## 6.4.5. Biomonitoring: in situ bioassays and contaminant concentrations in organisms

Author: Michiel Kraak

Reviewers: Ad Ragas, Suzanne Stuijfzand, Lieven Bervoets

Learning objectives:

You should be able to

- name tools specifically designed for ecological risk assessment in the field.
- define biomonitoring and to describe biomonitoring procedures.
- list the characteristics of suitable biomonitoring organisms.
- list the most commonly used biomonitoring organisms per environmental compartment.
- argue the advantages and disadvantages of in situ bioassays.
- argue the advantages and disadvantages of measuring contaminant concentrations in organisms.

Key words: Biomonitoring, test organisms, in situ bioassays, contaminant concentrations in organisms, environmental quality

Introduction

Several approaches and tools are available for diagnostic risk assessment. Tools specially developed for field assessments include the TRIAD approach (see section on TRIAD approach), in situ bioassays and biomonitoring. In ecotoxicology, biomonitoring is defined as the use of living organisms for the in situ assessment of environmental quality. Passive biomonitoring and active biomonitoring are distinguished. For passive biomonitoring, organisms are collected at the site of interest and their condition is assessed or the concentrations of specific target compounds in their tissues are analysed, or both. By comparing individuals from reference and contaminated sites an indication of the impact on local biota at the site of interest is obtained. For active biomonitoring, organisms are collected from reference sites and exposed in cages or artificial substrates at the study sites. Ideally, reference organisms are simultaneously exposed at the site of origin to control for potential effects of the experimental set-up on the test organisms. As an alternative to field collected animals, laboratory cultured organisms may be employed. After exposure at the study sites for a certain period of time, the organisms are recollected and either their condition is analysed (in situ bioassay) or the concentrations of specific target compounds are measured in the organisms, or both.

The results of biomonitoring studies may be used for management decisions, e.g. when accumulation of contaminants has been demonstrated in the field and especially when the sources of the pollution have been identified. However, the use of biomonitoring studies in environmental management has not been captured in formal protocols or guidelines like those of the Water Framework Directive (WFD) or – to a lesser extent – the TRIAD approach and effect-based quality assessments. Biomonitoring studies are typically applied on an case-by-case basis and their application therefore strongly depends on the expertise and resources available for the assessment. The text below explains and discusses the most important aspects of biomonitoring techniques used in diagnostic risk assessment.

Selection of biomonitoring test organisms

The selection of adequate organisms for biomonitoring partly follows the selection of test organisms for toxicity tests (see section on the Selection of test organisms). Suitable biomonitoring organisms:

- Are sedentary, since sedentary organisms may adapt more easily to the in situ experimental setup than more mobile organisms, for which caging may be an additional stress factor. Moreover, for sedentary organisms the relationship between the accumulated compounds and the environmental quality at the exposure site is straightforward, although this is more relevant to passive than to active biomonitoring.
- Are representative for the community of interest and native to the study sites, since this will ensure that the biomonitoring organisms tolerate local conditions other than contamination, preventing that stressors other than poor environmental conditions may affect their performance. Obviously, it is also undesirable to introduce exotic species into new environments.
- Are long living, at least substantially longer than the exposure duration and preferably large enough to obtain sufficient material for chemical analysis.
- Are easy to handle.
- Respond to a gradient of environmental quality, if the purpose of the biomonitoring study is to analyse the condition of the organisms after recollection (in situ bioassay).
- Accumulate contaminants without being killed, if the purpose of the biomonitoring study is to measure contaminant concentrations in the organisms after recollection.
- Are large enough to obtain sufficient biomass for the analysis of the target compounds above the limits of detection, if the purpose of the biomonitoring study is to measure contaminant concentrations in the organisms after recollection.

Based on the above listed criteria, in the marine environment mussels belonging to the genus Mytilus are predominantly selected. The genus Mytilus has the additional advantage of a global distribution, although represented by different species. This facilitates the comparison of contaminant concentrations in the organisms all around the globe. Lugworms have occasionally also been used for biomonitoring in marine systems. For freshwater, the cladoceran Daphnia magna is most frequently employed, although occasionally other species are selected, including mayflies, snails, worms, amphipods, isopods, caddisflies and fish. Given the positive experience with marine mussels, freshwater bivalves are also employed as biomonitoring organisms. Sometimes primary producers have been used, mainly periphyton. Due to the complexity of the sediment and soil compartments, few attempts have been made to expose organisms in situ, mainly restricted to chironomids on sediment.

In situ exposure devices

An obvious requirement of the in situ exposure devices is that the test organisms do not suffer from (sub)lethal effects of the experimental setup. If the organisms are large enough, cages may be used, like for freshwater and marine mussels. For daphnids, a simple glass jar with a permeable lid suffices. For riverine insects, the device should allow the natural flow of the stream to pass, but meanwhile prevent the organisms from escaping. In the device shown in Figure 1a, caddisfly larvae containing tubes are connected to floating tubes, maintaining the larvae at a constant depth of 65 cm. In the tubes, the caddisfly larvae are able to settle and build nets on artificial substrate, a plastic doormat with bristles standing out.

An elegant device for in situ colonization of periphyton was developed by Blanck (1985)(Figure 1b). Sand-blasted glass discs (1.5 cm2 surface) are used as artificial substratum for algal attachment. Substrata are placed vertically in the water, parallel to the current, by means of polyethylene racks, each rack supporting a total of 170 discs. After the colonization period, the periphyton containing glass discs can be harvested, offering the unique possibility to perform laboratory or field experiments with entire algal and microbial communities, replicated 170 times.

Figure 1. Left: Experimental set-up for in situ exposure of caddisfly larvae according to Stuijfzand et al. (1999, derived from Vuori (1995)). Right: Experimental set-up for in situ colonization of periphyton according to Ivorra et al. (1999), derived from Blanck (1985). Drawn by Wilma IJzerman.

In situ bioassays

After exposure at the study sites for a certain period of time, the organisms are recollected and their condition can be analysed (Figure 2). The endpoint is mostly survival, especially in routine monitoring programs. If the in situ exposure lasts long enough, also effects on species specific sublethal endpoints can be assessed. For daphnids and snails, this is reproduction and for isopods growth. For aquatic insects (mayflies, caddisflies, damselflies, chironomids), emergence has been assessed as a sensitive ecological relevant endpoint (Barmentlo et al., 2018).

Figure 2. In situ exposure experiment. (A). Preparing damselfly containing jars. (B) Exposure of the in situ jars in ditches. (C). Retrieved jar containing a single damselfly larva. (D). Close up of the damselfly larva ready for inspection. Photos by Henrik Barmentlo.

In situ bioassays come closest to the actual field situation. Organisms are directly exposed at the site of interest and respond to all joint stressors present. Yet, this is also the limitation of the approach. If organisms do respond it remains unknown what causes the observed adverse effects. This could be (the combination of) any natural or anthropogenic physical or chemical stress factor. In situ bioassays can therefore be best combined with laboratory bioassays (see section on Bioassays) and the analysis of physico-chemical parameters, conform the TRIAD approach (see section on TRIAD approach). If the adverse effects are also observed in the bioassays under controlled laboratory conditions, then poor water quality is most likely the cause. The water sample may then be subjected to suspected target analysis, non-target analysis or effect directed analysis (EDA). If adverse effects are observed in situ but not in the laboratory, then the presence of hazardous compounds is most likely not the cause. Instead, the effects may be attributable to e.g. low pH, low oxygen concentrations, high temperatures etc, which may be verified by physico-chemical analysis in the field.

Online biomonitoring

A specific application of in situ bioassays are the online systems for continuous water quality monitoring. In these systems, behaviour is generally the endpoint (see section on Endpoints). Organisms are exposed in a laboratory setting in situ (on shore or on a boat) in an experimental device to a continuous flow of surface water. If the water quality changes, the organisms respond by changing their behaviour. Above a certain threshold an alarm may go off and, for instance, the intake of surface water for drinking water preparation can be temporarily stopped.

Contaminant concentrations in organisms

As an addition or as an alternative to analysing the condition of the exposed biomonitoring organisms upon retrieval, contaminant concentrations in organisms can be analysed. This has several advantages over chemical analysis of environmental samples: biomonitoring organisms may be exposed for days to weeks at the site of interest, providing time integrated measurements of contaminant concentrations, in contrast to the chemical analysis of grab samples. This way, biomonitoring organisms actually serve as ‘biological passive samplers’ (see to section on Experimental methods of assessing available concentrations of organic chemicals). Another advantage of measuring contaminant concentrations in organisms is that they only take up the bioavailable (fraction of) substances, ecologically very relevant information, that remains unknown if chemical analysis is performed on water, sediment, soil or air samples. Yet, elevated concentrations in organisms do not necessarily imply toxic effects, and therefore these measurements are best complemented with determining their condition, as described above. Moreover, analysing contaminants in organisms may be more expensive than measurements of environmental samples, due to a more complex sample preparation. Weighing the advantages and disadvantages, the explicit strength of biomonitoring programs is that they provide insight into the spatial and temporal variation in bioavailable contaminant concentrations. In Figure 3 two examples are given. The left panel shows the concentrations of PCBs in zebra mussels at different sampling sites in Flanders, Belgium (Bervoets et al., 2004). The right panel shows the rapid (within 2 wk) Cd accumulation and depuration in biofilms translocated from a reference to a polluted site and from a polluted to a reference site, respectively (Ivorra et al., 1999).

Figure 3. Left panel: Mean concentration of PCBs in 25 pooled zebra mussels at different sampling sites in Flanders, Belgium. Comparison between indigenous (black bars) and transplanted mussels (grey bars), from Bervoets et al. (2004). Right panel: Cd concentrations in local and translocated biofilms (R: Reference site; P: polluted site) from Ivorra et al. (1999). Drawn by Wilma IJzerman.

References

Barmentlo, S.H., Parmentier, E.M., De Snoo, G.R., Vijver, M.G. (2018). Thiacloprid-induced toxicity influenced by nutrients: evidence from in situ bioassays in experimental ditches. Environmental Toxicology and Chemistry 37, 1907-1915.

Bervoets, L., Voets, J., Chu, S.G., Covaci, A., Schepens, P., Blust, R. (2004). Comparison of accumulation of micropollutants between indigenous and transplanted zebra mussels (Dreissena polymorpha). Environmental Toxicology and Chemistry 23, 1973-1983.

Blanck, H. (1985). A simple, community level, ecotoxicological test systemusing samples of periphyton. Hydrobiologia 124, 251-261.

Ivorra, N., Hettelaar, J., Tubbing, G.M.J., Kraak, M.H.S., Sabater, S., Admiraal, W. (1999). Translocation of microbenthic algal assemblages used for in situ analysis of metal pollution in rivers. Archives of Environmental Contamination and Toxicology 37, 19-28.

Stuijfzand, S.C., Engels, S., Van Ammelrooy, E., Jonker, M. (1999). Caddisflies (Trichoptera: Hydropsychidae) used for evaluating water quality of large European rivers. Archives of Environmental Contamination and Toxicology 36, 186-192.

Vuori, K.M. (1995). Species- and population-specific responses of translocated hydropsychid larvae (Trichoptera, Hydropsychidae) to runoff from acid sulphate soils in the River Kyronjoki, western Finland. Freshwater Biology 33, 305-318.