## 6.4.3. Effect-based monitoring: In vivo bioassays

Effect based monitoring: in vivo bioassays

Authors: Michiel Kraak, Carlos Barata

Reviewers: Kees van Gestel, Jörg Römbke

Learning objectives:

You should be able to:

- define in vivo bioassays and to explain how in vivo bioassays are performed.
- give examples of the most commonly used in vivo bioassays per environmental compartment.
- motivate the necessity to incorporate several in vivo bioassays into a bioassay battery.

Key words: risk assessment, diagnosis, effect based monitoring, in vivo bioassays, environmental compartment, bioassay battery

Introduction

To determine whether organisms are at risk when exposed to hazardous compounds present at contaminated field sites, the toxicity of environmental samples can be analysed. To this purpose, several diagnostic tools have been developed, including a wide variety of in vitro, in vivo and in situ bioassays (see sections on In vitro bioassays and on In situ bioassays). In vivo bioassays make use of whole organisms (in vivo means “while living”). The species selected as test organisms for in vivo bioassays are generally the same as the ones selected for single species toxicity tests (see sections 4.3.4, 4.3.5, 4.3.6 and 4.3.7 on the Selection of ecotoxicity test organisms). Likewise, also the endpoints measured in in vivo bioassays are the same as those in single species ecotoxicity tests (see section on Endpoints). I n vivo bioassays therefore have a relatively high ecological relevance, as they provide information on the survival, reproduction, growth, or behaviour of the species tested. A major difference between toxicity tests and bioassays is the selection of the controls. In laboratory toxicity experiments the controls consist of non-spiked ‘clean’ test medium (see section on Concentration response relationships). In bioassays the choice of the controls is more complicated though. Non-treated test medium may be incorporated as a control in bioassays to check for the health and quality of the test organisms. But control media, like standard test water or artificial soil and sediment may differ in numerous aspects from natural environmental samples. Therefore, the control should preferably be a test medium that has exactly the same physicochemical properties as the contaminated sample, except for the chemical pollutants being present. This ideal situation, however, hardly ever exists. Hence, it is recommended to also incorporate environmental samples from less or non-contaminated reference sites into the bioassay and to compare the response of the organism to samples from contaminated sites with those from reference sites. Alternatively, controls can be selected as the least contaminated environmental samples from a gradient of pollution or as the dilution required to obtain no effect. As dilution medium artificial control medium can be used or medium from a reference site.

The most commonly used in vivo bioassays

For the soil compartment, the earthworms Eisenia fetida, E. andrei and Lumbricus rubellus, the enchytraeid Enchytraeus crypticus and the collembolan Folsomia candida are most frequently selected as in vivo bioassay test organisms. An example of employing earthworms to assess the ecotoxicological effects of Pb contaminated soils is given in Figure 1. The figure shows the total Pb concentrations in different field soils taken from a soccer field (S), a bullet plot (B), grassland (G1, G3) and a forest (F1-F3) site near a shooting range. The pH of the grassland soils was near neutral (pHCaCl2 = 6.5-6.8), but the pH was rather low (3.2-3.7) for all other field sites. Earthworms exposed to these soils showed a significantly reduced reproductive output (Figure 1) at the most contaminated sites. At the less contaminated sites, earthworm responses were also affected by the difference in soil pH, leading to low juvenile numbers in the acid soil F0 but high numbers in the near neutral reference R3 and the field soil G3. In fact, earthworm reproduction was highest in the latter soil, even though it did contain an elevated concentration of 355 ± 54 mg Pb/kg dry soil. In soil G1, which contained almost twice as much Pb (656 ± 60 mg Pb/kg dry soil), reproduction was much lower and also reduced compared to the control, suggesting the presence of additional, unknown stressor (Luo et al., 2014).

Figure 1. Reproduction of the earthworm Eisenia andrei after 4 weeks of exposure to control soils (LF2.2, R1, R2, R3) and field soils (S, B0, G1, G3, F0, F1, F3) from a Pb pollution gradient near a shooting range. Shown are the mean relative numbers of juveniles ± SD (n=4-5), compared to the control Lufa 2.2 (LF2.2) soil, as a function of average total Pb concentrations in the soils. Data from Luo et al. (2014).

For water, predominantly daphnids are employed, mainly Daphnia magna, but sometimes also other daphnid species or other aquatic invertebrates are selected. Also bioassays with several primary producers are available. An example of exposing daphnids (Chydorus sphaericus) to water samples is shown in Figure 2. The bars show the toxicity of the water samples and the diamonds the concentrations of cholinesterase inhibitors, as a proxy for the presence of insecticides. The toxicity of the water samples was higher when also the concentrations of insecticides were higher. Hence, in this case, the observed toxicity is well explained by the measured compounds. Yet, it has to be realized that this is an exception rather than a rule, since mostly a large portion of the toxic effects observed in surface waters cannot be attributed to compounds measured by water authorities and moreover, interactions are also not covered by such analytical data (see section on Effect based water quality assessment).

Figure 2. Toxicity of water samples to daphnids (Chydorus sphaericus)(bars) and the concentrations of cholinesterase inhibitors, as a proxy for the presence of insecticides (diamonds). Data from Pieters et al. (2008).

For sediments, oligochaetes and chironomids are selected as test organisms, but sometimes also rooting macrophytes and benthic diatoms. An example of exposing chironomids (Chironomus riparius) to contaminated sediments is shown in Figure 3. Whole sediment bioassays with chironomids allow the assessment of sensitive species-specific sublethal endpoints (see section on Chronic toxicity), in this case emergence. Figure 3 shows that more chironomids emerged on the reference sediment than on the contaminated sediment and that the chironomids on the reference sediment also emerged faster than on the contaminated sediment.

Figure 3. Emergence of chironomids (Chironomus riparius) on a reference (blue line) and a contaminated sediment (red line). Data from Nienke Wieringa.

For sediment, also benthic diatoms are selected as in vivo bioassay test organisms. Figure 4 shows the growth of the benthic diatom Nitzschia perminuta after 4 days of exposure to 160 sediment samples. The dotted line represents control growth. The growth of the diatoms ranged from higher than the controls to no growth at all, raising the question which deviation from the control should be considered a significant adverse effect.

Figure 4. Growth of the benthic diatom Nitzschia perminuta after 4 days of exposure to 160 sediment samples. The dotted line represents control growth. Data from Harm van der Geest.

In vivo bioassay batteries

Environmental quality assessments are often performed with a single test species, like the four examples given above. Yet, toxicity is species and compound specific and this may therefore result in large margins of uncertainty in the environmental quality assessments, consequently leading to over- or underestimation of environmental risks. Obvious examples include the presence of herbicides that only would induce responses in bioassays with primary producers and the other way around, the presence of insecticides that induces strong effects on insects and to a lesser extent on other animals, but that would be completely overlooked in bioassays with primary producers. To reduce these uncertainties and to increase ecological relevance it is therefore advised to incorporate more test species belonging to different taxa in a bioassay battery (see section on Effect based water quality assessment).

References

Luo, W., Verweij, R.A., Van Gestel, C.A.M. (2014). Determining the bioavailability and toxicity of lead to earthworms in shooting range soils using a combination of physicochemical and biological assays. Environmental Pollution 185, 1-9.

Pieters, B.J., Bosman-Meijerman, D., Steenbergen, E., Van den Brandhof, E.-J., Van Beelen, P., Van der Grinten, E., Verweij, W., Kraak, M.H.S. (2008). Ecological quality assessment of Dutch surface waters using a new bioassay with the cladoceran Chydorus sphaericus. Proceedings Netherlands Entomological Society Meetings 19, 157-164.