Non-animal Methods for Toxicity Testing


Last Updated: December 6, 2007

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Ecotoxicity involves the identification of chemical hazards to the environment. "Ecotoxicity studies measure the effects of chemicals on fish, wildlife, plants, and other wild organisms" (US EPA, 2007). Chemical and pesticide manufacturers submit ecotoxicity studies to regulatory authorities to support the registration and/or approval of their products. Testing on animals or plants to determine whether environmental samples such as soil, sediment, or effluents contain toxic compounds is also called ecotoxicity testing.

Although ecotoxicity, in general, refers to hazards to both aquatic and terrestrial animals and plants, this brief review will cover only methods for predicting hazards to the aquatic environment.

Three specific properties of a chemical are used to describe its potential hazard to the aquatic environment (UNECE, 2004b):

  • Aquatic toxicity: The hazard of a substance to living organisms, based on toxicity tests to aquatic animals and plants
  • Degradability: The persistence of the substance in the environment, based on molecular structure or analytical testing
  • Bioaccumulation/bioconcentration: The accumulation of a substance in living organisms (from water sources for bioconcentration), which may or may not lead to a toxic effect; based on calculations or bioconcentration factor (BCF) studies using fish

The Animal Test(s)

The assessment of aquatic toxicity for the classification of chemicals and environmental risk assessments is typically based on toxicity test data for fish, crustacea (daphnids), and algae/aquatic plants (UNECE, 2004b). Toxicity data from freshwater and marine species are considered equivalent, although this is not true for all substances. The data showing the highest toxicity (the lowest acute toxicity values) is to be used in classifying a substance.

Fish are used to test for both acute and chronic toxic effects. The fish 96-hour LC50 test (Organisation for Economic Cooperation and Development [OECD] Test Guideline (TG) 203) is the standard acute toxicity test. The LC50 (lethal concentration 50%) refers to the concentration of test substance that is lethal to 50% of the fish within 96 hours. Chronic fish tests may start with eggs, embryos, or juveniles and last from 7 to more than 200 days (OECD TG 210; US EPA OPPTS 850.1500; other equivalent assay). Test endpoints include "hatching success, growth, spawning success, and survival" (UNECE, 2004b).

Acute and chronic tests are also conducted using crustacea: daphnids, mysids, or others. A 96-hour test with lethality as the endpoint is used for acute toxicity (OECD TG 202, part 1; US EPA OPPTS 850.1035; other equivalent assay). Longer term testing through maturation and production is used to assess chronic toxic effects (OECD TG 202, part 2; US EPA OPPTS 850.1350; other equivalent assay). The chronic testing endpoints include "time to first brood, number of offspring produced per female, growth, and survival" (UNECE, 2004b).

Although the following are not animal tests, they are included here to illustrate the complete set of tests required by many regulatory authorities. The algal growth inhibition test (OECD TG 201) is typically used to determine an acute EC50 for algae. Several Lemna species of aquatic vascular plants can also be used to obtain an acute EC50 (US EPA OPPTS 850.4400). The EC50 (Effective Concentration 50%) is the concentration causing an adverse effect in 50% of the test organisms.

Bioconcentration factor (BCF) studies in fish are conducted using the OECD TG 305. Experimentally-derived BCF values are preferred for classification purposes (UNECE, 2004b).

The OECD recently developed a draft TG for an alternative test method that would provide a reduction in the number of fish used in testing: the Fish Embryo Toxicity Test (FET). In the FET test, newly fertilized zebrafish eggs are exposed to a chemical for up to 48 hours and lethality to the embryos is evaluated after 24 and 48 hours.

The European Centre for the Validation of Alternative Methods' (ECVAM) Scientific Advisory Committee (ESAC) (2006) recently reviewed and recommended the Upper Threshold Concentration (UTC) Step Down Approach for implementation "as a valid strategy to significantly reduce the number of fish used in the assessment of acute aquatic toxicity for hazard classification." The UTC is a tiered testing strategy with the potential for reducing the number of fish used by at least 65%. The UTC is based on pharmaceutical industry studies showing that algae and daphnid acute EC50 tests were more sensitive than fish LC50 tests about 80% of the time (Hutchinson, et al., 2003).

Determining the endocrine disruption (ED) potential of pesticides and chemicals is a relatively new testing requirement in the US and the EU. ED assays for aquatic toxicity testing include the Fish Screen assay, the Fish Lifecycle assay, and the Invertebrate Lifecycle assay using mysid shrimp.

Regulatory Requirements & Test Guidelines

Most regulatory authorities still require the submission of in vivo fish lethality test data. OECD deleted the analogous lethality assays for assessing mammalian toxicity (LD50 or lethal dose to 50% of the animals) in 2002. In addition to the significant ethical concerns over this type of toxicity testing, most scientists also question the technical and scientific merit of fish lethality assays for determining the environmental impact and fate of chemicals, pesticides, and pharmaceuticals released into the aquatic environment (Castaño, et al., 2003). The time and costs of conducting animal experiments for estimating ecotoxicity effects for the many existing and new chemicals are prohibitive. To protect the environment and its inhabitants, faster and cheaper alternative methods are needed. In the near term, reduction strategies using tiered testing schemes and/or tests using fish embryos provide the greatest opportunity for reducing the numbers of fish used for ecotoxicity testing. However, these strategies alone are not sufficient, scientifically, ethically, or economically, for meeting testing needs. Considerable effort is being invested in cellular, genomic, and computational models to meet this need.

The Globally Harmonized System for Classification and Labelling of Chemicals (GHS) describes testing for hazards to the aquatic environment in Part 3, Chapter 3.10 (UNECE, 2004a). The GHS criteria for determining environmental hazards does not specify test methods but rather indicates the need to use methods considered to be valid such as the ecotoxicity test guidelines of the OECD and US Environmental Protection Agency (EPA). Further GHS guidance is provided in Annex 8, Guidance on Hazards to the Aquatic Environment, which describes the harmonized classification scheme, aquatic toxicity testing, degradation, bioaccumulation, and the use of (quantitative) structure-activity relationships [(Q)SARs] in aquatic toxicology (UNECE, 2004b).

The purpose of obtaining aquatic toxicity data for chemicals is to use it in the hazard classification of the chemicals. GHS classifications are as follows (UNECE, 2004b):

Acute Toxicity Classification
< 1 part per million Class I
1-10 parts per million Class II
10-100 parts per million Class III

Chronic toxicity may include a number and range of endpoints. However, the data is often not available, and a combination of acute toxicity, degradability, and bioaccumulation may be used to determine the potential for chronic aquatic toxicity. The GHS criteria for specific values of chronic toxicity are not yet set. (An OECD scientific issue paper on this topic was developed in 2006).

The OECD provides a number of TGs for aquatic and terrestrial toxicity testing. The aquatic toxicity TGs (201, 202, 203, 204, 210, 211, 212, 215, and 221) involve the testing of chemicals on fish and other aquatic organisms. The OECD also provides a number of guidance documents on ecotoxicity testing methods.

Most regulatory authorities require acute toxicity testing using a similar minimal base set of organisms: 1) fish, 2) an aquatic invertebrate, and 3) an algal species. The following are links to environmental assessment guidance of several U.S. agencies:

The report from the ECVAM workshop on the use of fish cells in ecotoxicology describes the regulatory testing uses of fish in ecotoxicology and provides estimates of the number of fish used for this testing in the EU (Castaño, et al., 2003). Chemical testing required by the OECD High Production Volume programme requires data from the base set of organisms, and the recently enacted EU REACH (Registration, Evaluation and Authorization of Chemicals) policy is expected to significantly increase the number of fish used for ecotoxicity testing.

Legislation in the US and proposed legislation in the EU and Japan will require the testing of pesticides and chemicals to determine their effect on the endocrine system of humans and animals. The US EPA has developed the Endocrine Disruptor Screening Program (EDSP) to meet this new testing requirement and is working with other organizations to validate in vivo and in vitro endocrine disruptor (ED) tests. The OECD is validating animal ED tests, including a 21-day fish assay for the detection of endocrine disruptor substances and has posted a draft TG for a fish ED test, The Fish Screening Assay for Endocrine Active Substances.

Cronin, et al. (2003), and Comber, et al. (2003) summarized the regulatory uses of (Q)SARs to predict chemicals' ecological effects. The US EPA has significant experience in using (Q)SAR and structure activity relationship (SAR) models. Cronin, et al. (2003), report that the EPA's Office of Pollution Prevention and Toxics (OPPT) has been using (Q)SARs for more than two decades for predicting effects such as ecological hazard and fate and assessing new chemical risk and testing needs. The GHS describes how QSARs can be used to predict the acute toxicity for fish, daphnia, and algae for certain classes of chemicals (UNECE, 2004b, p. 356 and Chapter A8.6), and to predict bioconcentration (p. 376). Greater testing demands in the EU, due to the cosmetic directive and the REACH initiative, have also stimulated efforts there for the validation and greater use of (Q)SAR prediction models to meet regulatory testing needs.

Non-animal Alternative Methods

Cell-based assays, toxicogenomic microarrays, and (Q)SAR models are used to predict toxic effects to aquatic organisms. However, current in vitro methods for acute aquatic toxicity are neither standardized nor validated. New strategies for addressing the major limitations of cell-based assays have been proposed; see Emerging Research, Methods & Policies. Current in vitro/in silico approaches to replacing animals in aquatic toxicity testing include:

  • Fish cell-based cytotoxicity assays
  • Fish cell-based assays with other mechanisms of toxicity as endpoints
  • Mammalian cell assays using cytotoxicity or other endpoints
  • Bacterial cell assays, usually based on luminescent reporter genes (primarily used for detection)
  • Fish embryo assays based on embryo survival and pathophysiological changes
  • In vitro endocrine disruptor assays
  • Genomic microarrays (toxicogenomics)
  • (Q)SAR and other computational programs
  • Test batteries and/or tiered testing schemes incorporating the above types of assays

Validation and Acceptance of Non-animal Alternative Methods

The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has not reviewed any alternatives to the fish or crustacean tests for acute aquatic toxicity testing. ICCVAM has been participating since 2002 in international activities to validate several endocrine disruptor assays. The in vitro ED assays being reviewed by ICCVAM are human cell assays and are covered in Toxicity Endpoints & Tests: Reproductive & Developmental Toxicity.

ECVAM issued an ESAC Statement "that the UTC approach should be implemented as a valid strategy to significantly reduce the number of fish used in the assessment of acute aquatic toxicity for hazard classification." However, ECVAM has not validated any non-animal methods for ecotoxicity testing. Several binding and transcriptional assays for detecting endocrine disrupting chemicals are under consideration at ECVAM, but their applicability to testing for aquatic species is not known.

In vitro endocrine disruptor assays relevant to aquatic species have been developed and are in the pipeline for prevalidation and validation within the US EPA's endocrine program.

In the near term, reduction strategies using tiered testing schemes and/or tests using fish embryos provide the greatest opportunity for reducing the numbers of fish used for ecotoxicity testing. However, these strategies alone are not sufficient, scientifically, ethically, or economically, for meeting testing needs. There is considerable effort being invested in cellular, genomic, and computational models to meet this need.