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The Effects of Selected Pyrethroids on Embryos of Bombina bombina during different Culture and Semi-field Conditions
2 Materials and methods
Briefly, the present study includes work related to maintaining and breeding the fire-bellied toad, Bombina bombina, toxicity test in 120 h or 216 h (depending on the temperature), a
study of the recovery of the embryos and larvae after transferring to fresh pesticide free medium, and a prolongation of the study to include possibly effects up to 6-7 weeks after the
exposure was ended.
2.1 Principles and methods for maintaining and breeding the fire-bellied toads, Bombina bombina
2.1.1 Principles and methods for maintaining adults Bombina bombina
Adults Bombina bombina 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 from 7 am to 7 pm daily, 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 two times a week) during November-February when the animals showed minimal activity.
Temperature
Adults were kept at 19-21°C in the winter and at room temperature in the summer period.
2.1.2 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 to 2 islands of floating cork enabled the animals to leave the water if desired.
Artificial lighting was switched on from 5 am to 9 pm daily, 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 the 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 higher 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 from aquatic plants.
2.2 Toxicity tests using embryos of Bombina bombina
2.2.1 Principle and design of the toxicity test
The new Bombina bombina test is a 120 h or 216 h, (depending on the temperature) renewal whole embryo assay that can be used to evaluate the developmental toxicity of a test
material. Exposure was continuous throughout the test. For each concentration, two dishes each containing 5 embryos and 10 ml test solution were used. For each control, four dishes of
5 embryos each were used in the standard experiments with Bombina bombina. Embryos were randomly assigned to test dishes. Dishes were randomly assigned to positions in the
incubator. Data were recorded to evaluate developmental toxicity, mortality, malformation, and growth inhibition properly. In most tests, it was possible to generate
concentration-response curves for mortality, malformation, and growth inhibition. The mortality and malformation concentration-response curves were then used to estimate the
concentration that affected 50% of the exposed embryos. It was assured that according to Nieuwkoop and Faber (1975) at least 90% of the controls had attained stage 46 at the end
of the experiments.
Test organisms
The fire-bellied toad, Bombina bombina has been kept at laboratory facilities for more than two years.
Egg manipulation
To assess the influence of esfenvalerate on the embryos of Bombina bombina as realistic as possible the jelly coats were not removed. The jelly coat is normally removed before
starting an experiment in an ordinary FETAX test.
2.2.2 Test substances
Esfenvalerate (CAS no. 66230-04-4), a pyrethroid insecticide, was added as the formulated product Sumi Alfa 5 FW (contains 5% esfenvalerate as the active ingredient). Selected
physico-chemical properties are shown in table 2.1.
Table 2.1
Selected physico-chemical properties of esfenvalerate.
Udvalgte fysisk-kemiske egenskaber for esfenvalerat.
| Parameter |
Esfenvalerate |
| Mol.wt. |
419.9 |
| Mol. Formula |
C25H22 Cl NO3 |
| M.p. |
59.0 - 60.2°C |
| V.p. |
0.0002 mPa (25°C) |
| Kow |
logP = 6.22 (25°C) |
| Solubility in water |
0.002 mg/l |
| Hydrolysis |
Fairly stable to hydrolysis at pH 5 and 7 |
| Photo degradation * |
Some photo degradation in water |
* Aqueous photolysis studies with esfenvalerate indicated that the half-lives 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.
Esfenvalerate is an insecticide with contact and stomach action and is a voltage dependent sodium channel agonist. It is used as a potent contact and ingested insecticide with a very
broad range of activity, especially effective against Coleoptera, Diptera, Hemiptera, Lepidoptera, and Orthoptera on cotton, fruit, vegetables, and other crops at 5-25 g a.i./ha. It is
effective against strains resistant to organochlorine, organophosphorus and carbamate insecticides.
2.2.3 Preparation of test solutions
Stock solutions of esfenvalerate were prepared daily. The pH of the stock solutions was adjusted to 7.5 ±0.5.
2.2.4 Embryos
Staging of embryos
Staging of embryos was done according to Gosner (1960) and Nieuwkoop and Faber (1975).
Embryo selection
Normally cleaving embryos were selected for use in testing. 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 selected. Abnormal pigmentation was viewed as an indicator of bad
embryos. Both Nieuwkoop and Faber (1975) and the "Atlas of Abnormalities" [1] were used as references to determine whether the cleavage patterns were normal. Midblastula (stage
8) to early gastrula (stage 11) was used to start the test. At this stage, normal cleavage and development can be ascertained. Embryos, chosen prior to stage 8, might develop abnormal
cleavage patterns later, whereas embryos selected after stage 11 have commenced organogenesis. The sorting was done in Petri dishes.
2.2.5 Culture medium
The culture medium used for the test was FETAX solution or pond water.
FETAX solution
FETAX solution was composed of 625 mg NaCl, 96 mg NaHCO3, 30 mg KCl, 15 mg CaCl2, 60 mg CaSO4 2H2O, and 75 mg MgSO4 per litre of deionized or distilled water. The
pH of the final solution was controlled to be between 7.6 and 7.9. All chemicals were reagent-grade or better.
Pond water
Pond water was collected in experimental ponds at National Environment Research Institute. GF/C filtrated water was stored at 4°C. Before use the water was warmed up to test
temperature.
2.2.6 Experimental design
Tests with embryos of Bombina bombina were conducted in an incubator at 20 or 24 ±0.5°C. The tests chambers were covered by 60 mm glass Petri dishes (before use all glass wares
were treated with silylation reagent solution and thoroughly washed in water) with an initial culture volume of 10 ml.
A binocular dissection microscope capable of magnifications up to 30 was used to count and evaluate abnormal embryos. The embryo length (head-tail length measurements) was
measured with a map measurer or an ocular micrometer.
Maintenance of separate clutches
It is 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.
Esfenvalerate concentrations
The effect of esfenvalerate was tested at 1, 2.5, 5, 10, 50, 100, 150, and 300 μg/l (a.i.) and the results were compared with the control group.
Renewal
Renewal of the medium was performed every 24 h during the test. Just prior to medium renewal the pH was measured in the control and in the highest test concentrations to determine if
significant changes had occurred. The test solution was removed with a Pasteur pipette. The orifice of the Pasteur pipette was enlarged and fire-polished to accommodate embryos
without damage in case the embryos were accidentally picked up. This is standard procedure for FETAX. New medium was added immediately after.
Duration of the test
The standard exposure time for Bombina bombina was 120 h at 24°C and 216 h at 20°C, at which time stage 46 was attained.
2.2.7 Determination of the effects of esfenvalerate on embryos of Bombina bombina
In vivo observations
Dead embryos were removed at the end of each 24 h period when the solutions were renewed and the mortality data were recorded. If dead embryos were not removed, microbial
growth could occur which may kill living embryos. Death during the first 24-72 h, depending on the temperature and exposure, was ascertained by the embryo's skin pigmentation,
structural integrity, and irritability (measured as lack of response on physical stimulations). After 72 h the lack of heartbeat served as an unambiguous sign of death. At 120 h (at 24°C)
or 216 h (at 20°C) of exposure or on stage 46 of controls, the total number of dead embryos was recorded during the test (mortality was registered after 24, 48, 72, 96,120, 144, 168,
192, and 216 h of exposure). Dead embryos were removed while the remaining living embryos were fixed in 3% formaldehyde solution.
Malformations were recorded at the end of the test. Embryos exposed to the test material were compared with appropriate controls. The number of malformations in each category was
reported in standard format to ease interlaboratory comparison (cf. Appendix A).
Malformation
Head-tail length (growth) was measured at the end of each test. If the embryo was curved or kinked, the measurement was made as if the embryo was straight. The embryos were
measured after fixation in 3% formaldehyde solution.
Teratogenic Index
Teratogenic Index (TI) was determined after 120 h or 216 h, depending of temperature and of exposure. TI is defined as 120 h or 216 h LC50 (mortality) divided with 120 h or 216 h
EC50 (malformations). TI values higher than 1.5 signify large separation of mortality and malformation concentration ranges and therefore a large potential for all embryos to be
malformed in the absence of significant embryo mortality (ASTM 1991).
2.2.8 Replicates and controls
Number of tests and data collection
Three definitive tests were conducted in a random block design. Because it is necessary to acquire data on mortality, malformation, and growth inhibition, the concentration series were
adjusted to the expected 120 or 216 h LC50, 120 h or 216 h EC50 (malformation) values, and the minimum concentration needed to inhibit growth (MCIG).
Reference toxicant
For a positive control or reference toxicant, 6-aminonicotinamide presented a mortality and malformation database convenient for reference purpose. For each test, the positive control
consisted of two dishes of five embryos each exposed to 2500 mg 6-aminonicotinamide/l and two dishes of five embryos each exposed to 5.5 mg 6-aminonicotinamide/l. The mortality
and malformation observed should be between 40 and 60%. For example, at 2500 mg/l 4 to 6 of the 10 embryos should have died by 120 or 216 h (depending on the temperature).
Only biological responses related to mortality and malformation were considered in this analysis. Growth inhibition was not considered with regard to aminonicotinamide.
2.2.9 Data treatment and reporting
With the Probit analysis it was possible to obtain concentration-response curves to determine the values of 120 h or 216 h LC50 and 120 h or 216 h EC50.
The comparison of measurement results (head-tail length) between controls and treated embryos was obtained with the ANOVA statistical analysis.
The minimum concentration to inhibit growth (MCIG) is the minimum concentration of test material that significantly inhibits growth as determined by measurement of head-tail length. A
significant difference in growth should be determined by the t-Test for grouped observations at the p = 0.05 level (Dawson et al. 1989).
2.3 Principle and design of recovery experiments using embryos of Bombina bombina
2.3.1 Exposure of the embryos to esfenvalerate
The recovery test included two independent tests (on different days). The test consisted of two sublethal concentrations of the test substance, 20 and 125 μg/l esfenvalerate, and an
untreated control group. The selected concentrations were based on concentrations-response curves for both mortality and malformation determined in a toxicity test with Bombina
bombina. For each concentration, at least two dishes each containing five embryos of Bombina bombina and 10 ml of test solution were used. For each control, at least four dishes of
5 embryos were used. Each test used early embryos derived from a single mating pair. The toxicity test were otherwise carried out exactly as described previously in FETAX solution at
20°C and according to Nieuwkoop and Faber (1975) the embryos were at stage 46 at the end of this part of the test.
2.3.2 Principles and methods for maintaining embryos and larvae (tadpoles) of Bombina bombina during the recovery period
Transfer of embryos to fresh medium
At the end of the toxicity test (after 216 h) dead embryos were removed and the remaining living embryos were transferred to three different glass dishes each containing 200 ml FETAX
solution without pesticides. The lack of heartbeat served as an unambiguous sign of death. All the control embryos were transferred to one glass dish, and embryos, which previously
had been exposed to the same concentration of pesticide, were collected in the same dish. The maximum number of embryos in each glass dish was about 20. 8 h after the embryos
were transferred to pesticide free medium, half of the FETAX solution was withdrawn and replaced with aerated tap water supplemented with green algae. After 24 h the medium was
exchanged with pure tap water containing algae.
Culture conditions
After transfer to a pesticide free medium, the embryos and larvae were kept in an animal room at 21-22°C so that a photoperiod of 15 h day/9 h night could be maintained. During the
recovery period the embryos and larvae were moved from a smaller aquaria to a larger aquaria serveral times to maintain a sufficient amount of food and space. After 1 week each of
the three groups were transferred from the 200 ml glass dishes to 3 l aquaria containing aquatic plants and small stones on the bottom. After two weeks of recovery the tadpoles were
transferred to 11 l aquaria with more aquatic plants. After four weeks of recovery the tadpoles were transferred to 45 l aquria each containing 25 l of water and many aquatic plants and
stones on the bottom where they were maintained for additionally another week.
The water was in all cases aerated by the use of air stones, and the pH of the water was about 7. A suspension of green algae like Mougeotia and Oedogonium was added every day
to each glass dishes and aquaria together with Spirulina discs (algae pills from Wordley) to ensure a sufficient food supply. Renewal of the water was performed every day during the
first two weeks (in the glass dishes and the 3 l aquaria) and twice a week in the 11 l and 45 l aquaria.
Determination of the effects on embryos and larvae
In vivo observations were performed every day during the first two weeks and twice a week during the rest of the test. Dead, malformation, and development stages were followed.
Growth was measured during the period.
2.3.3 Principles and methods for maintaining Bombina bombina just before and during metamorphosis
Culture conditions
Five weeks after the embryos of Bombina bombina were transferred to a pesticide free medium, the tadpoles were transferred to a 50 30 cm vivarium with 70% water, about 8 cm
deep, and 1 to 2 islands of floating cork enabling the animals to leave the water if desired. The land area was dark-coloured with hiding places and a feeding place. The vivarium was
fitted with a mesh to prevent escape and was placed in the room as previously described. Natural aquatic plants and broken pots created hiding places.
Food consisted primarily of flies (with crumpled wings) and later, mealworms, small earthworms, and other suitable invertebrates supplemented with a special vitamin supplement.
Duration of the test
This part of the test had duration of three weeks starting from five weeks after the embryos were transferred to a pesticide free medium to about eight weeks after this transfer, or until
the metamorphosis of most of the tadpoles had been completed. At the end of the test all animals, which had previously been in contact with the test substance, were anaesthetized with
benzokain, killed and fixed in 25% glutaraldehyde. The same number tadpoles of the control group was also killed and fixed using the same procedure whereas the rest of the control
groups were kept in laboratory facilities.
Determination of the effects on larvae
In vivo observations were performed five times a week. Dead, malformation, and development stages were followed. In addition the head-tail length data (growth) were recorded every
week. Different stages of the Bombina bombina are illustrated figure 2.1.
Click here to see the Figure.
Figure 2.1
Different stages of Bombina bombina.
Forskellige stadier af Bombina bombina.
Footnotes
[1] Availabe from John A. Bantle, Dept. of Zoology, 430 LSW, Oklahoma State University, Stillwater OK 74078.
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