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

4 Discussions

4.1 The use of amphibians as an ecotoxicological test organism

Populations of many amphibians have declined and some species have disappeared from certain regions around the world, a phenomenon that appears to have accelerated during the last years (Blaustein & Wake, 1990). While there is evidence for widespread reduction in numbers of amphibians, the reasons for the declines are less clear. Much of the amphibian life cycle occurs in ponds, streams, and temporary pools that are often associated with agricultural areas receiving pesticide applications. In addition, breeding and larval development of amphibian occur in spring and summer at the same time that heavy application of pesticides on agricultural lands occurs. At present it is suggested that agricultural pesticides may contribute to the decline in the amphibian population (Phillips, 1990; Berrill et al. 1994). Therefore effects of the pyrethroid insecticide esfenvalerate on amphibians have been investigated in the present study. The main objectives in the project have been to establish data for evaluating the possible effects of esfenvalerate on amphibians in Danish aquatic environments.

Bombina bombina as test organism

In the present study effects of esfenvalerate on the fire-bellied toad Bombina bombina have been studied. A new developed test guideline for ecotoxicologcal tests with Bombina bombina (Larsen and Sørensen 2004) have been used.

In relation to the standard FETAX (Frog Embryo Teratogenesis Assay-Xenopus) test the present test have been prolonged to include recovery up to 6-7 weeks after the exposure by esfenvalerate has been ended. In FETAX the South African clawed frog (Xenopus laevis) has been used as test organism. Since Xenopus laevis only live in water and are resticted to only very special limited ecosystems in Africa it makes it less suited for ecotoxicological risk assessment in Denmark and other European countries. However, it is found that Bombina bombina and Xenopus laevis have almost the same sensitivity for esfenvalerate (Larsen and Sørensen, 2004).

Successful tests

In the present study, the test with Bombina bombina has been very successful and realistic. After injection of human chorionic gonadotropin each female bred between 75-400 eggs, which is within the normal range. By selection the best fertilised eggs and only normally cleaving embryos the mortality in control systems could be kept below 5%, which is satisfactory low in ecotoxicology tests with amphibians (ASTM 1991). Also the embryo development was satisfactory. Developmental stage 46 was reached after 120 h at 24°C and after 216 h at 20 °C. At both temperatures the larvae were about 9.5 mm in length at stage 46. In addition the test with a reference substance (6-aminonicotinamid) resulted in the effects expected from the ASTM standard with Xenopus laevis at both temperatures and in both pond water and FETAX media.

4.2 Effects of esfenvalerate during the exposure period

As found in others studies with embryos of amphibians, esfenvalerate affected the embryos gradually during the exposure period (Larsen & Sørensen 2004, ref). Very fine concentration-response relationships were found in the present study. The initial response was a decrease in activity followed by spasmodic twisting and immobilisation at the highest concentrations. At the end of the exposure period after 216 h spasmodic twisting was seen in the embryos exposed to concentration as low as 1 μg/l esfenvalerate. At concentrations above 50 μg/l, the embryos were immobilised caused by constant spasmodic twisting and blistering and edema were often seen. At concentrations above 100 μg/l, blistering and edema were frequently seen as well as head and brain malformations. The heartbeat was very slow and severe heart malformations were seen at 100, 150, and 300 μg/l.

Neither growth nor mortality was significant affected by esfenvalerate at any of the tested concentrations. This corresponds to the results of other studies where it has been found that mortality and growth inhibition did not seem to be sensitive effect parameters (e.g. Materna et al., 1995, Larsen & Sørensen, 2004). Detection of malformations during the development revealed effects at concentrations as low as 2.5 μg/l esfenvalerate. However, in vivo observations revealed effect as low as 1 μg/l esfenvalerate, which indicates the importance of in vivo observation in test with amphibians. Such sublethal effects are likely to have serious implications on the long-term success of the exposed individuals and such effects need to be included in risk evaluation of toxicants in the environment.

However, such risk evaluation is not straightforward since exposure condition in toxicity test and in the environment may differ. Normally is the concentration of the toxicant kept constant in standard test. In the present study, the medium was renewed every day keeping the concentration of esfenvalerate constant. Especially for toxicants with fast decline in the environment it is uncertain how risk evaluation should be done. Typically it is found that esfenvalerate will decline relatively fast in pond after spraying (Mogensen et al., 2004). At the moment our knowledge on the relation between toxicity and duration of exposure is not adequate. Analogous the usability of time weighted average concentration in risk assessment as suggested by EU is uncertain and currently insufficient tested.

4.3 Recovery and delayed effects

After the end of the toxicity test (after 216 h) the surviving embryos treated with 20 and 125 μg/l esfenvalerate, and untreated controls were transferred to fresh medium without esfenvalerate. At the same time algae was supplied to feed the larvae. Immediately after transfer, the embryos in the control group started to eat algae and moved around searching for food. In the groups pre-exposed to 20 μg/l esfenvalerate about 70% of the organisms had recovered after 24 h, while only 20% of the embryos pre-exposed to 125 μg/l esfenvalerate had recovered. After two days all surviving larvae in all the three groups were swimming around eating algae. The recovery seemed completed and fast. And the conclusion could be that the effect of esfenvalerate on amphibians in the environment is insignificant as the concentrations of esfenvalerate typically decline relatively fast after spraying. However, such a conclusion does not lay open all the life stages of an amphibian and if the ecological realism of the test is low as well, it is necessary to be very causious when evaluating such ecotoxicological tests. A distinct effect of esfenvalerate was seen six weeks after transfer to the fresh medium without esfenvalerate. During the development it got more and more evident that esfenvalerate at both concentrations induced malformed forelimbs still six weeks after the exposure was ended. The malformed forelimbs was fatal because the toads tumbled around when they tried to jump. This malformation is detrimental, since these toads will not be able to escape from predators, catch flies, and probably not reproduce themselves.

The present study is special in the context to its thoroughness and in duration. The majority of the tests with amphibians are limited to test effects during a few days (ref) and prolonged studies as the present are uncommon. There may exist studies on effects of esfenvalerate tested in prolonged experiments although it has not been possible to find such references for the present study.

In this connection especially one study deserves to be mentioned. In the study delayed effects of pre- and early-life time exposure to PCB on tadpoles of Xenopus laevis and Rana temporaria were investigated (Gutleb et al., 1999). The study is unique in extent and it includes effects of PCB exposure in amphibians, such as mortality, number and pattern of malformations, or body weight and successful metamorphosis into tadpoles, depended exposure route, the point of time of exposure during the complex life cycle of amphibians, and the length of the observation period. The conclusion in the study by Gutleb et al. (1999) is that presently used early-life test systems such as the FETAX assay may underestimate toxic effects of compounds due to long-term effects such as PCBs on amphibians. This conclusion agrees with the conclusion of the present study.

4.4 Effects of different media used

The results revealed that the embryo development was almost identical in the two media, FETAX and pond water. The embryos reached stage 46 according to Nieuwkoop and Faber (1975) after 120 h and 216 h of exposure at 24°C and 20°C, respectively, in both media, and this correspond to the development in the standard FETAX test with Xenopus laevis. It indicates that the test design with Bombina bombina was optimal, as previously concluded (Larsen & Sørensen, 2004; Larsen, Sørensen & Gustavson, 2004), and that the jelly coat, which was not removed in the experiments, did not effect the embryo development.

Effects of esfenvalerate

The effects of esfenvalerate in the two different media, although comparable, occurred at different concentration of esfenvalerate. The first spasmodic twisting started in FETAX solution at 5 μg/l and in higher concentrations after 96 h, while in pond water spasmodic twisting started at 50 μg/l. The same pattern of malformations was found at the end of the experiments in both media, however, the embryos in FETAX medium had more malformations and these malformations were also more distinct. In FETAX solution about 30% of the embryos had malformations at 10 μg/l and about 90% of the embryos exposed to 100 μg/l and higher concentrations had malformations. In pond water a significant effect of esfenvalerate was seen at 50 μg/l where about 15% of the embryos had malformations.

Ionic characteristic of the water

It has previously been shown that differences in water hardness alter the toxicity of pyrethroids to aquatic organisms (Coats et al., 1989). Besides affecting the nervous system (Tippe, 1987), pyrethroids also cause an osmoregulatory imbalance and ionic characteristics of the water have been demonstrated to influence the toxicity of fenvalerate to bluegill (Coats et al., 1989).

Dissolved organic matter

Another explanation to the low toxicity of esfenvalerate in the pond water may be that the dissolved organic matter strongly absorb the esfenvalerate and thereby reduce the bioavaliability. FETAX solution is not added any organic substance so it is evident that it may be an explanation. It is well-known that pyrethroids strongly adsorb to the organic matters causing a decrease in bioavalaibility.

Using natural water as medium

Too low ion concentrations can, however, severely affect embryo survival, growth and occurrence of malformations (Tietge et al., 2000). This was found to be the case in the Northern USA, where three different species of frogs developed limb abnormalities which was found to be caused by low ion concentrations in the lake used in the study (Tietge et al., 2000). The occurrence of common metals as Al and Fe in high concentrations may, however, also be toxic to amphibians (Horne & Dunson, 1995). Furthermore, some acidic lakes and ponds are unsuitable for amphibian breeding, since amphibians embryos and larvae are highly sensitive to low pH (Freda, 1986). Careful consideration should therefore be taken before applying the FETAX protocol to natural samples when using water from ponds of unknown water chemistry, and initial tests should preferable be run prior to testing for e.g. toxicity of substances. The present study shows that test medium FETAX prepared in laboratory facilities with a controlled content of ions and free of toxicants is very suitable for Bombina bombina breeding and can be used without further testing. Furthermore, it shows that the FETAX medium produces reliable results that perhaps shows low effects concentrations for some soft water lakes, but takes into account that some surface waters have high concentrations of ions.

Effects in sprayed ponds and lakes

In the present study the media with esfenvalerate was renewed every day. In natural environments the insecticides which end up in aquatic environments are reduced in concentration in the water phase quite dramatically within a few days especially due to absorption by the sediments and aquatic plants (Mogensen et al., 2004, Larsen, Sørensen & Gustavson, 2004), since pyrethroids are highly hydrophobic. The tadpoles, however, feed on phytoplankton, epiphytes and filamentous algae, and may therefore increase their exposure to and uptake of pyrethroids through their feeding behaviour even though the concentration of pyrethroid in the water column is undetectable. The procedure of renewing the media containing the test substance is therefore sensible, for the purpose of detecting reliable effects of exposure to toxic substances.

4.5 Effects of different temperatures

It is well-known that effects of toxic substances strongly depend on the temperature. In the present study the embryo development was tested in FETAX solution at 24°C corresponding to the temperature used in the normal FETAX test with Xenopus laevis and at 20°C corresponding to the temperature in the new guideline and more often found in Danish ponds. Since amphibians absorb heat from the environment and an increase in temperature increases their metabolic activity, it was expected that the embryos reached stage 46 faster at 24°C than embryos reared at 20°C. Accordingly, they reached stage 46 only 120 h after the start of the test at 24°C, which was 96 h earlier than the embryos at 20°C (Table 3.1).

Effects of esfenvalerate

The effects of esfenvalerate added in different concentrations were comparable in the two different temperatures, although displaced in time. Spasmodic twisting of embryos was seen down to 1 μg/l esfenvalerate and all embryos were immobilised at concentrations above 50 μg/l at 120 and 216 h at 24°C and 20°C, respectively. The same pattern of malformations was found at both temperatures, however, the embryos at 20°C had more malformations and lower EC50 (32.7 and 24.7 μg/l at 24 and 20°C, respectively) and these malformations were also more distinct. This agrees with the effects of temperature found in the study of Materna et al. (1994), where lower EC50 was found for tadpoles of Leopard frogs (Rana spp) exposed to esfenvalerate at 18°C compared with 22°C (3.40 and 6.14 μg/l, respectively). The lower EC50 values found for Leopard frogs compared to this study with Bombina bombina can be due to the use of tadpoles in the toxicity tests of Materna et al. (1994). There appears to be differences in sensitivity between embryonic stages, where older embryos show higher sensitivity to pyrethroids than younger stages (Berrill et al., 1993), and tadpoles could possibly be more sensitive than embryos. Furthermore, there is also interspecies variation in tolerance towards pyrethroids and toxic substances, since some amphibians are more sensitive than others are (Berrill et al., 1993; Berrill et al., 1995; Freda, 1986).

Pyrethroids have generally been found to be more toxic at low temperatures for several aquatic organisms (After Coats et al., 1989 and Berrill et al. 1993). The sensitivity of embryos and larvae of amphibians in natural environments may therefore vary with the season and latitude. The temperature in Danish ponds will most likely not exceed 20°C. In spring, which is the breeding season for amphibians, the temperature is most likely 10-15°C, this will apparently affect the development of embryos even more than the effects observed in the present study if they are exposed to pyrethroids. In addition it should be noticed that the exposure time in the present study is almost twice as long at 20°C (216 h) than at 24°C (120 h).

4.6 Developmental effects of esfenvalerate in relation to other studies

The malformations found in the larvae at the end of the experiments in this study as a result of treatment with esfenvalerate were many. The most significant malformations observed included cardiac malformations, severe lateral flexure, oedema, notochord, brain and gut malformations. Especially lateral flexure or axial malformations have been often reported in developing amphibian larvae exposed to different toxicants in the laboratory (Cooke, 1981; Bantle et al., 1991; Hatch et al., 1998; Joffre et al., 1999). They have also been found in bullfrogs larvae in nature from coal ash-polluted sites, where lateral curvature of the spine occurred in high rates due to high concentrations of trace elements (As, Cd, Se, Cu, Cr, and V) (Hopkins et al., 2000). Axial malformation has a negative impact on the swimming performance, which is related to foraging, predator avoidance and thermoregulation and has accordingly serious implications for survival of the larvae.

Uncoiling of the gut, brain and heart malformations, and edema were malformations also found in this study as effects of esfenvalerate. These malformations have been less often reported in other studies with amphibians and other toxicants than pyrethroids. Perkins et al. (2000) did not observe any malformations at any concentrations of two herbicides, glyphosate and triclopyt that were not also lethal to the embryos in Xenopus laevis. And edema was the only sublethal effect of the herbicide atrazine in two amphibians (Howe et al., 1998). Lordoscoliosis occurred as a malformation beside tail curvature in a FETAX experiment with lead (Sobotka & Rahwan, 1995).

The many malformations recorded in this study as well as the above mentioned results of the recovery experiment as a consequence of exposure to sublethal concentrations of esfenvalerate emphasize the severe impact that this toxicant can have on populations of amphibians in natural environments. Other studies on toxic effects of pyrethroids on amphibians have also showed that several malformations and behavioural abnormalities developed in the larvae. Furthermore, the amphibians are generally very sensitive to sublethal exposures of pyrethroids and will probably die as a consequence of the developmental effects caused by pyrethroids (Berrill et al., 1993; Materna et al., 1995).

4.7 Further aspect

Many different chemicals are applied to arable farmland and some are known to occur in freshwater ponds, perhaps at concentration that may have effects on amphibians. The present study shows that pesticides may have significant effect on amphibian and that prolongation of standard test are absolutely relevant and important in the future both in relation to risk and hazard evaluation and in monitoring for effect on amphibians in the environment. Trial is described in which the incidence of deformities was unusually high in caged tadpoles of Rana temporaria beside potato fields after application of oxamyl, carbamate nematicide and insecticide (Cook 1981). Effects on amphibians in the environment may be monitored by caging technique (e.g. Hopkins et al., 2000, Cook 1981 and Larsen, Sørensen & Gustavson 2004), releasing of tadpoles into the environment (Hopkins et al., 2000, Cook 1981), reciprocal transplanting or detecting e.g. malformation on natural populations if such effects occur.

In view of the current world-wide decline in amphibian populations it has bring into focus to study the potential for environmental contaminants to act as endocrine disrupters of the amphibian reproductive system. Recently it has been shown that DDT induces endocrine disruption in Tiger Salamander (Clark et al., 1998). OECD has therefore recently called to some meeting inside test for endocrine disrupters in amphibian. At present no papers have yet been prepared on subject by OECD (pers. comment, Ms. Marie Chantal Huet, OECD).

 

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Version 1.0 September 2004, © Danish Environmental Protection Agency