## 5.7.2. Community ecotoxicology in practice

Author: Martina G. Vijver

Reviewers: Paul J. van den Brink, Kees van Gestel

Learning objectives:

To be able to

- describe the variety of ecotoxicological test systems available to address different research questions.
- explain what type of information is gained from low as well as higher level ecotoxicological tests.
- explain the advantages and disadvantages of different higher level ecotoxicological test systems

Keywords: microcosms, mesocosms, realism, different biological levels

Introduction: Linking effects at different levels of biological organization

It is generally anticipated that ecotoxicological tests should provide data useful for making realistic predictions of the fate and effects of chemicals in natural ecosystems (Landner et al., 1989). The ecotoxicological test, if used in an appropriate way, should be able to identify the potential environmental impact of a chemical before it has caused any damage to the ecosystem. In spite of the considerable amount of work devoted to this problem and the plethora of test methods being published, there is still reason to question whether current procedures for testing and assessing the hazard of chemicals in the environment do answer the questions we have asked. Most biologists agree that at each succeeding level of biological organization new properties appear that would not have been evident even by the most intense and careful examination of lower levels of organization (Cairns Jr., 1983).

These levels of biological hierarchy might be crudely characterized as subcellular, cellular, organ, organism, population, multispecies, community, and ecosystem (Figure 1). At the lower biological level, responses are faster than those occurring at higher levels of organization.

Figure 1: Tests at different biological levels, from molecule to ecosystem scale (modified from Newman, 2008). Each biological level is of equal importance in environmental toxicology, but has a different implication to the ecosystem health. If the fitness at the gene to the individual species level is affected due to exposure, this can be seen as a warning for the ecosystem health. If the impact of exposure can be detected at individual species (e.g. reproductive output) to population levels, this can be seen as an incident. If the impact of exposure is detectable at the community (structure and functioning) level, this is considered a disaster for ecosystem health. Measurements performed at the different biological level inform us differently: at the higher biological levels in general more ecological realism regarding exposure as well as species-interactions is gained, whereas at the lower biological levels causality and tractability of the link between the response and the dose of chemicals is achieved. Drawn by Wilma IJzerman.

Experiments executed at the lower biological level often are performed under standard laboratory conditions (see Section on Toxicity testing). The laboratory setting has advantages like allowing for replication, the use of relatively easy and simplified conditions that enable outcomes that are rather robust across different laboratories, the stressor of interest being more traceable under optimal stable conditions, and easy repetition of experiments. As a consequence, at the lower biological level the responses of organisms to chemical stressors tend to be more tractable, or more causal, than those identified when studying effects at higher tiered levels.

The merit to perform cosm studies, so at the higher biological level (see Figure 1), is to investigate the impact of a stressor on a variety of species, all having interactions with each other. This enables detecting both direct and indirect effects on the structure of species assemblages due to the chemicals. Indirect effects can become manifest as disruptions of species interactions, e.g. competition, predator-prey interactions and the like. A second important reason for conducting cosm studies is that abiotic interactions at the level of the ecosystem can be accounted for, allowing for measurement of effects of chemicals under more environmentally realistic exposure conditions. Conditions that likely influence the fate and behavior of chemical are sorption to sediments and plants, photolysis, changes in pH (see section on Bioavailability for a more detailed description), and other natural fluctuations.

What are cosm studies?

Microcosm or mesocosm (or cosm) studies represent a bridge between the laboratory and the natural world (examples of aquatic cosms are given in Figure 2). The difference between micro- and mesocosms is mostly restricted to size (Cooper and Barmuta, 1993). Aquatic microcosms are 10-3 to 10 m3 in size, while mesocosms are 10 to 104 m3 or even larger equivalent to whole or natural systems. The originality of cosms is mainly based on the combination of ecological realism, achieved by the introduction of the basic components of natural ecosystems, and facilitated access to a number of physicochemical, biological, and toxicological parameters that can be controlled to some extent. The cosm approach also makes it possible to work with treatments that can be replicated, so enabling the study of multiple environmental factors which can be manipulated. The system allows the establishment of food webs, the assessment of direct and indirect effects, and the evaluation of effects of contamination on multiple trophic and taxonomic levels in an ecologically relevant context. Cosm studies make it possible to assess effects of contaminants by looking at the parts (individuals, populations, communities) and the whole (ecosystems) simultaneously.

Figure 2. Different aquatic ecotoxicological testing facilities: A) indoor microcosms (water-sediment interface, at Leiden University), B) in situ (or caged) outdoor enclosures (at Wageningen Environmental Research), and C) cosms or experimental ditches (at Living Lab, Leiden University).

As given in the OECD guidance document (OECD, 2004), the size to be selected for a meso- or microcosm study will depend on the objectives of the study and the type of ecosystem that is to be simulated. In general, studies in smaller systems are more suitable for short-term studies of up to three to six months and studies with smaller organisms (e.g. planktonic species). Larger systems are more appropriate for long-term studies (e.g. 6 months or longer). Numerous ecosystem-level manipulations have been conducted since the early 1970s (Hurlbert et al., 1972). The Experimental Lakes Area (ELA) situated in Ontario, Canada deserves special attention because of its significant contributions to the understanding of how natural communities respond to chemical stressors. This ELA consists of 46 natural, relatively undisturbed lakes, which were designated specially for ecosystem-level research. Many different questions have been tackled here, e.g. manipulations with nutrients (amongst others Levine and Schindler, 1999), synthetic estrogens (e.g. Kidd et al., 2014) and Wallace with pesticides in the Coweeta district (Wallace et al., 1999). It is nowadays realized that there is a need for testing more than just individual species and to take into account ecosystem elements such as fluctuations of abiotic conditions and biotic interactions when trying to understand the ecological effects of chemicals. Therefore a selection of study parameters is often considered as given by OECD (2004):

- Regarding treatment regime:
  - dosing regime, duration, frequency, loading rates, preparation of application
  - solutions, application of test substance, etc.;
  - meteorological records for outdoor cosms;
  - physicochemical water parameters (temperature, oxygen saturation, pH, etc.);

- Regarding biological levels it should be recorded what sampling methods and taxonomic identification methods are used;
  - phytoplankton: chlorophyll-a; total cell density; abundance of individual dominant taxa; taxa (preferably species) richness, biomass;
  - periphyton: chlorophyll-a; total cell density; density of dominant species; species richness, biomass;
  - zooplankton: total density per unit volume; total density of dominant orders (Cladocera, Rotifera and Copepoda); species abundance; taxa richness, biomass;
  - macrophytes: biomass, species composition and % surface covering of individual plants;
  - emergent insects: total number emerging per unit of time; abundance of individual dominant taxa; taxa richness; biomass; density; life stages;
  - benthic macroinvertebrates: total density per unit area; species richness, abundance of individual dominant species; life stages;
  - fish: total biomass at test termination; individual fish weights and lengths for adults or marked juveniles; condition index; general behaviour; gross pathology; fecundity, if necessary.

Two typical examples of results obtained in an aquatic cosm study

A cosm approach assist in identifying and quantifying direct as well as indirect effects. Here two different types of responses are described, for more examples it is referred to the Section on Multistress.

Joint interactions: Barmentlo et al. (2018) used an outdoor mesocosm system consisting of 65 L ponds. Using a full factorial design, they investigated the population responses of macroinvertebrate species assemblages exposed for 35 days to environmentally relevant concentrations of three commonly used agrochemicals (imidacloprid, terbuthylazine, and NPK fertilizers). A detrivorous food chain as well as an algal-driven food chain were inoculated into the cosms. At environmentally realistic concentrations of binary mixtures, the species responses could be predicted based on concentration addition (see Section on Mixture toxicity). Overall, the effects of trinary mixtures were much more variable and counterintuitive. This was nicely illustrated by how the mayfly Cloeon dipterum reacted to the various combinations of the pesticides. Compared to single substance exposures and binary mixtures, extreme low recovery of C. dipterum (3.6% of control recovery for both mixtures) was seen. However, after exposure to the trinary mixture, recovery of C. dipterum no longer deviated from the control, and therefore was was higher than expected. Unexpected effects of the mixtures were also obtained for both zooplankton species (Daphnia magna and Cyclops sp.) As expected, the abundance of both zooplankton species was positively affected by nutrient applications, but pesticide addition did not lower their recovery. These type of unexpected results can only been identified when multiple species and multiple stressors are tested and cannot be detected in a lab-test with single species.

Indirect cascading effects: Van den Brink et al. (2009) studied the effects of chronic applications of a mixture of the herbicide atrazine and the insecticide lindane in indoor freshwater plankton-dominated microcosms. Both top-down and bottom-up regulation mechanisms of the species assemblage selected were affected by the pesticide mixture. Lindane exposure also caused a decrease in sensitive detritivorous macro-arthropods and herbivore arthropods. This allowed insensitive food competitors like worms, rotifers and snails to increase in abundance (although not always significantly). Atrazine inhibited algal growth and hence also affected the herbivores. A direct result of the inhibition of photosynthesis by atrazine exposure were lower dissolved oxygen and pH levels and an increase in alkalinity, nitrogen and electrical conductivity. See Figure 3 for a synthesis of all interactions observed in the study of Van den Brink et al. (2009).

Figure 3. Ecological effect chain as observed in a microcosm experiments using atrazine and lindane as stressors. The arrows indicate the hypothesis-driven relationships between species. The red colors (with -) represent negative feedbacks, the green colors (with +) positive feedbacks. See the text for further explanation. Adapted from Van den Brink et al. (2009) by Wilma IJzerman.

Realism of cosm studies

There is a conceptual conflict between realism and replicability when applied to mesocosms. Replicability may be achieved, in part, by a relative simplification of the system. The crucial point in designing a model system may not be to maximize the realism, but rather to make sure that ecologically relevant information can be obtained. Reliability of information on ecotoxicological effects of chemicals tested in mesocosms closely depends on the representativeness of biological processes or structures that are likely to be affected. This means that within cosms key features at both structural and functional levels should be preserved as they ensure ecological representativeness. Extrapolation from small experimental systems to the real world seems generally more problematic than the use of larger systems in which more complex interactions can be studied experimentally as well. For that reason, Caquet et al. (2000) claim that testing chemicals using mesocosms refines the classical methods of ecotoxicological risk assessment because they provide conditions for a better understanding of environmentally relevant effects of chemicals.

References

Barmentlo S.H., Schrama M., Hunting E.R., Heutink R., Van Bodegom P.M., De Snoo G.R., Vijver M.G. (2018). Assessing combined impacts of agrochemicals: Aquatic macroinvertebrate population responses in outdoor mesocosms, Science of the Total Environment 631-632, 341-347.

Caquet, T., Lagadic, L., Sheffield, S.R. (2000) Mesocosm in ecotoxicology: outdoor aquatic systems. Reviews of Environmental Contamination and Toxicology 165, 1-38.

Cairns Jr. J. (1983). Are single species toxicity tests alone adequate for estimating environmental hazard? Hydrobiologica 100, 47-57.

Cooper, S.D., Barmuta, L.A. (1993) Field experiments in biomonitoring. In Rosenberg, D.M., Resh, V.H. (Eds.) Freshwater Biomonitoring and Benthic Macroinvertebrates. Chapman and Hall, New York, pp. 399–441.

OECD (2004). Draft Guidance Document on Simulated Freshwater Lentic Field Tests (Outdoor Microcosms and Mesocosms) (July 2004). Organization for Economic Cooperation and Development, Paris. http://www.oecd.org/fr/securitechimique/essais/32612239.pdf

Hurlbert, S.H., Mulla, M.S., Willson, H.R. (1972) Effects of an organophosphorus insecticide on the phytoplankton, zooplankton, and insect populations of fresh-water ponds. Ecological Monographs 42, 269-299.

Kidd, K.A., Paterson, M.J., Rennie, M.D., Podemski, C.L., Findlay, D.L., Blanchfield, P.J., Liber, K. (2014). Direct and indirect responses of a freshwater food web to a potent synthetic oestrogen. Philosophical Transactions of the Royal Society B Biological Sciences 369, Article AR 20130578, DOI:10.1098/rstb.2013.0578

Landner, L., Blanck, H., Heyman, U., Lundgren, A., Notini, M., Rosemarin, A., Sundelin, B. (1989) Community Testing, Microcosm and Mesocosm Experiments: Ecotoxicological Tools with High Ecological Realism. Chemicals in the Aquatic Environment. Springer, pp. 216-254.

Levine, S.N., Schindler, D.W. (1999). Influence of nitrogen to phosphorus supply ratios and physicochemical conditions on cyanobacteria and phytoplankton species composition in the Experimental Lakes Area, Canada. Canadian Journal of Fisheries and Aquatic Sciences 56, 451-466.

Newman, M.C. (2008). Ecotoxicology: The History and Present Directions. In Jørgensen, S.E., Fath, B.D. (Eds.), Ecotoxicology. Vol. 2 of Encyclopedia of Ecology, 5 vols. Oxford: Elsevier, pp.1195-1201.

Van den Brink, P.J., Crum, S.J.H., Gylstra, R., Bransen, F., Cuppen, J.G.M., Brock, (2009). Effects of a herbicide – insecticide mixture in freshwater microcosms: risk assessment and ecological effect chain. Environmental Pollution 157, 237-249.

Wallace, J.B., Grubaugh, J.W., Whiles, M.R. (1996). Biotic indices and stream ecosystem processes: Results from an experimental study. Ecological Applications 6, 140-151.