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Mesocosm experiments in the approval procedure for pesticides

Summary and conclusions

Objective

Based on a critical analysis of already published results of mesocosm experiments, the objective of the project was to elaborate a checklist for evaluating mesocosms in connection with the approval procedure.

Methods

The checklist has been elaborated on the basis of

1. a thorough examination of existing literature,
2. a critical review of investigations based on objective criteria,
3. construction of a database containing all relevant data,
4. statistical analyses elucidating the effects of pesticides on the various groups of organisms, the influence of mesocosm system characteristics on pesticide impact, etc.

Two different approaches have been applied in the study. We have used a multivariate statistical method (PLS, partial least squares) to examine relationships between toxic effects of pesticides and system characteristics such as mesocosm design, season and location of study. This analysis has been carried out at a rather high level of taxonomy and organism functionality. These analyses have been supplemented by more detailed analysis using traditional statistics to examine differences in sensitivity, potentials of recovery etc. within taxonomic groups.

Database

Selected studies were entered into a database provided that they met certain criteria for documentation and quality. The generated database is based on 112 publications and includes 91 experiments covering 3,635 effect concentrations for 31 different pesticides. Of a total of 3,635 effect concentrations 410 focus on flow-through systems. The majority of the effect concentrations are on zooplankton, followed by effects on macroinvertebrates, phytoplankton and periphyton.

The database encompasses mesocosm studies with 8 different herbicides (2,4-D, Alachlor, Atrazine, Glufosinate-ammonium, Glyphosate, Hexazinone, Linuron, Triclopyr-ester), 22 insecticides (Aminocarb, Azinphos-methyl, Bifenthrin, Carbaryl, Carbofuran, Chlorpyrifos, Cyfluthrin, Deltamethrin, Diazonon, Diflubenzuron, Dimethoate, Endosulfan, Esfenvalerate, Fenitrothioun, Fenvalerate, Lambda-cyhalothrin, Lindan, Methoxychlor, Mexacarbate, Permethrin, Tebufenozide, Tralomethrin) and 1 fungicide: Propiconazole.

Relationships between toxic effect of pesticides and system characteristics - PLS (partial least squares)

PLS is a regression technique that is used to describe the relationship between two sets of variables, X: system characteristics (season, mesocosm size, single species toxicity, log K_ow) and Y: toxic effect to each group in the mesocosm. Each substance thus makes up an observation, and the various physical-chemical characteristics and the toxic effects on the various test organisms function as individual variables.

Separate PLS models were developed for macroinvertebrates, zooplankton and micro algae (periphyton and phytoplankton). For all communities the lowest effect concentrations observed for each functional or taxonomic group in each mesocosm experiment were used as Y variables, expressing the toxic response of the organisms in the mesocosms.

When appropriate PLS models are developed it is possible to use the models for prediction of effect concentrations for the organisms in the mesocosms and to associate the predicted effect concentrations with for instance a 95 % confidence interval. The PLS models even allow the effect concentrations with associated confidence interval to be predicted for experiments where toxicity data for certain groups of organisms were missing. Since the PLS models are based on all the appropriate data in the database it is thus possible to develop en evaluation procedure taking all the available information into account, rather than basing the evaluation on a restricted use of a single or a few mesocosm experiments for each pesticide. Thus with the aid of the PLS models it is possible to evaluate all mesocosm experiments with pesticides on a common basis.

The amount of data available for the different communities was quite variable, and a direct comparison of PLS models should therefore be conducted with caution. However, important conclusions are

Macroinvertebrates

To obtain a PLS model with a reasonable predictability of the toxic effects to various macroinvertebrate groups, mesocosms should contain sediment and preferably macrophytes in the test system. Overall, the model developed was able to predict 63 % of the observed effects among macroinvertebrates.

In summary, the PLS analysis showed that

1. All macroinvertebrate groups in the mesocosms seem to be most sensitive when the experiments are conducted at high latitudes. Therefore, toxic effects at lower concentrations are expected with increasing distance from Equator, which may be due to a slower turn-over of populations at high latitudes, i.e. fewer generations each year at lower temperatures. Therefore, recovery of populations affected by pesticide exposure takes longer time at northern latitudes.
2. Macroinvertebrates living within the sediment (i.e. infauna) were less sensitive the pesticides than macroinvertebrates living on the sediment surface.
3. At a given total dose the effect of pesticides decreases with number of pesticide additions. Therefore, a low but persistent pesticide concentration will have a lower effect on the macroinvertebrates than a high but temporary pesticide concentration.
4. The toxic effects of pesticides are most pronounced in shallow mesocosms. At decreasing mesocosm depth a larger fraction of the pesticides will end up in the sediment compartment and thus increase the exposure to the sediment living macroinvertebrates. This interpretation is further reinforced by the inverse relation between Log K_D of pesticides and toxicity to invertebrates.

Zooplankton

The PLS model with the highest predictability for zooplankton was obtained when the pesticides were applied as single addition and the analysis was restricted to insecticides only.

The PLS analysis showed that

Microalgae

The highest predictability of pesticide effects to microalgae was obtained when only field mesocosm experiments were included in the analysis.

The PLS analysis showed that

1. Hydrophobic and adsorpable pesticides with high single species toxicity were the most toxic to the micro algae in the mesoscosmos.
2. At a given total dose pesticides added over a short period were more toxic to the algae in mesocosmos than pesticides dosed at longer intervals. Frequent dosings will prevent microalgae to recover, while microalgae characterised by short generation times will be able to recover in between dosings applied at longer intervals.

Comparison of sensitivity among different groups of organisms

Zooplankton

Direct effects of insecticides on zooplankton were examined and quantified by relating the dosing of insecticides to changes in abundance relative to corresponding controls (without insecticide dosing). For comparison the average decrease in abundance within the period 3-14 days after the first application of insecticide was used.

Zooplankters are very sensitive to insecticide exposure. At the group level:

1. Cladocerans and Chaoborus are the most sensitive followed by copepod nauplii and adult copepods.
2. At a given concentration the cladoceran population on average will show larger reductions (20 %) than the copepod population.
3. Copepod nauplii on average will show 10 % larger reductions than the adult population. Observed reductions in one group are a very good predictor of the reductions the other group.

The variation in sensitivity within each zooplankton group as demonstrated in mesocosm studies is considerable. For esfenvalerate LOEC varied 2.5 orders of magnitude for the different species among cladocerans. This variation is probably related to the size of the different species, their habitat and/or feeding mode.

Recovery within zooplankton was dependent on the maximal impact by insecticides on the population. For cladocerans the time elapsed for full recovery after the insecticide dosage varied between 10 and 120 days. In mesocosm experiments where cladocerans had been reduced severely (i.e. > 95 %) it took more than 12-15 weeks for full recovery. At reductions below 80 % of the initial population size recovery was fast, less than 20 days. Still, even at population reductions close to 100 % full recovery of cladocerans was observed in all mesocosms (where the length of observation period was sufficient). For copepods an almost identical relation between initial decrease and recovery was obtained.

Indirect effects of insecticides on plankton communities

The most prominent indirect effect of insecticides in the plankton community includes increases in phytoplankton and rotifers. Following a decrease in population size of crustacean zooplankton, phytoplankton biomass generally will increase due to relaxation of grazing control. In addition, planktonic rotifers that are less sensitive to insecticides will increase in abundance due to increased food availability and reduced competition from crustacean zooplankton. Generally, low impacts on the crustacean zooplankton will not result in increased growth of phytoplankton. If however, zooplankton becomes reduced by more than 50 % dramatic increases in phytoplankton (>100 %) must be expected.

The indirect effects of insecticides on the plankton community are at least as sensitive as direct effects, e.g. a 75 % reduction in crustacean zooplankton on average will be followed by a 500 % increase in rotifer abundance and a 200 % increase in phytoplankton biomass. However, indirect effects are very variable in both magnitude and direction and thus less robust compared to direct effects.

Macroinvertebrates

In the data base direct effects of insecticides on macroinvertebrates were examined and quantified by relating the dosing of insecticides to changes in abundance relative to corresponding controls (without insecticide dosing). For comparison the average decrease in abundance within the period 28-56 days after the first application of insecticide was used. The sensitivity of alternative end-points such as increase in drift in artificial streams and emergence of imago insects was compared to sensitivity of abundance.

The analysis showed that:

1. The sublethal effects drift in stream macroinvertebrates generally appears to be a more sensitive endpoint than changes in as abundance.
2. The endpoint emergence of adult insects generally is as sensitive as changes in abundance of larvae. Insecticides may increase the mortality of larvae and reduce growth rate. In effect, emergence will decrease or be delayed.
3. The insect order Tricoptera consistently was the most sensitive macroinvertebrate group to insecticides, followed by Plecoptera/Hemiptera/Ephemeroptera/Coleoptera/Amfipoda/Isopoda (no particular order). Chironomidae as a very diverse group (individual size, mode of feeding etc.) showed a rather large variation in sensitivity (1-2 orders of magnitude). Odonata and Gastropoda consistently were the groups with the lowest sensitivity to insecticides.

In macroinvertebrates recovery may take place by invasion from non-affected populations (e.g. by drift in streams, reproduction from insects) and reproduction by surviving individuals. In order to evaluate recovery mesocosm studies need to be carried out in the field (to allow flying insects to lay eggs) and should at the minimum extend a full life cycle length of the organisms studied after insecticide dosage. Very few studies in the database fulfilled the criteria. Chironomids and Isopoda were the most important taxonomic groups in the "slight recovery group" whereas Chironomids and Ephemeropterans dominated the "moderate recovery group". Both Chironomids and Ephemeropterans in general are considered as good colonisers with short life cycles and this probably explains why they show the most rapid recovery.

Comparison of extrapolated Hazard Concentrations and Observed Effects in mesososms

Only a limited number of "high quality" mesocosm experiments 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. The mostly used calculation of hazard concentration, HC_5,50% aim to protect 95% of the organisms in an ecosystem with a 50% probability. A alternative approach adopted by the OECD procedure by multiplying the lowest LC(EC)50 observed among all standardised tests by 0.1 (application factor of 10).

To compare the "validity" of extrapolated Hazard Concentrations in protecting complex ecosystems we used the ratio HC_5,50/LOEC or OECD/LOEC. In 14 out of 66 experiments the widely used approach failed to protect all organisms in the ecosystem. Even using the more conservative OECD approach the hazard concentration failed to protect the organisms in 6 experiments. In about half of the experiments where HC_5,50/LOEC exceeded 1, NOEC could not be established for the most sensitive parameter, hence the ratio HC_5,50/LOEC calculated for these experiments represent a minimum.

The vast majority of examples of "failures" of extrapolated hazard concentrations were found in experiments, where LOECs were recorded for macroinvertebrates and insects, while LOECs for phytoplankton and zooplankton except for two occasions occurred in experiments with ratios HC_5,50/LOEC well below 1. 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 was further demonstrated by an ANOVA. If macroinvertebrates were monitored in mesocosms the risk that extrapolated hazard concentrations would fail to protect the whole ecosystem would be substantial.

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