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Mesocosm experiments in the approval procedure for pesticides

7 Comparison of extrapolated hazard concentration and observed effects in mesocosms

As shown in this study only a limited number of "high quality" mesocosm experiments (see Chapter 4 for selection criteria) examining the effects of pesticides in freshwater systems have been reported. As a consequence, an alternative approach using the results from numerous standardised single species tests has been developed. Hazard concentrations for ecosystems may be calculated from distribution-based extrapolation of single species toxicity data (EC50, LC50) using (slightly) different statistical methods (e.g. Wagner & Løkke 1991, Miljøstyrelsen 1994, Emans 1994). The widely used calculation of hazard concentration, HC_5,50% aims to protect 95% of the organisms in an ecosystem with a 50% probability. Others consider that a 90% protection of ecosystem species is adequate to avoid adverse effects on the natural ecosystem, i.e. HC_10,50% (Hall et al., 1998). A alternative approach adopted by OECD multiplies the lowest effect concentration observed among all standardised tests by 0.1 (application factor of 10) (see Chapter 4 for more details).

The major limitation of these approaches is the availability of single species test results, as they are biased towards dominance of cladocerans, planktonic algae and fish. Thus, data on effect concentrations of insect larvae are scarce or not available for several pesticides (see Table 3 and Annex 1). For macrophytes, no standardised test results were available for the pesticides included in the data base! As standardised tests usually are short-term (48-96 hours) they may fail to reveal long-term effects caused by pesticides accumulated in organisms. And relying solely on standardised single species tests’ extrapolation methods will never be able to account for behavioural effects and interactions between populations and trophic groups (i.e. indirect effects).

In Table 20 we have summarised a comparison of extrapolated hazard concentrations and the lowest observed effect concentrations in the mesocosm experiments contained in the data base. An extended version of the comparison including 66 mesocosm experiments is shown in Annex 3. Within single mesocosm experiments LOECs for different organisms can vary 2-3 fold (see Chapter 6 & 7), hence LOEC for one group can be NOEC for several others groups. Still, we have selected the lowest observed effect concentrations within an Order, genus or species and tested effects for significance (i.e. persistence) (see chapter 5).

We have used the ratio HC_5,50/LOEC or OECD/LOEC as a measure of the success of the extrapolated hazard concentration (HC_5,50 or OECD) to "protect the species" in an aquatic ecosystem. Table 20 only includes experiments, where the ratio HC_5,50/LOEC is above 1. With exception of experiment 57flm (that included 25 different taxons) the experiments in Table 20 included less than 20 taxons (range 1-13). Hence, in 13 out of 66 experiments the widely used approach failed to protect 95 % organisms in the ecosystem (see Annex 3). Even using the more conservative OECD approach the hazard concentration failed to protect 95% of the organisms in 5 experiments. In about half of the experiments contained in Table 20 (i.e. 8 experiments) NOEC was not be established for the most sensitive parameter, because sufficient low concentrations were not tested. Hence the ratio HC_5,50/LOEC calculated for these experiments represents a minimum.

It is noticeable, that the vast majority of examples of "failures" of extrapolated hazard concentrations to protect sensitive species are found in experiments, where LOECs were recorded for macroinvertebrates and insects, while LOECs for phytoplankton and zooplankton except for two occasions (see Table 20) result in ratios of HC_5,50/LOEC well below 1 (see Annex 3). Therefore, extrapolated hazard concentrations generally will protect the plankton environment in ecosystems, which hardly is surprising as the extrapolated values primarily rely on standardised tests with cladocerans and phytoplankton. On the other hand, extrapolated hazard concentrations are much less successful in protecting the macroinvertebrate community.

The importance of including macroinvertebrates in mesocosm experiments is further demonstrated by an ANOVA, where the "failure" of extrapolated hazard concentrations (HC_5,50) to protect the aquatic ecosystem was explained by number of organism groups monitored, inclusion of macroinvertebrates and the number of insecticide doses (Table 21).

Intuitively, one would expect that the chance of "failure" would increase with increasing number of organism groups monitored and when the insecticide was dosed several times. In the analysis neither variable was important. However, if macroinvertebrates were monitored in mesocosms the risk that extrapolated hazard concentrations would fail to protect the whole ecosystem was substantial (significance level – p = 0.017) (see Table 21). Average ratio HC_5,50/LOEC in experiments with macroinvertebrates was 9.0 (1.10 if the high value of 80 in exp. 76tll was omitted) but much lower at 0.26 in experiments without macroinvertebrates.

Table 20.
Comparison of extrapolated hazard concentrations and the lowest observed effect concentrations in the mesocosm experiments. NOEC: Yes = lowest test concentration were lower than the lowest effect concentration observed in mesocosm; No = effect was observed at the lowest test concentration applied. HC_5,50/LOEC = ratio between extrapolated hazard concentration (see Table 1) and lowest observed effect concentration. OECD/LOEC = ratio between hazard concentration (OECD₁₀ approach) and lowest observed effect concentration. HC_5,50/low = ratio between hazard concentration and the lowest test concentration applied. OECD/low = ratio between extrapolated hazard concentration (OECD₁₀ approach) and the lowest test concentration applied. Observed effects at lowest concentration: ß decrease (mostly in abundance);
Ý increase.

* Only significant effects included (see chapter 4).

** 2,4 D are toxic to higher plants only, while extrapolated hazard concentrations were based on single species test with algae and zooplankton, only.

The failure of extrapolated hazard concentrations in protecting 95% of organisms and especially macroinvertebrates, against insecticides in the aquatic environment probably occurs because

	macroinvertebrates are the most sensitive organisms to insecticide exposure, probably related to the high Kd of most insecticides. Hence, under natural conditions the exposure of sediment-dwellers will be higher than plankters.
	macroinvertebrates are underrepresented in the single-species tests used for extrapolation of hazard concentrations
	the duration of standardised single species tests is too short to reveal the potential effects on macroinvertebrates as the maximum effects on macroinvertebrates are recorded 2-8 weeks after exposure start in mesocosm experiments.

Table 21.
Result of 1-way ANOVA for effect of number of organism groups (phytoplankton, periphytes, macrophytes, zooplankton, macroinvertebrates, fish) monitored, inclusion of macroinvertebrates (yes, no) and number of insecticide doses (1-10) during the experiment on the ratio HC_5,50/LOEC (HC_5,50/LOEC >1 ® value = failure; HC_5,50/LOEC <1 ® value = success). The analysis was restricted to experiments with insecticids, where NOEC was recorded for the most sensitive organism (n=23). Mean squared effect, mean squared error, F statistics and level of significance shown.

In conclusion, long-term (abundance and emergence) and short-term effects (drift in streams) of insecticides on macroinvertebrates are among the most sensitive effect parameters recorded in mesocosms. Such effects cannot be explained in sufficient detail by extrapolations based on calculations of Hazard Concentrations from standardised single species test. Therefore, the data bases used for extrapolation ought to be extended with tests on macroinvertebrates and preferentially the duration of these test should be increased. Alternatively, mesocosm experiments should be carried out. To arrive at environmentally realistic effect concentrations and protect the whole ecosystem, mesocosms need to include a benthic compartment encompassing a diverse fauna including important and sensitive taxonomic groups such as Tricoptera, Ephmeroptera and Amphipoda.

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