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

7 Results

7.1 Embryo development of Bombina bombina
- 7.1.1 Embryo development in laboratory control
7.2 Effects of selected pyrethroids on Bombina bombina in a semi-field test
- 7.2.1 In vivo observations
- 7.2.2 Recordings of malformation, growth inhibition and mortality at the end of the test (nine days)

7.1 Embryo development of Bombina bombina.

Egg deposition of Bombina bombina occurred about 9-12 h after injection of human chorionic gonadotropin. About 150-300 eggs were found in the breeding aquarium, and about 120–150 eggs were found normal cleavaged after selection. Laboratory control mortality was kept to less than 5% by only using the best eggs in the tests and only normally cleaving embryos.

7.1.1 Embryo development in laboratory control

Laboratory control

Most of the embryos of Bombina bombina were still in the jelly coat after four days and were not free until after about six days. At 20°C, the control group of Bombina bombina reached stage 46 after nine days and all embryos were normal and showed no malformations. At stage 46, larvae were recognised by the appearance of the hind limb buds, the coiling of the gut, and the shape of the operculum covering the gills. The best indicator that the larva stage had been attained at stage 46 was the appearance of the hind limb buds. Gut coiling was also easily observed at stage 46 (at stage 45 embryos do not display complete tight gut coiling). At stage 46, the larvae were about 9.5 mm in length.

7.2 Effects of selected pyrethroids on Bombina bombina in a semi-field test

Control pond

The development of the control groups of Bombina bombina in the control pond is shown in table 7.1 (stage according to Gosner (1960) and selected stage according to Nieuwkoop and Faber (1975) are included), and corresponds to the development in the laboratory experiments.

Table 7.1
The development of embryos of Bombina bombina in an artificial pond in Denmark. Staging of embryos according to Gosner (1960).

Udviklingen af embryoer af Bombnina bombina i et kunstigt vandhul i Danmark. Embryo-stadier blev bestemt efter Gosner (1960).

Days after the start of the test	1	2	3	4	5	6	7	8	9
In the control pond	12-13	18-19	19-20	20	20-21	21-23	23	23-24	25 46*

* stage according to Nieuwkoop and Faber (1975) for comparison with Xenopus l

7.2.1 In vivo observations

Treated pond

Pyrethroids affected gradually the embryos during the exposure. After 6h no effects of the pyrethroids were seen on the embryos compared with the controls. After 1 day, beginning twisting of the embryos was seen now and then. The spasmodic twisting increased in intensity with increased time of exposure and many embryos had constant spasmodic twisting two days after application of the pyrethroids. The spasmodic twisting of the embryos caused that some of the embryos had already left the jelly coat at this stage. In the control group, only a few embryos had begun to leave the jelly coat.

After three days, all embryos exposed to pyrethroids were free of the jelly coat. Spasmodic twisting was seen in most of the embryos and constant spasmodic twisting was seen in many embryos. Occasionally, blistering and cardiac edema appeared. About 40% of the living embryos were immobilised by constant spasmodic twisting. Several of the embryos in the control group began to leave the jelly coat and move around. In the control group from the pond all embryos were free from the jelly coat and moved around after four days.

After five days, blistering and cardiac edema were frequently seen in embryos exposed to pyrethroids. The embryos were often immobilised by constant spasmodic twisting. The heartbeat was very slow and severe heart malformations were often seen.

After six, seven, eight and nine days the same picture was seen, however, the effects of pyrethroids seemed to be less pronounced. The intensity of the spasmodic twisting seemed to decrease and several of the surviving embryos started to swim around. All embryos in the control group were swimming around and had attained the development stage 46 according to Nieuwkoop and Faber (1975) after nine days.

7.2.2 Recordings of malformation, growth inhibition and mortality at the end of the test (nine days)

Malformation

Embryos from the control pond posed no malformations and the larvae were at the normal stage 46 according to Nieuwkoop and Faber (1975) and comparable with the laboratory controls in all respects. 9% of the embryos had malformation in the control pond whereas 5% of the embryos had malformation in the laboratory control culture. In embryos from the pond, sprayed with pesticides, gut abnormalities were often seen, indicated by a loose gut coiling. In addition to gut abnormalities, a relatively mild flexure of the tail was normally seen and heart malformations were also observed. 85% of the embryos had malformations. Most abnormal embryos possessed multiple malformations. The most significant malformations observed included cardiac, severe lateral flexure, edema, notochord, and gut malformations.

Growth inhibition

After nine days of exposure to pyrethroids, the mean length of the embryos was compared with the length of the embryos from the control pond. The mean length of embryos exposed to pyrethroids was 9.0 mm (S.D. 0.7) while in the control pond the mean length of the embryos was 10.3 mm. (S.D. 0.6). The mean length of embryos from the control cultures in the laboratory was 9.5 mm (S.D. 0.6).

Mortality

No significant mortality was seen in the control pond as compared with the laboratory experiments: the mortality of the embryos at the end of the experiments was 5 and 10%, respectively. A mortality of 51% was found for embryos exposed to pyrethroids.