## 4.4.6. Multigeneration toxicity testing - Eco

Author: Michiel Kraak

Reviewers: Kees van Gestel, Miriam Leon Paumen

Learning objectives:

You should be able to

· explain how effects of toxicants may propagate during multigeneration exposure.

· describe the experimental challenges and limitations of multigeneration toxicity testing and to be able to design multigeneration tests.

· explain the implications of multigeneration testing for ecological risk assessment.

Key words: Multigeneration exposure, extinction, adaptation, test design

Introduction

It is generally assumed that chronic life cycle toxicity tests are indicative of the actual risk that populations suffer from long-term exposure. Yet, at contaminated sites, organisms may be exposed during multiple generations and the shorter the life cycle of the organism, the more realistic this scenario becomes. There are, however, only few multigeneration studies performed, due to the obvious time and cost constraints. Since both aquatic and terrestrial life cycle toxicity tests generally last for 28 days (see section on Chronic toxicity), multigeneration testing will take approximately one month per generation. Moreover, the test compound often affects the life cycle of the test species in a dose-dependent manner. Consequently, the control population, for example, could already be in the 9th generation, while an exposed population could still be in the 8th generation due to chemical exposure related delay in growth and/or development. On top of these experimental challenges, multigeneration experiments are extremely error prone, simply because the chance that an experiment fails increases with increasing exposure time.

Experimental considerations

Designing a multigeneration toxicity experiment is challenging. First of all, there is the choice of how many generations the experiment should last, which is most frequently, but completely arbitrarily, set at approximately 10. Test concentrations have to be chosen as well, mostly based on chronic life cycle EC50 and EC10 values (Leon Paumen et al. 2008). Yet, it cannot be anticipated if, and to what extent, toxicity increases (or decreases) during multigeneration exposure. Hence, testing only one or two exposure concentrations increases the risk that the observed effects are not dose related, but are simply due to stochasticity. If the test concentrations chosen are too high, many treatments may go extinct after few generations. In contrast, too low test concentrations may show no effect at all. The latter was observed by Marinkovic at al. (2012), who had to increase the exposure concentrations during the experiment (see Figure 1). Finally, since a single experimental treatment often consists of an entire population, treatment replication is also challenging.

Figure 1. Experimental design of a multigeneration toxicity experiment with the non-biting midge Chironomus riparius. After six generations the exposure concentrations were increased due to lack of effect. To evaluate if multigeneration led to adaptation, after 3, 6 and 9 generations the sensitivity of the test organisms to the test compound was determined. Redrawn from Marinkovic et al. (2012) by Evelin Karsten-Meessen.

Once the experiment is running, choices have to be made on the transition from generation to generation. If a replicate is maintained in a single jar, vessel or aquarium, generations may overlap and exposure concentrations may decrease with time. Therefore, most often a new generation is started by exposing offspring from the previous exposed parental generation in a freshly spiked experimental unit.

If the aim is to determine how a population recovers when the concentration of the toxicant decreases with time, exposure to a single spiked medium also is an option, which seems most applicable to soils (Ernst et al., 2016; van Gestel et al., 2017). To assess recovery after several generations of (continuous) exposure to contaminated media, offspring from previous exposed generations may be maintained under control conditions.

A wide variety of endpoints can be selected in multigeneration experiments. In case of aquatic insects like the non-biting midge Chironomus riparius these include survival, larval development time, emergence, emergence time, adult life span and reproduction. For terrestrial invertebrates survival, growth and reproduction can be selected. Only a very limited number of studies evaluated actual population endpoints like population growth rate (Postma and Davids, 1995).

To persist or to perish

If organisms are exposed for multiple generations the effects tend to worsen, ultimately leading to extinction, first of the population exposed to the highest concentration, followed by populations exposed to lower concentrations in later generations (Leon Paumen et al. 2008). Yet, it cannot be excluded that extinction occurs due to the relatively small population sizes in multigeneration experiments, while larger populations may pass a bottleneck and recover during later generations.

Thresholds have also been reported, as shown in Figure 2 (Leon Paumen et al. 2008). Below certain exposure concentrations the exposed populations perform equally well as the controls, generation after generation. Hence, these concentrations may be considered as the ‘infinite no effect concentration’. A mechanistic explanation may be that the metabolic machinery of the organism is capable of detoxifying or excreting the toxicants and that this takes so little energy that there is no trade off regarding growth and reproduction.

Figure 2. Transition from dose-response relationships to threshold concentrations during a multigeneration toxicity experiment with the collembolan Folsomia candida. Redrawn from Leon Paumen et al. (2008) by Wilma IJzerman.

It is concluded that the frequently reported worsening of effects during multigeneration toxicant exposure raises concerns about the use of single-generation studies in risk assessment to tackle long-term population effects of environmental toxicants.

Figure 3. Extinction at a relatively high exposure concentration and adaptation at a relatively low exposure concentration during a multigeneration toxicity experiment with the non-biting midge Chironomus riparius. Redrawn from Postma & Davids (1995) by Wilma IJzerman.

If populations exposed for multiple generations do not get extinct and persist, they may have developed resistance or adaptation (Figure 3). Regular sensitivity testing can therefore be included in multigeneration experiments, as depicted in Figure 1. Yet, it is still under debate whether this lower sensitivity is due to genetic adaptation, epigenetics or phenotypic plasticity (Marinkovic et al., 2012).

References

Ernst, G., Kabouw, P., Barth, M., Marx, M.T., Frommholz, U., Royer, S., Friedrich, S. (2016). Assessing the potential for intrinsic recovery in a Collembola two-generation study: possible implementation in a tiered soil risk assessment approach for plant protection products. Ecotoxicology 25, 1–14.

Leon Paumen, M., Steenbergen, E., Kraak, M.H.S., Van Straalen, N. M., Van Gestel, C.A.M. (2008). Multigeneration exposure of the springtail Folsomia candida to phenanthrene: from dose-response relationships to threshold concentrations. Environmental Science and Technology 42, 6985-6990.

Marinkovic, M., De Bruijn, K., Asselman, M., Bogaert, M., Jonker, M.J., Kraak, M.H.S., Admiraal, W. (2012). Response of the nonbiting midge Chironomus riparius to multigeneration toxicant exposure. Environmental Science and Technology 46, 12105−12111.

Postma. J.F., Davids, C. (1995). Tolerance induction and life-cycle changes in cadmium-exposed Chironomus riparius (Diptera) during consecutive generations. Ecotoxicology and Environmental Safety 30, 195-202.

Van Gestel, C.A.M., De Lima e Silva, C., Lam, T., Koekkoek, J.C. Lamoree, M.H., Verwei, R.A. (2017). Multigeneration toxicity of imidacloprid and thiacloprid to Folsomia candida. Ecotoxicology 26, 320–328.