 	United States

Environmental Protection Agency	Office of Chemical Safety

and Pollution Prevention

(7101)	EPA 730-C-11-003

June 19, 2012



	

Product Performance

Test Guidelines





OCSPP 810.2600:

	Disinfectants and Sanitizers for Use in Water – Efficacy Data
Recommendations 





NOTICE

This guideline is one of a series of test guidelines established by the
United States Environmental Protection Agency’s Office of Chemical
Safety and Pollution Prevention (OCSPP) for use in testing pesticides
and chemical substances to develop data for submission to the Agency
under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.),
the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C.
136, et seq.), and section 408 of the Federal Food, Drug and Cosmetic
Act (FFDCA) (21 U.S.C. 346a).  Prior to April 22, 2010, OCSPP was known
as the Office of Prevention, Pesticides and Toxic Substances (OPPTS). 
To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically
included OPPTS as part of the guideline’s number.  Any test guidelines
developed after April 22, 2010 will use the new acronym (OCSPP) in their
title.

	 The OCSPP test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform
regulatory decisions under TSCA, FIFRA and/or FFDCA.  This document
provides guidance for conducting the test, and is also used by EPA, the
public and the companies that are subject to data submission
requirements under TSCA, FIFRA and/or the FFDCA.  As a guidance
document, these guidelines are not binding on either EPA or any outside
parties, and the EPA may depart from the guidelines where circumstances
warrant and without prior notice.  At places in this guidance, the
Agency uses the word “should.”  In this guidance, the use of
“should” with regard to an action means that the action is
recommended rather than mandatory.  The procedures contained in this
guideline are strongly recommended for generating the data that are the
subject of the guideline, but EPA recognizes that departures may be
appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will
assess them for appropriateness on a case-by-case basis.  

	For additional information about these test guidelines and to access
these guidelines electronically, please go to http://www.epa.gov/ocspp
and select “Test Methods & Guidelines” on the left side navigation
menu.  You may also access the guidelines in   HYPERLINK
"http://www.regulations.gov"  http://www.regulations.gov  grouped by
Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through
EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.



OCSPP 810.2600:  Disinfectants and sanitizers for treating water –
efficacy data recommendations. 

	(a)	Scope.—

	(1) Applicability.  This guideline describes test methods that EPA
believes will generally satisfy testing requirements of the Federal
Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.) and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C.
346a).  It addresses testing to demonstrate the effectiveness of
antimicrobial pesticides bearing label claims for the disinfection or
sanitization of water.

(2) Background.  The source materials used in developing this OCSPP test
guideline are OPP guidelines 91-8 Products for treating water systems
and 91-30 Acceptable methods (Pesticide Assessment Guidelines,
Subdivision G, Product Performance, EPA report 540/9-82-026, October
1982).

	   (b) Purpose.  This guideline addresses efficacy testing for
antimicrobial pesticides intended to be used in water systems, namely
drinking water and swimming pools, as disinfectants or sanitizers. 

	  (c) General considerations. –(1)  This guideline recommends methods
for use in tests to be conducted to address the data requirements for
pesticide registration.  Good Laboratory Practice Standards (GLP) as
defined in 40 CFR Part 160 apply to studies of disinfectants for water.
According to 40 CFR §160.17: “EPA may refuse to consider reliable for
purposes of supporting an application for a research or marketing permit
any data from a study which was not conducted in accordance with this
part.” 40 CFR §160.12(b) requires with any submitted research data
“[a] A statement that the study was conducted in accordance with this
part; [b] A statement describing in detail all differences between the
practices used in the study and those required by this part; or [c] A
statement that the person was not a sponsor of the study, did not
conduct the study, and does not know whether the study was conducted in
accordance with this part.” Note:  The Association of Official
Analytical Chemists (AOAC) recommended tests are designed to be
conducted as written. For deviations (e.g., cultures grown with shaking
instead of static, dilution of culture prior to drying on carriers)
proposed to be used in the conduct of these tests, obtain written
approval from the Agency and document such deviations in the study
reports submitted to the Agency. The Agency may consult with the AOAC
prior to accepting modifications to their standardized methods. Refer to
OCSPP Test Guideline 810.2000 for general testing recommendations prior
to initiating tests.

		(d)  Drinking water for humans—(1) Public water systems. Applicants
should contact EPA’s Antimicrobials Division prior to conducting
studies to support label claims for disinfection of public water
systems. Evidence should be submitted that the chemical intended for use
as a drinking water disinfectant is in compliance with the EPA’s
Office of Drinking Water’s regulations.

		(2)  Emergency water supplies. This section addresses efficacy testing
for additives such as solutions, powders, or tablets intended for
emergency disinfection of small quantities of drinking water of
questionable potability (e.g., water obtained from lakes, streams, etc),
by the general public, in the absence of bacteriological monitoring
facilities.

	(i) Test procedure.  The Agency recommends the use of The Guide
Standard and Protocol for Testing Microbiological Water Purifiers (Ref.
1).  Three samples of product, representing three different batches, one
of which should be at least 60 days old, should be tested in triplicate.
 The test organisms should be Raoultella terrigena – American Type
Culture Collection (ATCC) 33257 (R. terrigena) (formerly designated as
Klebsiella terrigena), Polio 1 (LSa) (ATCC VR-59), or Chat (ATCC
VR-1562) Rotavirus (Wa or SA-11) (ATCC VR-2018 or VR-1565),
Cryptosporidium parvum (C. parvum) and Giardia intestinalis (G.
intestinalis) (formerly Giardia lamblia). Giardia muris may be tested in
place of Giardia intestinalis.

	(ii) Evaluation of success—(A)  Bacteria. A 6-log10 (99.9999 percent)
reduction of an influent challenge of 1 x107 organisms per 100 mL of the
bacterial challenge organism R. terrigena should be demonstrated by the
testing.

(B)  Viruses.  A 4-log10 (99.99 percent) reduction of a mixed influent
challenge of 1 x 107 organisms per liter of each of the following
viruses: Poliovirus (LSa) and Rotavirus (Wa) should be demonstrated by
the testing. Virus types should be mixed in roughly equal 1 x 107/L
concentrations and a joint 4-log10 reduction should be demonstrated. MS2
coliphage virus may also be used as a surrogate for Poliovirus and
Rotavirus.

 

		(C)  Cysts (Protozoan).  A 3-log10 (99.9 percent) reduction of an
influent challenge of 1 x 106 organisms per liter of C. parvum and G.
intestinalis (or G. muris if tested) or a 3-log10 reduction of an
influent challenge of 1 x 107 particles or spheres of 4 to 6 um diameter
(as an option for units or components based on occlusion filtration,
where disinfection or other active processes are not involved) should be
demonstrated by the testing.

	

		(3) Water purifier units.  This section addresses efficacy testing for
products bearing label claims for microbiological water purification
(removal of pathogenic microorganisms).  This section does not apply to
products only used to treat water in order to achieve specific chemical
removal from water or other non-microbiological claims.  National
Sanitation Foundation (NSF) Standard 42 (Aesthetic Effects) provides
partial guidance for chemical removal.

		(i)  Test procedure.  The Agency recommends the use of The Guide
Standard and Protocol for Testing Microbiological Water Purifiers (Ref.
1).  This is a general guide and in some cases may present only the
minimum features and framework for the testing of microbiological water
purifiers. Users of the Guide Standard and Protocol should include
pre-testing of their units in a testing rig, including the sampling
techniques to be used. In some cases, it may be appropriate to alter
and/or add to the guide in order to meet specific operational problems,
to use an alternative organism or laboratory procedure, or to respond to
innovative treatment units. However, these alterations and/or additions
should not decrease the level of testing or alter the intent of the
protocol and should be submitted to the Antimicrobials Division for
approval prior to initiation of testing. 

		(ii)  Evaluation of microbiological water purifier success—(A) 
Bacteria.  A 6-log10 (99.9999 percent) reduction of an influent
challenge of 1 x 107 organisms per 100 mL of the bacterial challenge
organism Raoultella terrigena (ATCC) 33257) should be demonstrated by
the testing.

	(B)  Viruses.  A 4-log10 (99.99 percent) reduction of a mixed influent
challenge of 1 x 107 organisms per liter of each of the following
viruses: Poliovirus (LSa) (ATCC-VR-59) or Chat (ATCC 1562 and Rotavirus
(Wa) (ATCC-VR-2018) or SA11 (ATCC 1565) should be demonstrated by the
testing. Virus types should be mixed in roughly equal 1 x 107/L
concentrations and a joint 4-log10 reduction should be demonstrated. MS2
coliphage virus may also be used as a surrogate for Poliovirus and
Rotavirus.

	(C)   Cysts (Protozoan).  A 3-log10 (99.9 percent) reduction of an
influent challenge of 1 x 106 organisms per liter of Cryptosporidium
parvum and Giardia intestinalis (formerly Giardia lamblia) or Giardia
muris (if tested) or a 3-log10 reduction of an influent challenge of 1 x
107 particles or spheres of 4 to 6 um diameter (as an option for units
or components based on occlusion filtration, where disinfection or other
active processes are not involved) should be demonstrated by the
testing. Note: When using spheres as surrogates for oocysts, the charge
of the surrogate particle, the shape of the particle, the size range of
the particle, as well as the actual method of sample collection, are
critical to the accurate and conservative evaluation of the reduction
efficiency of size occlusion technologies.

	(D) Other Optional Protocols. The Agency will consider protocols
developed by the National Sanitation Foundation (NSF) International to
support microbiological water purifier label claims. Specifically, NSF
53, NSF 55, NSF 58 and NSF 62 may be used where appropriate. NSF
protocols P231 and P248 may also be used to generate data to support
label claims as microbiological water purifiers. A pre-registration
consultation is recommended prior to the use of any of these NSF
standards to ensure the data will support the label claims.

	(e)  Swimming pool, spa, and hot tub water. This section addresses
efficacy testing for disinfectants bearing label claims for use in
swimming pools, spas, and hot tubs. Numerous factors influence the
concentrations necessary for disinfection of swimming pool water in
practical applications: the number of swimmers/bathers in the pool;
frequency of use; frequency with which water is changed; general weather
conditions; and types and degree of organic contamination of the water
by the swimmers/bathers themselves (e.g., suntan lotion and oils) and by
various debris.  Therefore, a two-phased study, consisting of a
laboratory test and confirmatory field test, is recommended. 

(1)  Test procedure - Laboratory test.  The Agency recommends the use of
the AOAC International Disinfectants (Water) for Swimming Pools (Ref.
2).  The test organisms should be Escherichia coli (E.coli) (ATCC 11229)
and Enterococcus faecium (E. faecium) (ATCC 6569) (formerly designated
as Streptococcus faecalis).

(i)  Evaluation of success.  The lowest concentration of the test
substance providing results equivalent to those of the sodium
hypochlorite reference standard is the lowest concentration of the
product that should be considered effective.

	(2)  Test procedure - Field test. The Agency recommends efficacy data
to be derived from in-use tests under an experimental use permit (EUP)
in at least two swimming pools, using the following components.

	(i)  Bacteriologic monitoring.  Bacteriologic monitoring of swimming
pool water following treatment with disinfectant product during the
field test should be conducted in the following manner: Water samples
for bacteriologic monitoring should be taken on opposite sides of the
pool in the shallow area and as remote as possible from the inlets,
preferably at the midpoint between the inlets.  A minimum of 144 samples
(72 samples per pool) should be collected during the test period.
Samples should be taken just below the surface of the water and
preferably at such times when the number of persons using the pool
during the preceding hour has been at least equal to 50 percent of the
maximum bather load of the pool and the number of persons in the pool
water at the time the samples are collected is at least 25 percent of
the maximum bather load (as defined in Suggested Ordinance and
Regulations Covering Public Swimming Pools, APHA Joint Committee on
Swimming Pools and Bathing Places) of the pool (Ref. 3).  Pertinent
chemical characteristics (e.g., pH, nitrogenous substances, metals, and
water hardness) of the pool water at the sampling site should be
determined at the time of sampling. (Ref. 4)

	(ii)  Field test.  Field tests should be conducted for an entire
swimming season (4 to 12 months).  Reports should include (but are not
limited to) the following information.

	(A)  The daily swimmer/bather load.

	(B)  The design of the pool, the recirculation and filter system, and
water capacity.

	(C)  The amount and identification of all chemicals added daily to the
swimming pool water (including the time, site, and method).

	(D)  The range of chemical characteristics of the swimming pool water
such as:  pH, nitrogenous substances, metals, and hardness.

	(E) The physical characteristics of the swimming pool water, including
temperature and clarity, determined at least daily.

	(F)  Meteorological data, including air temperature, rainfall, and
number of hours of sunlight (determined daily) for outdoor pools.

	(G)  Bacteriologic monitoring, conducted daily, in accordance with the
suggested Ordinance and Regulations Covering Public Swimming Pools of
the American Public Health Association. (Ref. 3)

	(H) The concentration of the antimicrobial agent in the swimming pool
monitored daily at the same time intervals that the bacteriological
assay samples are obtained.

	(I) The method that the product user will employ for monitoring the
level (concentration in ppm) of antimicrobial agent contained in the
pool water.

	(iii)  Evaluation of field testing success.  The product, when used as
recommended in swimming pool water, should demonstrate that not more
than 15 percent of the samples collected fail to meet the following
bacterial indices (Ref. 3):

(A) The standard plate count at 35°C not to exceed 200 colonies/1.0 mL.

(B) The most probable number of coliform bacteria to be less than 2.2
organisms/100.0 mL.  When the membrane filter test is used, no more than
1.0 coliform organism/50 mL.

		(C)  The most probable number of enterococcal organisms to be less
than 2.2 organisms/100 mL.  When the membrane filter test is used, no
more than 1.0 enterococcal organism/50 mL.

	(3) Optional Test Method. The Agency recommends use of NSF/ANSI
Standard 50. The test organisms should be Enterococcus faecium (ATCC
6569 and Pseudomonas aeruginosa (ATCC 27313).

	(i)  Evaluation of success. The product, when used as directed in
swimming pool water should demonstrate a 3-log kill of the two target
microorganisms, Enterococcus faecium and Pseudomonas aeruginosa.

		(f) Data collection and reporting—(1) General.  To assist in the
proper review and evaluation of product performance, complete
descriptions of the test employed and the results obtained should be
submitted to the Agency.  All test reports should include, at the least,
the following information:

		(i) Study title;

		(ii)  Product Identity;

		(iii)  Guideline number/Data Requirement; 

		(iv)  Identification of the testing laboratory or organization;

		(v)  Location where the test was performed;

		(vi)  Name(s) of the person(s) responsible for the test;

		(vii)  Statement of Confidentiality Claims;

		(viii)  Statement of 40 CFR Part 160 Good Laboratory Practice
compliance and Quality Assurance Statement;

		(ix)  Purpose of the study;

		(x)  Date and time of the start and end of the test;

		(xi)  Test employed and any modifications (e.g., organic soil, hard
water, etc), when using standard tests (e.g., AOAC, ASTM, etc) all
deviations to the test methods should be reported;

		(xii)  Test microorganisms employed, including identification of the
specific strain (ATCC or other);

		(xiii)  Description of the test substance, including the percent of
active ingredient;

		(xiv)  Concentration or dilution of the product tested and how
prepared;

		(xv)  Number of samples, batches and replicates tested;

		(xvi)  Manufacture date of each product batch; 

		(xvii)  Identification of all material or procedural options employed,
where such choice is provided for or recommended in the test method
selected (e.g., growth media, drying time for inoculated carriers,
neutralization confirmation and/or subculture media, secondary
subculturing);

		(xviii)  Test exposure conditions (e.g., contact time, temperature,
and relative humidity);

		(xix)  Complete reports of results obtained for each replication;

		(xx)  Any control data essential to establish the validity of the
test;

		(xxi)  Carrier counts;

		(xxii)  Statistical treatment of the data;

		(xxiii)  Conclusions;

		(xxiv)  References;

		(xxv)  Appendices, including study protocol and all raw data reports
associated with the conduct of the study;

The applicant is encouraged to use the EPA’s standard efficacy report
format, which may be found at  HYPERLINK
"http://www.epa.gov/oppad001/efficacystudystandards.htm"
http://www.epa.gov/oppad001/efficacystudystandards.htm .

		(2)  Data for modifications of recommended methods.  When recommended
methods are modified for testing conducted to support specific claims
and/or use patterns for a product, the protocol, identifying and
describing each modification, should be provided with the study report. 
The applicant is encouraged to submit the proposed modification to the
Agency for review and evaluation prior to initiation of the test.

		(3)  Data for other methods.  When recommended methods, or
modifications thereto, are not employed to develop efficacy data (such
as actual in-use or many kinds of simulated-use testing), complete
testing protocols should be submitted with the test reports.  All
materials and procedures employed in testing should be described in a
manner consistent with original research reports published in technical
or scientific journals.  Where references to published reports or papers
are made, copies or reprints of such references should be provided with
the test reports.  The applicant should submit the proposed testing
protocols for in-use or simulated-use studies (with a proposed label to
show the claims to be supported by the protocol) to the Agency for
review and evaluation prior to initiation of the test.

		(g)  References.  The references in this paragraph may be consulted
for additional background information.		

			(1)	Guide Standard and Protocol for Testing Microbiological Water
Purifiers.  United States Environmental Protection Agency, Registration
Division, Office of Pesticide Programs and Criteria and Standards
Division, Office of Drinking Water, April 1987.

			(2) Official Methods of Analysis of the Association of Official
Analytical Chemists, Use-Dilution Methods, Chapter 6, Disinfectants;
Current Edition.  Association of Official Analytical Chemists (AOAC),
2200 Wilson Boulevard Arlington, VA 22201.

			(3)	Suggested Ordinance and Regulations Covering Public Swimming
Pools.  Joint Committee on swimming Pools of the American Public Health
Association, in Cooperation with the United States Public Health
Service, 1964. The American Public Health Association, New York, NY.		

			(4)	Standard Methods for the Examination of Water and Wastewater,
Current Edition, Standard Methods Online: 
http://www.standardmethods.org.		

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