Toxicity Endpoints & Tests


Last updated: August 22, 2015

Toxicity Endpoint

An ecological risk assessment is the scientific process for assessing risk based on the following factors:

  • amount of a chemical present in an environmental medium (e.g., soil, water, air),
  • exposure of an ecological receptor (e.g., birds, fish, wildlife)  with the contaminated environmental medium, and
  • inherent toxicity of the chemical.

National regulatory authorities involved in protecting human and animal health and the environment use ecological risk assessments “to characterize the nature and magnitude of health risks to humans…and ecological receptors…from chemical contaminants and other [environmental] stressors.”

Ecotoxicity involves the identification of chemical hazards to the environment, and can be defined as “the study of toxic effects on nonhuman organisms, populations, or communities.” Chemical and pesticide manufacturers submit ecotoxicity study data to regulatory authorities to support the registration and marketing approval of their products. Ecotoxicity testing also involves assessing the toxicity of contaminated environmental samples (e.g., soil, sediment, or effluents) on aquatic organisms. 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.

The assessment of aquatic toxicity, conducted for the hazard classification of chemicals and environmental risk assessments, is based typically on toxicity test data from three trophic levels of aquatic organisms: fish (vertebrates), invertebrates such as daphnids, and algae (aquatic plants).

The specific properties of a chemical/substance used to describe its potential hazard to the aquatic environment are (UNECE, Part 4, 2013):

  • Acute aquatic toxicity: The hazard of a substance to living aquatic organisms based on short-term exposures of aquatic animals and plants
  • Chronic aquatic toxicity: The hazard of a substance to aquatic animals and plants during exposures determined in relation to the life-cycle of the organism
  • Degradability: The persistence of a substance in the environment with “rapid degradability” as the desired characteristic; based on molecular structure or analytical testing
  • Bioaccumulation/bioconcentration: The accumulation of a substance in living organisms (from all routes of exposure for bioaccumulation; from water sources for bioconcentration), which may or may not lead to a toxic effect; based on a bioconcentration factor (BCF) from calculations (log Kow, (Q)SAR, or other computational-based model), or from in vivo studies using fish
The Animal Tests

Most regulatory authorities require aquatic toxicity testing using a similar base set of organisms: 1) fish, 2) an aquatic invertebrate, and 3) an algal species.

For example, specific organisms, tests, and toxicity endpoints are recommended by the US Environmental Protection Agency (EPA) to calculate risk from plant protection products such as pesticides, and ecological risk assessments are developed to assess the likelihood of wildlife/plant toxicity and environmental fate. “EPA estimates the toxicity or hazard of a pesticide by evaluating ecological effects tests that vary from short-term (acute) to long-term (chronic) laboratory studies and may also include field studies. In these tests, animals and plants are exposed to different amounts of pesticides, and their responses to these varying concentrations are measured. The results of these tests may be used to establish a dose-response or cause-and-effect relationship between the amount of pesticide to which the organism is exposed and the effects on the organism.” In addition to pesticides, many other substances are subject to ecotoxicity tests due to specific regulatory requirements, including human and veterinary pharmaceuticals, industrial chemicals, biocides, and others.

The Globally Harmonized System for Classification and Labelling of Chemicals (GHS) describes testing for hazards to the aquatic environment in Part 4: Environmental Hazards. GHS does not specify test methods but rather indicates the preferred use of methods considered to be scientifically valid and internationally harmonized such as test guidelines of the Organisation for Economic Cooperation and Development (OECD), or national methods considered as equivalent such as those of the US EPA. GHS describes a harmonized classification scheme, aquatic toxicity testing, degradation, bioaccumulation, and the use of (quantitative) structure-activity relationships ((Q)SARs) in aquatic toxicology. Two additional GHS guidance documents are needed for data interpretation and special types of substances: Annex 9, Guidance on Hazards to the Aquatic Environment, and Annex 10, Guidance on transformation/dissolution of metals and metal compounds.

The OECD provides a number of Test Guidelines (TGs) and Guidance Documents (GDs) for aquatic and terrestrial toxicity testing. Fish are used to test for both acute and chronic toxic effects. Due to the large number of fish tests with protocols that overlap in terms of exposure scenarios, fish species, and life-stage, OECD published the Fish Toxicity Testing Framework (2012) that describes each OECD test guideline and the conditions under which it may be useful. The Fish Acute Toxicity Test, a 96-hour LC50 test, is the standard acute toxicity test (OECD TG 203). LC50 (lethal concentration 50%) refers to the concentration of test substance that is lethal to 50% of the fish. The ECVAM Scientific Advisory Committee (ESAC) reviewed and recommended the Upper Threshold Concentration (UTC) Step Down Approach for implementation in 2006 “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). The OECD Guidance Document, GD 126 (2010), describes the UTC method. More recently, OECD adopted the Fish Embryo Acute Toxicity (FET) Test (OECD TG 236) (2013) as an alternative test method that could provide a reduction in the number of fish used in acute aquatic toxicity testing (see discussion of this method in section below).

Chronic fish tests may start with eggs, embryos, or juveniles, and last from 7 to more than 200 days, depending on the reproductive cycle of the organism (OECD TG 210; US EPA OPPTS 850.1500; other equivalent assay). Test endpoints include hatching success, growth, spawning success, and survival. Other fish tests that may be used are OECD TG 212 and OECD TG 215.

OECD TGs for degradation and bioaccumulation are part of the Section 3 Guidelines. Degradability studies are analytical tests and do not involve the use of animals. Bioconcentration factor (BCF) studies (bioaccumulation) are conducted in fish using OECD TG 305 (updated 2012), where fish are exposed to dietary or aqueous uptake of the test substance for usually 28 days. OECD TG 315 “describes a method to assess bioaccumulation of sediment-associated chemicals in endobenthic oligochaetes worms.” The Section 3 Introduction (OECD, 2006) explains a stepwise approach to biodegradation testing, including substances for which the n-octanol/water partition coefficient can be considered a good and conservative predictor of bioaccumulation.

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). Chronic testing endpoints may include number of offspring, growth, and survival.

Although the following are not animal tests, they are included here to illustrate the types 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.

Legislation in the US spurred the EPA to create a program to evaluate the testing of pesticides and some other chemicals, primarily those likely to be found in drinking water, to determine their effect on the endocrine system of humans and animals. The EPA established the Endocrine Disruptor Screening Program (EDSP) consisting of two batteries or “tiers” of tests – the first tier is intended as a screen for activity, the second as a collection of tests that measure adverse effects on a variety of species. Tier 1 contains eleven in vitro and in vivo tests, two of which have relevance to ecotoxicity; the Amphibian Metamorphosis Assay (AMA), and the short-term fish reproduction assay. There are EPA and OECD TGs for both of these assays. In addition, OECD has published a series of guidance and review documents covering endocrine assessment. EPA is currently in the process of validating multi-generation tests for fish, birds, and amphibians. For further information on testing for endocrine active substances see the section Endocrine Disruptors.

Validated Non-animal Methods


Test Purpose

Validation Authority

International Acceptance

Fish Embryo Acute Toxicity Test (FET)
Zebrafish Embryo Acute Toxicity Test (ZFET)

Acute aquatic toxicity


OECD TG 236 (2013)**

* Validated reduction, refinement, and replacement methods are listed in AltTox’s Table of Validated & Accepted Alternative Methods. Validated methods for endocrine disruptors are described at: Toxicity Endpoints & Tests: Endocrine Disruptors.
** “OECD TG 236 does not indicate whether the fish embryo acute toxicity test can be used as an alternative to the OECD TG 203; however, several recently published papers demonstrate that the LC50 values produced with the fish embryo acute toxicity test correlate well with those observed in juvenile or adult fish (Lammer et al., 2009; Knobel et al., 2012; Belanger et al., 2013).”

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. Two methods currently considered as scientifically valid that can be used in approaches for acute aquatic toxicity testing are:

  1. Upper Threshold Concentration (UTC) Step Down approach (OECD GD 126; ESAC Statement): a fish method that reduces the number of fish used in acute aquatic toxicity testing (this reduction method is discussed in the previous section)
  2. Zebrafish Embryo Acute Toxicity (FET or ZFET) Test ( OECD TG 236): uses Zebrafish embryo for acute aquatic toxicity testing

The OECD adopted the Fish Embryo Acute Toxicity (FET) Test (OECD TG 236) (2013) as an alternative method that would reduce the number of fish used in testing. In the EU, the FET is not considered an animal test since non-independently feeding larval forms do not fall under the scope of EU Directive 2010/63/EU. In the FET test, newly fertilized zebrafish eggs are exposed to a chemical for up to 96 hours, and four indicators of lethality to the embryos are evaluated every 24 hours. EURL ECVAM coordinated the validation study of FET for OECD (Busquet et al., 2014), and commented that: “TG236 does not indicate whether the fish embryo acute toxicity test can be used as an alternative to the OECD TG203; however, several recently published papers demonstrate that the LC50 values produced with the fish embryo acute toxicity test correlate well with those observed in juvenile or adult fish.” More recently, EURL ECVAM revised the name of this test method to Zebrafish Embryo Acute Toxicity Test Method (ZFET) for Acute Aquatic Toxicity Testing.

EURL ECVAM, in collaboration with other organizations, is also working to optimize and validate the in vitro “Trout S9 assay” for predicting bioaccumulation/bioconcentration in fish. “Since bioaccumulation is the result of absorption, distribution, metabolism and excretion (ADME) processes, information on ADME processes derived with in vitro methods is used to improve the prediction of BCF models.”

The US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has not reviewed alternatives to the fish or crustacean tests for acute aquatic toxicity testing. ICCVAM has, however, participated since 2002 in international activities to validate several cell-based endocrine disruptor assays. Endocrine disruptor test methods are covered in this section of AltTox: Toxicity Endpoints & Tests: Endocrine Disruptors.

Other in vitro and in silico methods for acute aquatic toxicity testing, although there are many, have not been formally validated and may not be standardized. Cell-based assays, toxicogenomic microarrays, physiologically-based toxicokinetic (PBTK) models, threshold of toxicological concern (TTC), and (Quantitative) Structure-Activity Relationship ((Q)SAR) models are being used, however, to predict toxic effects to aquatic organisms.

The GHS, Part 4 (p. 226), says that “While experimentally derived test data are preferred, where no experimental data are available, validated QSARs for aquatic toxicity and log Kow may be used in the classification process.” (Q)SARs are defined by the OECD as “methods for estimating properties of a chemical from its molecular structure and have the potential to provide information on hazards of chemicals, while reducing time, monetary cost and animal testing currently needed.” Cronin, et al. (2003), and Comber, et al. (2003) summarized the regulatory uses of (Q)SARs to predict chemicals’ ecological effects. The 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. GHS, Annex 9 (p. 475), summarizes the development and publications on QSARs by other organizations, and describes how QSARs can be used to predict various aquatic toxicity endpoints, including bioconcentration. Greater testing demands in the EU, due to the cosmetic directive and the REACH legislation, have also stimulated efforts for the validation and greater use of (Q)SAR prediction models to meet regulatory testing needs. The “Non-test Approaches” section of AltTox provides further information on QSAR and Read-across methods.

Pharmacokinetics/toxicokinetics is “defined as the study of the rates of absorption, distribution, metabolism, and excretion [ADME] of toxic substances or substances under toxicological study” (OECD, n.d.). Non-animal alternatives for assessing pharmacokinetics/toxicokinetics consist of mathematical models that describe rates of ADME. Metabolism generally refers to the biotransformation of a substance within the body to other molecular species, which may have a different toxicity than the original substance. The lack of metabolism of a substance can result in its bioaccumulation within the body. The EPA report, Approaches for the application of physiologically based pharmacokinetic (PBPK) models and supporting data in risk assessment (2006), is described as the definitive reference and learning tool for risk assessors using PBPK models. “The report gives an overview of PBPK modeling and the data needed to develop these models, as well as provides considerations for evaluating these models prior to using them to perform interspecies, intraspecies, and other extrapolations needed in risk assessment.” The EPA report is not considered to be guidance, but the International Programme on Chemical Safety (IPCS) document, Characterization and application of physiologically based pharmacokinetic models in risk assessment (2010), is presented as guidance. The European Food Safety Authority (EFSA) report, Modern methodologies and tools for human hazard assessment of chemicals (2014), explains emerging methods and tools for using toxicokinetic and toxicodynamic processes in hazard assessment.

EFSA revised its guidance on aquatic toxicology in 2013 to cover more aquatic organisms as well as initiate including mechanistic effect modelling approaches in aquatic risk assessment.

Recent reports that provide useful overviews of regulatory frameworks for ecotoxicology, as well as approaches to reduce the numbers of fish used in testing, are the following:

Other new and emerging ecotoxicity test methods and approaches are covered in the next section, Ecotoxicity: Emerging Science & Policy.

Additional sources of information on in vitro methods being developed for ecotoxicity testing:

Ecotoxicity Tools and Databases:

Environmental Law and Regulations:


The information provided here is intended only as an overview, and is neither guidance or a comprehensive review of the laws and regulations on ecotoxicity testing. Individual countries/ regions and their regulatory authorities usually provide specific guidance on hazard/ toxicity testing requirements.

Self Assessment Quiz
1. Why would a company conduct toxicity tests on fish, aquatic invertebrates, and algae?

2. What four properties of a substance are used to explain its potential hazard to aquatic organisms?

a. acute and chronic aquatic toxicity, degradation, and bioaccumulation
b. effects on survival, growth, reproductive cycle, and number of offspring
c. chemical composition, degradability, solubility, and pH

3. What kinds of ecotoxicity tests are conducted for regulatory submissions?

a. tests on mice
b. in silico and QSAR computational methods
c. tests on fish and other aquatic organisms
d. a risk assessment

4. Tests conducted using early stage (non-free swimming) Zebrafish larvae are considered to be animal tests in the US.

a. True
b. False
c. Not defined

5. Which validated test methods, if used, have the potential to reduce the numbers of fish used for regulatory testing purposes?

a. acute fish toxicity test; chronic fish toxicity test
b. fish embryo acute toxicity test; upper threshold concentration step down approach
c. QSAR; fish cell cytotoxicity assay
d. QSAR; fish acute toxicity test

6. Currently, there are only two scientifically validated alternative test methods that have the potential to reduce the number of fish used in ecotoxicity testing for hazard assessment. What are they, and what organisms are used in these assays?

7. Which one of the alternative methods from your response to question 6 is considered a non-animal method, and why?

8. What other methods/ approaches/strategies might further reduce/replace fish in ecotoxicity testing?