Effekter af cypermethrin, azoxystrobin og bentazon på limniske invertebrater

Effects of Cypermethrin, Azoxystrobin and Bentazone on Limnic Invertebrates

Summary and conclusions

PURPOSE

Pesticides are found in almost all investigated Danish lakes and ponds. Measurements have shown that the concentrations are modest compared with known lethal effect concentrations, so to evaluate whether impacts can be expected to occur under the present conditions it is of essential importance to focus on the sublethal effects. Hence, the purpose of the project has been, using concentration-response experiments and subsequent sublethal effect experiments, to evaluate sublethal effects on a number of limnic invertebrates, including 4 typical zooplankton species: Daphnia magna, D. galeata, Eudiap-tomus graciloides and Cyclops vicinus, and 4 macroinvertebrates: Chaoborus flavicans, Chironomus plumosus, Cloeon dipterum-gr. and Hydropsyche angusti-pennis.

Methods

The immediate effects of pesticides on experimental organisms were investigated via concentration-response experiments with immobility as effect parameter. The sublethal effects were studied by means of i) physiological, ii) reproduction, and iii) swimming behaviour experiments. Thus, the physiological effects were investigated via analyses of video recordings of a fixed and an exposed animal to observe for effects on antennae, pectoral limbs, heart and hind claw. The reproduction experiments were undertaken using, respectively, egg and nauplii development time and clutch size as effect parameters, while swimming behaviour investigations were made using swimming velocity as effect parameter.

Pesticides

In the project the pyrethroid-insecticide ”cypermethrin”, the strobilurin-fungicide ”azoxystrobin” and the triazin-herbicide ”bentazone” were used. These pesticides were selected in consultation with the Danish Environmental Protection Agency on the basis of the present knowledge of use, activity and effects of pesticides on the aquatic environment. Cypermethrin acts as a neu-rotoxin, influencing sodium-channels of the nervous system of insects, azox-ystrobin inhibits mitochondrial respiration and thus the formation of ATP in eukaryote cells, and bentazone is an electron transport inhibitor blocking the photosynthesis of weeds. Generally, nominal concentrations were used in the experiments, but twice during the project period samples were taken for analyses of actual concentrations. The samples were analysed using gas chromatography coupled with mass spectrometry (GC-MS) and fluid chromatography coupled with mass spectrometry (LC-MS) by the company Miljøkemi a/s (now EUROFINS).

Sample survey

First, concentration-response experiments were made for cypermethrin and azoxystrobin on 7 of the 8 experimental organisms. Bentazone was only tested on D. magna. Based on the concentration-response results, experiments were undertaken at sublethal concentrations. As for cypermethrin and azoxystrobin the reproduction experiments were made for the 4 zooplankton species. Effects on physiology using cypermethrin and azoxystrobin were investigated as for D. magna. Using data from the physiology investigations, effect experiments of cypermethrin and azoxystrobin were made of D. magna swimming behaviour.

Results

The concentration-response experiments using cypermethrin showed that D. magna was the most tolerant test organism, E. graciloides and D. galeata being the most sensitive zooplankton species. Overall, the May fly nymph C. dip-terum-gr. was the most sensitive organism. A similar pattern was found as to that of azoxystrobin; D. magna being the most tolerant and E. graciloides and D. galeata the most sensitive zooplankton organism. H. angustipennis was generally the most tolerant of the tested species. For both cypermethrin and azox-ystrobin neonate D. magna were more sensitive than adult D. magna, and D. galeata the laboratory culture was more sensitive than the field culture. Benta-zone was only tested for D. magna and E. graciloides, and effects were only found at very high concentrations (> 3000 × worst case situation), and, for the same reason, no further experiments were made with bentazone.

The reproduction experiments with cypermethrin showed that the eggs of egg-laying zooplankton developed into hatched nauplii. As to egg development time and development of live nauplii, D. galeata turned out to be the most sensitive of the tested zooplankton, while C. vicinus was the most tolerant species. When using a different effect parameter, e.g. clutch size, C. vici-nus was as sensitive as the Daphnia species. Thus, the most sensitive effect parameter need not be the same for all species. The experiments with azox-ystrobin generally showed that the development from egg into nauplii was hampered for Daphnia species and when using this as an effect parameter, D. galeata appeared to be more sensitive than D. magna. When including other effect parameters E. graciloides turned out to be the most sensitive zooplankton species. Overall, D. magna was the most tolerant species.

The physiological experiments with cypermethrin and D. magna showed that the hind claw was the most sensitive response parameter, the most tolerant parameter being the mandibles. Over time the pectoral limb activity changed most significantly, while heart activity changed the least. For all parameters the maximum effect of the pesticide occurred within 3 hours, but already after 15 minutes of exposure to > 1 µg cypermethrin l^-1 effects could be observed on pectoral limbs, mandibles and hind claw. Exposure of azoxystrobin to D. magna led to reduced activity of all 4 response parameters. Pectoral limb activity was the most sensitive parameter relative to azoxystrobin exposure, hind claw activity being the most tolerant parameter. Mandible activity changed most significantly over time, while the hind claw activity changed the least.

The physiological experiments clearly demonstrated that cypermethrin and azoxystrobin effect zooplankton in different ways. For instance, there was a tendency to increasing activity of response parameters at increased cyperme-thrin concentrations, while the tendency was opposite at increased azoxystro-bin concentrations.

Conclusions

The most frequently applied zooplankton test organism in eco-toxicological tests is D. magna. In this project E. graciloides and D. galeata proved to be significantly more sensitive than D. magna when exposed to cypermethrin and azoxystrobin. In connection with the approval procedure for a pesticide such as cypermethrin, it cannot on the background of the toxicological tests undertaken on D. magna be concluded that no effects will be seen on the zooplankton community in a given freshwater system unless a safety factor > 100 is used. Despite a marked difference in the size of organisms, the concentration-response experiments showed that also the macroinvertebrates C. plumosus and C. flavicans (mosquito larvae) and C. dipterum-gr. (May fly nymph) were more sensitive than D. magna when exposed to cypermethrin.

On the background of the pesticide measurements made in Danish lakes and ponds and the effect results obtained in this project, it seems unlikely that bentazon and azoxystrobin should have an impact on the zooplankton community. However, if a field sprinkler holding azoxystrobin passes over or partly over a pond, a concentration impeding Daphnia reproduction can be reached (33 – 83 µg azoxystrobin l^-1). A cypermethrin concentration of, say, 0.5 µg l^-l (which is realistic in a Danish freshwater system) will negatively influence the food intake, reproduction and swimming capacity of D. magna. In addition it will influence the survival and reproduction of D. galeata as well as the survival of the tested macroinvertebrates.