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

6 Materials and methods

• 6.1 Principles and methods for maintaining and breeding the fire-bellied toads, Bombina bombina
  • 6.1.1 Principles and methods for maintaining adults Bombina bombina
  • 6.1.2 Principles and methods for breeding Bombina bombina
• 6.2 Semi-field toxicity tests using embryos of Bombina bombina
  • 6.2.1 Principle and design of the semi-field toxicity test
  • 6.2.2 Establishing of experimental ponds, mesocosms
  • 6.2.3 Embryo selection for the test
  • 6.2.4 Experimental design of the semi-field toxicity test
  • 6.2.5 Test substances
  • 6.2.6 Spraying method and estimated initial concentrations
  • 6.2.7 Physical state of the ponds
  • 6.2.8 Determination of the effects of pesticide application on embryos of Bombina bombina

6.1 Principles and methods for maintaining and breeding the fire-bellied toads, Bombina bombina

6.1.1 Principles and methods for maintaining adults Bombina bombina

Adult Bombina bombina

Adult Bombina bombina used in the present study were at least two years of age and weighed about 6.5 to 8.5 g (males and females are about the same size). 1-2 pairs were kept together in a 50 30 cm vivarium. The floor area consisted of 60% water, with a depth of 8 cm, and the land area was dark-coloured with hiding areas and a feeding place. The vivarium was fitted with a mesh to prevent escape. Artificial lighting was switched on daily from 7 am to 7 pm, however, the vivarium was not protected from natural light so the animals were subjected to natural fluctuations in day length. Slowly growing natural aquatic plants and plants of plastic and broken pots were creating hiding places.

Diet

Food consisted of crickets, meal worms, small earthworms, flies (with crumpled wings) and other suitable invertebrates supplemented with a special vitamin supplement. Feeding was continued throughout the year, although at a reduced rate (about 2 times a week) during November-February when the animals showed minimal activity.

Temperature

Adults were kept at 19-21°C in the winter period and at room temperature in the summer period.

6.1.2 Principles and methods for breeding Bombina bombina

Males and females were bred as a single pair and the animals were moved to a 45 l glass aquarium with a water depth of about 20 cm. The aquarium was richly furnished with both natural and plastic plants. In addition, 2 mm round sticks of wood were placed in the aquarium, and 1-2 islands of floating cork enabled the animals to leave the water if desired. Artificial lighting was switched on daily from 5 am to 9 pm, however, the vivarium was not protected from natural light so the animals were subjected to natural fluctuations in day length.

Water temperature was adjusted to 22 ±0.5°C.

Breeding

To mature the oocyttes, the females received about 14 IU of Pregnant Mare Gonadotropin (PMG) two days before the animals were moved to the breeding aquarium. Just before the animals were moved to the breeding aquarium both males and females received 55 to 100 IU of human chorionic gonadotropin to induce breeding.

The amount of hormones injected depended on the size of the animals and the time of year. Low doses were usually used in spring and high doses in autumn. Amplexus normally ensued within 2 to 6 h and eggs deposition about 9 to 12 h after injection. The eggs were immediately inspected for fertility and quality. The fertility rate should be > 75% before a toxicity test was performed. Examination of the aquarium showed that the eggs were often found on vegetation and on the round sticks of wood, which were placed in the breeding aquarium to imitate straw and stems.

6.2 Semi-field toxicity tests using embryos of Bombina bombina

6.2.1 Principle and design of the semi-field toxicity test

In situ experiment

The tests were carried out at semi-field conditions from 12 to 21 June 1996 in two artificial ponds, at the mesocosm facilities at the National Environmental Research Institute, Denmark. One pond was sprayed with pesticides and one pond served as a control. Air and water temperatures were measured daily. Just before spraying, 8 glass Pyrex dishes (9 cm in diameter and 5 cm high) each containing 10 embryos were placed in two baskets of stainless steel in the pond about 20 cm below the water surface at opposite banks before spraying with pesticides. Four dishes containing 25 embryos each were simultaneously placed in the control pond also about 20 cm below the water surface. The cages were placed in the ponds on June 11 at 23.00 pm. The test ponds were sprayed with pesticides on June 12 at 9.25 a.m.

To evaluate developmental toxicity, mortality, malformation, and growth inhibition properly, data were collected every day until at least 90% of the controls had attained the development stage 46 according to Nieuwkoop and Faber (1975).

In addition to the control group in the control pond, a control group grown under laboratory condition was included in the test design. For this purpose, four dishes of five embryos each were used.

Test organisms and egg manipulation

The fire-bellied toads, Bombina bombina had been kept in laboratory facilities for more than two years. This study was carried out with eggs in which the jelly coat had not been removed. The jelly coat is normally removed before starting an experiment in the standard FETAX test.

Test chemicals

The test chemicals were esfenvalerate, deltamethrine, fenpropathrin, which were added as formulated products, and permathrin, which was added as an analytical grade active ingredient.

6.2.2 Establishing of experimental ponds, mesocosms.

Ponds

The mesocosm facilities at National Environmental Research Institute (50 km west of Copenhagen, Denmark) were kindly provided for the present study. The facilities consist of four ponds with a bottom area of about 130 m² and a depth of about 0.75 m, which were established in November-December 1994. The mesocosms were established in an area with heavy clay making it possible to retain water in the ponds without assistance of an artificial membrane. A pipe from each pond leads to a ditch outside the bank. In case of surplus rainfall it is possible to adjust the water level in all ponds to 1.5 m by draining surplus water into the ditch.

From a natural pond in a landscape similar to the test facilities, natural sediment was transported to the mesocosms. The sludge was uniformly sprayed onto the bottom of the ponds in a layer of about 2 cm. The natural sediment was introduced to initiate aquatic flora and fauna in the ponds and a variety of plants, crustaceans and insects developed, which created an ecosystem resembling the ecosystem of a natural pond. For further details, cf. Mogensen et al. (2004).

6.2.3 Embryo selection for the test

Embryo selection

Normally cleaving embryos were selected for the tests. Two levels of selection were used. Normally cleaving embryos were first sorted into dishes containing fresh water. After a short period during which cleavage continued embryos were sorted again to ensure that only normal embryos were used. Abnormal pigmentation was viewed as an indicator of bad embryos. Both Nieuwkoop and Faber (1975) and the "Atlas of Abnormalities" ^[2] were used as references to determine whether the cleavage pattern was normal. Midblastula (stage 8) to early gastrula (stage 11) was used to start the tests. By this stage, normal cleavage and development can be ascertained. Embryos chosen prior to stage 8 may develop abnormal cleavage patterns later whereas embryos selected after stage 11 have commenced organogenesis. The sorting was done in Petri dishes. Staging of embryos was done according to Gosner (1960) and Nieuwkoop and Faber (1975). After the last sorting the embryos were transferred to test chambers containing 40 ml of pond water.

6.2.4 Experimental design of the semi-field toxicity test

In the present study embryos from two mating pairs were used to ensure a sufficient number of embryos. Embryos from each mating pair were randomly divided in 3 groups: one group was used in the pond sprayed with the pyrethroid insecticides, the second group was placed in the control pond, and the third group was used as the laboratory control test. It was necessary to keep clutches separate because embryos of a particular mating pair may develop poorly although they initially appear acceptable. This would cause all the embryos to be discarded if embryos were mixed from different mating pairs.

Determination of the effects on embryos

To evaluate developmental toxicity, mortality, malformation, and growth inhibition properly, in vivo observations were performed 6 h after spraying and then every day after the start of the test. Each day the embryos were picked up from the test chamber using a Pasteur pipette, and malformation, mortality and growth inhibition data were recorded. After investigation all living embryos were taken back to the test chambers and returned to the respective ponds or to the incubator in the laboratory.

Duration of the semi-field test

The experimental period was 11 days (including the day of spraying) after which stage 46 was attained in the controls.

Laboratory control experiments

The test design of the laboratory control experiments was the same as previously described for toxicity bioassay with Bombina bombina (Larsen et al., 2004). The culture medium used for the laboratory control test was pond water taken from the control pond. Tests with embryos of Bombina bombina were conducted in an incubator at 20 ±0.5 °C. The tests chambers were covered with 60 mm glass Petri dishes and had a culture volume of 10 ml. The medium was renewed every 24 h during the laboratory tests. It took 216 h to attain stage 46 under these conditions.

6.2.5 Test substances

The spraying liquid was prepared from three formulated products shown in table 6.1. Active ingredients are given in brackets: Decis from Hoechst (deltamethrin), Sumirody 10 FW from Du Pont (fenpropathrin) and Sumi-Alpha 5 FW from Du Pont (esfenvalerate). A formulated product with permethrin was not available so the active ingredient was dissolved in Decis prior to dilution with water.

Table 6.1*
Selected physico-chemical properties of the test substances used in the experiment.

Udvalgte fysisk-kemiske egenskaber for de anvendte forsøgsstoffer.

* Aqueous photolysis studies with esfenvalerate and prochloraz indicated that the half-lives of both pesticides are about 10 days, however, the half-lives are of course dependent on the intensity of the light.

Data from The Pesticide Manual, tenth edition and the Danish EPA.

6.2.6 Spraying method and estimated initial concentrations

Special equipment was constructed to make it possible to spray pesticides uniformly upon the water surface (Mogensen et al., 2004). Two-three persons carried the spraying boom during spraying. The amount of pesticide sprayed per ha depends on the pressure and walking speed. In the present study the pressure was 2.5 bar, spraying time 49 s. corresponding to application of approximately 5 L of spraying liquid according to Mogensen et al.; (2004). All four pyrethroids were sprayed simultaneously. Permethrin dissolved in Decis was mixed with Sumirody 10 FW and Sumi-Alpha 5 FW. Table 6.2 shows the composition of the spraying liquid and the approximate application rate.

Table 6.2
Concentration of pyrethroids in spraying liquid and amount of pyrethroids applied to the pond surface, mg/m² (After Mogensen et al. 2004).

Koncentrationen af pyrethroider i sprøjtevæsken og mængden af pyrethroid, der er udsprøjtet på overfladen af vandhullet, mg/m² (Efter Mogensen et al. 2004).

6.2.7 Physical state of the ponds

The physical state of the ponds was controlled during the experiments by measuring a number of parameters like chlorophyll a, alkalinity, nitrogen and phosphorus, oxygen, temperature, pH, etc. The results from these measurements are reported in Mogensen et al. (2004).

6.2.8 Determination of the effects of pesticide application on embryos of Bombina bombina

6 hours after spraying all baskets were carefully hoisted up from their respective ponds (by way of a fishing line attached to the handle of each basket) and the embryos were examined every day by picking up the embryos from the test chambers using a Pasteur pipette. The orifice of the Pasteur pipette was enlarged and fire-polished to accommodate embryos without damage. After examination, the embryos were placed in the test chambers and returned to the respective ponds or incubator in the laboratory. The dishes in the ponds were covered with a 500 μm plankton net. The plankton net was mounted on the dishes with a rubber band made of silicone 6 hours after the spraying to prevent the embryos to escape.

In vivo observations

A binocular dissection microscope capable of magnifications up to 30 was used to evaluate the abnormal embryos. The embryo lengths (head-tail length measurements) were measured by using a map measurer or an ocular micrometer. Staging of the embryos was done according to Gosner (1960) and Nieuwkoop and Faber (1975).

Mortality

Dead embryos were removed at the end of each 24 h period and the mortality data were recorded. Death during the first three days was ascertained by the embryo's skin pigmentation, structural integrity, and irritability (measured by lack of response on physical stimulations). After four days the lack of heartbeat served as an unambiguous sign of death. After nine days of exposure or at stage 46 for the controls, the total number of dead embryos was recorded during the test. Dead embryos were removed and the remaining living embryos were fixed in 3% formaldehyde solution.

Malformation

Malformation and other sublethal end points are normally more sensitive than mortality and are therefore included in the present test protocol. Malformation was recorded at the end of the test. Embryos exposed to the test material were compared with controls.

Growth inhibition

The ability of a material to inhibit embryonic growth is often the most sensitive indicator of developmental toxicity. Head-tail length data (growth) were likewise tested at the end of each test. If the embryo was curved or kinked, the measurement was made as if the embryo was straight. Measurements were made after the embryos were fixed in 3% formaldehyde solution.

Footnotes

[2] Availabe from John A. Bantle, Dept. of Zoology, 430 LSW, Oklahoma State University, Stillwater OK 74078.

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Version 1.0 September 2004, © Danish Environmental Protection Agency