## 4.3.3. Endpoints

Author: Michiel Kraak

Reviewers: Kees van Gestel, Carlos Barata

Learning objectives:

You should be able to

- list the available whole organism endpoints in toxicity tests.
- motivate the importance of sublethal endpoints in acute and chronic toxicity tests.
- describe how sublethal endpoints in acute and chronic toxicity tests are measured.

Keywords: Mortality, survival, sublethal endpoints, growth, reproduction, behaviour, photosynthesis

Introduction

Most toxicity tests performed are short-term high-dose experiments, acute tests in which mortality is often the only endpoint. Mortality, however, is a crude parameter in response to relatively high and therefore often environmentally irrelevant toxicant concentrations. At much lower and therefore environmentally more relevant toxicant concentrations, organisms may suffer from a wide variety of sublethal effects. Hence, toxicity tests gain ecological realism if sublethal endpoints are addressed in addition to mortality.

Mortality

Mortality can be determined in both acute and chronic toxicity tests. In acute tests, mortality is often the only feasible endpoint, although some acute tests take long enough to also measure sublethal endpoints, especially growth. Generally though, this is restricted to chronic toxicity tests, in which a wide variety of sublethal endpoints can be assessed in addition to mortality (Table 1).

Mortality at the end of the exposure period is assessed by simply counting the number of surviving individuals, but it can also be expressed either as percentage of the initial number of individuals or as percentage of the corresponding control. The increasing mortality with increasing toxicant concentrations can be plotted in a dose-response relationship from which the LC50 can be derived (see section on Concentration-response relationship). If assessing mortality is non-destructive, for instance if this can be done by visual inspection, it can be scored at different time intervals during a toxicity test. Although repeated observations may take some effort, they generally do generate valuable insights in the course of the intoxication process over time.

Sublethal endpoints in acute toxicity tests

In acute toxicity tests it is difficult to assess other endpoints than mortality, since effects of toxicants on sublethal endpoints like growth and reproduction need much longer exposure times to become expressed (see section on Chronic toxicity). Incorporating sublethal endpoints in acute toxicity tests thus requires rapid responses to toxicant exposure. Photosynthesis of plants and behaviour of animals are elegant, sensitive and rapidly responding endpoints that can be incorporated into acute toxicity tests (Table 1).

Behavioural endpoints

Behaviour is an understudied but sensitive and ecologically relevant endpoint in ecotoxicity testing, since subtle changes in animal behaviour may affect trophic interactions and ecosystem functioning. Several studies reported effects on animal behaviour at concentrations orders of magnitudes lower than lethal concentrations. Van der Geest et al. (1999) showed that changes in ventilation behaviour of fifth instar larvae of the caddisfly Hydropsyche angustipennis occurred at approximately 150 times lower Cu concentrations than mortality of first instar larvae. Avoidance behaviour of the amphipod Corophium volutator to contaminated sediments was 1,000 times more sensitive than survival (Hellou et al., 2008). Chevalier et al. (2015) tested the effect of twelve compounds covering different modes of action on the swimming behaviour of daphnids and observed that most compounds induced an early and signiﬁcant swimming speed increase at concentrations near or below the 10% effective concentration (48-h EC10) of the acute immobilization test. Barata et al. (2008) reported that the short term (24 h) D. magna feeding inhibition assay was on average 50 times more sensitive than acute standardized tests when assessing the toxicity of a mixture of 16 chemicals in different water types combinations. These and many other examples all show that organisms may exhibit altered behaviour at relatively low and therefore often environmentally relevant toxicant concentrations.

Behavioural responses to toxicant exposure can also be very fast, allowing organisms to avoid further exposure and subsequent bioaccumulation and toxicity. A wide array of such avoidance responses have been incorporated in ecotoxicity testing (Araújo et al., 2016), including the avoidance of contaminated soil by earthworms (Eisenia fetida) (Rastetter & Gerhardt; 2018), feeding inhibition of mussels (Corbicula fluminea) (Castro et al., 2018), aversive swimming response to silver nanoparticles by the unicellular green alga Chlamydomonas reinhardtii (Mitzel et al., 2017) and by daphnids to twelve compounds covering different modes of toxic action (Chevalier et al., 2015).

Photosynthesis

Photosynthesis is a sensitive and well-studied endpoint that can be applied to identify hazardous effects of herbicides on primary producers. In bioassays with plants or algae, photosynthesis is often quantified using pulse amplitude modulation (PAM) fluorometry, a rapid measurement technique suitable for quick screening purposes. Algal photosynthesis is preferably quantified in light adapted cells as effective photosystem II (PSII) efficiency (ΦPSII) (Ralph et al., 2007; Sjollema et al., 2014). This endpoint responds most sensitively to herbicide activity, as the most commonly applied herbicides either directly or indirectly affect PSII (see section on Herbicide toxicity).

Sublethal endpoints in chronic toxicity tests

Besides mortality, growth and reproduction are the most commonly assessed endpoints in ecotoxicity tests (Table 1). Growth can be measured in two ways, as an increase in length and as an increase in weight. Often only the length or weight at the end of the exposure period is determined. This, however, includes both the growth before and during exposure. It is therefore more distinctive to measure length or weight at the beginning as well as at the end of the exposure, and then subtract the individual or average initial length or weight from the final individual length or weight. Growth during the exposure period may subsequently be expressed as percentage of the initial lengths or weight. Ideally the initial length or weight is measured from the same individuals that will be exposed. When organisms are sacrificed to measure the initial length or weight, which is especially the case for dry weight, this is not feasible. In that case a subsample from the individuals is taken apart at the beginning of the test.

Reproduction is a sensitive and ecological relevant endpoint in chronic toxicity tests. It is an integrated parameter, incorporating many different aspects of the process, that can be assessed one by one. The first reproduction parameter is the day of first reproduction. This is an ecologically very relevant parameter, as delayed reproduction obviously has strong implications for population growth. The next reproduction parameter is the amount of offspring. In this case the number of eggs, seeds, neonates or juveniles can be counted. For organisms that produce egg ropes or egg masses, both the number of egg masses as well as the number of eggs per mass can be determined. Lastly the quality of the offspring can be quantified. This can be achieved by determining their physiological status (e.g. fat content), their size, survival and finally their chance or reaching adulthood.

Table 1. Whole organism endpoints often used in toxicity tests. Quantal refers to a yes/no endpoint, while graded refers to a continuous endpoint (see section on Concentration-response relationship).

Endpoint | Acute/Chronic | Quantal/Graded
mortality | both | quantal
behaviour | acute | graded
avoidance | acute | quantal
photosynthesis | acute | graded
growth (length and weight) | mostly chronic | graded
reproduction | chronic | graded

A wide variety of other, less commonly applied sublethal whole organism endpoints can be assessed upon chronic exposure. The possibilities are endless, with some specific endpoints being designed for the effect of a single compound only, or species specific endpoints, sometimes described for only one organism. Sub-organismal endpoints are described in a separate chapter (see section on Molecular endpoints in toxicity tests).

References

Araujo, C.V.M., Moreira-Santos, M., Ribeiro, R. (2016). Active and passive spatial avoidance by aquatic organisms from environmental stressors: A complementary perspective and a critical review. Environment International 92-93, 405-415.

Barata, C., Alanon, P., Gutierrez-Alonso, S., Riva, M.C., Fernandez, C., Tarazona, J.V. (2008). A Daphnia magna feeding bioassay as a cost effective and ecological relevant sublethal toxicity test for environmental risk assessment of toxic effluents. Science of the Total Environment 405(1-3), 78-86.

Castro, B.B., Silva, C., Macario, I.P.E., Oliveira, B., Concalves, F., Pereira, J.L. (2018). Feeding inhibition in Corbicula fluminea (OF Muller, 1774) as an effect criterion to pollutant exposure: Perspectives for ecotoxicity screening and refinement of chemical control. Aquatic Toxicology 196, 25-34.

Chevalier, J., Harscoët, E., Keller, M., Pandard, P., Cachot, J., Grote, M. (2015). Exploration of Daphnia behavioral effect profiles induced by a broad range of toxicants with different modes of action. Environmental Toxicology and Chemistry 34, 1760-1769.

Hellou J., Cheeseman, K., Desnoyers, E., Johnston, D., Jouvenelle, M.L., Leonard, J., Robertson, S., Walker, P. (2008). A non-lethal chemically based approach to investigate the quality of harbor sediments. Science of the Total Environment 389, 178-187.

Mitzel, M.R., Lin, N., Whalen, J.K., Tufenkji, N. (2017). Chlamydomonas reinhardtii displays aversive swimming response to silver nanoparticles Environmental Science: Nano 4, 1328-1338.

Ralph, P.J., Smith, R.A., Macinnis-Ng, C.M.O., Seery, C.R. (2007). Use of fluorescence-based ecotoxicological bioassays in monitoring toxicants and pollution in aquatic systems: Review. Toxicological and Environmental Chemistry 89, 589–607.

Rastetter, N., Gerhardt, A. (2018). Continuous monitoring of avoidance behaviour with the earthworm Eisenia fetida. Journal of Soils and Sediments 18, 957-967.

Sjollema, S.B., Van Beusekom, S.A.M., Van der Geest, H.G., Booij, P., De Zwart, D., Vethaak, A.D., Admiraal, W. (2014). Laboratory algal bioassays using PAM fluorometry: Effects of test conditions on the determination of herbicide and field sample toxicity. Environmental Toxicology and Chemistry 33, 1017–1022.

Van der Geest, H.G., Greve, G.D., De Haas, E.M., Scheper, B.B., Kraak, M.H.S., Stuijfzand, S.C., Augustijn, C.H., Admiraal, W. (1999). Survival and behavioural responses of larvae of the caddisfly Hydropsyche angustipennis to copper and diazinon. Environmental Toxicology and Chemistry 18, 1965-1971.