Pesticides Research, 84 – The Effects of Selected Pyrethroids on Embryos of Bombina bombina during different Culture and Semi-field Conditions

The Effects of Selected Pyrethroids on Embryos of Bombina bombina during different Culture and Semi-field Conditions

8 Discussions

8.1 The use of amphibians as an ecotoxicological test organism
8.2 Development in the controls and in the sprayed pond

8.1 The use of amphibians as an ecotoxicological test organism

FETAX was first developed by Dumont et al. (1983) and is useful for screening for the potential developmental toxicity of both single chemicals, and complex chemical mixtures (Bantle et al., 1991) on embryos of Xenopus laevis. Recently, a new test guideline, related to FETAX with the fire-bellied toad Bombina bombina, was developed (Larsen & Sørensen, 2004).

Besides using Bombina bombina embryos instead of Xenopus laevis embryos, the new guideline differs by not removing the jelly coat before the start of the test. Previous experiments indicate that the removal of the jelly coat did not affect the development of the embryos (Larsen & Sørensen, 2004). However, since the jelly coat may influence the transport and exposure to toxic compounds from the water to the embryos it was chosen not to remove the jelly coat in the new guideline. Tests where the jelly coat is intact may, other things being equal, be more ecological relevant.

Standard toxicity tests where chemical substances are tested for toxicity on one organism at a time have been criticized for not being realistic, since they are carried out in a laboratory under artificial conditions. In the present study, effects of four pyrethroids, applied to an artificial pond, on caged embryos of Bombina bombina, were studied for the purpose of examining the developmental toxicity effects under more realistic conditions and to establish a test protocol for using Bombina bombina in semi-field investigations.

8.2 Development in the controls and in the sprayed pond

The development of the embryos of Bombina bombina in the control pond corresponded to the embryo development in the laboratory control in which the embryos were kept under constant conditions. The develop-ment under these conditions from embryo to larvae stage took in both experiments nine days. Since the mortality of the embryos in the control was rather low, 5 and 10 % in the pond and in the laboratory, respectively, and the appearances of malformations were few, 9 and 5%, respectively, the development of the embryos was comparable with the two controls. It can therefore be concluded that the test ponds were very suitable for out-planting Bombina bombina embryos.

The pesticides applied

Four different pesticides were sprayed simultaneously on the surface of the test pond. This procedure does not mimic normal agricultural practice where usually only one pesticide is sprayed at the time. However, the present study was only a minor part of a large project where the objective was primarily to study the general fate of the pesticides and to generate data for calibration of a distribution model, and not to study effects to aquatic life (Mogensen et al. 2004). The results from the present experiment can therefore not be used to predict the effects of the individual pyrethroids used in this study on the development of embryos of Bombina bombina. The developmental abnormalities found by the end of the experiment are the results of the concerted actions of the four pyrethroids.

In situ observations of the effects of the pyrethroids

Spasmodic twisting was seen as an effect of the pyrethroids and caused the embryos exposed to pyrethroids to leave their jelly coats one day before the control group. The constant spasmodic twisting caused immobilisation of more than half of the embryos at day three, but this effect seemed to decrease after six days, where the surviving embryos started to swim around. The concentration of the total amount of the four pyrethroids was measured in an accompanying experiment by Mogensen et al. (2004) and was around 8 μgl^-1 in the surface layer 2-4 h after spraying. The concentration then decreased to around 3 μgl^-1 after 24 h, to concentration below 0.5 μgl-1 after six days. At the time where the surviving embryos started to swim around the concentration of the pyrethroids had therefore decreased to less than 1/10 of the water concentration 2-4 hours after spraying, due to absorption by the sediment and macrophytes (Mogensen et al., 2004). This might have caused the increased swimming activity. It is important to realize that the pyrethroid concentration used in the present study is almost 5 times higher than compared with the concentration that is expected to occur in ponds in agricultural areas. Fairchild et al. (1992) found that a pyrethroid concentration of 1.7 μg/l represents the worst case exposure condition caused by direct overspray or extensive drift during aerial application adjacent to a pond.

Mortality

The pyrethroids sprayed onto the pond had a direct effect by increasing the mortality from 5-10 % in the controls to 51% in ponds sprayed with the pyrethroids. However, this high mortality is not likely to occur under natural conditions, due to the high pyrethroid concentration applied in this study.

Malformations

The most significant malformations observed were heart malformations, severe lateral flexure, edema, notochord and gut malformations, and since 85 % of the embryos possessed multiple malformations the pyrethroids had severe effects. These results confirm the results found in other studies of toxic effects of pyrethroids on amphibians (Larsen & Sørensen, 2004; Berrill et al., 1993).

Growth

The ability of toxicants to inhibit embryonic growth is often a sensitive indicator of developmental toxicity. Thus, Berrill et al. (1993) exposed five species of amphibians to pyrethroid insecticide at concentrations between 10 and 200 g/l with no results of mortality, but with a notable reduction in growth rates. The applied pyrethroids did not inhibit the final length of the larvae in the present study. Although the larvae were not weighed after the experiments, the larvae which were exposed to pyrethroids did not appear smaller in any way compared with the control organisms, and the growth rates were therefore not notable affected by pyrethroids in this study. This agrees well with results of experiments on effects of pyrethroids on amphibian embryos (Larsen & Sørensen, 2004; Larsen, et al., 2004), but a reduction of the growth has been found in other studies (Berrill et al., 1993; Materna et al., 1995).

While the growth of the surviving embryos was not affected by the pyrethroids, the embryos were seriously affected by the pyrethroid treatment. Although embryos were surviving the exposure to the initial high concentrations of pyrethroids, it does not indicate that the development of the embryos will result in surviving adult toads, capable of reproducing. Larsen et al. (2004) found in a accompanying study with recovery experiments where larvae, previously exposed to 20 or 125 μg/l esfenvalerate, were transferred to pure medium, that the larvae, which survived the treatment, developed severe malformations during metamorphosis. The most obvious deformity was their forelimbs which were bent in over the body and prevented them to jump. Instead they tumbled around when they tried to jump on land or catch flies and they were therefore unfit to survive in the nature. Survival and growth of embryos and larvae and progress of metamorphosis can therefore not be used as a success criterion in investigations on effects of toxic substances on amphibians. Furthermore, amphibians are not likely to be exposed to lethal concentrations of pyrethroids in the environment, due to short half-life and the low concentration likely to occur in ponds and lakes (Berrill et al., 1993). It is therefore important to identify sublethal effects.

From the present study it can be concluded that the new toxicity test guideline using Bombina bombina as test organism can be used under in situ conditions for testing developmental toxicity of toxicants. When applying the test into natural ponds or lakes the realism of such tests increases by including natural food webs, decomposers, sediment and natural physical conditions. These kinds of caging experiments are therefore very suited for evaluating the fitness of a pond or lake to inhabit populations of amphibians as well as for evaluation of effects of toxicants such as insecticides sprayed adjacent to ponds or lakes. Compared with monitoring on natural population caging has several advantages. Time for out-planting is known, dispersion and predation are prevented, time-integrated monitoring can occur and a real control can be established. Time-integrated monitoring has the benefit that possibly effects of short-term pulses may be recorded.