United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 730-C-11-003

May 30, 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.” You may also access the guidelines in 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.
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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.
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(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 x107organisms 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 

107organisms 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 106organisms 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 
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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 107organisms 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 107organisms per liter of each of the following viruses: Poliovirus (LSa) (ATCC-VR59) 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 106organisms 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 107particles 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 
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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.
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(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;
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(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;
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The applicant is encouraged to use the EPA’s standard efficacy report format, which 

may be found at 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.