------------------                                                    
 Slow Sand                                                              
 Filtration:                                                            
  Surface loading                                                       
   at design flow                                                       
   (gpd/ft\2\)                                                          
                                                                        
--------------------                                                    
 Ion Exchange:                                                          
  Purpose (one or                                                       
   more of the                                                          
   following)                                                           
    1 Softening                                                         
    2 Contaminant                                                       
     removal                                                            
  Contaminant name                                                      
  Media type (Name)                                                     
  Design exchange                                                       
   capacity (equ/                                                       
   ft\3\)                                                               
  Surface loading                                                       
   at design flow                                                       
   (gpm/ft\2\)                                                          
  Bed depth (ft)                                                        
  Regenerant Name                                                       
   (one of the                                                          
   following)                                                           
    1 Sodium                                                            
     Chloride                                                           
     (NaCl)                                                             
    2 Sulfuric Acid                                                     
     (H2SO4)                                                            
    3 Sodium                                                            
     Hydroxide                                                          
     (NaOH)                                                             
    4 Other                                                             
  If other: Name                                                        
   and formula                                                          
  Operating                                                             
   regeneration                                                         
   frequency (hr)                                                       
  Regenerant                                                            
   concentration                                                        
   (%)                                                                  
  Regenerant Used                                                       
   (lb/day)                                                             
                                                                        
--------------------                                                    
 Other treatment:                                                       
  Name                                                                  
  Purpose                                                               
  Design Parameters                                                     
                                                                        


                            Table 7.--Analytical Methods Approved for Monitoring Rule                           
----------------------------------------------------------------------------------------------------------------
                                                                     Methodology                                
              Analyte              -----------------------------------------------------------------------------
                                       40 CFR reference\1\           EPA method            Standard method\2\   
----------------------------------------------------------------------------------------------------------------
pH................................  141.74(a)(7), 141.89(a)   ........................  4500-H+                 
Alkalinity........................  141.89(a)                 ........................  2320 B                  
Turbidity.........................  141.22(a), 141.74(a)(4)   180.1\3\                  2130 B                  
Temperature.......................  141.74(a)(6), 141.89(a)   ........................  2550 B                  
Calcium Hardness..................  141.89(a)                 200.7\4\                  3111 B, 3120 B, 3500-Ca 
                                                                                         D                      
Free Residual Chlorine............  141.74(a)(5)              ........................  4500-Cl D, 4500-Cl F,   
                                                                                         4500-Cl G, 4500-Cl H   
Total Residual Chlorine...........  141.74(a)(5)              ........................  4500-Cl D, 4500-Cl E,   
                                                                                         4500-Cl F, 4500-Cl G,  
                                                                                         4500-Cl I              
Chlorine Dioxide Residual.........  141.74(a)(5)              ........................  4500-ClO2 C, 4500-ClO2  
                                                                                         D, 4500-ClO2 E         
Ozone Residual....................  141.74(a)(5)              ........................  4500-O3 B               
Chloroform........................  141 Subpt C, App. C       502.2\5\, 524.2\5\,\6\,,  ........................
                                                               551\7\,\8\                                       
Bromodichloromethane..............  141 Subpt C, App. C       502.2\5\, 524.2\5\,\6\,,  ........................
                                                               551\7\,\8\                                       
Dibromochloromethane..............  141 Subpt C, App. C       502.2\5\, 524.2\5\,\6\,,  ........................
                                                               551\7\,\8\                                       
Bromoform.........................  141 Subpt C, App. C       502.2\5\, 524.2\5\,\6\,,  ........................
                                                               551\7\,\8\                                       
Monochloroacetic Acid.............  ........................  55.1\6\                   6233 B                  
Dichloroacetic Acid...............  ........................  552.1\6\                  6233 B                  
Trichloroacetic Acid..............  ........................  552.1\6\                  6233 B                  
Monobromoacetic Acid..............  ........................  552.1\6\                  6233 B                  
Dibromoacetic Acid................  ........................  552.1\6\                  6233 B                  
Bromochloroacetic Acid............  ........................  552.1\6\                  6233 B\9\               
Chloral Hydrate...................  ........................  551\7\                    ........................
Trichloroacetonitrile.............  ........................  551\7\,\8\                ........................
Dichloroacetonitrile..............  ........................  551\7\,\8\                ........................
Bromochloroacetonitrile...........  ........................  551\7\,\8\                ........................
Dibromoacetonitrile...............  ........................  551\7\,\8\                ........................
1,1-Dichloropropanone.............  ........................  551\7\,\8\                ........................
1,1,1,-Trichloropropanone.........  ........................  551\7\,\8\                ........................
Chloropicrin......................  ........................  551\7\,\8\                ........................
Chlorite..........................  ........................  300.0\10\                 ........................
Chlorate..........................  ........................  300.0\10\                 ........................
Bromide...........................  ........................  300.0\10\                 ........................
Bromate...........................  ........................  300.0\10\                 ........................
Cyanogen Chloride.................  ........................  524.2\6\                  ........................
Aldehydes.........................  ........................  ........................  Draft method submitted  
                                                                                         to 19th Edition        
Total Organic Halide (TOX)........  ........................  ........................  5320 B                  
Total Organic Carbon..............  ........................  ........................  5310C, 5310 D           
UV absorbance at 254 nm (method     ........................  ........................  ........................
 described in preamble--protocol                                                                                
 will be developed).                                                                                            
Simulated Distribution System Test  ........................  ........................  5710 E                  
 (SDS).                                                                                                         
Total Hardness....................  ........................  ........................  2340 B, 2340 C          
Ammonia...........................  ........................  ........................  4500-NH3 D, 4500-NH3 F  
Oxidant Demand/Requirement          ........................  ........................  2350 B, 2350 C, 2350 D  
 (optional).                                                                                                    
AOC/BDOC (optional)...............  ........................  ........................  9217 B/                 
----------------------------------------------------------------------------------------------------------------
\1\Currently approved methodology for drinking water compliance monitoring is listed in Title 40 of the Code of 
  Federal Regulations in the sections referenced in this column.                                                
\2\Standard Methods for the Examination of Water and Wastewater, 18th ed., American Public Health Association,  
  American Water Works Association, Water Pollution Control Federation, 1992.                                   
\3\``Methods of Chemical Analysis of Water and Wastes,'' EPA Environmental Monitoring Systems Laboratory,       
  Cincinnati, OH EPA-600/4-79-020, Revised March 1983.                                                          
\4\Methods for the Determination of Metals in Environmental Samples. Available from National Technical          
  Information Service (NTIS), U.S. Department of Commerce, Springfield, Virginia, PB91-231498, June 1991.       
\5\USEPA, ``Methods for the Determination of Organic Compounds in Drinking Water,'' EPA/600/4-88/039, PB91-     
  231480, National Technical Information Service (NTIS), December 1988 (revised July 1991).                     
\6\USEPA, ``Methods for the Determination of Organic Compounds in Drinking Water--Supplement II,'' EPA/600/R-92/
  129, PB92-207703, NTIS, August 1992.                                                                          
\7\USEPA, ``Methods for the Determination of Organic Compounds in Drinking Water--Supplement I,'' EPA/600/4-90- 
  020, PB91-146027, NTIS, July 1990.                                                                            
\8\Pentane may be used as the extraction solvent for this analyte, if the quality control criteria of the method
  are met.                                                                                                      
\9\Although this analyte is not currently included in the method, EPA has reviewed data demonstrating it can be 
  added to the method. The method is being revised and will be included in the 19th edition of Standard Methods.
\10\USEPA, ``Methods for the Determination of Inorganic Substances in Environmental Samples,'' EPA/600/R/93/100-
  Draft, June 1993.                                                                                             


             Table 8.--Sampling of GAC Bench-scale Systems              
------------------------------------------------------------------------
    Sampling point              Analyses             Sample frequency   
------------------------------------------------------------------------
GAC Influent...........  Alkalinity, total &      Two samples per batch 
                          calcium hardness,        of influent evenly   
                          ammonia and bromide.     spaced over the RSSCT
                                                   run.                 
GAC Influent...........  pH, turbidity,           Three samples per     
                          temperature, TOC and     batch of influent    
                          UV254. SDS\1\ for        evenly spaced over   
                          THMs, HAA6, TOX, and     the RSSCT run.       
                          chlorine demand.                              
GAC Effluent @ EBCT=10   pH, temperature, TOC,    A minimum of 12       
 min (scaled).            and UV254. SDS\1\ for    samples. One after   
                          THMs, HAA6, TOX, and     one hour, and        
                          chlorine demand.         thereafter at 5% to  
                                                   8% increments of the 
                                                   average influent TOC.
GAC Effluent @ EBCT=20   pH, temperature, TOC     A minimum of 12       
 min (scaled).            and UV254. SDS\1\ for    samples. One after   
                          THMs, HAA6, TOX, and     one hour, and        
                          chlorine demand.         thereafter at 5% to  
                                                   8% increments of the 
                                                   average influent TOC.
                                                                        
------------------------------------------------------------------------
\1\--SDS conditions are defined in Sec. 141.142(b)(4).                  


           Table 9.--Sampling of Bench-scale Membrane Systems           
------------------------------------------------------------------------
     Sampling point              Analyses           Sample frequency\2\ 
------------------------------------------------------------------------
Membrane Influent......  Alkalinity, total        Two samples per batch 
                          dissolved solids,        of influent evenly   
                          total & calcium          spaced over the      
                          hardness and bromide.    membrane run. If a   
                                                   continuous flow (non-
                                                   batch) influent is   
                                                   used then samples are
                                                   taken at the same    
                                                   time as the membrane 
                                                   effluent samples.    
Membrane Influent......  pH, turbidity,           Three samples per     
                          temperature, HPC, TOC    batch of influent    
                          and UV254. SDS\1\ for    evenly spaced over   
                          THMs, HAA6, TOX, and     the membrane run. If 
                          chlorine demand.         a continuous flow    
                                                   (non-batch) influent 
                                                   is used then samples 
                                                   are taken at the same
                                                   time as the membrane 
                                                   effluent samples.    
Membrane Permeate for    pH, alkalinity, total    A minimum of 8 samples
 each membrane tested.    dissolved solids,        evenly spaced over   
                          turbidity,               the membrane run.    
                          temperature, total &                          
                          calcium hardness,                             
                          bromide, HPC, TOC and                         
                          UV254. SDS\1\ for                             
                          THMs, HAA6, TOX, and                          
                          chlorine demand.                              
------------------------------------------------------------------------
\1\--SDS conditions are defined in Sec. 141.142(b)(4).                  
\2\--More frequent monitoring of flow rate and pressure will be required
  to accurately assess flux loss.                                       


             Table 10.--Sampling of GAC Pilot-scale Systems             
------------------------------------------------------------------------
      Sampling point               Analyses           Sample frequency  
------------------------------------------------------------------------
GAC Influent...............  pH, alkalinity,       A minimum of 15      
                              turbidity,            samples taken at the
                              temperature, total    same time as the    
                              & calcium hardness,   samples for GAC     
                              ammonia, bromide,     effluent at EBCT=20 
                              TOC and UV254. SDS1   min.                
                              for THMs, HAA6,                           
                              TOX, and chlorine                         
                              demand.                                   
GAC Effluent EBCT=10 min...  pH, turbidity,        A minimum of 15      
                              temperature,          samples. One after  
                              ammonia2, TOC and     one day, and        
                              UV254. SDS1 for       thereafter at 3% to 
                              THMs, HAA6, TOX,      7% increments of the
                              and chlorine          average influent    
                              demand.               TOC.                
GAC Effluent @ EBCT=20 min.  pH, turbidity,        A minimum of 15      
                              temperature,          samples. One after  
                              ammonia2, TOC and     one day, and        
                              UV254. SDS1 for       thereafter at 3% to 
                              THMs, HAA6, TOX,      7% increments of the
                              and chlorine          average influent    
                              demand.               TOC.                
------------------------------------------------------------------------
1--SDS conditions are defined in Sec. 141.142(b)(4).                    
2--If present in the influent.                                          
                                                                        
Note: More frequent effluent monitoring may be necessary to predict the 
  3% to 7% increments of average influent TOC.                          


          Table 11.--Sampling of Pilot-scale Membrane Systems           
------------------------------------------------------------------------
    Sampling point              Analyses             Sample frequency3  
------------------------------------------------------------------------
Membrane Influent......  pH, alkalinity, total    A minimum of 15       
                          dissolved solids,        samples to be taken  
                          turbidity,               at the same time as  
                          temperature, total &     the membrane effluent
                          calcium hardness,        samples.             
                          ammonia, bromide, HPC,                        
                          TOC and UV254. SDS1                           
                          for THMs, HAA6, TOX,                          
                          and chlorine demand..                         
Membrane Permeate......  pH, alkalinity, total    A minimum of 15       
                          dissolved solids,        samples evenly spaced
                          turbidity,               over the membrane    
                          temperature, total &     run.                 
                          calcium hardness,                             
                          ammonia2, bromide,                            
                          HPC, TOC and UV254.                           
                          SDS1 for THMs, HAA6,                          
                          TOX, and chlorine                             
                          demand..                                      
------------------------------------------------------------------------
1--SDS conditions are defined in Sec. 141.142(b.4).                     
2--If present in the influent.                                          
3--More frequent monitoring of flow rate and pressure will be required  
  to accurately assess flux loss.                                       


      Appendix A to Subpart M--Monitoring Scheme For Microorganisms     
------------------------------------------------------------------------
                        Source  Finished  Source  Finished  Source      
      Data needed        water    water    water    water    water  etc.
------------------------------------------------------------------------
Sample collection date                                                  
Plant id..............                                                  
------------------------------------------------------------------------
                                                                        
                       Giardia and Cryptosporidium                      
                                                                        
------------------------------------------------------------------------
Sample analysis date..                                                  
Sample volume                                                           
 collected (liters).                                                    
Sample volume examined                                                  
 (liters).                                                              
------------------------------------------------------------------------
                                                                        
                                 Giardia                                
                                                                        
------------------------------------------------------------------------
Presumptive count\1\..                                                  
Total density/100                                                       
 liter\2\ (based on                                                     
 presumptive count).                                                    
Confirmed count\1\....                                                  
Density/100 liters\2\                                                   
 (confirmed count).                                                     
------------------------------------------------------------------------
                                                                        
                             Cryptosporidium                            
                                                                        
------------------------------------------------------------------------
Presumptive count\1\..                                                  
Total density/100                                                       
 liter\2\ (based on                                                     
 presumptive count).                                                    
Confirmed count\1\....                                                  
Density/100 liters\2\                                                   
 (confirmed count).                                                     
-----------------------                                                 
                                                                        
           Total culturable viruses (systems >100,000 people)           
                                                                        
------------------------------------------------------------------------
Sample analysis date..                                                  
Sample volume                                                           
 collected.                                                             
% of total volume of                                                    
 concentrate examined.                                                  
MPN density/liter\2\..                                                  
Upper 95% confidence                                                    
 bound (of MPN).                                                        
Lower 95% confidence                                                    
 bound (of MPN).                                                        
------------------------------------------------------------------------
                                                                        
                             Total Coliforms                            
                                                                        
------------------------------------------------------------------------
Confirmed or validated                                                  
 counts per 100 ml.                                                     
------------------------------------------------------------------------
                                                                        
                         Fecal Coliforms/E. coli                        
                                                                        
------------------------------------------------------------------------
Counts per 100 ml.....                                                  
------------------------------------------------------------------------
\1\Alternate terms being considered are ``total count'' for             
  ``presumptive count'' and ``count with internal structures'' for      
  ``confirmed count''. ``Presumptive'' and ``total count'' are semantic 
  equals. However, ``confirmed'' Giardia cysts, unlike Cryptosporidium  
  oocysts, require demonstration of two internal structures, while      
  ``count with internal structures only requires the identification of  
  one internal structure in Giardia cysts.                              
\2\If organism is not detected, report data as < the detection limit per
  volume examined. For example, if no organism is detected in 200 L,    
  report as < 0.5/100L. If no organism is detected in 50L, report as < 2/
  100L.                                                                 

Appendix B to SubPart M--Treatment process information for systems 
serving at least 10,000 but less than 100,000 population

    Instructions:
    Unit Processes
    1. Indicate existing treatment process(es) and corresponding 
hydraulic loading rates at design flow in gallons per minute per square 
foot.
    2. Indicate liquid volume in gallons.
    3. Indicate baffling type, and T10/T during average flow if 
known, as defined in Appendix C of the guidance manual to the Surface 
water Treatment Rule.\1\
---------------------------------------------------------------------------

    \1\U.S.Environmental Protection Agency. 1991. Guidance manual 
for compliance with the filtration and disinfection requirements for 
public water systems using surface water sources. Office of Ground 
Water and Drinking Water, Washington, DC.
---------------------------------------------------------------------------

Chemical Additions
    1. Indicate the name of chemical coagulants and disinfectants and 
the applied dose in mg/L.
    2. If a chemical is not added at an indicated step then enter 
``None'' for the chemical name.

1. Plant Information:
    Design Flow ________ (MGD)
    Average Monthly Flow ________ (MGD)
    Maximum Daily Flow ________ (MGD)
    Average Water Temperature ________ (C)
    Minimum Water Temperature ________ (C)
2. Chemical Addition:
    Name ________
    Dose ________ (mg/L)
3. Presedimentation Processes
    Design Liquid Loading ________ (gpm/ft2)
    Liquid Volume ________ (gallons)
    Baffling (Check one of the following)
    ________ Unbaffled ____ Poor ____ Average ____ Superior ____ 
Perfect Ratio of T10/T ________ during average flow
4. Chemical Addition:
    Name ________ Dose ________ (mg/L)
5. Clarification/Sedimentation Processes
    Design Liquid Loading ---------- (gpm/ft2)
    Liquid Volume ---------- (gallons)
    Baffling (Check one of the following) ____ Unbaffled ____ Poor ____ 
Average ____ Superior ____ Perfect
    Ratio of T10/T ---------- during average flow
    Check all that apply:
    ____ Gravity Settling Basin
    ____ Upflow Solids Contact Basin
    ____ Adsorption Clarification
    ____ Dissolved Air Flotation
    ____ Tubes Installed
    ____ Lamella Plates Installed
6. Chemical Addition:
    Name ________ Dose ________ (mg/L)
7. Filtration
    Design Liquid Loading ________ (gpm/ft2)
    Liquid Volume ________ (gallons)
    Baffling (Check one of the following)
    ____ Unbaffled ____ Poor ____ Average ____ Superior ____ Perfect
    Ratio of T10/T ________ during average flow
    Filter Type. Check one of the following:
    ____ Rapid Sand Filter
    ____ Direct Filtration
    ____ Roughing Filter
    ____ Slow Sand Filtration
    ____ Diatomaceous Earth
    ____ Membrane Filtration
    Media Type. Check all that apply
    ____ Sand
    ____ Anthracite
    ____ Garnet
    ____ Granular Activated Carbon
8. Chemical Addition:
    Name ________ Dose ________ (mg/L)
9. Contact Tank and/or Clearwell
    Liquid Volume ________ (gallons)
    Baffling (Check one of the following)
    ____ Unbaffled ____ Poor ____ Average ____ Superior ____ Perfect 
Ratio of T10/T ________ during average flow

Appendix C to Subpart M--Proposed ICR Protozoan Method for 
Detecting Giardia Cysts and Cryptosporidium Oocysts in Water by a 
Fluorescent Antibody Procedure

1. Scope

    1.1  This test method describes the detection and enumeration of 
Giardia cysts and Cryptosporidium oocysts in ground, surface, and 
finished waters by a fluorescent antibody procedure. These 
pathogenic intestinal protozoa occur in domestic and wild animals as 
well as in humans. The environment may become contaminated through 
direct deposit of human and animal feces or through sewage and 
wastewater discharges to receiving waters. Ingestion of water 
containing these organisms may cause the disease.
    1.2  It is the user's responsibility to ensure the validity of 
this test method for waters of untested matrices. Results obtained 
by this method should be interpreted with extreme caution. Samples 
with high turbidity are not recommended with this procedure. A 
negative count and low detection limit does not ensure pathogen-free 
water.
    1.3  This method does not purport to address all of the safety 
problems associated with its use. It is the responsibility of the 
user of this method to establish appropriate safety and health 
practices and determine the applicability of regulatory limitations 
prior to use.

2. Terminology

    2.1  Description of Terms Specific to this Method:
    2.1.1  axoneme--an internal flagellar structure which occurs in 
some protozoa, e.g., Giardia, Spironucleus, and Trichomonas.
    2.1.2  cyst--a phase or a form of an organism produced either in 
response to environmental conditions or as a normal part of the life 
cycle of the organism. It is characterized by a thick and 
environmentally-resistant cell wall.
    2.1.3  median bodies--prominent, dark-staining, paired 
organelles consisting of microtubules and found in the posterior 
half of Giardia. In G. lamblia (from humans), these structures often 
have a claw-hammer shape while in G. muris (from mice), the median 
bodies are round.
    2.1.4  oocyst--the encysted zygote of some Sporozoa, e.g., 
Cryptosporidium. This is a phase or a form of the organism produced 
either in response to environmental conditions or as a normal part 
of the life cycle of the organism. It is characterized by a thick 
and environmentally-resistant cell wall.
    2.1.5  sporozoite--a motile, infective, asexual stage of certain 
sporozoans, e.g., Cryptosporidium. There are four sporozoites in 
each Cryptosporidium oocyst, and they are generally banana-shaped.
    2.1.6  nucleus--a prominent internal structure seen both in 
Giardia cysts and Cryptosporidium oocysts. Sometimes 2 to 4 nuclei 
can be seen in Giardia cysts. In Cryptosporidium oocysts there is 
one nucleus per sporozoite.

3. Summary of Test Method

    3.1  Pathogenic intestinal protozoa are concentrated from a 
large volume of water sample by retention on a yarn-wound filter. 
Retained particulates are eluted from the filter with a eluting 
solution and are concentrated by centrifugation. Giardia cysts and 
Cryptosporidium oocysts are separated to some extent from other 
particulate debris by flotation on a Percoll-sucrose solution with a 
specific gravity of 1.1. A monolayer of the water layer/Percoll-
sucrose interface is placed on a membrane filter, indirectly stained 
with fluorescent antibody, and examined under a microscope. Cysts 
and oocysts are classified as presumptive and confirmed,\1\ 
according to specific criteria (immunofluorescence, size, shape, and 
internal morphological characteristics), and the results are 
reported in terms of the number per 100 L. The confirmed number of 
cysts and/or oocysts is a subset of the presumptive number of cysts 
and/or oocysts.
---------------------------------------------------------------------------

    \1\Alternate terms being considered are ``total count'' and 
``count with internal structures'', respectively. ``Presumptive'' 
and ``total count'' are semantic equals. However, ``confirmed'' 
Giardia cysts, unlike Cryptosporidium oocysts, require demonstration 
of 2 internal structures, while ``count with internal structures'' 
only requires the identification of 1 internal structure in Giardia 
cysts.
---------------------------------------------------------------------------

4. Significance and Use

    4.1  This test method will provide a quantitative indication of 
the level of contamination in raw and treated drinking waters with 
the environmentally resistant stages of two genera of pathogenic 
intestinal protozoa: Giardia and Cryptosporidium.
    4.2  This test method will not identify the species of protozoa, 
it will not identify the host species of origin, it cannot determine 
the viability status, nor can it determine the infectivity status of 
detected cysts and oocysts.
    4.3  This test method may be useful in determining the source or 
sources of contamination of water supplies, the occurrence and 
distribution of protozoa in water supplies, and in evaluating the 
effectiveness of treatment practices.

5. Interferences

    5.1  Turbidity due to inorganic and organic debris and other 
organisms, can interfere with the concentration, purification and 
examination of the sample for Giardia cysts and Cryptosporidium 
oocysts.
    5.2  Inorganic and organic debris may be naturally-occurring, 
e.g., clays and algae, or may be added to water in the treatment 
process, e.g., iron and alum coagulants and polymers.
    5.3  Organisms and debris that autofluoresce or demonstrate non-
specific fluorescence, e.g., algal and yeast cells and Spironucleus 
(Hexamita) sp.\2\, when examined by epifluorescent microscopy could 
interfere with the detection of cysts and oocysts and contribute to 
false positive values.
---------------------------------------------------------------------------

    \2\Januschka, M.M., et al. 1988. A Comparison of Giardia microti 
and Spironucleus muris cysts in the vole: an immunocytochemical, 
light, and electron microscopic study. Journal of Parasitology 
74(3):452-458.
---------------------------------------------------------------------------

    5.4  Chlorine compounds, and perhaps other chemicals used to 
disinfect or treat drinking water and wastewater, may interfere with 
the visualization of internal structures of Giardia cysts and 
Cryptosporidium oocysts.
    5.5  Freezing filter samples, eluates or concentrates could 
interfere with the detection and/or identification of cysts and 
oocysts originally present in the sample.

6. Apparatus

    6.1  Sample Collection.
    6.1.1  Filter and filter holder, a 25.4 cm (10 in.) long 1 
m nominal porosity, yarn-wound polypropylene cartridge 
Commercial honeycomb fFilter tube (M39R10A; Commercial Filters 
Parker H annifin Corp., P.O. Box 1300, Lebanon, IN) or Filterite 
(Filterite Corporation, Timmonium, MD), with VIH # 10 Clear w/pr 
(with pressure relief) (Ametek part # 150163; Ametek, Plymouth 
Products Division, P.O. Box 1047, Sheboygan, WI) should be used.
    6.1.2  Water meter.
    6.1.3  Fluid proportioner (or proportioning injector) for 
chlorinated water.
    6.1.4  Flow control valve, 4 L/min.
    6.1.5  Pump, electric or gasoline powered.
    6.1.6  Ice chest or cooler.
    6.2  Sample Processing.
    6.2.1  Centrifuge, with swinging bucket rotors having a capacity 
of 15 to 250 mL per conical tube or bottle.
    6.2.2  Mixer, vortexer.
    6.2.3  Vacuum source.
    6.2.4  Membrane filter holder, Hoefer manifold, model FH 
225V,\3\ 10 place holder for 25 mm diameter filters.
---------------------------------------------------------------------------

    \3\Hoefer Scientific Instruments, 654 Minnesota Street, Box 
77387, San Francisco, California 94107.
---------------------------------------------------------------------------

    6.2.5  Slide warming tray, or incubator, 37 deg.C.
    6.2.6  pH meter.
    6.2.7  Rubber policeman.
    6.3  Sample Examination.
    6.3.1  Microscope, capable of epifluorescence and D.I.C. or 
Hoffman modulation optics, with stage and ocular 
micrometers and 20X (N.A. = 0.6) to 100X (N.A. = 1.3) objectives. 
Equip the microscope with appropriate excitation and band pass 
filters for examining fluorescein isothiocyanate-labeled specimens 
(exciter filter: 450-490 nm; dichroic beam-splitting mirror: 510 nm; 
barrier or suppression filter: 515-520 nm).

7. Reagents and Materials

    7.1  Purity of Reagents--Reagent grade chemicals shall be used 
in all tests. Unless otherwise indicated, it is intended that all 
reagents shall conform to the specifications of the committee on 
Analytical Reagents of the American Chemical Society where such 
specifications are available.\4\
---------------------------------------------------------------------------

    \4\``Reagent Chemicals, American Chemical Society 
Specifications,'' American Chemical Society, Washington, DC. For 
suggestion on the testing or reagents not listed by the American 
Chemical Society, see ``Analar Standards for Laboratory Chemicals,'' 
BDH, Poole, Dorset, U.K. and the ``United States Pharmacopeia.''
---------------------------------------------------------------------------

    7.2  Preparation of Reagents--Prepare reagents as specified by 
the formulations.
    7.3  Purity of Water--Use distilled deionized or double 
distilled water.
    7.4  Sample Collection.
    7.4.1  Sodium Thiosulfate Solution (0.5 %)--Dissolve 0.5 g of 
sodium thiosulfate (Na2S2O35H2O) in 50 
mL water and then adjust to a final volume of 100 mL.
    7.5  Sample Processing.
    7.5.1  Neutral Buffered Formalin Solution (10 %)--Dissolve 0.762 
g disodium hydrogen phosphate (Na2HPO4), 0.019 g sodium 
dihydrogen phosphate (NaH2PO4), and 100 mL formalin in 
water to a final volume of 1 L.
    7.5.2  Phosphate Buffered Saline (PBS)--Prepare a 10X stock 
solution by dissolving 80 g sodium chloride (NaCl), 2 g potassium 
dihydrogen phosphate (KH2PO4), 29 g hydrated disodium 
hydrogen phosphate (Na2HPO412 H2O) and 2 g 
potassium chloride (KCl) in water to a final volume of 1 L. The 10X 
solution is used to prepare 1X PBS by diluting one volume of the 10X 
solution with 9 volumes of water and adjust the pH with a pH meter 
to 7.4 with 0.1 N HCl or 0.1 N NaOH before use.
    7.5.3  Sodium Dodecyl Sulfate Stock Solution (1%)--Prepare 
solution by dissolving 1.0 g of sodium dodecyl sulfate (SDS) in 
water to a final volume of 100 mL.
    7.5.3  Tween 80 Stock Solution (1%)--Mix 1.0 mL of 
polyoxyethylenesorbitan monooleate 80 (Tween 80) stock solution with 
99 mL of water.
    7.5.4  Eluting Solution (Buffered Detergent Solution)--Prepare 
solution by mixing 100 mL 1% SDS, 100 mL 1% Tween 80, 100 mL 10X 
PBS, and 0.1 mL Sigma Antifoam A with 500 mL water. Adjust the pH to 
7.4 using a pH meter. Adjust the final volume to 1 L with additional 
water. Use within one week of preparation.
    7.5.5  Sucrose Solution (2.5 M)--Dissolve 85.58 g of sucrose in 
40 mL prewarmed water then adjust the final volume to 100 mL with 
water.
    7.5.6  Percoll-Sucrose Flotation Solution, Sp. Gr. 1.10--Mix 45 
mL Percoll (sp. gr. 1.13; Sigma), 45 mL water and 10 mL 2.5 M 
sucrose solution. Check the specific gravity with a hydrometer. The 
specific gravity should be between 1.09 and 1.10 (do not use if less 
than 1.09). Store at 4 deg.C and use within a week. Allow to reach 
room temperature before use.

7.6  Sample Examination

    7.6.1  Meridian Hydrofluor-Combo kit\5\ (cat. no. 240025) for 
detecting Giardia cysts and Cryptosporidium oocysts in water 
samples. The expiration date for the reagents is printed on the 
Hydroflour-Combo kit label. Discard the kit once the expiration date 
is reached. Store the kit at 2-8 deg.C and return it promptly to 
this temperature range after each use. The labeling reagent should 
be protected from exposure to light. Do not freeze any of the 
reagents in this kit. Diluted, unused working reagents should be 
discarded after 48 hours.
---------------------------------------------------------------------------

    \5\Meridian Diagnostics, Inc., 3471 River Hills Drive, 
Cincinnati, Ohio 45244.
---------------------------------------------------------------------------

    7.6.2  Ethanol, (95%).
    7.6.3  Glycerol.
    7.6.4  Ethanol/Glycerol Series--Prepare a series of solutions 
according to the following table:

------------------------------------------------------------------------
                                                                 Final %
  95% ethanol       Glycerol      Reagent water   Final volume   ethanol
                                                                        
------------------------------------------------------------------------
10 mL            5 mL            80 mL           95 mL                10
20 mL            5 mL            70 mL           95 mL                20
40 mL            5 mL            50 mL           95 mL                40
80 mL            5 mL            10 mL           95 mL                80
95 mL            5 mL            0 mL            95 mL                95
------------------------------------------------------------------------

    7.6.5  DABCO-Glycerol Mounting Medium (2%)--Prewarm 95 mL 
glycerol using a magnetic stir bar on a heating stir plate. Add 2 g 
1,4 diazabicyclo [2.2.2] octane (DABCO, Sigma #D-2522) to the warm 
glycerol with continuous stirring until it dissolves. (CAUTION: 
hygroscopic; causes burns; avoid inhalation, as well as skin and eye 
contact.) Adjust the final volume to 100 mL with additional 
glycerol. Store at room temperature and discard after 6 months.
    7.6.6  Bovine Serum Albumin (1%)--Sprinkle 1.0 g bovine serum 
albumin (BSA) crystals over 85 mL 1X PBS, pH 7.4. Allow crystals to 
fall before stirring into solution with a magnetic stir bar. After 
the BSA is dissolved, adjust the volume to 100 mL with PBS. For 
prolong storage, sterilize by filtering through a 0.22 m 
membrane filter into a sterile tube or bottle. Store at 4 deg.C and 
discard after 6 months.
    7.7  Sample Collection Materials.
    7.7.1  Filters, a 25.4 cm (10 in.) long 1 m nominal 
porosity, yarn-wound polypropylene cartridge commercial Honeycomb 
Filter Tube (M39R10A) or Filterite (Filterite Corporation, 
Timmonium, MD).
    7.7.2  Garden hose and connectors.
    7.7.3  Whirl-pak or zip-loc bags, 15 in. (38 cm) x 15 in (38 
cm).
    7.7.4  Cold packs or wet ice.
    7.8  Sample Processing Materials.
    7.8.1  Pans or trays, stainless steel or glass trays, approx. 
16.5 in. (41.91 cm) x 10 in. (25.4 cm) x 2 in. (5.08 cm) deep.
    7.8.2  Knife/cutting tool, for cutting the polypropylene filter 
fibers off filter core.
    7.8.3  Hydrometer, for liquids heavier than water (range: 1.000-
1.225), for adjusting specific gravity of flotation solutions.
    7.9  Sample Examination Materials.
    7.9.1  Slides, glass microscope, 1 in. (2.54 cm.) x 3 in. (7.62 
cm) or 2 in. (5.08 cm.) x 3 in. (7.62 cm.).
    7.9.2  Cover slips, 25 mm\2\, No. 1\1/2\.
    7.9.3  Filters, Sartorius brand cellulose acetate, either 0.45 
or 0.2 m pore size, 25 mm diameter.
    7.9.4  Support Filters, ethanol-compatible membrane, any pore 
size, 25 mm.
    7.9.5  Fingernail polish, clear or clear fixative (cat. no. 60-
4890; PGC Scientifics).
    7.9.6  Splinter forceps, fine tip.
    7.9.7  Blunt-end filter forceps.
    8. Precautions.
    8.1  The analyst/technician must know and observe the normal 
safety procedures required in a microbiology laboratory while 
preparing, using and disposing of sample concentrates, reagents and 
materials and while operating sterilization equipment.
    8.2  Do not mouthpipet in any portion of this procedure.
    9.  Sampling.
    9.1  Sampling Apparatus Preparation and Assembly.
    9.1.1  The sampling apparatus (Fig. 1) consists of an inlet 
hose, filter holder, a 1 m nominal porosity filter, an 
outlet hose, a water meter, and a flow control valve or device (4 L/
min). A pump will be needed for unpressurized sources and a fluid 
proportioner or proportioning injector will be needed for 
chlorinated or other disinfectant treated waters.
    9.1.2  The sampling apparatus does not have to be sterile but it 
must be clean and uncontaminated by cysts and/or oocysts. Thoroughly 
clean the apparatus, including filter holder, hoses and pumps, and 
rinse between samples. If multiple samples are to be collected with 
the same apparatus (but using different filters and, preferably, 
different filter holders), arrange the sampling sequence to begin 
with the least contaminated water (e.g., treated drinking water) and 
end with the most contaminated water (e.g., source water). If field 
conditions preclude complete disassembly and thorough cleaning of 
apparatus components between samples, thoroughly rinse all surfaces 
that will come in contact with the water with at least 50 gal (190 
L) of the water to be sampled prior to the installation of the 
filter cartridge.
    9.1.3  Filter Holder.
    9.1.3.1  Thoroughly wash the filter holder with a stiff brush in 
hot water containing detergent.
    9.1.3.2  Rinse the filter holder with tap water until the soap 
residue is gone. Follow with a thorough rinse in reagent water and 
air dry.
    9.1.3.3  Attach a water-resistant label containing the following 
information to the filter holder:

Start Time: ______ Meter Reading: ______ Turbidity: ______
Stop Time: ______ Meter Reading: ______ Turbidity: ______
Operator's Name: ______________ Total Volume Filtered: ____________
Date: ____________________ Sampling Location: ____________________
    9.1.3.4  The turbidity value should be recorded, if available.
    9.1.4  Hoses.
    9.1.4.1  Inlet and outlet hoses for the filter holder consist of 
standard garden hoses and fittings. It is helpful to use different 
colors for inlet and outlet hoses.
    9.1.4.2  Outlet hoses may be used repeatedly without washing but 
inlet hoses are considered contaminated after one use. Use the 
shortest length of inlet hose necessary for collecting the sample 
and discard the inlet hose after use. If this is not practical, 
rinse the inlet hose thoroughly with at least 50 gal (190 L) of the 
water to be sampled prior to connecting the filter holder.
    9.1.5  Pump.
    9.1.5.1  If a pump must be used to collect the sample, it is 
recommended that the pump be installed on the outlet end of the 
sampling apparatus. In this manner, the sample will be pulled 
through the filter and the pump may be used repeatedly without the 
fear of contamination and without the need for washing.
    9.1.5.2  If the pump is installed on the inlet side of the 
sampling apparatus, thoroughly clean and rinse all parts that come 
in contact with the sampled water prior to collection of the next 
sample. If pump disassembly is not practical between samples, rinse 
thoroughly with at least 50 gal (190 L) of the water to be sampled 
prior to connecting the filter holder.
    9.1.6  Fluid Proportioner or Proportioning Injector.
    9.1.6.1  If the water to be sampled is chlorinated or 
disinfected by any other chemicals, the disinfectant must be 
neutralized during sample collection. While the assay system allows 
detection of disinfected cysts and oocysts, exposure to disinfectant 
may interfere with the visualization of internal morphologies of 
these organisms.
    9.1.6.2  Use sodium thiosulfate solution to neutralize the 
disinfectant in water samples. Add the sodium thiosulfate solution 
to the water during sample collection with a mechanical fluid 
proportioner pump or an in-line Venturi-operated injector.\6\
---------------------------------------------------------------------------

    \6\Details on the operation and use of proportioner pumps and 
injectors can be found in Standard Methods for the Examination Water 
and Wastewater, Section 9510C, ``Virus Concentration from Large 
Sample Volumes by Adsorption to and Elution from Microporous Filters 
(PROPOSED),'' 18th ed., 1989, pp. 9-105 to 9-109.
---------------------------------------------------------------------------

    9.2  Sample Collection.
    9.2.1  Connect inlet end of sampling apparatus to a pressurized 
water tap or follow pump manufacturer's instructions for priming the 
pump if an unpressurized source is being sampled.
    9.2.2  Use a water-resistant marking pen to record the start 
time, meter reading, name of person collecting the sample, 
turbidity, date and sampling location on the filter holder label.
    9.2.3  Start water flow through the filter. The flow rate should 
not exceed 4 L/min.
    9.2.4  A minimum sample size of 140 L of raw water and 1400 L of 
finished water is required.
    9.2.5  If the water must be neutralized, add sodium thiosulfate 
solution via the proportioner system to produce a final 
concentration in the sampled water of 50 mg/L. One L of 0.5% sodium 
thiosulfate solution will be needed for each 100 L of water sampled. 
Periodically check a sample of effluent to be certain that no 
residual chlorine remains after the addition of the thiosulfate. 
Measure chlorine using Test Method D1253.\7\
---------------------------------------------------------------------------

    \7\Annual Book of ASTM Standards, Vol. 11.01.
---------------------------------------------------------------------------

    9.2.6  After the required volume of water has passed through the 
filter, shut off the water flow, record the stop time, final meter 
reading and turbidity of the water at the end of filtration on the 
filter holder label.
    9.2.7  Disconnect sampling apparatus while maintaining the inlet 
hose level above the level of the opening on the outlet hose in 
order to prevent backwashing and the loss of particulate matter from 
the filter.
    9.2.8  Pour the residual water remaining in the filter holder 
into a 15 in. (38 cm.) x 15 in. (38 cm.) whirl pack or zip-lock bag.
    9.2.9  Aseptically remove the filter from the holder and 
transfer the filter to the bag containing the residual water.
    9.2.10  Seal the bag and place it inside a second 15 in. (38 
cm.) x 15 in. (38 cm.) whirl pack or zip-lock bag. Transfer the 
label or label information from the filter holder to the outside of 
this second bag.
    9.2.11  Transport the sample to the laboratory on wet ice or 
cold packs and refrigerate at 2-5  deg.C. Do not freeze during 
transport or storage.

10. Procedure

    10.1  Filter Elution. The initiation of sample collection and 
elution from the collection filter must be performed within 96 hrs. 
Two approaches to eluting the particulates from the filter may be 
used: either washing by hand or using a stomacher.
    10.1.1  Handwashing.
    10.1.1.1  Pour the residual solution in the bag into a beaker, 
rinse the bag with eluting solution, add the rinse solution to the 
beaker and discard the bag.
    10.1.1.2  Using a razor knife or other appropriate cutting 
instrument, cut the filter fibers lengthwise down to the core. 
Divide the filter fibers into a minimum of three equal portions with 
one-third consisting of those cleanest fibers nearest the core; the 
second one-third being the middle layer of fibers, and the final 
one-third consisting of the outer-most filter fibers (the dirtiest 
fibers).
    10.1.1.3  Beginning with the cleanest fibers (the one-third 
nearest the core), hand wash the fibers in three consecutive 1.0 L 
volumes of eluting solution. Wash the fibers by kneading them in the 
eluting solution contained either in a beaker or a plastic bag. 
Wring the fibers to express as much of the liquid as possible before 
discarding. Main- tain the three 1.0 L volumes of eluate separate 
throughout the washing procedure.
    10.1.1.4  Using the three 1.0 L volumes of eluate used in the 
above section (11.1.4), repeat the washing procedure on the middle 
one-third layer of fibers and then on the final outer one-third 
layer of fibers.
    10.1.1.5  The minimum total wash time of fibers should be 30 
min. After all the fibers have been washed, combine the three 1.0 L 
volumes of eluate with the residual filter water obtained in 10.1.1 
and discard the fibers.
    10.1.2  Stomacher Washing.
    10.1.2.1  Use a stomacher with a bag capacity of 3500 mL. Using 
a razor knife or other appropriate cutting instrument, cut the 
filter fibers lengthwise down to the core.
    10.1.2.2  After loosening the fibers, place all the filter 
fibers in a stomacher bag. To insure against bag breakage and sample 
loss, place the filter fibers in the first stomacher bag into a 
second stomacher bag.
    10.1.2.3  Add 1.75 L of eluting solution to the fibers. 
Homogenenize for 2 five minute intervals. Between each 
homogenization period, hand kneed the filter material to 
redistribute the fibers in the bag.
    10.1.2.4  Wring the fibers out to express as much of the liquid 
as possible before discarding.
    10.1.3  Concentrate the combined eluate and residual water into 
a single pellet by centrifugation at 1,050 x g for 10 min using a 
swinging bucket rotor and plastic conical centrifuge bottles. 
Carefully aspirate and discard the supernatant fluid and resuspend 
the pellet by vortexing. After pooling the particulates in one 
conical bottle, record the packed pellet volume. Resuspend the 
packed pellet in an equal volume of 10% neutral buffered formalin 
solution. If the packed pellet volume is less than 0.5 mL, add 
enough buffered formalin solution to bring the resuspended pellet 
volume to 1.0 mL.
    10.1.4  All raw water sample particulates must be archived. A 
minimum of 25% or a maximum of 5 ml packed pellet volume of the raw 
water sample should be transferred to 15 ml conical, plastic 
centrifuge tube. The tube size is manditory due to storage 
considerations. Attach a water resistant label containing the 
following information to the tube:

Start Time: ______ Meter Reading: ______ Turbidity: ______
Stop Time: ______ Meter Reading: ______ Turbidity: ______
Operator's Name: ____________ Total Volume Filtered: ____________
Date: ____________ Sampling Location: ____________
    10.2  Flotation Purification.
    10.2.1  In a clear plastic 50 mL conical centrifuge tube(s), 
vortex a volume of resuspended pellet equivalent to not more than 1 
mL of packed pellet volume with a sufficient volume of eluting 
solution to make a final volume of 20 mL.
    10.2.2  Using a 50 mL syringe and 14 gauge cannula, underlay the 
20 mL vortexed suspension of particulates with 30 mL Percoll-sucrose 
floatation solution (sp. gr. 1.1). An alternate procedure would be 
to overlay the 30 mL of Percoll-sucrose floatation solution with the 
20 mL of suspended particulates.
    10.2.3  Without disturbing the pellet suspension/Percoll-sucrose 
interface, centrifuge the preparation at 1,050 x g for 10 min using 
a swinging bucket rotor. Slowly accelerate the centrifuge over a 30-
sec interval up to the speed where the tubes are horizontal in order 
to avoid disrupting the interface. Similarly, at the end of 
centrifugation, decelerate slowly. DO NOT USE THE BRAKE.
    10.2.4  Using a polystyrene 25 mL pipet rinsed with eluting 
solution, draw off the top 20 mL particulate suspension layer, the 
interface, and 5 mL of the Percoll-sucrose below the interface. 
Place all these volumes in a plastic 50 mL conical centrifuge tube.
    10.2.5  Add additional eluting solution to the plastic conical 
centrifuge tube (10.2.4) to a final volume of 50 mL. Centrifuge at 
1,050 x g for 10 min.
    10.2.6  Aspirate and discard the supernatant fluid down to 5 mL 
(plus pellet). Resuspend the pellet by vortexing and save this 
suspension for further processing with fluorescent antibody 
reagents.
    10.2.7  At this point, a break may be inserted if the procedure 
is not going to progress immediately to the Indirect fluorescent 
Antibody procedure (10.3) below. If a break is inserted, then the 
pellet from 10.2.6 should be washed with eluting solution to ensure 
eliminating osmotic stress to cysts and oocysts from residual 
Percoll-sucrose floatation solution. Wash the pellet two or more 
times by resuspending it in 50 mL of eluting solution, centrifuging 
at 1,050 X g for 10 min, and aspirating the supernatant down to 5 mL 
above the pellet. Store the pellet at 4  deg.C.
    10.3  Indirect Fluorescent Antibody (IFA) Procedure.
    10.3.1  Determining Sample Volume per Filter.
    10.3.1.1  Determine the volume of sample concentrate (from 
10.2.7) that may be applied to each 25-mm diameter membrane filter 
used in the IFA assay.
    10.3.1.2  Vortex the sample concentrate and apply 40 L 
to one 5-mm diameter well of a 12-well red heavy teflon-coated 
slide.\8\
---------------------------------------------------------------------------

    \8\Cel-line Associates, Inc., 33 Gorgo Lane, Newfield, NJ 08344, 
Cat. #10-111.
---------------------------------------------------------------------------

    10.3.1.3  Allow the sample to sit approximately 2 min at room 
temperature.
    10.3.1.4  Examine the flooded well at 200X total magnification. 
If the particulates are distributed evenly over the well surface 
area and are not crowded or touching, then apply 1 mL of the 
undiluted sample to a 25-mm diameter membrane filter in 10.3.4.6.
    10.3.1.5  Adjust the volume of the sample accordingly if the 
particulates are too dense or are widely spread. Retest on another 
well. Always adjust the sample concentrate volume so that the 
density of the particulates is just a little sparse. If the layer of 
sample particulates on the membrane filters is too dense, any cysts 
or oocysts present in the sample may be obscured during microscopic 
examination. Make sure the dilution factor, if any, from this step 
is recorded.
    10.3.2  Preparing the Filtration Manifold.
    10.3.2.1  See Fig. 2 for a diagram of the filtration manifold 
assembly.
    10.3.2.2  Connect the filtration manifold to the vacuum supply 
using a vacuum tube containing a ``T''-shaped tubing connector. 
Attach a Hoffman screw clamp to 4-6 cm of latex tubing and then 
attach the latex tubing to the stem of the ``T'' connector. The 
screw clamp is used as a bleeder valve to regulate the vacuum to 2-4 
in Hg.
    10.3.2.3  Close all the manifold valves and open the vacuum all 
the way. Using the bleeder valve on the vacuum tubing, adjust the 
applied vacuum to 2-4 in. of Hg. Once adjusted, do not readjust the 
bleeder valve during filtration. If necessary, turn the vacuum on 
and off during filtration at the vacuum source.
    10.3.3  Membrane Filter Preparation.
    10.3.3.1  One Sartorius 25 mm diameter cellulose acetate filter, 
0.2-0.45 m pore size\9\ and one 25-mm diameter ethanol 
compatible membrane support filter,\10\ any porosity, are required 
for each 1 mL of adjusted suspension obtained in 10.3.1.5. Soak the 
required number of each type of filter separately in Petri dishes 
filled with 1X PBS. Drop the filters, handling them with blunt-end 
filter forceps, one by one flat on the surface of the buffer. Once 
the filters are wetted, push the filters under the fluid surface 
with the forceps. Allow filters to soak for a minimum of 1 min 
before use.
---------------------------------------------------------------------------

    \9\Sartorius Corp., Filter div., 30940 San Clemente, Hayward, CA 
94544.
    \10\Nitrocellulose, 8 m porosity, Cat. No. SCWP 025, 
Millipore Corp., Bedford, MA, or equivalent.
---------------------------------------------------------------------------

    10.3.3.2  Turn the filtration manifold vacuum source on. Leaving 
all the manifold well support valves closed, place one support 
filter on each manifold support screen. This filter ensures even 
distribution of sample.
    10.3.3.3  Place one Sartorius 25-mm diameter cellulose acetate 
filter on top of each support filter. Use a rubber policeman to 
adjust the cellulose acetate filter, if necessary. Open the manifold 
well support valves to flatten the filter membranes. Make sure that 
no bubbles are trapped and that there are no creases or wrinkles on 
any of the filter membranes.
    10.3.3.4  Use as many filter positions as there are sample 
volumes to be assayed. Record the number of sample 25-mm membrane 
filters prepared and the volume of floated pellet (10.3.1) 
represented by these membranes. In addition, include at least one 
positive control for Giardia cysts and Cryptosporidium oocysts and 
one negative control each time the manifold is used.
    10.3.3.5  Position the 1 lb (454 g) stainless steel wells firmly 
over each filter.
    10.3.3.6  Label each sample and control well appropriately with 
little pieces of tape on the top of the stainless steel wells.
    10.3.4  Sample Application.
    10.3.4.1  Open the manifold support valve for each well 
containing filters.
    10.3.4.2  Rinse the inside of each stainless steel well and 
membrane filter with 2 mL 1% BSA applied with a Pasteur pipet. Drain 
the BSA solution completely from the membrane.
    10.3.4.3  Close the manifold valves under each membrane filter.
    10.3.4.4  For the positive controls, add 500-1000 Giardia 
lamblia cysts and 500-1000 Cryptosporidium parvum oocysts or use the 
Meridian diagnostic positive control antigen as specified in the kit 
to a well.
    10.3.4.5  For a negative control, add 1.0 mL 1X PBS to one well.
    10.3.4.6  Add 1.0 mL of vortexed, adjusted water sample from 
10.3.1.5 to a well.
    10.3.4.7  Open the manifold valve under each membrane filter to 
drain the wells. Rinse each stainless steel well with 2 mL 1% BSA. 
Do not touch the pipet to the membrane filter or to the well. Close 
the manifold valve under each membrane filter.
    10.3.5  Indirect Fluorescent Antibody Staining.
    10.3.5.1  Dilute the primary antibody mixture and labeling 
reagent according to the manufacturer's instructions using 1X PBS.
    10.3.5.2  Pipet 0.5 mL of the diluted primary antibody mixture 
onto each membrane and allow to remain in contact with the filter 
for 25 min at room temperature.
    10.3.5.3  At the end of the contact period, open the manifold 
valve to drain the antisera.
    10.3.5.4  Rinse each well and filter 5 times with 2 mL 1X PBS. 
Do not touch the tip of the pipet to the membrane filter or to the 
stainless steel wells. Close all manifold valves after the last wash 
is completed.
    10.3.5.5  Pipet 0.5 mL labeling reagent onto each membrane and 
allow to remain in contact with the filter for 25 min at room 
temperature. Cover all wells with aluminum foil to shield the 
reagents from light and to prevent dehydration and crystallization 
of the fluorescein isothiocyanate dye during the contact period.
    10.3.5.6  At this point start the 10.3.6. procedure.
    10.3.5.7  At the end of the contact period, open the manifold 
valves to drain the labeling reagent.
    10.3.5.8  Rinse each well and filter 5 times with 2 mL 1X PBS. 
Do not touch the tip of the pipet to the membrane filter or to the 
stainless steel wells. Close all manifold valves after the last wash 
is completed.
    10.3.5.9  Dehydrate the membrane filters in each well by 
sequentially applying 1.0 mL of 10, 20, 40, 80 and 95% ethanol 
solutions containing 5% glycerol. Allow each solution to drain 
thoroughly before applying the next in the series.
    10.3.6  Filter Mounting.
    10.3.6.1  Label glass slides for each filter and place them on a 
slide warmer or in an incubator calibrated to 37  deg.C.
    10.3.6.2  Add 75 L 2% DABCO-glycerol mounting medium to 
each slide on the slide warmer or in the incubator and allow to warm 
for 20-30 min.
    10.3.6.3  Remove the top cellulose acetate filter with fine-tip 
forceps and layer it over the correspondingly labeled DABCO-glycerol 
mounting medium prepared slide. Make sure the sample application 
side is up. If the entire filter is not wetted by the DABCO-glycerol 
mounting medium, pick up the membrane filter with the same forceps 
and add a little more DABCO-glycerol mounting medium to the slide 
under the filter.
    10.3.6.4  Use a clean pair of forceps to handle each membrane 
filter. Soak used forceps in a beaker of diluted detergent cleaning 
solution.
    10.3.6.5  After a 20 min clearing period on the slide warmer, 
the filter should become transparent and appear drier. After 
clearing, if the membrane starts to turn white, apply a small amount 
of DABCO-glycerol mounting medium under the filter.
    10.3.6.6  After the 20 min clearing period, apply 20 L 
DABCO-glycerol mounting medium to the center of each membrane filter 
and cover with a 25 mm x 25 mm cover glass. Tap out air bubbles with 
the handle end of a pair of forceps. Wipe off excess DABCO-glycerol 
mounting medium from the edge of each cover glass with a slightly 
moistened Kimwipe.
    10.3.6.7  Seal the edge of each cover glass to the slide with 
clear fingernail polish.
    10.3.6.8  Store the slides in a ``dry box''. A dry box can be 
constructed from a covered Tupperware container to which a thick 
layer of Drierite has been added. Cover the dessicant with paper 
towels and the slides should be laid flat on the top of the paper 
towels. Place the lid on the dry box and store at 4  deg.C.
    10.3.6.9  Examine the slides microscopically as soon as possible 
but within 5 days of preparation, because they may become opaque if 
stored longer, and D.I.C. or Hoffman modulation optical 
examination would then no longer be possible.
    10.4  Microscopic Examination.
    10.4.1  General--Microscopic work by a single analyst should not 
exceed 4 hours/day nor more than 5 consecutive days/week. 
Intermittent rest periods during the 4 hours/day are encouraged.
    10.4.1.1  Remove the dry box from 4  deg.C storage and allow it 
to warm to room temperature before opening.
    10.4.1.2  Adjust the microscope to assure that the 
epifluorescence and Hoffman modulation or differential 
interference contrast optics are in optimal working order. Make sure 
that the fluorescein isothiocyanate cube is in place in the 
epifluorescent portion of the microscope (see 6.3.1). Detailed 
procedures required for adjusting and aligning the microscope are 
found in appendix X4.
    10.4.2  Assay Controls.
    10.4.2.1  The purpose of these controls is to assure that the 
assay reagents are functioning, that the assay procedures have been 
properly performed, and that the microscope has been adjusted and 
aligned properly.
    10.4.2.2  Assay Giardia/Cryptosporidium Control
    (a) Using epifluorescence, scan the positive control slide at no 
less than 200X total magnification for apple-green fluorescence of 
Giardia cyst and Cryptosporidium oocyst shapes. Background 
fluorescence of the membrane should be either very dim or non-
existent.
    (b) If no apple-green fluorescing Giardia cyst or 
Cryptosporidium oocyst shapes are observed, then the fluorescent 
staining did not work or the positive control cyst preparation was 
faulty. Do not examine the water sample slides for Giardia cysts and 
Cryptosporidium oocysts. Recheck reagents and procedures to 
determine the problem.
    (c) If apple-green fluorescing cyst and oocyst shapes are 
observed, change the microscope from epifluorescence to the 100X oil 
immersion Hoffman modulation or differential interference 
contrast objective.
    (d) At no less than 1000X total oil immersion magnification, 
examine Giardia cyst shapes and Cryptosporidium oocyst shapes for 
internal morphology.
    (e) The Giardia cyst internal morphological characteristics 
include 1-4 nuclei, axonemes, and median bodies. Giardia cysts 
should be measured to the nearest 0.5 m with a calibrated 
ocular micrometer. Record the length and width of cysts. Also record 
the morphological characteristics observed. Continue until at least 
3 Giardia cysts have been detected and measured in this manner.
    (f) The Cryptosporidium oocyst internal morphological 
characteristics include 1-4 sporozoites. Examine the Cryptosporidium 
oocyst shapes for sporozoites and measure the oocyst diameter to the 
nearest 0.5 m with a calibrated ocular micrometer. Record 
the size of the oocysts. Also record the number, if any, of the 
sporozoites observed. Sometimes a single nucleus is observed per 
sporozoite. Continue until at least 3 oocysts have been detected and 
measured in this manner.
    10.4.2.3  Assay Negative Control.
    (a) Using epifluorescence, scan the negative control membrane at 
no less than 200X total magnification for apple-green fluorescence 
of Giardia cyst and Cryptosporidium oocyst shapes.
    (b) If no apple-green fluorescing cyst or oocyst shapes are 
found, and if background fluorescence of the membrane is very dim or 
non-existent, continue with examination of the water sample slides.
    (c) If apple-green fluorescing cyst or oocyst shapes are found, 
discontinue examination since possible contamination of the other 
slides is indicated. Clean the equipment (see Appendix X1), recheck 
the reagents and procedure and repeat using additional aliquots of 
the sample.
    10.4.3  Sample Examination.
    10.4.3.1  Scanning Technique.
    (a) Scan each membrane in a systematic fashion beginning with 
one edge of the mount and covering the entire membrane. An up-and-
down or a side- to-side scanning pattern may be used. See Fig. 3 for 
an illustration of 2 alternatives for systematic slide scanning.
    10.4.3.2  Presumptive Count and Confirmed Count
    (a) When appropriate responses have been obtained for the 
positive and negative controls, use epifluorescence to scan the 
entire membrane from each sample at not less than 200X total 
magnification for apple-green fluorescence of cyst and oocyst 
shapes.
    (b) When brilliant apple-green fluorescing round to oval objects 
(8 to 18 m long by 5 to 15 m wide) are observed, 
switch the microscope to either Hoffman modulation or 
differential interference contrast optics. Look for external or 
internal morphological characteristics atypical of Giardia cysts 
(e.g., spikes, stalks, appendages, pores, one or two large nuclei 
filling the cell, red fluorescing chloroplasts, crystals, spores, 
etc.). If these atypical structures are not observed, then identify 
such apple-green fluorescing objects of the aforementioned size and 
shape as presumptive Giardia cysts. Record the shape and 
measurements (to the nearest 0.5 m at 1000X) for each such 
object as the part of the presumptive count. If two or more internal 
morphological structures are observed at this point, record this as 
a comfirmed Giardia cyst as well. Counts with internal structures 
must be confirmed by a senior analyst.
    (c) When brilliant apple-green fluorescing ovoid or spherical 
objects (3 to 7 m in diameter) are observed, switch the 
microscope to either Hoffman modulation or differential 
interference contrast optics. Look for external or internal 
morphological characteristics atypical of Cryptosporidium oocyst 
(e.g., spikes, stalks, appendages, pores, one or two large nuclei 
filling the cell, red fluorescing chloroplasts, crystals, spores, 
etc.). If these atypical structures are not observed, then identify 
such apple-green fluorescing objects of the aforementioned size and 
shape as presumptive Cryptosporidium oocysts. Record the shape and 
measurements (to the nearest 0.5 m at 1000X) for each such 
object as part of the presumptive count. Although not a defining 
characteristic, surface oocyst folds may be observed in some 
specimens. If one or more sporozoites are observed at this point, 
record this as a comfirmed Cryptosporidium oocyst as well. Counts 
with internal structures must be confirmed by a senior analyst.

11.  Calculation

    11.1 Percentage of Floated Sample Examined.
    11.1.1 Record the percentage of floated sediment examined 
microscopically. [Calculate this value from the total volume of 
floated pellet obtained (10.1.8), the number of 25-mm membrane 
filters prepared together with the volume of floated pellet 
represented by these membrane filters (10.3.1.6), and the number of 
membrane filters examined.]
11.2 The following values are used in calculations:
    V=volume (liters) of original water sample (9.2.2 and 9.2.6)
    P=eluate packed pellet volume (10.1.8), (mL),
    F=fraction of eluate packed pellet volume (P) subjected to 
flotation, determined as

TP10FE94.013

R=Percentage (expressed as a decimal) of floated sediment examined 
(11.1.1)
PRG=Presumptive no. of Giardia cysts detected (10.4.3.2b)
PRC=Presumptive no. of Cryptosporidium oocysts detected (10.4.3.2c)
CG=Confirmed number of Giardia cysts detected with internal 
structures (10.4.3.2b)
CC=Confirmed number of Cryptosporidium oocysts detected with 
internal structures (10.4.3.2c)
    11.3 For positive samples, calculate the number of cysts or 
oocysts per 100 liters of sample as follows:

TP10FE94.014

    A sample calculation is shown in Appendix X2.
    11.4 For samples in which no cysts or oocysts are detected, (PRG 
or PRC or CG or CC) = <1. Calculate the detection limit as follows:

TP10FE94.015

    A sample calculation is shown in Appendix X2.
    11.5 Reporting.
    11.5.1 Report results as presumptive count and confirmed count 
for Giardia cysts or Cryptosporidium oocysts per 100 L of sample. 
Report negative results in terms of the detection limit. 
Representative reporting forms are given in Appendix X3.
    11.5.2 Enter all data into the computer spreadsheet provided 
with this protocol.

12. Water Sample Controls

    12.1 Water Sample Negative Control.
    12.1.1 This control is a check on equipment, materials, reagents 
and technique. It involves collecting a sample from water known to 
be free of cysts and oocysts and processing and examining that 
sample as if it were an unknown. Every 10th sample processed in the 
laboratory should be a negative control.
    12.1.2 Using the procedures detailed in 10.2 through 10.4, 
collect, process, and examine a 40 L (10 gal) or larger sample of 
reagent water or tap water that has first been passed through a 
filter of not more than 1 m absolute porosity.
    12.1.3 The entire concentrate from this sample should be 
examined. If any cysts or oocysts are detected, do not process any 
unknown samples until the source of the contamination is located and 
corrected.
    12.2 Water Sample Positive Control.
    12.2.1 The purpose of this control is to assure that the 
laboratory can recover cysts and oocysts when they are spiked into a 
sample at a known level.
    12.2.2 It is recommended that, once every three months, or when 
a sample outside the norm is encountered, the eluate packed pellet 
(10.1.8) from an actual sample be split in half.
    12.2.3 One half should be processed as an unknown; the second 
half should be spiked with 1,000 cysts and 1,000 oocysts/ mL of 
eluate packed pellet. Process and examine the sample using the 
procedures detailed in 10.2 through 10.4.
    12.2.4 Calculate the recovery efficiency in the spiked aliquot 
after substracting any cysts and oocysts observed in the unspiked 
aliquot. If cysts and oocysts are not recovered in the spiked 
sample, do not process any more unknown samples until the laboratory 
can demonstrate recovery in spiked samples.

13. Education, Training and Proficiency

    To be added at a later date.

14. Key Words

    14.1 Antibody, Cryptosporidium, cysts, fluorescence, Giardia, 
immunoassay, oocysts, protozoa.

Appendices

X1.  Cleaning the Manifold and Stainless Steel Wells
X1.1  Manifold
X1.1.1  After all the membrane filters have been mounted on glass 
slides (10.3.6), remove the support filters and discard them.
X1.1.2  Open all the manifold valves and increase the vacuum 
pressure to the manifold by closing the bleeder valve associated 
with the vacuum tubing.
X1.1.3  Rinse each manifold filter support screen with 10-20 mL of 
0.01% Tween 80 solution.
X1.1.4  Rinse each manifold filter support screen with 10-20 mL 
water.
X1.1.5  Disconnect the manifold from the vacuum and wash the cover 
and fluid collection box in warm detergent solution. Rinse with tap 
water and reagent water.
X1.2  Stainless Steel Wells
X1.2.1  Place a cloth on the bottom of an autoclavable container 
which is large enough to accommodate all 10 stainless steel wells in 
a single layer.
X1.2.2  Put the stainless steel wells top side down on the cloth. 
The rim on the underside of the well is fragile. Care must be taken 
to avoid scratching and denting the rim.
X1.2.3  Add enough reagent water containing detergent to cover the 
stainless steel wells by an inch or more.
X1.2.4  Autoclave the stainless steel container with the stainless 
steel wells for 15 min at 15 lbs/in\2\ and 121  deg.C. Use the slow 
exhaust mode at the completion of the autoclave cycle.
X1.2.5  Transfer the wells to a pan of hot detergent cleaning 
solution.
X1.2.6  Individually scrub the inside and bottom of stainless steel 
wells with a sponge.
X1.2.7  Rinse each well with tap water followed by reagent water. 
Drain and air dry the wells.
X1.2.8  Always check the bottom ridge of each stainless steel well 
for dents and scratches.
X1.2.9  If dents or scratches are found on the bottom of a stainless 
steel well, do not use it until it is properly reground.
X2.  Sample Calculation
X2.1  Positive Samples
X2.1.1  Assume that a 100 gal (380 L) water sample was collected. 
The sample was eluted resulting in 5 mL of sediment. Fifty percent 
(2.5 mL) of the sediment was purified by Percoll-sucrose flotation. 
Forty percent of the floated material was examined microscopically. 
A total of 8 presumptive and 3 confirmed Giardia cysts were found. 
No presumptive or confirmed Cryptosporidium oocysts were observed. 
Using the formula in 12.1:

TP10FE94.011


X2.2  Negative Samples
X2.2.1  Using the description given in X2.1.1, no Cryptosporidium 
oocysts were observed. The calculated detection limit per 100 liters 
would be:

TP10FE94.012

      
X3.1  Giardia Report Form
Slide prepared by:-----------------------------------------------------
Date prepared:---------------------------------------------------------
Analyst:---------------------------------------------------------------
Date analyzed: --------------------------------------------------------

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
                                                     Morphological Characteristics                                                                                                                 
   Object    Shape (oval or     Size LxW    ------------------------------------------------   Presumptive      Confirmed                                                                          
 located by      round)       (m)                     Median body      Axonemes          Count           Count                                                                            
  IFA No.                                     Nucleus (#)     ()     ()     ()     ()                                                                          
---------------------------------------------------------------------------------------------------------------------------                                                                        
1            ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
2            ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
3            ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
4            ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
5            ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
6            ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
7            ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
8            ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
9            ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
10           ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
11           ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
12           ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
13           ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
14           ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
15           ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
    Total    ..............  ..............  ..............  ..............  ..............  ..............  ..............                                                                        
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Calculated number of presumptive cysts/100 liters----------------------
Calculated number of confirmed cysts/100 liters------------------------
X3.2  Cryptosporidium Report Form
Slide prepared by:-----------------------------------------------------
Date prepared:---------------------------------------------------------
Analyst:---------------------------------------------------------------
Date analyzed: --------------------------------------------------------

------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
                                                Morphological                                                                                                                                                                                                                                   
    Object                                     characteristic    Presumptive      Confirmed                                                                                                                                                                                                     
  located by   Shape (oval or     Size LxW                          count           count                                                                                                                                                                                                       
   IFA No.         round)       (m)  ----------------  ()      ()                                                                                                                                                                                                    
                                               Sporozoite (#)                                                                                                                                                                                                                                   
---------------------------------------------------------------------------------------------                                                                                                                                                                                                   
1              ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
2              ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
3              ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
4              ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
5              ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
6              ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
7              ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
8              ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
9              ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
10             ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
11             ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
12             ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
13             ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
14             ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
15             ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
    Total      ..............  ..............  ..............  ..............  ..............                                                                                                                                                                                                   
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Calculated number of presumptive oocysts/100 liters--------------------
Calculated number of confirmed oocysts/100 liters----------------------
X4.  Microscope Adjustments\12\
---------------------------------------------------------------------------

    \12\Smith, R.F. 1982. Microscopy and Photomicrography: A 
Practical Guide. Appelton-Century-Crofts, New York.
---------------------------------------------------------------------------

    The microscopic portion of this procedure depends upon very 
sophisticated optics. Without proper alignment and adjustment of the 
microscope the instrument will not function at maximal efficiency 
and the probability of obtaining the desired image (information) 
will not be possible. Consequently, it is imperative that all 
portions of the microscope from the light sources to the oculars are 
properly adjusted.
    While microscopes from various vendors are configured somewhat 
differently, they all operate on the same general physical 
principles. Therefore, slight deviations or adjustments may be 
required to make these guidelines work for the particular instrument 
at hand.
X4.1.  Adjustment of the Epifluorescent Mercury Bulb and Transmitted 
Light Bulb Filament. The sole purpose of these procedures is to 
insure even field illumination.
X4.1.1  Mercury Bulb Adjustment. This section assumes that you have 
successfully replaced the mercury bulb in your particular lamp 
socket and reconnected the lamp socket to the lamp house. These 
instructions also assume the condenser has been adjusted to produce 
Kohler illumination. Make sure that you have not touched any glass 
portion of the mercury bulb with your bare fingers while installing 
it. Warning: Never look at the ultraviolet light coming out of the 
mercury lamp house or the ultraviolet light image without a barrier 
filter in place.
X4.1.1.1.  Usually there is a diffuser lens between the lamp and the 
microscope which either must be removed or swung out of the light 
path.
X4.1.1.2.  Using a prepared microscope slide, adjust the focus so 
the image in the oculars is sharply defined.
X4.1.1.3.  Replace the slide with a business card or a piece of lens 
paper.
X4.1.1.4.  Close the field diaphragm (iris diaphragm in the 
microscope base) so only a small point of light is visible on the 
card. This dot of light tells you where the center of the field of 
view is.
X4.1.1.5.  Mount the mercury lamp house on the microscope without 
the diffuser lens in place and turn on the mercury bulb.
X4.1.1.6.  Remove the objective in the light path from the 
nosepiece. You should see a primary (brighter) and secondary image 
(dimmer) of the mercury bulb arc on the card after focusing the 
image with the appropriate adjustment.
X4.1.1.7.  Using the other lamp house adjustments, adjust the 
primary and secondary mercury bulb images so they are side by side 
(parallel to each other) with the transmitted light dot in between 
them.
X4.1.1.8.  Reattach the objective to the nosepiece.
X4.1.1.9.  Insert the diffuser lens into the light path between the 
mercury lamp house and the microscope.
X4.1.1.10.  Turn off the transmitted light, remove the card from the 
stage, and replace it with a slide of fluorescent material. Check 
the field for even fluorescent illumination. Adjustment of the 
diffuser lens will most likely be required. Additional slight 
adjustments as in step 6 above may be required.
X4.1.1.11.  Maintain a log of the number of hours the U.V. bulb has 
been used. Never use the bulb for longer than it has been rated. 
Fifty watt bulbs should not be used longer than 100 hours; 100 watt 
bulbs should not be used longer than 200 hours.
X4.1.2.  Transmitted Bulb Adjustment. This section assumes that you 
have successfully replaced the transmitted bulb in your particular 
lamp socket and reconnect the lamp socket to the lamp house. Make 
sure that you have not touched any glass portion of the transmitted 
light bulb with your bare fingers while installing it. These 
instructions also assume the condenser has been adjusted to produce 
Kohler illumination.
X4.1.2.1.  Usually there is a diffuser lens between the lamp and the 
microscope which either must be removed or swung out of the light 
path. Reattach the lamp house to the microscope.
X4.1.2.2.  Using a prepared microscope slide and a 40X objective (or 
similar), adjust the focus so the image in the oculars is sharply 
defined.
X4.1.2.3.  Without the ocular or Bertrand optics in place the pupil 
and filament image inside can be seen at the bottom of the tube.
X4.1.2.4.  Focus the lamp filament image with the appropriate 
adjustment on your lamp house.
X4.1.2.5.  Similarly, center the lamp filament image within the 
pupil with the appropriate adjustment(s) on your lamp house.
X4.1.2.6.   Insert the diffuser lens into the light path between the 
transmitted lamp house and the microscope.
X4.2.  Adjustment of Interpupillary Distance and Oculars for Each 
Eye. These adjustments are necessary, so eye strain is reduced to a 
minimum. These adjustment must be made for each individual using the 
microscope. This section assumes the use of a binocular microscope.
X4.2.1.  Interpupillary Distance. The spacing between the eyes 
varies from person to person and must be adjusted for each 
individual using the microscope.
X4.2.1.1.   Place a prepared slide on the microscope stage, turn on 
the transmitted light, and focus the specimen image using the course 
and fine adjustment knobs.
X4.2.1.2.   Using both hands, adjust the oculars in and out until a 
single circle of light is observed while looking through the two 
oculars with both eyes.
X4.2.2.  Ocular Adjustment for Each Eye. This section assumes a 
focusing ocular(s). This adjustment can be made two ways, depending 
upon whether or not the microscope is capable of photomicrography 
and whether it is equipped with a photographic frame which can be 
seen through the binoculars. Precaution: Persons with astigmatic 
eyes should always wear their contact lenses or glasses when using 
the microscope.
X4.2.2.1.  For microscopes not capable of photomicrography. This 
section assumes only the right ocular is capable of adjustment.
    (a) Place a prepared slide on the microscope stage, turn on the 
transmitted light, and focus the specimen image using the course and 
fine adjustment knobs.
    (b) Place a card between the right ocular and eye keeping both 
eyes open. Using the fine adjustment, focus the image for the left 
eye to its sharpest point.
    (c) Now transfer the card to between the left eye and ocular. 
Without touching the course or fine adjustment and with keeping both 
eyes open, bring the image for the left eye into sharp focus by 
adjusting the ocular collar at the top of the ocular.
X4.2.2.2.  For microscopes capable of viewing a photographic frame 
through the viewing binoculars. This section assumes both oculars 
are adjustable.
    (a) Place a prepared slide on the microscope stage, turn on the 
transmitted light, and focus the specimen image using the course and 
fine adjustment knobs.
    (b) After activating the photographic frame, place a card 
between the right ocular and eye keeping both eyes open. Using the 
correction (focusing) collar on the left ocular focus the left 
ocular until the double lines in the center of the frame are as 
sharply focused as possible.
    (c) Now transfer the card to between the left eye and ocular. 
Again keeping both eyes open, bring the image of the double lines in 
the center of the photographic frame into as sharp a focus for the 
right eye as possible by adjusting the ocular correction (focusing) 
collar at the top of the right ocular.
X4.3.  Calibration of an Ocular Micrometer\13\--This section assumes 
that an ocular reticle has been installed in one of the oculars by a 
microscopy specialist and that a stage micrometer is available for 
calibrating the ocular micrometer (reticle). Once installed the 
ocular reticle should be left in place. The more an ocular is 
manipulated the greater the probability is for it to become 
contaminated with dust particles. This calibration should be done 
for each objective in use on the microscope. If there is an 
optivar\14\ on the microscope, then the calibration procedure must 
be done for the respective objective at each optivar setting.
---------------------------------------------------------------------------

    \13\Melvin, D.M. and M.M. Brooke. 1982. Laboratory Procedures 
for the Diagnosis of Intestinal Parasites. U.S. Department of Health 
and Human Services, HHS Publication No. (CDC) 82-8282.
    \14\A device between the objectives and the oculars that is 
capable of adjusting the total magnification.
---------------------------------------------------------------------------

X4.3.1.  Place the stage micrometer on the microscope stage, turn on 
the transmitted light, and focus the micrometer image using the 
course and fine adjustment knobs for the objective to be calibrated. 
Continue adjusting the focus on the stage micrometer so you can 
distinguish between the large (0.1 mm) and the small (0.01 mm) 
divisions.
X4.3.2.  Adjust the stage and ocular with the micrometer so the 0 
line on the ocular micrometer is exactly superimposed on the 0 line 
on the stage micrometer.
X4.3.3.  Without changing the stage adjustment, find a point as 
distant as possible from the two 0 lines where two other lines are 
exactly superimposed.
X4.3.4.  Determine the number ocular micrometer spaces as well as 
the number of millimeters on the stage micrometer between the two 
points of superimposition.
    For example: Suppose 48 ocular micrometer spaces equal 0.6 mm.
X4.3.5.  Calculate the number of mm/ocular micrometer space.
    For example: 0.6 mm/48 ocular micrometer spaces = 0.0125 mm/
ocular micrometer space
X4.3.6.  Since most measurements of microorganisms are given in 
m rather than mm, the value calculated above must be 
converted to m by multiplying it by 1000 m/mm.
    For example: 

                                                                                                                                                        
                                           0.0125 mm                   1000                                                                             
                                -------------------------------     m                                                                          
                                                                x                  =12.5 m/ocular micrometer                                   
                                    ocular micrometer space        ------------                  space                                                  
                                                                        mm                                                                              
                                                                                                                                                        

X4.3.7.  Follow steps A through F for each objective. It is helpful 
to record this information in a tabular format, like the example 
below, which can be kept near the microscope.

--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                        m/                                                                                                                                                         
               Obj.                          Ocular     Stage microm.      Ocular                                                                                                                                                           
  Item #      power      Description     microm. space  space (mm)\1\    micrometer                                                                                                                                                         
                                                                          space\2\                                                                                                                                                          
------------------------------------------------------------------------------------                                                                                                                                                        
1           10X        N.A.\3\ =                                                                                                                                                                                                            
2           20X        N.A. =                                                                                                                                                                                                               
3           40X        N.A. =                                                                                                                                                                                                               
4           100X       N.A. =                                                                                                                                                                                                               
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
\1\1000 m/mm                                                                                                                                                                                                                       
\2\Stage micrometer length in mm X 1000/# of Ocular Micrometer Spaces                                                                                                                                                                       
\3\N.A. stands for numerical aperture. The numerical aperture value is engraved on the barrel of the objective.                                                                                                                             

X4.4.  Kohler Illumination. This section assumes that Kohler 
illumination will be established for only the 100X oil differential 
interference contrast or Hoffman modulation objective 
which will be used to identify internal morphological 
characteristics in Giardia cysts and Cryptosporidium oocysts. If by 
chance more than one objective is to be used for either differential 
interference contrast or Hoffman modulation optics, then 
each time the objective is changed, Kohler illumination must be 
reestablished for the new objective lens. Previous sections have 
adjusted oculars and light sources. This section aligns and focuses 
the light going through the condenser underneath the stage at the 
specimen to be observed. If Kohler illumination is not properly 
established, then differential interference contrast or Hoffman 
modulation optics will not work to their maximal 
potential. These steps need to become second nature and must be 
practiced regularly until they are a matter of reflex rather than a 
chore.
X4.4.1.  Place a prepared slide on the microscope stage, place oil 
on the slide, move the 100X oil objective into place, turn on the 
transmitted light, and focus the specimen image using the coarse and 
fine adjustment knobs.
X4.4.2.  At this point both the radiant field diaphragm in the 
microscope base and the aperture diaphragm in the condenser should 
be wide open. Now close down the radiant field diaphragm in the 
microscope base until the lighted field is reduced to a small 
opening.
X4.4.3.  Using the condenser centering screws on the front right and 
left of the condenser, move the small lighted portion of the field 
to the center of the visual field.
X4.4.4.  Now look to see whether the leaves of the iris field 
diaphragm are sharply defined (focused) or not. If they are not 
sharply defined, then they can be focused distinctly by changing the 
height of the condenser up and down with the condenser focusing knob 
while you are looking through the binoculars. Once you have 
accomplished the precise focusing of the radiant field diaphragm 
leaves, open the radiant field diaphragm until the leaves just 
disappear from view.
X4.4.5.  The aperture diaphragm of the condenser is adjusted now to 
make it compatible with the total numerical aperture of the optical 
system. This is done by removing an ocular, looking into the tube at 
the rear focal plane of the objective, and stopping down the 
aperture diaphragm iris leaves until they are visible just inside 
the rear plane of the objective.
X4.4.6.  After completing the adjustment of the aperture diaphragm 
in the condenser, return the ocular to its tube and proceed with the 
adjustments required to establish either differential interference 
contrast or Hoffman modulation optics.

BILLING CODE 6560-50-P

TP10FE94.000


TP10FE94.001


TP10FE94.002


BILLING CODE 6560-50-C

Appendix D to Subpart M--Proposed Virus Monitoring Protocol

Foreword

    The surface water treatment rule (40 CFR part 141) established 
the maximum contam-ination level for enteric virus in public water 
systems by requiring that systems using surface water or ground 
water under the influence of surface water reduce the amount of 
virus in source water by 99.99%. The rule requirements are currently 
met on basis of treatment alone (i.e., disinfection and/or 
filtration), and thus the degree of actual protection against 
waterborne viral disease depends upon the source water quality. 
Utilities using virus-free source water or source water with low 
virus levels may be overtreating their water, while utilities using 
highly contaminated water may not be providing adequate protection. 
In order to more adequately determine the degree of protection and 
to reduce the levels of disinfection and disinfection byproducts, 
where appropriate, EPA is requiring all utilities serving a 
population of over 100,000 to monitor their source water for viruses 
monthly for a period of 18 months. Systems finding greater than one 
infectious enteric virus particle per liter of source water must 
also monitor their finished water on a monthly basis. The authority 
for this requirement is Section 1445(a)(1) of the Safe Drinking 
Water Act, as amended in 1986.
    The presence of coliphage in water in temperate climates is 
perceived as an indicator of fecal pollution, as a practical model 
to be applied in the evaluation of treatment processes, and as a 
possible indicator of the presence of enteric viruses. As a 
secondary approach in the establishment of water quality criteria in 
public water systems serving a population of over 100,000, the U.S. 
EPA recommends that coliphage be surveyed along with human enteric 
viruses. These studies are to generate and provide specific 
monitoring data and other information characterizing water 
utilities.
    This protocol was developed by virologists at the U.S. 
Environmental Protection Agency and modified to reflect the 
consensus agreements from national experts attending a Virus 
Monitoring Workshop held in Cincinnati, Ohio, on August 12, 1993. 
The protocol was subsequently revised to reflect comments obtained 
from many of the Workshop attendees in light of the consensus 
agreements. The procedures contained herein do not preclude the use 
of additional tests for research purposes (e.g., polymerase chain 
reaction-based detection methods for non-cytopathic viruses).
    The concentrated water samples to be monitored may contain 
pathogenic human enteric viruses. Laboratories performing virus and 
coliphage analyses are responsible for establishing an adequate 
safety plan and must rigorously follow the guidelines on 
sterilization and aseptic techniques given in Part 5.
    Analytical Reagent or ACS grade chemicals (unless specified 
otherwise) and deionized, distilled water (dH2O) should be used 
to prepare all media and reagents. The dH2O must have a 
resistance of greater than 0.5 megohms-cm, but water with a 
resistance of 18 megohms-cm is preferred. Water and other reagent 
solutions may be available commercially. For any given section of 
this protocol only apparatus, materials, media and reagents which 
are not described in previous sections are listed, except where 
deemed necessary. The amount of media prepared for each Part of the 
Protocol may be increased proportionally to the number of samples to 
be analyzed.

Virus Monitoring Protocol

Table of Contents

Foreword
Table of Contents
Part 1--Sample Collection Procedure
    Apparatus and Materials
    Media and Reagents
    Procedure
Part 2--Processing of Collected Sample
    Elution Procedure
    Apparatus and Materials
    Media and Reagents
    Procedure
    Organic Flocculation Concentration Procedure
    Apparatus and Materials
    Media and Reagents
    Procedure
Part 3--Total Culturable Virus Assay
    Quantal Assay
    Apparatus and Materials
    Media and Reagents
    Sample Inoculation and CPE Development
    Virus Quantitation:
    Reduction of Cytotoxicity in Sample Concentrates
    Media and Reagents
    Procedure for Cytotoxicity Reduction
Part 4--Coliphage Assay of Processed Sample
    Plaque Assay Procedure
    Apparatus and Materials
    Media and Reagents
    Sample Processing
    Storage of E. coli C Host Culture for Somatic Coliphage Assay
    Preparation of Host for Somatic Coliphage Assay
    Preparation of X174 Positive Control
    Procedure for Somatic Coliphage Assay
    Storage of E. coli C-3000 Host Culture for Male-Specific 
Coliphage Assay
    Preparation of Host for Male-Specific Coliphage Assay
    Preparation of MS2 Positive Control
    Procedure for Male-Specific Coliphage Assay
Part 5--Sterilization and Disinfection
    General Guidelines
    Sterilization Techniques
    Solutions
    Autoclavable Glassware, Plasticware, and Equipment
    Chlorine Sterilization
    Procedure for Verifying Sterility of Liquids
    Media and Reagents
    Verifying Sterility of Small Volumes of Liquids
    Visual Evaluation of Media for Microbial Contaminants
    Contaminated Materials
Part 6--Bibliography and Suggested Reading
Part 7--Vendors
Part 8--Data Sheets

Part 1--Sample Collection Procedure

Apparatus and Materials

    It is recommended that apparatus and materials be provided to 
sample collectors by the approved laboratory contracted to analyze 
samples for viruses. Several configurations are given below for the 
assembly of filter apparatus. Combinations of these configurations can 
be prepared by combining the directions of two or more configurations.
    1. Standard filter apparatus containing 1MDS positively charged 
filter (see Figure 1).
    a. Parts needed:
    i. BR--Backflow Regulator (Watts Regulator\1\ Product Series 8--\3/
4\'' Hose Connection Vacuum Breaker).
---------------------------------------------------------------------------

    \1\See Part 7 for addresses of the vendors listed. The vendors 
listed in this protocol represents one possible source for required 
products. Other vendors may supply the same or equivalent products.
---------------------------------------------------------------------------

    ii. SF--Swivel Female insert with garden hose threads (United 
States Plastic Product No. 63003).
    iii. BT--Braided Tubing, \1/2\'' clear (Fisher Scientific Product 
No. 14-169-10C).
    iv. HC--Hose Clamp (Cole-Parmer Product No. G-06403-20).
    v. HF1--Hose Fitting, nylon, \3/4\'' male NPT x \1/2\'' tubing ID 
(United States Plastic Product No. 61143).
    vi. CH--Cartridge Housing (Cuno Product No. AP11T).
    vii. FC--Filter Cartridge, positively charged 1MDS, ZetaPor 
Virosorb (Cuno Product No. 45144-01-1MDS).
    viii. WM--Water Meter (Neptune Equipment Product No. \5/8\'' 
Trident 10). Meters are normally rated in cubic feet (a cubic foot of 
water is 7.481 gallons or 28.316 liters).
    b. Apparatus assembly (to be performed by the approved laboratory 
contracted to analyze samples for viruses)--in order, as shown in 
Figure 1, connect the backflow regulator (BR) to a swivel female insert 
(SF). Clamp a  6-18'' piece of tubing (BT) onto the tubing connector of 
the insert using a hose clamp (HC). Attach the other end onto a \3/
4\ x \1/2\'' fitting (HF1) connected with the inlet of the cartridge 
housing (CH). Attach another \3/4\ x \1/2\'' fitting to the outlet of 
the housing. The entire assembly to this point should be sterilized 
with chlorine as described in Part 5. Presterilize a 1MDS filter 
cartridge (FC) as described in Part 5 and place into the housing using 
aseptic technique. Seal the openings into the apparatus with sterile 
aluminum foil. Prepare the discharge portion of the assembly by 
attaching a swivel female insert to another piece of tubing and 
connecting the insert to the inlet of the water meter (WM). Attach 
another swivel female insert to the outlet of the meter and connect a 
piece of tubing for discharge. This discharge portion does not have to 
be sterilized and should be attached to the filter housing after 
flushing of the system.
    Teflon tape (Cole Parmer Product No. G-08782-27) must be used on 
all fittings.
    2. Filter apparatus for waters exceeding 100 NTU (see Figure 2).
    a. Additional parts needed: PP--10 m Polypropylene 
Prefilter (Parker Hannifin Product No. M19R10-A).
    b. Apparatus assembly--connect a second cartridge housing to the 
standard apparatus by connecting a short piece of tubing between the 
two housings via additional HF1 hose fittings and clamps. Add a 
presterilized prefilter (see Part 5) using aseptic technique.
    3. Filter apparatus for water pressures exceeding 50 psi (see 
Figure 3).
    a. Additional parts needed:
    i. HF2--Hose Fitting, nylon, \3/8\'' male NPT x \1/2\'' tubing ID 
(United States Plastic Product No. 61141).
    ii. PR--Pressure Regulator (Watts Regulator Product No. \3/8\'' 
26A, Suffix C).
    iii. PN--PVC Nipple, \3/8\'' x 2'' (Ryan Herco Product No. 3861-
057).
    iv. TE--PVC TEE with \3/8\'' female NPT ports (Ryan Herco Product 
No. 3805-003).
    v. RB--Reducing Bushing, \3/8\'' NPT(M) x \1/4\'' NPT(F) (Cole 
Parmer Product No. G-06349-32).
    vi. PG--Pressure Gauge 0-30 psi (Cole Parmer Product No. G-68004-
03).
    b. Apparatus assembly--assemble as described for the standard 
apparatus, except clamp the other end of the tubing with the backflow 
regulator and swivel female insert to a \3/8\'' x \1/2\'' fitting 
(HF2). Screw the fitting into the inlet of the pressure regulator (PR). 
Connect the outlet of the pressure regulator to the PVC TEE (TE) via 
the 2'' nipple (PN). Connect the pressure gauge (PG) to the top of the 
TEE using the bushing (RB). Attach a \3/8\'' x \1/2\'' fitting to the 
other end of the TEE. Clamp a piece of tubing to the fitting and 
connect the other end to the HF1 fitting on the cartridge housing.
    4. Filter apparatus for finished waters requiring dechlorination 
(see Figure 4).\2\
---------------------------------------------------------------------------

    \2\The standard filter apparatus may be used as an alternative 
to the apparatus described here if thiosulfate is added to a water 
sample in a calibrated container as described in Step 5 of the 
Sample Collection Procedure.
---------------------------------------------------------------------------

    a. Additional parts needed:
    i. IN--In-line INjector (DEMA Engineering Product No. 204B \1/2\'' 
NPT).
    ii. HF3--Hose Fitting, nylon, \1/2\'' male NPT  x  \1/2\'' tubing 
ID (United States Plastic Product No. 62142).
    b. Apparatus assembly--assemble as described for the standard 
apparatus, except clamp the other end of the tubing with the backflow 
regulator and swivel female insert to a \1/2\'' x \1/2\'' fitting 
(HF3). Attach the water inlet of the injector (IN) to the HF3 fitting. 
Attach another \1/2\'' x \1/2\'' fitting to the outlet of the injector 
and connect this fitting to the inlet of the cartridge housing with a 
short piece of tubing. Connect a piece of sterile standard Tygon tubing 
(TT) to the injection port of the injector.
    5. Portable pH probe (Omega Product No. PHH-1X).
    6. Portable temperature probe (Omega Product No. HH110).
    7. Commercial ice packs (Cole Parmer Product No. L-06346-85).
    8. 1 liter polypropylene wide-mouth bottles (Nalge Product No. 
2104-0032).
    9. 17'' x 17'' x 13'' styrofoam shipping box with carrying strap 
(Cole Parmer Product No. L-03748-00 and L-03742-30).
    10. Miscellaneous--aluminum foil, data card (see Part 8), surgical 
gloves, screwdriver or pliers for clamps, waterproof marker.
    11. Chemical resistant pump and appropriate connectors (if a garden 
hose-type pressurized faucets for the source or finished water to be 
monitored are unavailable).

Media and Reagents

    1. 10% sodium thiosulfate (Na2S2O3)--dissolve 100 g 
of Na2S2O3 in a total of 1000 ml dH2O to prepare a 
stock solution. Autoclave for 15 minutes at 121 deg.C.

Procedure

    Operators must wear surgical gloves and avoid conditions which can 
contaminate a sample with virus.
    Step 1. Purge the water tap to be sampled for at least one minute 
prior to connecting the filter apparatus.
    Surface water sampling must be conducted at the plant intake, prior 
to impoundment or any other treatment. Finished water sampling must be 
conducted at the point of entry into the distribution system.
    Step 2. Remove the foil and connect the backflow regulator of the 
inlet hose to the tap. Loosen the clamp on the tubing at the inlet side 
of the cartridge housing (1MDS filter housing or, if used, the inlet 
side of the prefilter housing). Remove the housing(s) and cover the 
inlet with sterile foil. Place the tubing removed from the housing into 
a 1 liter plastic bottle. Flush the system for at least ten minutes 
with the water to be sampled. While the system is being flushed, 
measure and record onto the Sample Data Sheet (see Part 8) the pH and 
temperature values from the water collecting in and overflowing from 
the 1 liter plastic bottle. The pH meter should be calibrated prior to 
each use for the pH range of the water to be sampled.
    Step 3. After flushing the system, turn off the flow of water at 
the sample tap and reconnect the filter housing to the inlet hose. 
Connect the discharge hose (with water meter) to the filter housing 
outlet.
    Step 4. Record the sample number, location, date, time of day and 
initial cubic feet (or gallon) reading from the water meter onto the 
sample data sheet.
    A consistent system for assigning unique utility-specific sample 
numbers will be developed prior to the start of the monitoring period.
    Step 5. Slowly turn on the water with the filter housing placed in 
an upright position, while pushing the red vent button on top of the 
filter housing to expel air. When the air is totally expelled from the 
housing, release the button, and open the sample tap completely.
    For taps with pressures exceeding 50 psi, use an apparatus with a 
pressure regulator (Figure 3) and adjust the pressure to below 50 psi.
    For sampling chlorinated finished water place the sterile end of 
the tubing from the injection port of the injector into a graduated 
container containing the 10% sodium thiosulfate solution and adjust the 
injector to add thiosulfate at a rate of 0.5 ml per liter of water 
sample. Alternatively, place the water sample into a sterile calibrated 
polyethylene (e.g., garbage container) or polypropylene container, add 
0.5 ml per liter thiosulfate, mix and pump the dechlorinated solution 
through a standard apparatus.
    Step 6. Sample a minimum volume for surface water of 200 liters 
(7.1 ft3, 52.9 gallons) and for finished water of 1200 liters 
(42.4 ft3, 317.0 gallons). For surface water the flow rate and the 
total amount of sample that can be passed before the filter clogs will 
depend upon water quality and will have to be determined from 
experience.
    It may be convenient to start the sampling in the afternoon and 
sample overnight so that the sample can be shipped to the testing 
laboratory during the morning. Sampling should not be performed 
throughout the night if experience shows that the filters may clog 
during the collection period, unless it can be monitored.
    Step 7. Turn off the flow of water at the sample tap at the end of 
the sampling period and record the date, time of day, and cubic feet 
(or gallon) reading from the water meter onto the Sample Data Sheet.
    Step 8. Disconnect the filter housing(s) from the inlet and outlet 
hoses. Turn the filter housing(s) upside down and allow excess water to 
flow out as waste water. Turn the housing(s) upright and cover 
completely with aluminum foil, making sure to cover the inlet and 
outlet ports.
    Step 9. Pack the filter housing(s) and all apparatus components 
prior to the housing(s) into an insulated shipping box. Add 
refrigerated ice packs to keep the sample cool in transit (the number 
of ice packs may have to be adjusted based upon experience to ensure 
that the samples remain cold). Place the Sample Data Sheet (protected 
with a closable plastic bag) in with the sample and ship by overnight 
mail to the contracted, approved laboratory for virus analysis. Notify 
the laboratory by phone upon the shipment of sample.
    The approved laboratory will elute virus from the 1MDS filter (and 
prefilter, if appropriate) and analyze the eluates as described in 
Parts 2, 3, and 4. After removing the filter, the laboratory will 
sterilize the apparatus components with chlorine and dechlorinate with 
sodium thiosulfate as described in Part 5. After flushing with sterile 
dH2O, a new 1MDS cartridge (and prefilter, if appropriate) will be 
added, the openings sealed with sterile aluminum foil, and the 
apparatus returned to the utility for the next sample. The discharge 
hoses with water meter can be stored at the utility between samplings. 
Openings should be covered with aluminum foil during storage.

Part 2--Processing of Collected Sample

    The cartridge filters must arrive at the approved laboratory in a 
refrigerated, but not frozen, condition. The arrival condition should 
be recorded on the Sample Data Sheet (Part 8). Filters should be 
refrigerated upon arrival and eluted within 72 hours of the start of 
the sample collection.
Elution Procedure
Apparatus and Materials
    1. Positive pressure air or nitrogen source equipped with a 
pressure gauge.
    If the pressure source is a laboratory air line or pump, it must be 
equipped with an oil filter.
    2. Dispensing pressure vessels--5 or 20 liter capacity (Millipore 
Corp. Product No. XX67 00P 05 and XX67 00P 20).
    3. pH meter, measuring to an accuracy of at least 0.1 pH unit, 
equipped with a combination-type electrode.
    4. Autoclavable inner-braided tubing with screw clamps for 
connecting tubing to equipment.
    5. Magnetic stirrer and stir bars.
Media and Reagents
    1. Sodium hydroxide (NaOH)--prepare 1 M and 5 M solutions by 
dissolving 4 or 20 g of NaOH in a final volume of 100 ml of dH2O, 
respectively.
    NaOH solutions may be stored for several months at room 
temperature.
    2. Beef extract V powder (BBL Microbiology Systems Product No. 
97531) prepare buffered 1.5% beef extract by dissolving 30 g of beef 
extract powder and 7.5 g of glycine (final glycine concentration = 0.05 
M) in 1.9 liters of dH2O. Adjust the pH to 9.5 with 1 or 5 M NaOH 
and bring the final volume to 2 liters with dH2O. Autoclave at 
121 deg.C for 15 min and use at room temperature.
    When used in the organic flocculation concentration step, each beef 
extract lot must be screened prior to use to determine adequate virus 
recoveries (mean recovery of 50% with poliovirus in 3 trials). Beef 
extract solutions may be stored for one week at 4 deg.C or for longer 
periods at -20 deg.C. A 3% beef extract solution may be prepared by 
doubling the amount of beef extract and used if the 1.5% solution fails 
the proficiency testing.
Procedure
    Step 1. Attach sections of inner-braided tubing (sterilized on 
inside and outside surfaces with chlorine and dechlorinated with 
thiosulfate as described in Part 5) to the inlet and outlet ports of a 
cartridge filter housing containing a 1MDS filter to be tested for 
viruses. If a prefilter was used, keep the prefilter and 1MDS housing 
connected and attach the tubing to the inlet of the prefilter housing 
and to the outlet of the 1MDS housing.
    Step 2. Place the sterile end of the tubing connected to the outlet 
of the 1MDS housing into a sterile 2 liter glass or polypropylene 
beaker.
    Step 3. Connect the free end of the tubing from the inlet port of 
the filter housing to the outlet port of a sterile pressure vessel and 
connect the inlet port of the pressure vessel to a positive air 
pressure source.
    Sterile tubing and a peristaltic pump may be used as an alternative 
to the pressure vessel.
    Step 4. Remove the top of the pressure vessel and pour 1000 mL of 
buffered 1.5% beef extract (pH 9.5) into the vessel.
    Step 5. Replace the top of the pressure vessel and close its vent/
relief valve.
    Step 6. Open the vent/relief valve(s) on the cartridge filter 
housing(s). Apply sufficient pressure to purge the trapped air from the 
filter housing(s). Close the vent/relief valve(s) as soon as the 
buffered beef extract solution begins to flow from it.
    Wipe up spilled liquid with laboratory disinfectant.
    Step 7. Increase the pressure to force the buffered beef extract 
solution through the filter(s).
    The solution should pass through the cartridge filter(s) slowly to 
maximize the elution contact period. When air enters the line from the 
pressure vessel, elevate and invert the filter housing to permit 
complete evacuation of the solution from the filters.
    Step 8. Turn off the pressure at the source and open the vent/
relief valve on the pressure vessel. Place the buffered beef extract 
from the 2 liter beaker back into the pressure vessel. Repeat Steps 5-
7.
    Step 9. Thoroughly mix the eluate and adjust the pH to 7.0-7.5 with 
1 N HCl. Measure and record the volume of the eluate onto the Virus 
Data Sheet. Remove exactly one tenth of the eluate, freeze at -70 deg.C 
and ship to the laboratory designated for archiving. Remove 40 ml of 
the eluate for coliphage analysis as described in Part 4.
    Proceed to the organic flocculation concentration procedure 
immediately. If the concentration of enteric virus cannot be undertaken 
immediately, store the eluate for up to 24 hours before concentration 
at 4 deg.C or for longer periods at -70 deg.C.

Organic Flocculation Concentration Procedure

Apparatus and Materials
    1. Refrigerated centrifuge capable of attaining 2,500-10,000  x  g 
and screw-capped centrifuge bottles with 100 to 1000 ml capacity.
    Each bottle must be rated for the relevant centrifugal force.
Media and Reagents:
    1. Hydrochloric acid (HCl)--Prepare 1 and 5 M solutions by mixing 
10 or 50 ml of concentrated HCl with 90 or 50 ml of dH2O, 
respectively.
    2. Sodium phosphate, dibasic (Na2HPO4  
7H2O)--0.15 M.
    Dissolve 40.2 g of sodium phosphate in a final volume of 1000 ml. 
The pH should be checked to ensure that it is between 9.0-9.5 and 
adjusted with NaOH, if necessary. Autoclave at 121 deg.C for 15 
minutes.
Procedure
    Step 1. Place a sterile stir bar into the beaker containing the 
buffered beef extract eluate from the cartridge filter(s). Place the 
beaker onto a magnetic stirrer, and stir at a speed sufficient to 
develop a vortex.
    To minimize foaming (which may inactivate viruses), do not mix 
faster than necessary to develop a vortex.
    Step 2. Insert a combination-type pH electrode into beef extract 
eluate. Add 1 M HCl to the flask slowly until pH of beef extract 
reaches 3.5  0.1. Continue to stir slowly for 30 minutes at 
room temperature.
    The pH meter must be standardized at pH 4 and 7. Electrodes must be 
sterilized before and after each use as described in Part 5.
    A precipitate will form. If pH is accidentally reduced below 3.4, 
add 1 M NaOH to bring it back to 3.5 # 0.1. Exposure to a pH below 3.4 
may result in some virus inactivation.
    Step 3. Remove the electrode from the beaker, and pour the contents 
of the beaker into a centrifuge bottle. Cap the bottle and centrifuge 
the precipitated beef extract suspensions at 2,500  x  g for 15 minutes 
at 4 deg.C. Remove and discard the supernatant.
    To prevent the transfer of the stir bar into a centrifuge bottle, 
hold another stir bar or magnet against the bottom of the beaker when 
decanting the contents. The beef extract suspension will usually have 
to be divided into several centrifuge bottles.
    Step 4. Place a stir bar into the centrifuge bottle that contains 
the precipitate. Add 30 ml of 0.15 M sodium phosphate. Place the bottle 
onto a magnetic stirrer, and stir slowly until the precipitate has 
dissolved completely.
    Support the bottle as necessary to prevent toppling. Avoid foaming, 
which may inactivate or aerosolize viruses. The precipitate may be 
partially dissipated with a spatula before or during the stirring 
procedure or may be dissolved by repeated pipetting in place of 
stirring. When the centrifugation was performed in more than one 
bottle, dissolve the precipitates in a total of 30 ml and combine into 
one bottle. If the precipitate is not completely dissolved before 
proceeding, significant virus loss may occur in Step 5. Virus loss may 
also occur by prolonged exposure to pH 9.0-9.5, thus, for some samples 
it may be beneficial to resuspend the precipitate initially in 0.15 M 
sodium phosphate that has been adjusted to pH 7.5 with 1 M HCl. After 
the precipitate is completely dissolved, the pH should be adjusted to 
9.0-9.5 with 1 M NaOH and mixed for 10 minutes at room temperature 
before proceeding to Step 5.
    Step 5. Check the pH and readjust to 9.0-9.5 with 1 M NaOH, as 
necessary. Remove the stir bar and centrifuge the dissolved precipitate 
at 4,000 -10,000  x  g for 10 minutes at 4 deg.C. Remove the 
supernatant and discard the pellet. Adjust the pH of the supernatant 
(designated the final concentrated sample from this point on) to 7.0-
7.5 with 1 M HCl and record the final volume on the Virus Data Sheet 
(see Part 8).
    Step 6. Refrigerate the final concentrated sample immediately and 
hold at 4 deg.C until it is assayed in accordance with the instructions 
given below. If the virus assay cannot be undertaken within 24 hours, 
store at -70 deg.C.
    Final concentrated samples processed to this point by a laboratory 
not doing the virus assay must be frozen at -70 deg.C immediately and 
then shipped on dry ice to the laboratory approved for virus assay.

Part 3--Total Culturable Virus Assay

Quantal Assay

Apparatus and Materials
    1. Incubator capable of maintaining the temperature of cell 
cultures at 36.5 plus-minuse> 1 deg.C.
    2. Sterilizing filter--0.22 m (Costar Product No. 140666).
    Always pass about 10 ml of 1.5% beef extract adjusted to pH 7.0-7.5 
through the filter just prior to use to minimize virus adsorption to 
the filter.
Media and Reagents
    1. Prepare BGM cell culture test vessels using standard procedures.
    BGM cells are a continuous cell line derived from African Green 
monkey kidney cells and are highly susceptible to many enteric viruses 
(Dahling et al., 1984; Dahling and Wright, 1986). The characteristics 
of this line were described by Barron et al. (1970). The use of BGM 
cells for recovering viruses from environmental samples was described 
by Dahling et al. (1974). For laboratories with no experience with 
virus recovery from environmental samples, the media described by 
Dahling and Wright (1986) is recommended for maximum sensitivity.
    The U.S. Environmental Protection Agency will supply an initial 
culture of BGM cells to all laboratories seeking approval. Upon 
receipt, laboratories must prepare an adequate supply of frozen BGM 
cells using standard procedures to replace working cultures that become 
contaminated or lose virus sensitivity. BGM cells have been held at 
-70 deg.C for more than 15 years with a minimum loss in cell viability.
Sample Inoculation and CPE Development
    Cell cultures used for virus assay are generally found to be at 
their most sensitive level between the third and sixth days after their 
most recent passage. Those older than seven days should not be used.
    Step 1. Identify cell culture test vessels by coding them with an 
indelible marker. Return the cell culture test vessels to a 36.5 
plus-minuse> 1 deg.C incubator and hold at that temperature until 
the cell monolayer is to be inoculated.
    Step 2. Thaw the final concentrated sample from Step 6 of the 
Organic Flocculation Concentration Procedure in Part 2, if frozen, and 
hold at 4 deg.C for no more than 4 hours. Warm the sample to room 
temperature just prior to inoculation.
    Step 3. Decant and discard the medium from cell culture test 
vessels.
    Do not disturb the cell monolayer.
    Step 4. Inoculate each BGM cell monolayer with a volume of the 
final concentrated sample appropriate for the cell surface area of the 
cell culture test vessels used.
    Inoculum volume should be no greater than 0.04 ml/cm\2\ of surface 
area.
    Avoid touching either the cannula or the pipetting device to the 
inside rim of the cell culture test vessels to avert the possibility of 
transporting contaminants to the remaining culture vessels.
    a. Inoculate one or more BGM cultures with an appropriate volume of 
0.15 M Na2HPO4  7H2O (see the Media and Reagents 
section in the Organic Flocculation Concentration Procedures in Part 2) 
preadjusted to pH 7.0-7.5. These cultures will serve as negative 
controls.
    b. Inoculate one or more BGM cultures with an appropriate volume of 
0.15 M Na2HPO4  7H2O preadjusted to pH 7.0-7.5 
and spiked with 20-40 PFU of the Lederle Fox strain of poliovirus type 
3. These cultures will serve as a positive control for the quantal 
assay. Additional positive control samples may be prepared by adding 
virus to a small portion of the final concentrated sample and/or by 
using additional virus types.
    c. Using the same volume of inoculum per cell culture vessel, 
inoculate a portion of the final concentrated sample that represents at 
least 100 liters of surface water or 1,000 liters of finished water. 
Calculate the total amount of the original water sample assayed by 
multiplying the sample volume (in liters) from the Sample Data Sheet 
(Part 8) by the fraction of the total final concentrated sample 
inoculated. Record this value on the Virus Data Sheet (Part 8).
    It is advisable to inoculate a small subsample several days before 
inoculating the remaining samples as a control for cytotoxicity.
    The volume of the final concentrated sample that represents 100 or 
1,000 liters may be inoculated onto cultures at the same time or, 
preferably, inoculated in aliquots (i.e., a second half of the sample 
inoculated onto cultures that are at least one passage higher than the 
first half). If the latter approach is taken, the sample should be 
aliquoted before being frozen at -70 deg.C and the inoculation of the 
second half should not be done until it is clear from the results of 
the first inoculation that cytotoxicity is not a problem.
    Sufficient cultures must be inoculated to obtain the most probable 
number of infectious total culturable viruses (MPN) with acceptable 95% 
confidence limits. In order to demonstrate a total culturable virus 
level in source water of one per liter with an acceptable 95% 
confidence range, it is suggested that at least 20 cultures each be 
inoculated at the beginning of the monitoring period and during the 
Summer months with undiluted final concentrated sample and final 
concentrated sample diluted 1:5 and 1:25 in 0.15 M sodium phosphate, pH 
7.0-7.5. If the initial monitoring results demonstrate virus levels of 
less than 1.5 MPN units per liter, then the inoculation of 40 cultures 
with only undiluted final concentrated sample should be sufficient for 
the remaining non-Summer collection periods. Since finished waters 
should contain little or no virus, the inoculation of 20 cultures with 
only undiluted final concentrated sample from finished waters should be 
sufficient.
    Step 5. Rock the inoculated cell culture test vessels gently to 
achieve uniform distribution of inoculum over the surface of the cell 
monolayers. Place the cell culture test vessels on a level stationary 
surface at room temperature (22-25 deg.C) or at 36.5 plus-minuse> 
1 deg.C so that the inoculum will remain distributed evenly over the 
cell monolayer.
    Step 6. Continue incubating the inoculated cell cultures for 80-120 
minutes to permit viruses to adsorb onto and infect cells.
    It may be necessary to rock the vessels every 15-20 min or to keep 
them on a mechanical rocking platform during the adsorption period to 
prevent cell death in the middle of the vessels from dehydration.
    Step 7. Add liquid maintenance medium and incubate at 36.5 
plus-minuse> 1 deg.C.
    To reduce thermal shock to cells, warm the maintenance medium to 
36.5 plus-minuse> 1 deg.C before placing on the cell monolayer.
    To prevent disturbing cells with the force of liquid against the 
cell monolayer, add the medium to the side of the cell culture vessel 
opposite the cell monolayer. Also, if used, avoid touching either the 
cannula or syringe needle of the pipette or the pipetting device to the 
inside rim of the cell culture vessel to avert the possibility of 
transporting contaminants to the remaining culture vessels.
    Step 8. Examine each culture microscopically for the appearance of 
cytopathic effects (CPE) daily for the first three days and then every 
couple of days for a total of 14 days.
    CPE may be identified as cell disintegration or as changes in cell 
morphology. Rounding-up of infected cells is a typical effect seen with 
enterovirus infections. However, uninfected cells round-up during 
mitosis and a sample should not be considered positive unless there are 
significant clusters of rounded-up cells over and beyond what is 
observed in the uninfected controls. Photomicrographs demonstrating CPE 
appear in the reference by Malherbe and Strickland-Cholmley (1980).
    Step 9. Freeze cultures at -70 deg.C when more than 75% of the 
monolayer shows signs of CPE. Freeze all remaining negative cultures, 
including controls, after 14 days.
    Step 10. In order to confirm the results of the previous passage, 
thaw all the cultures. Filter at least 20% of the medium from each 
vessel through a 0.22 m sterilizing filter. Inoculate another 
BGM culture with a volume that represents 20% of the medium from the 
previous passage for each vessel. Repeat Steps 7 to 8.
    Confirmation passages may be performed in small vessels or 
multiwell trays, however, it may be necessary to distribute the 
inoculum into several vessels or wells to insure that the inoculum 
volume is less than or equal to 0.04 ml/cm\2\ of surface area.
    Step 11. Score cultures that developed CPE in both the first and 
second passages as confirmed positives. Cultures that show CPE in only 
the second passage must be passaged a third time along with the 
negative controls according to Steps 9-10. Score cultures that develop 
CPE in both the second and third passages as confirmed positives.
    Cultures with confirmed CPE may be stored in a -70 deg.C freezer 
for research purposes or for optional identification tests.\3\
---------------------------------------------------------------------------

    \3\For more information see Chapter 12 (May 1988 revision) of 
Berg et al. (1984).
---------------------------------------------------------------------------

Virus Quantitation
    Step 1. Determine the total number of confirmed positive and 
negative cultures and the volume which represents the amount of the 
original final concentrated sample for each dilution inoculated (e.g., 
if vessels are inoculated with 1 ml each of undiluted sample, sample 
diluted 1:5 and sample diluted 1:25, the volumes of the original final 
concentrated sample are 1 ml/vessel for undiluted sample, 0.2 ml/vessel 
for the 1:5 dilution and 0.04 ml/vessel for the 1:25 dilution). Record 
the values on the Virus Data Sheet (Part 8).
    Step 2. Calculate the MPN/ml value and 95% confidence limits using 
a computer program to be supplied by the U.S. Environmental Protection 
Agency. Calculate the MPN/liter value of the original water sample by 
multiplying the MPN/ml value by the total number of milliliters of the 
final concentrated sample (S) inoculated onto cultures and then 
dividing by the volume in liters of the original sample assayed (D). 
Record the value onto the Virus Data Sheet (Part 8).
    MPN values for samples assayed using several sample dilutions can 
be confirmed using the formula from Thomas: MPN/ml = P/(NQ)0.5, 
where P equals the total number of confirmed positive samples for all 
dilutions, N equals the total volume of the original final concentrated 
sample (in ml) inoculated for all dilutions, and Q equals the total 
volume (in ml) of sample inoculated onto cultures that remained CPE 
negative. Calculate the MPN/liter value of the original water sample as 
above. MPN values for the assay of undiluted samples can be confirmed 
with the formula: MPN = -ln (q/n), where q equals the number of CPE 
negative cultures and n equals the total number of cultures. Calculate 
the MPN/liter value of the original water sample by multiplying the MPN 
value by the number of milliliters of the final concentrated sample 
inoculated per culture, multiplying this value by S, and then dividing 
by D.
    Step 3. Calculate the upper and lower 95% confidence limit per 
liter values for each virus sample by multiplying the limit values 
obtained from the computer program by S and dividing by D. Record the 
limit per liter values on the Virus Data Sheet. Finished water must be 
tested for viruses following surface water samples which give a value 
of 1 or more per liter falling anywhere within the range of the 95% 
confidence limits.

Reduction of Cytotoxicity in Sample Concentrates

    The procedure described in this Section may result in a significant 
titer reduction and should be applied only to inocula known to be or 
expected to be toxic.
Media and Reagents
    1. Washing solution.
    a. To a flask containing a stir bar and an appropriate volume of 
dH2O, add NaCl to a final concentration of 0.85% (weight/volume; 
e.g., 0.85 g in 100 ml). Mix the contents of the flask on a magnetic 
stirrer at a speed sufficient to dissolve the salt. Remove the stir bar 
and autoclave the solution at 121 deg.C for 15 min. Cool to room 
temperature.
    The volume of the NaCl washing solution required will depend on the 
number of bottles to be processed and the cell surface area of the 
vessels used for the quantal assay.
    b. Add 2% (volume/volume, e.g., 2 ml per 100 ml) serum to the 
sterile salt solution. Mix thoroughly and store at 4 deg.C.
    Although the washing solution may be stored at 4 deg.C for an 
extended time period, it is advisable to prepare the solution on a 
weekly basis or to store it at -20 deg.C.
Procedure for Cytotoxicity Reduction
    Step 1. Decant and save the inoculum from inoculated cell culture 
vessels after the adsorption period (Step 6 of Sample Inoculation and 
CPE Development). Add 0.25 ml of the washing solution for each cm2 
of cell surface area into each vessel.
    To reduce thermal shock to cells, warm the washing solution to 36.5 
 1 deg.C before placing on cell monolayer.
    To prevent disturbing cells with the force of liquid against the 
cell monolayer, add washing solution to the side of the cell culture 
vessel opposite the cell monolayer. Also, if used, avoid touching 
either the cannula or syringe needle of the pipette or the pipetting 
device to the inside rim of the cell culture vessel to avert the 
possibility of transporting contaminants to the remaining culture 
vessels.
    The inocula saved after the adsorption period should be stored at 
-70 deg.C for subsequent treatment and may be discarded when 
cytotoxicity is successfully reduced.
    Step 2. Rock the washing solution gently across the cell monolayer 
a minimum of two times. Decant and discard the spent washing solution 
in a manner that will not disturb the cell monolayer.
    It may be necessary to gently rock the washing solution across the 
monolayer more than twice if sample is oily and difficult to remove 
from the cell monolayer surface.
    Step 3. Continue with Step 7 of the procedure for Sample 
Inoculation and CPE Development.
    If this procedure fails to reduce cytotoxicity with a particular 
type of water sample, backup samples may be diluted 1:2 to 1:4 before 
repeating the procedure. This dilution requires that two to four times 
more culture vessels be used. Dilution alone may sufficiently reduce 
cytotoxicity of some samples without washing. Alternatively, the 
changing of liquid maintenance medium at the first signs of 
cytotoxicity may prevent further development.
    Determine cytotoxicity from the initial daily macroscopic 
examination of the appearance of the cell culture monolayer by 
comparing the negative and positive controls from Steps 6a and 6b of 
the procedure for Sample Inoculation and CPE Development with the test 
samples from Step 6c). Cytotoxicity should be suspected when the cells 
in the test sample develop CPE prior to its development on the positive 
control.

Part 4--Coliphage Assay of Processed Sample

Plaque Assay Procedure

    This section outlines the procedures for coliphage detection by 
plaque assay. It should be noted that the samples to be analyzed may 
contain pathogenic human enteric viruses. Laboratories performing the 
coliphage analysis are responsible for establishing an adequate safety 
plan and must rigorously follow the guidelines on sterilization and 
aseptic techniques given in Part 5.
Apparatus and Materials
    1. Sterilizing filter--0.45 m (Costar Product No. 140667).
    Always pass about 10 ml of 3% beef extract through the filter just 
prior to use to minimize phage adsorption to the filter.
    2. Water bath set at 44.5  1 deg.C.
    3. Incubator set at 36.5  1 deg.C.
Media and Reagents
    1. Saline-calcium solution--dissolve 8.5 g of NaCl and 0.22 g of 
CaCl2 in a total of 1 liter of dH2O. Dispense in 9 ml 
aliquots in 16 x 150 mm screw-capped test tubes (Baxter Product No. 
T1356-6A) and sterilize by autoclaving at 121 deg.C for 15 min.
    2. Tryptone-yeast extract agar slants--add 1.0 g tryptone (Difco 
Product No. 0123), 0.1 g yeast extract (Difco Product No. 0127), 0.1 g 
glucose, 0.8 g NaCl, 0.022 g CaCl2, and 1.2 g of Bacto-agar (Difco 
Product No. 0140) to a total volume of 100 ml of dH2O in a 250 ml 
flask. Dissolve by autoclaving at 121 deg.C for 20 minutes and dispense 
8 ml aliquots into 16 x 150 mm test tubes with tube closures (Baxter 
Product Nos. T1311-16XX and T1291-16). Prepare slants by allowing the 
agar to solidify with the tubes at about a 20 deg. angle. Slants may be 
stored at 4 deg.C for up to two months.
    3. Tryptone-yeast extract bottom agar--Prepare one day prior to 
sample analysis using the ingredients and concentrations listed for 
tryptone-yeast extract agar slants, except use 1.5 g of Bacto-agar. 
After autoclaving, pipet 15 ml aliquots aseptically into sterile 100 x 
15 mm petri plates and allow the agar to harden. Store the plates at 
4 deg.C overnight or for up to one week in a sealed plastic bag and 
warm to room temperature for one hour before use.
    4. Tryptone-yeast extract top agar--Prepare the day of sample 
analysis using the ingredients and concentrations listed for tryptone-
yeast extract agar slants, except use 0.7 g of Bacto-agar. Autoclave 
and place in the 44.5  1 deg.C water bath.
    5. Tryptone-yeast extract broth--Prepare as for tryptone-yeast 
extract agar slants, except without agar.
Sample Processing
    Step 1. Filter the 40 ml eluate sample from Step 9 of the Elution 
Procedure through a 0.45 m sterilizing filter and store at 
4 deg.C.
    Step 2. Assay ten 1 ml volumes each for somatic and male-specific 
coliphage within 24 hours of elution. Store the remaining eluate at 
4 deg.C. This will serve as a reserve in the event of sample 
contamination or high coliphage densities. If the coliphage density is 
expected or demonstrated to be greater than 100 PFU/ml, dilute the 
original or remaining eluate with a serial 1:10 dilution series into 
saline-calcium solutions. Assay the dilutions which will result in 
plaque counts of 100 or less.
Storage of E. coli C Host Culture for Somatic Coliphage Assay
    1. For short term storage inoculate a Escherichia coli C (American 
Type Culture Collection Product No. 13706) host culture onto tryptone-
yeast extract agar slants with a sterile inoculating loop by spreading 
the inoculum evenly over entire slant surface. Incubate the culture 
overnight at 36.5  1 deg.C. Store at 4 deg.C for up to 2 
weeks.
    2. For long term storage inoculate a 5-10 ml tube of tryptone-yeast 
extract broth with the host culture. Incubate the broth culture 
overnight at 36.5  1 deg.C. Add \1/10\th volume of sterile 
glycerol. Dispense into 1 ml aliquots in cryovials (Baxter Product No. 
T4050-8) and store at -70 deg.C.
Preparation of Host for Somatic Coliphage Assay
    Step 1. Inoculate 5 ml of tryptone-yeast extract broth with E. coli 
C from a slant with an inoculating loop and incubate for 16 hours at 
36.5  1 deg.C.
    Step 2. Transfer 1.5 ml of the 16 hour culture to 30 ml of 
tryptone-yeast extract broth in a 125 ml flask and incubate for 4 hours 
at 36.5  1 deg.C with gentle shaking. The volume of 
inoculum and broth used in this step can be proportionally altered 
according to need.
Preparation of X174 Positive Control
    Step 1. Rehydrate a stock culture of X174 (American Type 
Culture Collection Product No. 13706-B1) and store at 4 deg.C.
    Step 2. Prepare a 30 ml culture of E. coli C as described in 
section titled Preparation of Host for Somatic Coliphage Assay. 
Incubate for 2 hours at 36.5  1 deg.C with shaking. Add 1 
ml of rehydrated phage stock and incubate for an additional 4 hours at 
36.5  1 deg.C.
    Step 3. Filter the culture through a 0.45 m sterilizing 
filter.
    Step 4. Prepare 10-7, 10-8 and 10-9 dilutions of the 
filtrate using saline-calcium solution tubes.
    These dilutions should be sufficient for most X174 
stocks. Some stocks may require higher or lower dilutions.
    Step 5. Add 1 ml of the 10-9 dilution into each of five 16 x 
150 mm test tubes. Using the same pipette, add 1 ml of the 10-8 
dilution into each of five additional tubes and then 1 ml of the 
10-7 dilution into five tubes. Label the tubes with the 
appropriate dilution.
    Step 6. Add 0.1 ml of the host culture into each of the 15 test 
tubes from Step 5.
    Step 7. Add 3 ml of the melted tryptone-yeast extract top agar held 
in the 44.5  1 deg.C water bath to one test tube at a time. 
Mix and immediately pour the contents of the tube over the bottom agar 
of a petri dish labeled with sample identification information. Rotate 
the dish to spread the suspension evenly over the surface of the bottom 
agar and place it onto a level surface to allow the agar to solidify.
    An alternative order of the procedural steps here and in the assay 
procedures described below is to add the top agar to the tubes first, 
then the host culture, followed by the sample.
    Step 8. Incubate the inoculated plates at 36.5  1 deg.C 
overnight and examine for plaques the following day.
    Step 9. Count the number of plaques on each of the 15 plates (don't 
count plates giving plaque counts significantly more than 100). The 
five plates from one of the dilutions should give plaque counts of 
about 20 to 100 plaques. Average the plaque counts on these five plates 
and multiply the result by the reciprocal of the dilution to obtain the 
titer of the undiluted stock.
    Step 10. Dilute the filtrate to 30 to 80 PFU/ml in tryptone-yeast 
extract broth for use in a positive control in the coliphage assay. 
Store the original filtrate and the diluted positive control at 
4 deg.C.
    Before using the positive control for the first time, place 1 ml 
each into ten 16 x 150 mm test tubes and assay using Steps 6-8. Count 
the plaques on all plates and divide by 10. If the result is not 30 to 
80, adjust the dilution of the positive control sample and assay again.
Procedure for Somatic Coliphage Assay
    Step 1. Add 1 ml of the water eluate to be tested to each of ten 16 
x 150 mm test tubes and 1 ml of the diluted X174 positive 
control to another tube.
    Step 2. Add 0.1 ml of the host culture to each test tube containing 
eluate or positive control.
    Step 3. Add 3 ml of the melted tryptone-yeast extract top agar held 
in the 44.5  1 deg.C water bath to one test tube at a time. 
Mix and immediately pour the contents of the tube over the bottom agar 
of a petri dish labeled with sample identification information. Tilt 
and rotate the dish to spread the suspension evenly over the surface of 
the bottom agar and place it onto a level surface to allow the agar to 
solidify.
    Step 4. Incubate the inoculated plates at 36.5  1 deg.C 
overnight and examine for plaques the following day.
    Step 5. Somatic coliphage enumeration.
    a. For each eluate sample count the total number of plaques on the 
ten plates receiving the water eluate and calculate the somatic 
coliphage titer (Vs) in PFU per liter according to the formula: 
Vs = ((P/I) x D x E)/C, where P is the total number of plaques in 
all test vessels for each sample, I is the volume (in ml) of the eluate 
sample assayed, D is the reciprocal of the dilution made on the 
inoculum before plating (D = 1 for undiluted samples), E is the total 
volume of eluate recovered (from the Virus Data Sheet) and C is the 
amount of water sample filtered in liters (from the Sample Data Sheet). 
Record the value of Vs on the Virus Data Sheet.
    b. Count the plaques on the positive control plate. Record the 
plaque count onto the Virus Data Sheet as a check on the virus 
sensitivity of the E. coli C host. Assay any water eluate samples again 
where the positive control counts are more than one log below their 
normal average.
Storage of E. coli C-3000 Host Culture for Male-Specific Coliphage 
Assay:\4\
---------------------------------------------------------------------------

    \4\The term ``male-specific'' refers to bacteriophages whose 
receptor sites are located on the bacterial F-pilus. In addition to 
E. coli C-3000, E. coli Famp and strains of Salmonella which contain 
the F gene are considered suitable as alternative hosts. However, 
all three of these hosts will support the replication of strain-
specific somatic bacteriophages in addition to the male-specific 
types. In addition, genetically modified Salmonella strains have the 
potential to support the replication of phages whose receptor is on 
other types of pili normally produced by Salmonella species.
---------------------------------------------------------------------------

    1. For short term storage inoculate a Escherichia coli C-3000 
(American Type Culture Collection Product No. 15597) host culture onto 
tryptone-yeast extract agar slants with a sterile inoculating loop by 
spreading the inoculum evenly over entire slant surface. Incubate the 
culture overnight at 36.5  1 deg.C. Store at 4 deg.C for up 
to 2 weeks.
    2. For long term storage inoculate a 5-10 ml tube of tryptone-yeast 
extract broth with the host culture. Incubate the broth culture 
overnight at 36.5  1 deg.C. Add \1/10\ volume of sterile 
glycerol. Dispense into 1 ml aliquots in cryovials (Baxter Product No. 
T4050-8) and store at -70 deg.C.
Preparation of Host for Male-Specific Coliphage Assay:
    Step 1. Inoculate 5 ml of tryptone-yeast extract broth with E. coli 
C-3000 from a slant with an inoculating loop and incubate for 16 hours 
at 36.5  1#C.
    Step 2. Transfer 1.5 ml of the 16 hour culture to 30 ml of 
tryptone-yeast extract broth in a 125 ml flask and incubate for 4 hours 
at 36.5  1#C with gentle shaking. The amount of inoculum 
and broth used in this step can be proportionally altered according to 
need.
Preparation of MS2 Positive Control:
    Step 1. Rehydrate a stock culture of MS2 (American Type Culture 
Collection Product No. 15597-B1) and store at 4 deg.C.
    Step 2. Prepare a 30 ml culture of E. coli C-3000 as described in 
section titled Preparation of Host for Male-Specific Coliphage Assay. 
Incubate for 2 hours at 36.5  1 deg.C with shaking. Add 1 
ml of rehydrated phage stock and incubate for an additional 4 hours at 
36.5  1 deg.C.
    Step 3. Filter the culture through a 0.45 m sterilizing 
filter.
    Step 4. Prepare 10-7, 10-8 and 10-9 dilutions of the 
filtrate using saline-calcium solution tubes.
    These dilutions should be sufficient for most MS2 stocks. Some 
stocks may require higher or lower dilutions.
    Step 5. Add 1 ml of the 10-9 dilution into each of five 16 x 
150 mm test tubes. Using the same pipette, add 1 ml of the 10-8 
dilution into each of five additional tubes and then 1 ml of the 
10-7 dilution into five tubes. Label the tubes with the 
appropriate dilution.
    Step 6. Add 0.1 ml of the host culture into each of the 15 test 
tubes from Step 5.
    Step 7. Add 3 ml of the melted tryptone-yeast extract top agar held 
in the 44.5 1 deg.C water bath to one test tube at a time. 
Mix and immediately pour the contents of the tube over the bottom agar 
of a petri dish labeled with sample identification information. Rotate 
the dish to spread the suspension evenly over the surface of the bottom 
agar and place it onto a level surface to allow the agar to solidify.
    Step 8. Incubate the inoculated plates at 36.5 1 deg.C 
overnight and examine for plaques the following day.
    Step 9. Count the number of plaques on each of the 15 plates (don't 
count plates giving plaque counts significantly more than 100). The 
five plates from one of the dilutions should give plaque counts of 
about 20 to 100 plaques. Average the plaque counts on these five plates 
and multiply the result by the reciprocal of the dilution to obtain the 
titer of the undiluted stock.
    Step 10. Dilute the filtrate to 30 to 80 PFU/ml in tryptone-yeast 
extract broth for use in a positive control in the coliphage assay. 
Store the original filtrate and the diluted positive control at 
4 deg.C.
    Before using the positive control for the first time, place 1 ml 
each into ten 16 x 150 mm test tubes and assay using Steps 6-8. Count 
the plaques on all plates and divide by 10. If the result is not 30 to 
80, adjust the dilution of the positive control sample and assay again.
Procedure for Male-Specific Coliphage Assay:
    Step 1. Add 1 ml of the water eluate to be tested to each of ten 16 
x 150 mm test tubes and 1 ml of the diluted MS2 positive control to 
another tube.
    Step 2. Add 0.1 ml of the host culture to each test tube containing 
eluate or positive control.
    Step 3. Add 3 ml of the melted tryptone-yeast extract top agar held 
in the 44.5 1 deg.C water bath to one test tube at a time. 
Mix and immediately pour the contents of the tube over the bottom agar 
of a petri dish labeled with sample identification information. Tilt 
and rotate the dish to spread the suspension evenly over the surface of 
the bottom agar and place it onto a level surface to allow the agar to 
solidify.
    Step 4. Incubate the inoculated plates at 36.5 1 deg.C 
overnight and examine for plaques the following day.
    Step 5. Coliphage enumeration.
    a. For each eluate sample count the total number of plaques on the 
ten plates receiving the water eluate and calculate the male specific 
phage titer (Vm) in PFU per liter according to the formula: 
Vm = ((P/I) x D x E)/C, where P is the total number of plaques in 
all test vessels for each sample, I is the volume (in ml) of the eluate 
sample assayed, D is the reciprocal of the dilution made on the 
inoculum before plating (D = 1 for undiluted samples), E is the total 
volume of eluate recovered (from the Virus Data Sheet) and C is the 
total number of liters of water sample filtered (from the Sample Data 
Sheet). Record this value on the Virus Data Sheet.
    b. Count the plaques on the positive control plate. Record the 
plaque count onto the Virus Data Sheet as a check on the virus 
sensitivity of the E. coli C-3000 host. Assay any water eluate samples 
again where the positive control counts are more than one log below 
their normal average.

Part 5--Sterilization and Disinfection

General Guidelines

    1. Use aseptic techniques for handling test waters, eluates and 
cell cultures.
    2. Sterilize apparatus and containers that will come into contact 
with test waters, all solutions that will be added to test waters 
unless otherwise indicated, and all eluants.
    3. Sterilize all contaminated materials before discarding.
    4. Disinfect all spills and splatters.

Sterilization Techniques

Solutions:
    1. Sterilize all solutions, except those used for cleansing, 
standard buffers, hydrochloric acid (HCl), sodium hydroxide (NaOH), and 
disinfectants by autoclaving them at 121 deg.C for 15 minutes.
    The HCl and NaOH solutions and disinfectants used are self-
sterilizing. When autoclaving buffered beef extract, use a vessel large 
enough to accommodate foaming.
Autoclavable Glassware, Plasticware, and Equipment:
    Water speeds the transfer of heat in larger vessels during 
autoclaving and thereby speeds the sterilization process. Add dH2O 
to vessels in quantities indicated in Table 1. Lay large vessels on 
sides in autoclave, if possible, to facilitate displacement of air in 
vessels by flowing steam.
    1. Cover the openings into autoclavable glassware, plasticware, and 
equipment loosely with aluminum foil before autoclaving. Autoclave at 
121 deg.C for one hour.
    Glassware may also be sterilized in a dry heat oven at a 
temperature of 170 deg.C for at least one hour.
    2. Sterilize stainless steel vessels (dispensing pressure vessel) 
in an autoclave at 121 deg.C for 30 minutes.
    Vent-relief valves on vessels so equipped must be open during 
autoclaving and closed immediately when vessels are removed from 
autoclave.
    3. Presterilize 1MDS filter cartridges and prefilter cartridges by 
wrapping the filters in Kraft paper and autoclaving at 121 deg.C for 30 
minutes.
    4. Sterilize working instruments, such as scissors and forceps, by 
immersing them in 95% ethanol and flaming them between uses.
    Table 1. Quantity of Water to be Added to Vessels during 
Autoclaving. 

------------------------------------------------------------------------
                                                             Quantity of
                    Vessel size (liter)                       dH2O (ml) 
------------------------------------------------------------------------
2 and 3....................................................           25
4..........................................................           50
8..........................................................          100
24.........................................................          500
54.........................................................        1000 
------------------------------------------------------------------------

Chlorine Sterilization:
    Sterilize plasticware (filter housings) and tubing that cannot 
withstand autoclaving or vessels that are too large for the autoclave 
by chlorination. Prefilters, but not 1MDS filters may be presterilized 
with chlorine as an alternative to autoclaving.
    1. Media and Reagents
    a. 0.1% chlorine (HOCl)--add 19 ml of household bleach (Clorox, The 
Clorox Co., or equivalent) to 981 ml of dH2O and adjust the pH of 
the solution to 6-7 with 1 M HCl.
    2. Procedures
    Ensure that the solutions come in full contact with all surfaces 
when performing these procedures.
    a. Sterilize the filter apparatus and tubing by recirculating or 
immersing in 0.1% chlorine for 30 minutes. Drain the chlorine solution 
from objects being sterilized. Dechlorinate using a solution containing 
0.5 ml of 10% sterile sodium thiosulfate per liter of dH2O. Rinse 
with sterile dH2O.
    b. Sterilize pH electrodes before and after each use by immersing 
the tip of the electrode in 0.1% chlorine for one min. Dechlorinate and 
rinse the electrode as in Step 2a above.

Procedure for Verifying Sterility of Liquids

    Do not add antibiotics to media or medium components until after 
sterility of the antibiotics, media and medium components has been 
demonstrated. The BGM cell line used should be checked every six months 
for mycoplasma contamination according to test kit instructions. Cells 
that are contaminated should be discarded.
Media and Reagents:
    1. Mycoplasma testing kit (Irvine Scientific Product No. T500-000). 
Use as directed by the manufacturer.
    2. Thioglycollate medium (Difco Laboratories Product No. 0257-01-
9). Prepare broth medium as directed by the manufacturer.
Verifying Sterility of Small Volumes of Liquids:
    Step 1. Inoculate 5 ml of the material to be tested for sterility 
into 5 ml of thioglycollate broth. Shake the mixture and incubate at 
36.5 plus-minuse> 1 deg.C.
    Step 2. Examine the inoculated broth daily for seven days to 
determine whether growth of contaminating organisms has occurred.
    Containers holding the thioglycollate medium must be tightly sealed 
before and after the medium is inoculated.
Visual Evaluation of Media for Microbial Contaminants:
    Step 1. Incubate either the entire stock of prepared media or 
aliquots taken during preparation which represent at least 5% of the 
final volume at 36.5 plus-minuse> 1 deg.C for at least one week 
prior to use.
    Step 2. Visually examine and discard any media that lose clarity.
    A clouded condition that develops in the media indicates the 
occurrence of contaminating organisms.

Contaminated Materials

    1. Autoclave contaminated materials for 30 minutes at 121 deg.C. Be 
sure that steam can enter contaminated materials freely.
    2. Many commercial disinfectants do not adequately kill enteric 
viruses. To ensure thorough disinfection, disinfect spills and other 
contamination on surfaces with either a solution of 0.5% iodine in 70% 
ethanol (5 g I2 per liter) or 0.1% chlorine. The iodine solution 
has the advantage of drying more rapidly on surfaces than chlorine, but 
may stain some surfaces.

Part 6--Biblography and Suggested Reading

Adams, M.H. 1959. Bacteriophages. John Wiley and Sons, Inc. New York.
ASTM. 1992. Standard Methods for the Examination of Water and 
Wastewater (A. E. Greenberg, L. S. Clesceri and A. D. Eaton, ed), 18th 
Edition. American Public Health Association, Washington, D.C.
Barron, A. L., C. Olshevsky and M. M. Cohen. 1970. Characteristics of 
the BGM line of cells from African green monkey kidney. Archiv. Gesam. 
Virusforsch. 32:389-392.
Berg, G., R. S. Safferman, D. R. Dahling, D. Berman and C. J. Hurst. 
1984. USEPA Manual of Methods for Virology. U.S. Environmental 
Protection Agency Publication No. EPA/600/4-84/013, Cincinnati, OH.
Chang, S. L., G. Berg, K. A. Busch, R. E. Stevenson, N. A. Clarke and 
P. W. Kabler. 1958. Application of the ``most probable number'' method 
for estimating concentration of animal viruses by the tissue culture 
technique. Virology 6:27-42.
Crow, E. L. 1956. Confidence intervals for a proportion. Biometrika. 
43:423-435.
Dahling, D. R. and B. A. Wright. 1986. Optimization of the BGM cell 
line culture and viral assay procedures for monitoring viruses in the 
environment. Appl. Environ. Microbiol. 51:790-812.
Dahling, D. R. and B. A. Wright. 1987. Comparison of the in-line 
injector and fluid proportioner used to condition water samples for 
virus monitoring. J. Virol. Meth. 18:67-71.
Dahling, D. R., G. Berg and D. Berman. 1974. BGM, a continuous cell 
line more sensitive than primary rhesus and African green kidney cells 
for the recovery of viruses from water. Health Lab. Sci. 11:275-282.
Dahling, D. R., R. S. Safferman and B. A. Wright. 1984. Results of a 
survey of BGM cell culture practices. Environ. Internat. 10:309-313.
Debartolomeis, J. and V. J. Cabelli. 1991. Evaluation of an Escherichia 
coli host strain for enumeration of F male-specific bacteriophages. 
Appl. Environ. Microbiol. 57:1301-1305.
Dutka, B. J., A. El Shaarawi, M. T. Martins and P. S. Sanchez. 1987. 
North and South American studies on the potential of coliphage as a 
water quality indicator. Water Res. 21:1127-1134.
Eagle, H. 1959. Amino acid metabolism in mammalian cell cultures. 
Science. 130:432-437.
EPA. 1989. Guidance manual for compliance with the filtration and 
disinfection requirements for public water systems using surface water 
sources. Office of Drinking Water, Washington, D.C.
Freshney, R. I. 1983. Culture of Animal Cells: A Manual of Basic 
Technique. Alan R. Liss, New York, NY.
Havelaar, A. H. and W. M. Hogeboom. 1984. A method for the enumeration 
of male-specific bacteriophages in sewage. J. Appl. Bacteriol. 56:439-
447.
Hay, R. J. 1985. ATCC Quality Control Methods for Cell Lines. American 
Type Culture Collection, Rockville, MD.
Hurst, C. J. 1990. Field method for concentrating viruses from water 
samples, pp. 285-295. In G. F. Craun (ed.), Methods for the 
Investigation and Prevention of Waterborne Disease Outbreaks. U.S. 
Environmental Protection Agency Publication No. EPA/600/1-90/005a, 
Washington, D.C.
Hurst, C. J. 1991. Presence of enteric viruses in freshwater and their 
removal by the conventional drinking water treatment process. Bull. 
W.H.O. 69:113-119.
Hurst, C. J. and T. Goyke. 1983. Reduction of interfering cytotoxicity 
associated with wastewater sludge concentrates assayed for indigenous 
enteric viruses. Appl. Environ. Microbiol. 46:133-139.
Katzenelson, E., B. Fattal and T. Hostovesky. 1976. Organic 
flocculation: an efficient second-step concentration method for the 
detection of viruses in tap water. Appl. Environ. Microbiol. 32:638-
639.
Laboratory Manual in Virology. 1974. 2nd Ed. Ontario Ministry of 
Health, Toronto, Ontario, Canada.
Leibovitz, A. 1963. The growth and maintenance of tissue-cell cultures 
in free gas exchange with the atmosphere. Amer. J. Hyg. 78:173-180.
Lennette, E. H. and N. J. Schmidt (ed.). 1979. Diagnostic Procedures 
for Viral, Rickettsial and Chlamydial Infections, 5th ed. American 
Public Health Association, Washington, D.C.
Malherbe, H. H. and M. Strickland-Cholmley. 1980. Viral Cytopathology. 
CRC Press. Boca Raton, FL.
Morris, R. and W. M. Waite. 1980. Evaluation of procedures for recovery 
of viruses from water--II detection systems. Water Res. 14:795-798.
O'Keefe, B. and J. Green. 1989. Coliphages as indicators of faecal 
pollution at three recreational beaches on the Firth of Forth. Water 
Res. 23:1027-1030.
Paul, J. 1975. Cell and Tissue Culture. 5th Ed. Churchill Livingstone, 
London, Great Britain.
Palmateer, G. A., B. J. Dutka, E. M. Janzen, S. M. Meissner and M. G. 
Sakellaris. 1991. Coliphage and bacteriophage as indicators of 
recreational water quality. Water Res. 25:355-357.
Payment, P. and M. Trudel. 1985. Influence of inoculum size, incubation 
temperature, and cell culture density on virus detection in 
environmental samples. Can. J. Microbiol. 31:977-980.
Ratto, A., B. J. Dutka, C. Vega, C. Lopez and A. El Shaarawi. 1989. 
Potable water safety assessed by coliphage and bacterial tests. Water 
Res. 23:253-255.
Rovozzo, G. C. and C. N. Burke. 1973. A Manual of Basic Virological 
Techniques. Prentice-Hall, Englewood Cliffs, NJ.
Simkova, A. and J. Cervenka. 1981. Coliphages as ecological indicators 
of enteroviruses in various water systems. Bull. W.H.O. 59:611-618.
Sobsey, M. D. 1976. Field monitoring techniques and data analysis, pp. 
87-96. In L. B. Baldwin, J. M. Davidson and J. F. Gerber (eds.), Virus 
Aspects of Applying Municipal Waste to Land. University of Florida, 
Gainesville, FL.
Stetler, R.E. 1984. Coliphages as indicators of enteroviruses. Appl. 
Environ. Microbiol. 48:668-670.
Thomas, H. A., Jr. 1942. Bacterial densities from fermentation tube 
tests. J. Amer. Water Works Assoc. 34:572-576.
Waymouth, C., R. G. Ham and P. J. Chapple. 1981. The Growth 
Requirements of Vertebrate Cells In Vitro. Cambridge University Press, 
Cambridge, Great Britain.

Part 7--Vendors

    The vendors listed below represents one possible source for 
required products. Other vendors may supply the same or equivalent 
products.

American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD 
20852, (800) 638-6597
Baxter Diagnostics, Scientific Products Div., 1430 Waukegan Rd., McGaw 
Park, IL 60085, (800) 234-5227
Becton Dickonson Microbiology Systems, 250 Schilling Circle, 
Cockeysville, MD 21030, (410) 771-0100 (Ask for a local distributor)
Cole-Parmer Instrument Co., 7425 N. Oak Park Ave., Niles, IL 60714, 
(800) 323-4340,
Costar Corp., 7035 Commerce Circle, Pleasanton, CA 94588, (800) 882-
7711
Cuno, Inc., 400 Research Parkway, Meriden, CT 06450, (800) 243-6894
DEMA Engineering Co., 10014 Big Bend Blvd., Kirkwood, MO 63122, (800) 
325-3362
Difco Laboratories, P.O. Box 331058, Detroit, MI 48232, (800) 521-0851 
(Ask for a local distributor)
Fisher Scientific, 711 Forbes Ave., Pittsburgh, PA 15219, (800) 766-
7000
Millipore Corp., 397 Williams St., Marlboro, MA 01752, (800) 225-1380
Nalge Co., P.O. Box 20365, Rochester, NY 14602, (716) 586-8800 (Ask for 
a local distributor)
Neptune Equipment Co., 520 W. Sharon Rd., Forest Park, OH 45240, (800) 
624-6975
OMEGA Engineering, Inc., P.O. Box 4047, Stamford, CT 06907, (800) 826-
6342
Parker Hannifin Corp., Commercial Filters Div., 1515 W. South St., 
Lebanon, IN 46052, (317) 482-3900
Ryan Herco, 2509 N. Naomi St., Burbank, CA 91504, (800) 848-1141
United States Plastic Corp., 1390 Neubrecht Rd., Lima, OH 45801, (800) 
537-9724
Watts Regulator, Box 628, Lawrence, MA 01845 , (508) 688-1811

Part 8--Data Sheets

Sample Data Sheet

Sample Number:---------------------------------------------------------
Water System Name:-----------------------------------------------------
System Location:-------------------------------------------------------
Sampler's Name:--------------------------------------------------------
Water pH: ______ Water Temperature: ______  deg.C
Initial Meter Reading: ______ (check units) ______ ft3 ______ 
gallons
Date: ______ Time: ______
Final Meter Reading: ______ (check units) ______ ft3 ______ 
gallons
Date: ______ Time: ______
Total sample volume: ______ liters
(Final--Initial meter readings x 28.316 (for readings in ft3 or 
x 7.481 (for readings in gallons))
Sample arrival condition:----------------------------------------------
Comments:

Virus Data Sheet

Sample Number:---------------------------------------------------------
Water System Name:-----------------------------------------------------
System Location:-------------------------------------------------------
Date Sample collected:-------------------------------------------------
Eluate volume recovered:-----------------------------------------------
Date eluted:-----------------------------------------------------------
Date concentrated:-----------------------------------------------------
Final concentrated sample volume: ______ ml
Date(s) assayed by CPE:------------------------------------------------
Original water sample volume assayed: ______ Liters
Coliphage Quantitation:
Date Assayed:----------------------------------------------------------
Somatic Coliphage Titer:-----------------------------------------------
No. Control plaques PFU/l----------------------------------------------
Male-Specific phage titer:---------------------------------------------
PFU/l------------------------------------------------------------------
Comments:

                                                           Total Culturable Virus Quantitation                                                          
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                     No. of     Volume on      No. of     Volume on                95% confidence limits
                       Sample                         Total No.     negative     negative     positive     positive     MPN/la   -----------------------
                                                       cultures     cultures     cultures     cultures     cultures                  Upper       Lower  
--------------------------------------------------------------------------------------------------------------------------------------------------------
1st Passage:                                                                                                                                            
    Negative Control...............................  ...........  ...........  ...........  ...........  ...........  NAb         NA          NA        
    Positive Control...............................  ...........  ...........  ...........  ...........  ...........  NA          NA          NA        
    Undiluted......................................  ...........  ...........  ...........  ...........  ...........  NA          NA          NA        
    1:5 Dilution...................................  ...........  ...........  ...........  ...........  ...........  NA          NA          NA        
    1:25 Dilution..................................  ...........  ...........  ...........  ...........  ...........  NA          NA          NA        
2nd Passage:c                                                                                                                                           
    Negative Control...............................  ...........  ...........  ...........  ...........  ...........  NA          NA          NA        
    Positive Control...............................  ...........  ...........  ...........  ...........  ...........  NA          NA          NA        
    Undiluted......................................  ...........  ...........  ...........  ...........  ...........  ..........  ..........  ..........
    1:5 Dilution...................................  ...........  ...........  ...........  ...........  ...........  ..........  ..........  ..........
    1:25 Dilution..................................  ...........  ...........  ...........  ...........  ...........  ..........  ..........  ..........
3rd Passage:d                                                                                                                                           
    Negative Control...............................  ...........  ...........  ...........  ...........  ...........  NA          NA          NA        
    Positive Control...............................  ...........  ...........  ...........  ...........  ...........  NA          NA          NA        
    Undiluted......................................  ...........  ...........  ...........  ...........  ...........  ..........  ..........  ..........
    1:5 Dilution...................................  ...........  ...........  ...........  ...........  ...........  ..........  ..........  ..........
    1:25 Dilution..................................  ...........  ...........  ...........  ...........  ...........  ..........  ..........  ..........
--------------------------------------------------------------------------------------------------------------------------------------------------------
aCompute MPN of confirmed samples only according to the Virus Quantitation Section of Part 3.                                                           
bNot applicable.                                                                                                                                        
cA portion of medium from each 1st passage vessel, including controls, must be repassaged for conformation. The terms ``Undiluted,'' ``1:5 Dilution''   
  and ``1:25 Dilution'' under the 2nd and 3rd Passage headings refer to the original sample dilutions for the 1st passage.                              
dSamples that were negative on the first passage and positive on the 2nd passage must be passaged a third time for conformation. If a third passage is  
  required, all controls must be passaged again.                                                                                                        

BILLING CODE 6560-50-P

TP10FE94.003


TP10FE94.004


TP10FE94.005


TP10FE94.006


[FR Doc. 94-2587 Filed 2-9-94; 8:45 am]
BILLING CODE 6560-50-C