# 4.3. Toxicity testing

Author: Kees van Gestel

Reviewer: Michiel Kraak

Learning objectives:

You should be able to

- Mention the two general types of endpoints in toxicity tests
- Mention the main groups of test organisms used in environmental toxicology
- Mention different criteria determining the validity of toxicity tests
- Explain why toxicity testing may need a negative and a positive control

Keywords: single-species toxicity tests, test species selection, concentration-response relationships, endpoints, bioaccumulation testing, epidemiology, standardization, quality control, transcriptomics, metabolomics,

Introduction

Laboratory toxicity tests may provide insight into the potential of chemicals to bioaccumulate in organisms and into their hazard, the latter usually being expressed as toxicity values derived from concentration-response relationships. Section 4.3.1 on Bioaccumulation testing describes how to perform tests to assess the bioaccumulation potential of chemicals in aquatic and terrestrial organisms, and under static and dynamic exposure conditions. Basic to toxicity testing is the establishment of a concentration-response relationship, which relates the endpoint measured in the test organisms to exposure concentrations. Section 4.3.2 on Concentration-response relationships elaborates on the calculation of the relevant toxicity parameters like the median lethal concentration (LC50) and the medium effective concentration (EC50) from such toxicity tests. It also discusses the pros and cons of different methods for analyzing data from toxicity tests.

Several issues have to be addressed when designing toxicity tests that should enable assessing the environmental or human health hazard of chemicals. This concerns among others the selection of test organisms (see section 4.3.4 on the Selection of test organisms for ecotoxicity testing), exposure media, test conditions, test duration and endpoints, but also requires clear criteria for checking the quality of toxicity tests performed (see below). Different whole organism endpoints that are commonly used in standard toxicity tests, like survival, growth, reproduction or avoidance behavior, are discussed in section 4.3.3 on Endpoints. The sections 4.3.4 to 4.3.7 are focusing on the selection and performance of tests with organisms representative of aquatic and terrestrial ecosystems. This includes microorganisms (section 4.3.6), plants (section 4.3.5), invertebrates (section 4.3.4) and vertebrate test organisms (e.g. fish: section 4.3.4 on ecotoxicity tests, and birds: section 4.3.7). Testing of vertebrates, including fish (section 4.3.4) and birds (section 4.3.7), is subject to strict regulations, aimed at reducing the use of test animals. Data on the potential hazard of chemicals to human health therefore preferably have to be obtained in other ways, like by using in vitro test methods (section 4.3.8), by using data from post-registration monitoring of exposed humans (section 4.3.9 on Human toxicity testing), or from epidemiological analysis on exposed humans (section 4.3.10).

Inclusion of novel endpoints in toxicity testing

Traditionally, toxicity tests focus on whole organism endpoints, with survival, growth and reproduction being the most measured parameters (section 4.3.3). In case of vertebrate toxicity testing, also other endpoints may be used addressing effects at the level of organs or tissues (section 4.3.9 on human toxicity testing). Behavioural (e.g. avoidance behavior) and biochemical endpoints, like enzyme activity, are also regularly included in toxicity testing with vertebrates and invertebrates (sections 4.3.3, 4.3.4, 4.3.7, 4.3.9).

With the rise of molecular biology, novel techniques have become available that may provide additional information on the effects of chemicals. Molecular tools may, for instance, be applied in molecular epidemiology (section 4.3.11) to find causal relationships between health effects and the exposure to chemicals. Toxicity testing may also use gene expression responses (transcriptomics; section 4.3.12) or changes in metabolism (metabolomics; section 4.3.13) in relation to chemical exposures to help unraveling the mechanism(s) of action of chemicals. A major challenge still is to explain whole organism effects from such molecular responses.

Standardization of tests

The standardization of tests is organized by international bodies like the Organization for Economic Co-operation and Development (OECD), the International Standardization Organization (ISO), and ASTM International (formerly known as the American Society for Testing and Materials). Standardization aims at reducing variation in test outcomes by carefully describing the methods for culturing and handling the test organisms, the procedures for performing the test, the properties and composition of test media, the exposure conditions and the analysis of the data. Standardized test guidelines are usually based on extensive testing of a method by different laboratories in a so-called round-robin test.

Regulatory bodies generally require that toxicity tests supporting the registration of new chemicals are performed according to internationally standardized test guidelines. In Europe, for instance, all toxicity tests submitted within the framework of REACH have to be performed according to the OECD guidelines for the testing of chemicals (see section on Regulation of chemicals).

Quality control of toxicity tests

Since toxicity tests are performed with living organisms, this inevitably leads to (biological) variation in outcomes. Coping with this variation requires the use of sufficient replication, careful test designs and good choice of endpoints (section 4.3.3) to enable proper estimates of relevant toxicity data.

In order to control the quality of the outcome of toxicity tests, several criteria have been developed, which mainly apply to the performance of the test organisms in the non-exposed controls. These criteria may e.g. require a minimum % survival of control organisms, a minimum growth rate or number of offspring being produced by the controls and limited variation (e.g. <30%) of the replicate control growth or reproduction data (sections 4.3.4, 4.3.5, 4.3.6, 4.3.7). When tests do not meet these criteria, the outcome is prone to doubts, as for instance a poor control survival will make it hard to draw sound conclusions on the effect of the test chemical on this endpoint. As a consequence, tests that do not meet these validity criteria may not be accepted by other scientists and by regulatory authorities.

In case the test chemical is added to the test medium using a solvent, toxicity tests should also include a solvent control, in addition to a regular non-exposed control (see section 4.3.4 on the selection of test organisms for ecotoxicity testing). In case the response in the solvent control differs significantly from that in the negative control, the solvent control will be used as the control for analyzing the effects of the test chemical. The negative control will then only be used to check if the validity criteria have been met and to monitor the condition of the test organisms. In case the responses in the negative control and the solvent control do not differ significantly, both controls can be pooled for the data analysis.

Most test guidelines also require frequent testing of a positive control, a chemical with known toxicity, to check if the long-term culturing of the test organisms does not lead to changes in their sensitivity.