Demonstrating efficacy of pool and spa sanitisers

Under agvet legislation pool and spa sanitisers are considered to be agricultural chemical products, and before new products can be registered and allowed into the marketplace, the Australian Pesticides and Veterinary Medicines Authority (APVMA) must be satisfied of certain statutory criteria that relate to their safety, efficacy, trade and labelling. This guideline sets out how applicants may demonstrate that a proposed new pool or spa sanitiser would satisfy the APVMA’s efficacy criteria.

For many years, disinfection of swimming pools and spa pools has relied mainly on chemical sanitisers based principally on chlorine and bromine. While the efficacy of these traditional sanitisers is well established with regard to different kinds of pathogenic microorganisms, new types of chemical sanitisers lack such an established record.

All new pool and spa sanitiser products need to demonstrate that they are efficacious, regardless of whether they are novel chemicals or traditional sanitisers with a long history of safe use. While you may choose to satisfy our efficacy criteria by demonstrating that your product meets the performance characteristics set out below, we are also prepared to consider alternative information and arguments aimed at satisfying the efficacy criteria.

There are APVMA standards for certain pool and spa products.

In addition to satisfying the efficacy criteria, a new sanitiser must meet our safety criteria relating to human health and to the environment. For example, there should be no adverse impact on bathers or toxic effect from the sanitiser or its by-products that exceeds health standards as a result of either short-term or extended immersion in water treated with the sanitiser. Information on toxicology data that may demonstrate that a sanitiser meets the safety criteria can be found in the toxicology guidelines of the APVMA’s data guidelines.

Efficacy criteria for pool and spa sanitisers

To satisfy the efficacy criteria, you should establish that the proposed new sanitiser product is effective against the key pathogens in the major classes of human pathogenic microorganisms commonly found in swimming-pool and spa-pool water, namely bacteria, protozoa and viruses. Where relevant, you should also establish that the new product has equivalent efficacy to a registered sanitiser based on hypochlorous acid or hypochlorite against these same classes of microorganisms.

You are encouraged to submit information from a combination of laboratory and field trials. Laboratory trials demonstrate efficacy against important human pathogens under a range of controlled conditions; field trials demonstrate that the product works as expected under actual use conditions. Both trial components are important in satisfying us of the efficacy criteria for this class of chemicals.

Data from laboratory and field trials may be generated within Australia or overseas. Data from overseas trials should be accompanied by a valid scientific argument that shows how the information provided is relevant to Australian conditions.

You are encouraged to seek our advice while planning laboratory and field trials to ensure that the efficacy against the most important pathogens will be addressed, and that field tests are planned at appropriate locations and under appropriate conditions. Results from other efficacy studies with other indicator organisms may be accepted by the APVMA, provided that additional scientific information and argument can satisfy us that those studies prove the product meets the efficacy criteria.

An APVMA research permit is required for the conduct of a field trial with an unregistered product because supply of an unregistered product without a permit is an offence under the Agvet Code. See further information about research permits.

Applications to register swimming-pool or spa-pool sanitiser or disinfecting processes should address the following criteria:

  • An effective residual concentration of sanitiser should be maintained in the body of the pool to provide continuous disinfection within the water at all times.
  • The concentration of the residual sanitiser (or its principal components if there is more than one active constituent) should be capable of being measured using a field test kit or another simple method that can be properly managed by an average home pool owner.
  • The sanitiser should be capable of supplementary dosing if measured levels are found to be below the recommended effective concentration.
  • A known safety margin of efficacy should be established for normal operating concentrations.
  • For sanitisers containing more than one active constituent, you should identify the relative contributions of each principal active constituent to the overall efficacy of the product.
  • If the sanitiser is to be used in conjunction with an algaecide, you should provide information that demonstrates that the two products are compatible and that their use together will not have any adverse effects on the performance of either product.

It is your responsibility to prove through your application that your proposed sanitiser or disinfecting process can meet these criteria.

Laboratory testing phase

As a first step, a sanitiser or disinfecting process should be shown to be effective under defined laboratory conditions against key indicator organisms within the major classes of pathogenic microorganisms associated with swimming and spa pools (see Table 1).

While there is no specific requirement for a parallel chlorine standard (control) to be incorporated into a laboratory test protocol, in testing a new pool or spa sanitiser, we expect you to show that the performance characteristics of the sanitiser being tested are essentially equivalent to recognised hypochlorous acid or hypochlorite antimicrobial efficacy characteristics. These established characteristics are detailed in Table 1.

If a parallel chlorine test is incorporated into the experimental protocol, the chlorine-testing methodology should follow established principles of controlling chlorine demand and verifying free chlorine concentration at the beginning and end of the exposure period. The Association of Official Analytical Chemists (AOAC) Official Method 965.13 can be used as a guide. A free chlorine starting concentration of one milligram per litre should be used (see also Target performance characteristics). As outlined in AOAC 965.13, a ratio of 199 to 1 for the chlorine test solution to the test organism suspension should provide a sufficient reserve of free chlorine during the test period. Free chlorine concentration should not drop below approximately 0.7 milligrams per litre by the end of the exposure period.

Satisfactory efficacy under laboratory conditions can be demonstrated by following the test design principles below. Design principles incorporated into laboratory testing and any associated validation results should be included in the final trial report.

Standards for testing

Tests, including preparation of materials and analysis of test samples, should be carried out by a National Association of Testing Authorities registered or similarly accredited laboratory with no affiliation with or commercial connection to the applicant. Assay methods for each type of test should be well established and reproducible by the host laboratory.

Test conditions should simulate use conditions

Tests should be carried out at 25 to 30 ˚C for swimming pools and 34 to 36 ˚C for spa pools and at a pH that is both consistent with good efficacy of the sanitiser being tested and acceptable for the comfort and safety of bathers. If parallel hypochlorite controls are incorporated into the testing protocol, these chlorine controls should be carried out at pH of 7.2 to 7.3.

During disinfection testing, no chemical with disinfecting properties other than the test sanitiser (which may be a mixture of two or more active constituents) should be present in the water.

Establishing a safety margin

The sanitiser needs to remain effective against pathogens at 50% of its recommended operating concentration. This efficacy margin can be established by testing against the single species Pseudomonas aeruginosa, according to the performance characteristics indicated in Table 1.

In relation to bather health, the sanitiser must have been independently demonstrated to be safe for bathers at 2 times the highest recommended concentration of the active constituent(s).

Establishing relative contributions of active constituents

All constituents that contribute to the disinfecting or sanitising properties of a product are included on the product label as ‘active constituents’. For products that have more than one active constituent with different modes of action (for example, metal ions and accompanying oxidisers) the independent contributions of the active constituents to overall efficacy should be demonstrated in satisfying the efficacy criterion. For an example test protocol, see Table 2.

Test organisms

The test organisms used in any testing should be recognised, standard strains of the species and should be derived from a recognised culture collection. You should include the reference identity number of the culture and its source in the test report. The suggested test species are identified in Table 1.

Contact times

The test contact times evaluated for specific indicator organisms should be in keeping with the recommended performance criteria in Table 1. Where a product is shown to be slower acting than free chlorine, it may still be acceptable, provided the data demonstrates that the difference in activity is not significant and efficacy against key indicator organisms is equivalent to or better than comparable features of chlorine.

Test volume to inoculum volume ratio

The test volume should have the capacity to act as a sufficient reservoir to maintain the recommended concentration of active constituent(s) when the volume of test inoculum is added. The inoculum volume and its concentration of excipients should not overwhelm the test system in such a way that the recommended concentration of active constituent(s) is substantially altered.

A test volume to inoculum volume ratio of 199 to 1, as described in AOAC 965.13, is satisfactory in most cases where the disinfectant demand of the system has been measured and accounted for. Inoculum suspensions may need to be checked for solutes that could interfere with the sanitiser.

Neutralisation of antimicrobial

The test protocol should incorporate a neutralisation step for the active constituent(s). At the end of each contact test period, aliquots of the test mixture intended for survival counts should be added immediately to a neutralisation diluent. You should validate the effectiveness of the neutralisation with appropriate controls or a separate test protocol.

The neutralisation broth should not exert any toxicity or antimicrobial or antiviral effects against the test organisms. Details of the neutralisation steps, including results from validation tests and broth analysis, should be included in the trial report.

Maintenance of active constituent(s) concentration

The active constituent(s) should be measured at the beginning and end of the biocidal test period to confirm that the concentration of active constituent(s) has been maintained within the correct concentration range for the duration of the experiment, as would occur in a swimming or spa pool under normal use conditions.

If chlorine is used as a comparative control, you should also determine the concentration of free chlorine at the beginning and end of the test contact period. One method is described in AOAC 965.13.

Inoculum density

The inoculum density of the test organism in the test mixture should be such that the appropriate kill factors presented in Table 1 can be measured. A microorganism density in the test mixture that is 100 times higher than the log reduction number (kill factor) being measured is usually practical. For example, with bacteria, a test organism count of 10per millilitre in the test volume is suitable and of a density that will minimise inoculum effects.

Inoculum preparation

Inoculum suspensions need to be in a carrier that will maintain the viability of the organisms, but one that does not contain solutes that interfere with the action of the sanitiser being tested.

In relation to virus suspensions, virus particles are often clustered and associated with cellular debris. Such clustering can protect some of the particles from exposure to the biocide being tested. Since the degree of aggregation and amount of debris cannot be precisely controlled from one test series to another, disaggregated and exposed virions may need to be tested in order to make valid comparisons. Therefore, you may need to treat virus suspensions prior to testing to ensure virions are disaggregated. You should confirm your nominated method of purification or disaggregation with the APVMA. Suitable methods for disaggregating adenovirus and rotavirus can be found in Thurston-Enriquez et al. (2003) and Vaughn et al. (1986), respectively.


The test protocol should incorporate at least duplicate trials for each set of conditions being evaluated for the product under test, in order to demonstrate that the results can be replicated. The recovery counts of the test organisms within each trial should also be performed at least in duplicate.

Appropriate controls should be incorporated into each trial to demonstrate the effect of the treatment compared with the absence of treatment.

Target performance characteristics

Table 1 shows the performance characteristics of an effective sanitiser against the recommended test organisms. The performance characteristics of one milligram per litre of free chlorine (from hypochlorous acid or hypochlorite) have been demonstrated in the scientific literature to be equivalent to the performance characteristics shown in Table 1.

Table 1: Performance characteristics of effective sanitisers against the recommended test organisms – swimming and spa pools
Test organism Number log10 reductions to be achieved Duration of exposure to test sanitiser (at normal concentration) in which reduction is to be achieved
Escherichia coli 4 30 seconds
Enterococcus faecium 4 2 minutes
Pseudomonas aeruginosa (for spa pools only) 4 2 minutes
Legionella pneumophila (for spa pools only) 4 2 minutes
Adenovirus (disaggregated)1 3 10 minutes
Rotavirus (disaggregated)1 3 2 minutes
Neagleria fowleri (cysts) 4 30 minutes
Giardia duodenalis2 (cysts)
or G. muris3 (cysts)
3 45 minutes


1 Prior to the test exposure, virus suspensions need to be treated to disassociate aggregated clusters of virus particles.

2 Older synonyms in the literature for this species are G. lamblia and G. intestinalis.

3 The animal pathogen G. muris can be used as a surrogate for the human pathogen.

General comments

Results from other efficacy studies with other indicator organisms may be accepted by the APVMA, provided that additional scientific information and argument can satisfy us that those studies prove the product meets the efficacy criteria.

Special instructions for testing silver- and copper-ion-based sanitisers

Phosphate buffers should not be used in disinfection tests, since phosphate complexes with copper ions will interfere with test results.

Disinfection test periods should not be terminated by using chelating agents to sequester copper and silver ions because this could cause test results to be invalidated. Chelating agents are not specific enough for copper or silver and will also react with other metal ions. Removing calcium ions, for example, is known to interfere with the infectivity of some viruses (including rotavirus), and there is evidence that Naegleria fowleri is adversely affected by chelating agents. As an alternative, it is recommended that you use at least a 100-fold dilution method with appropriate culture medium to terminate disinfection test periods, and that you progress the sample to the plating and incubation stage as quickly as possible to further dilute the concentration of metal ions. Other options are the use of a fresh, rapid-flow gel exclusion column for each sample of the longer test periods or centrifugation through sucrose cushions. Other scientifically valid procedures will also be considered.

Copper- and silver-ion-based sanitisers are necessarily used in conjunction with oxidisers, usually either chlorine or one or more of the peroxygen compounds. It is important to establish how much of the overall efficacy is contributed by the metal ions and how much by the oxidiser. In addition, it is important to demonstrate that the sanitiser is still effective at half its recommended operating concentration. This can be achieved by a series of experiments on Pseudomonas aeruginosa that test different ratios of the combined active constituents and different concentrations of the intended ratio of the active constituents. For example, if the proposed operating concentrations of the metal ions and oxidiser are M and O respectively, a suitable trial design is shown below in Table 2.

Table 2: Suitable trial designs using different ratios when active constituents are a combination of metal ions and oxidiser
Metal ion series Oxidiser series Efficacy threshold series
Nil M with O Nil O with M N/A
0.2 M with O 0.2 O with M 0.2 of (M with O)*
0.4 M with O 0.4 O with M 0.4 of (M with O)*
0.6 M with O 0.6 O with M 0.6 of (M with O)*
0.8 M with O 0.8 O with M 0.8 of (M with O)*
M with O O with M N/A
Control (nil M and O) Control (nil M and O) Control (nil M and O)

N/A = not applicable

* ie 0.2 or 0.4 etc. times the recommended operating concentrations of metal ions (M) and oxidiser (O)

For the trials suggested in Table 2, it may be necessary to complete a preliminary range-finding experiment to determine how many cells should be used for each test sample so that all are not killed and a reportable value is obtained. The reported value for each sample should be the log reduction in viable Pseudomonas aeruginosa cells after two minutes of exposure to the sanitiser.

When more than one type of metal ion is used in the system (for example, copper, silver and zinc), it is not necessary to test each metal ion separately. However, the mixture of metal ions in the intended ratio of the marketed product must be used. Similarly, if a mixture of oxidisers is formulated for the marketed product, the same mixture as intended for the marketed product must be used as the oxidiser in the tests.

Field testing in a full-sized swimming or spa pool

Field testing of swimming- and spa-pool sanitisers should be conducted after laboratory trials have been successfully completed. Efficacy trials in full-size swimming pools and spa pools should be conducted by an independent agency accredited by the Joint Accreditation System of Australia and New Zealand (JAS-ANZ) or an equivalent organisation with which JAS-ANZ has a memorandum of understanding. Results should be analysed and reported without intervention by the applicant. Pools to be used for these trials should be busy public pools that have a significant and variable bather load to ensure that the efficacy of the product is adequately tested.

The aim of the field test is to demonstrate the efficacy of the swimming-pool or spa-pool sanitiser or disinfection process under actual use conditions. You should design a suitable test protocol of not less than six months’ duration on the type of pool or spa in which the sanitiser or disinfecting process is to be used. The protocol should be designed to provide an accumulation of evidence that clearly shows compliance with relevant guidelines for the control of swimming-pool and spa pathogenic microorganisms under field conditions.

Because field studies such as these can be strongly affected by a pool’s location and use pattern, we recommend that you discuss the design of a field trial with us before committing to a particular test site and protocol.

Table 3 provides guidance on effective sanitiser performance characteristics during field testing.

Table 3: Guidance on effective sanitiser performance characteristics during field testing
Test organisms Test method Maximum count allowable (colony forming units)
Heterotrophic colony count Pour plate method. Australian Standard Method AS4276.3.1—2007 100CFU/mL
Thermotolerant coliforms Most probable number method. Australian Standard Method AS 4276.6—2007 or Membrane filtration method AS 4276.7—2007 Nil CFU/100mL
Pseudomonas aeruginosa Membrane filtration method. Australian Standard method AS 4276.13—2008 Nil CFU/100mL

CFU/mL = colony forming units per millilitre

The following minimum methodology and features should be incorporated into the trial design and reported in an applicant’s submission.

Features of the trial to be included and reported:

  • Pool design specifications – dimensions, volume and location (indoor or outdoor)
  • Water distribution and circulation pattern
  • Turnover rates of the pool(s) under test, and for spa pools, details of water dumping schedule and refill
  • Balance tank details
  • Method of dosing of the sanitiser (and if chlorine is part of the system, whether chlorine is stabilised or unstabilised)
  • Details of other chemicals used
  • Filtration, flocculation and backwashing details
  • Details of rainfall events (for outdoor pools)
  • Details of laboratories used
  • Methodology for all microorganism efficacy tests and key chemical assays
  • Appropriate safety data sheets for active constituents handled as concentrates

Test protocol aspects to be included and reported:

  • Water sampling location(s) for microorganisms and chemicals, sample replication and transport methodology
  • Sampling design and strategy (note that the number of samples planned per nominated time period and the number for the overall study should be clearly stated)
  • Details of other relevant parameters at sampling (such as water temperature and clarity)
  • Daily bather loads throughout the test
  • Bather load for the one-hour period prior to sampling (note that at least 50% of the total number of samples taken will need to be associated with operational bather loads). An operational bather load for this purpose is the number of bathers that would constitute 25 to 30% of the instantaneous maximum bathing load, multiplied by 12 (as per the United Kingdom Pool Water Treatment Advisory Group). A guideline for determining maximum bather load is shown in Table 4.
  • Concentration of sanitiser at time of sampling
  • Measurement of pH at time of sampling
  • Measurement of reserve (total) alkalinity
  • Concentration of any other relevant chemical
  • Millivolt equivalence of disinfection agent if it is proposed to control the sanitiser using redox potential
Table 4: Guideline for determining maximum bather load
Pool depth Surface water area per person (m2)
Shallow water (less than 1 metre deep) 2
Standing-depth water (1–1.5 metres deep) 3
Deep water (more than 1.5 metres deep) 4


Standards Australia 2007, Water microbiology—Heterotrophic colony count methods—Pour plate method using yeast extract agar (AS/NZS 4276.3.1:2007).

Standards Australia 2007, Water microbiology—Coliforms, Escherichia coli and thermotolerant coliforms—Determination of most probable number (MPN) (AS/NZS 4276.6:2007).

Standards Australia 2007, Water microbiology—Escherichia coli and thermotolerant coliforms—Membrane filtration method (AS 4276.7:2007).

Australian Standard 2008, Water microbiology—Pseudomonas aeruginosa—Membrane filtration method (AS/NZS 4276.13:2008)

Standards Australia 2002, Water management for public swimming pools and spas (HB 241-2002).

AOAC International 2000, Official methods of analysis, 17th edn, AOAC International, Gaithersburg, MD, United States.

Thurston-Enriquez, JA, Hass, CN, Jacangelo, J & Gerba, CP 2003, ‘Chlorine inactivation of adenovirus type 40 and feline calicivirus’, Applied and Environmental Microbiology, vol. 69, no. 7, pp. 3979–85.

Vaughn, JM, Chen, Y & Thomas, MZ 1986, ‘Inactivation of human and simian rotaviruses by chlorine’, Applied and Environmental Microbiology, vol. 51, no. 2, pp. 391–94.

PWTAG 2012, The Management and Treatment of Pool Water Code of Practice, Code of Practice 11.12, Pool Water Treatment Advisory Group.

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