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The Effect of Esfenvalerate and Prochloraz on Amphibians with special reference to Xenopus laevis and Bombina bombina

2 Materials and methods

2.1 Principles and methods for maintaining and breeding the South African clawed frog, Xenopus laevis and the fire-bellied toads, Bombina bombina
   2.1.1 Facilities for maintaining adults Xenopus
   2.1.2 Facilities for breeding Xenopus
   2.1.3 Principles and methods for maintaining adults Bombina bombina
   2.1.4 Principles and methods for breeding Bombina bombina
2.2 Toxicity tests using embryos of Xenopus laevis and Bombina bombina
   2.2.1 Principle and design of the toxicity test
   2.2.2 Test substances
   2.2.3 Preparation of test solutions
   2.2.4 Embryos
   2.2.5 Culture medium
   2.2.6 Experimental design
   2.2.7 Determination of the effects on embryo of Xenopus and Bombina
   bombina
   2.2.8 Replicates and controls
   2.2.9 Physical - analysis
   2.2.10 Chemical - analysis
   2.2.11 Data treatment and reporting

2.1 Principles and methods for maintaining and breeding the South African clawed frog, Xenopus laevis and the fire-bellied toads, Bombina bombina

2.1.1 Facilities for maintaining adults Xenopus

Adult Xenopus were kept in an animal room without any light-affection from outside, in such a way that a photoperiod of 12-h day/12-h night could be maintained. By keeping this photoperiod it was possible to breed Xenopus year-round. Adults were kept in large aquaria or in fibreglass raceways at densities of less than 6 per 1800 cm² of water surface area. The water depth was between 7 and 14 cm and the water was aerated by the use of air stones.

pH of the water was between 6.5 and 9.

Xenopus males were between 7.5 and 10 cm in crown-rump length and more than 2 years old. Adult males had dark pads on both side of every forearms and did not have cloacal labra. Adult females were between 10 and 12.5 cm in length and at least 3 years old. Females were larger than males and easy to identify by the presence of prominent cloacal labra.

Diet

The adults were fed three times per week with beef liver. Liquid multiple vitamins were added to the ground beef liver (AMSTM standard E 1439-91).

Temperature

Adults were kept at 23±3°C.

2.1.2 Facilities for breeding Xenopus

Males and females were bred as a single pair in a 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 deionised or distilled water. The pH of the final solution should be 7.6 to 7.9. All chemicals was reagent-grade or better.

A 45l glass aquarium fitted with a 1 cm mesh suspended about 3 cm above the bottom of the aquarium was used. This ensured that deposited eggs could lie undisturbed on the bottom of the aquarium and could be scraped into Petri dishes. The sides of the breeding aquarium were opaque and a bubbler fitted to oxygenate the water. The top of the aquarium was covered with opaque porous material.

Water temperature was adjusted to 23±0.5°C .

Breeding

To mature the oocyttes, the females received about 200 IU of Pregnant Mare Gonadotropin (PMG) 2 days before the animals were moved to the breeding aquarium. To induce breeding, the males and the females received 250 to 500 and 500 to 1000 IU, respectively, of human chorionic gonadotropin by way of injection into the dorsal lymph sac. The hormone concentration was about 1000 IU/ml in sterile 0.9% NaCl. The amount of human chorionic gonadotropin injected depended on the time of year and condition of the adults. Lower doses were usually used in spring and higher doses in autumn. Amplexus normally ensues 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. Eggs laid in “strings” or not perfectly round was not used because they develop abnormally.

2.1.3 Principles and methods for maintaining adults Bombina bombina

Adult Bombina was at least 2 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 x 30 cm vivarium, the floor area of which was 60% water, about 8 cm deep and the land area was dark-coloured with hiding 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. Slow growing natural aquatic plants and plants of plastic and broken pots were provided to create 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 and at room temperature in the summer period.

2.1.4 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) 2 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. Lower 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.

2.2 Toxicity tests using embryos of Xenopus laevis and Bombina bombina

2.2.1 Principle and design of the toxicity test

FETAX is a 96 h renewal whole embryo assay that can be used to evaluate the developmental toxicity of a test material. Exposure is continuous throughout the test. For each concentration, two dishes each containing 25 embryos and 10 ml of test solution are used. For each control, four dishes of 25 embryos each are used in the standard experiments with Xenopus, however, in experiments with Bombina only 5 embryos are placed in each dish. Embryos must be randomly assigned to test dishes. Dishes must be randomly assigned to their positions in the incubator. In order to evaluate developmental toxicity, mortality, malformation, and growth inhibition properly, data must be collected. In most tests, it will be possible to generate concentration-response curves for mortality, malformation, and growth inhibition. The mortality and malformation concentration-response curves should then be used to estimate the concentration that would affect 50% of the exposed embryos. At least 90% of the FETAX solution controls must have attained stage 46 at 96 h (Nieuwkoop and Faber, 1975).

Test organisms

The aim was to compare the results obtained from embryos of the South African clawed frog, Xenopus laevis with another species, which has a wide distribution in Europe, the fire-bellied toads, Bombina bombina.

Test chemicals

The aim was to determine the effect of two different pesticides with different mechanisms of toxic action and with different in physico-chemical properties.

Egg manipulation

The aim was to assess the influence of the jelly coat on the toxicity of pesticides. This part of the work was carried out because the jelly coat is normally removed before the starting on an experiment in an ordinary FETAX test.

2.2.2 Test substances

Two pesticides with different mechanism of toxic action and physicochemical properties were selected:

• Esfenvalerate (CAS no. 66230-04-4) pyrethroid insecticide
• Prochloraz (CAS no. 67747-09-5) conazole fungicide, steroid demethylation inhibitor.

Table 2-a
Selected physico-chemical properties of the two pesticides

Udvalgte fysisk-kemiske egenskaber for de to pesticider

* 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.

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.

Prochloraz is an ergosterol biosynthesis inhibitor. Prochloraz is a protectant and eradicant fungicide effective against a wide range of diseases affecting field crops, fruit, turf, and vegetables. An EC is normally recommended for use in cereals (400-600 g. a.i./ha).

2.2.3 Preparation of test solutions

The test material was reagent-grade or better unless for the commercial product, Sumi alfa (contains 5% esfenvalerate; active ingredients). Stock solutions were prepared daily. The pH of the stock solutions was 7.5±0.5.

Dimethyl sulfoxide and acetone were used for preparing stock solutions. Concentrations of dimethyl sulfoxide and acetone in test solutions were < 1.1% v/v. At these concentrations, no effect in the FETAX standard test has been observed (Fort et al., 1991).

2.2.4 Embryos

Removal of jelly coat

Dejellying of embryos should begin immediately after the end of egg laying. Dejellying of embryos was carried out by gentle swirling for 1 to 3 min in a 2% w/v L-cysteine (CAS #52-90-4) solution prepared in FETAX solution. The cysteine solution was adjusted to pH 8.1 with 1 N NaOH. The solution was made up immediately prior to use. Dejellying was monitored continuously and the process stopped just after all jelly was removed. Care should be taken not to treat the embryos too long because survival may be reduced.

Staging of embryos

Staging of embryos was done according to Nieuwkoop and Faber (1975).

Embryo selection

Normally cleaving embryos were selected for use in testing. Two levels of selection were used. In double selection, normally cleaving embryos were first sorted into dishes containing fresh FETAX solution. After a short period during which cleavage continues, 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 a reference to determine whether the cleavage pattern was normal. Midblastula (stage 8) to early gastrula (stage 11) was used to start the test. By 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. A large bore blood bank Pasteur pipette was used to transfer embryos at this stage without any harm. The sorting was done in Petri dishes.

2.2.5 Culture medium

The culture medium used for the test was FETAX solution. This medium was used for embryos of both Xenopus and Bombina.

2.2.6 Experimental design

All tests with embryos of Xenopus and Bombina were conducted in an incubator at 24±0.5°C. The tests chambers were covered 60 mm glass Petri dishes (before use all glass wares were treated with silylation reagent solution and thoroughly washed in water) or in a few cases in disposable 55 mm polystyrene Petri dishes with an initial culture volume of 10 ml. If a solvent other than dilution-water or FETAX solution was used for preparing test solutions, the concentration of solvent was the same in all test solutions that contained the test material and a solvent control was incorporated in the test which contained the same concentration of solvent.

The Petri dishes in the incubator were randomised by rows. 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 through the use of 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 might develop poorly although they initially appear acceptable. This would cause all the embryos to be discarded if embryos are mixed from different mating pairs.

Renewal

Renewal of the medium was performed every 24 h during the test. The renewal procedure entails fresh replacement of test material every 24 h during the test. Just prior to this change the pH was measured of the control and the highest test concentrations in order to determine if significant changes occurred. The pH of the test solutions was 7.5 +/- 0.5. Renewal is accomplished by removing the test solution with a Pasteur pipette. The orifice of the Pasteur pipette should be enlarged and fire-polished to accommodate embryos without damage in case the embryos are accidentally picked up. This is standard procedure for FETAX.

Duration of the test

The standard exposure time for FETAX is 96 h and the attainment of stage 46 in controls. Deviations from this standard exposure time must be reported as deviating from standard FETAX conditions. Thus in the Bombina experiments, the exposure time was 120 h.

2.2.7 Determination of the effects on embryo of Xenopus and Bombina bombina

In vivo observations

In vivo observations were performed every 24 h during the test. A binocular dissection microscope capable of magnifications up to 30 was used to evaluate the abnormal embryos.

Mortality

Dead embryos was 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 might kill living embryos. Death at 24 h (stage 27) was ascertained by the embryo's skin pigmentation, structural integrity, and irritability (measured on lack of response on physical stimulations). At 48 h (stage 35), 72 h (stage 42), and 96 h (stage 46) the lack of heartbeat serves as an unambiguous sign of death. At 96 h of exposure or stage 46 of controls, the total number of dead embryos was recorded during the test (mortality was registered after 24, 48, 72, and 96 h of exposure). 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. As abnormal embryos rarely survive in nature this test end point may also be used to estimate chronic toxicity to aquatic organisms.

Malformation was recorded at the end of 96 h. Embryos exposed to the test material was compared with appropriate controls. The number of malformations in each category was reported in standard format to ease interlaboratory comparison (cf. Appendix 1).

Growth inhibition

The ability of a material to inhibit embryonic growth is often the most sensitive indicator of developmental toxicity. Thus, Berrill et al. (1993) exposed five species of amphibians to pyrethroid insecticide at concentrations between 10 and 200 µg/l with no results of mortality, but with a notable reduction in growth rates.

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. Measurement was made after embryos were fixed in 3% formaldehyde solution.

Teratogenic Index

Teratogenic Index (TI) is determined after 96 h of exposure. TI is defined as 96 h LC₅₀ (mortality) divided with 96 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

The test design includes three independent tests (on different days). For each concentration, two dishes each containing 25 embryos of Xenopus and 5 embryos of Bombina and 10 ml of test solution are used. For each control, four dishes of 25 or 5 embryos are used.

Thus, a range-finding and three replicate tests are performed on each test material. A control, in which no test material has been added, is used to provide

1. a measure of the acceptance of the test by indication the quality of embryos and the suitability of the FETAX solution, test conditions and handling procedures, and
    
2. a basis for interpreting data from other treatments.

If a solvent other than dilution-water or FETAX solution is used, at least one solvent control, using solvent from the same batch used to make the stock solution, must be included in the test.

Range-finding

The range-finding test consists of a series of at least seven concentrations that differ by a factor of ten. This should be adequate to delineate the concentration range needed to establish the 96 h LC₅₀ and EC₅₀ (malformation). The more similar the range-finding and replicate-definitive test are, the more useful the range-finding test will be. Growth inhibition data are not collected from range-finding tests.

Replicate-definitive tests - number of tests and data collection

Three definitive tests were conducted on each test material in a random block design. Because it is necessary to acquire data on mortality, malformation, and growth inhibition, the concentration series needs to be adjusted to the expected 96 h LC₅₀, 96 h EC₅₀ (malformation), and the minimum concentration needed to inhibit growth (MCIG). To ensure an adequate supply of normal embryos for each test, two mating pairs were induced and clutches harvested, separately. Embryos were sorted to ensure viability prior to testing. Each test used early embryos derived from a single mating pair. Each individual test will yield data that will be used to generate concentration-response curves for mortality, malformation, and growth inhibition.

Experimental dilutions

Each test consisted of at least five concentrations for determining concentrations-response curves for both mortality and malformation.

Reference toxicant

For a positive control or reference toxicant, 6-aminonicotinamide presents a mortality and malformation database convenient for reference purpose. For each test, the positive control consisted of two dishes of 25 or 5 embryos each exposed to 2500 mg 6-aminonicotinamide/l and two dishes of 25 or 5 embryos each exposed to 5.5 mg 6-aminonicotina-mide/l. Only the biological responses related to mortality and malformation were considered in the analysis. Growth inhibition was not considered in regard to responses to 6-aminonicotinamide.

2.2.9 Physical - analysis

The temperature of the incubator was measured daily by a thermometer. The pH was daily checked before and after the addition of the test substance. The pH examination was made with a pH electrode. The pH of the stock and test solutions should be 7.5.

2.2.10 Chemical - analysis

Renewal of the medium was performed every 24 h during the test. Renewal was accomplished by removing the approximately 10 ml test solution (exact amount known) with a Pasteur pipette to a 10 ml glass flask. Determination of esfenvalerate and prochloraz in these solutions were carried out in samples after 24 h, 72 h, and after 96 h of exposure.

Esfenvalerate

The 10 ml sample was spiked with standard solutions of phenanthrene-d₁₀ and cyhalothrin in acetone. The spiked sample was then extracted with 1 ml toluene by shaking for 30 minutes on an “end-over-end” shaker. Finally, the organic phase was transferred to 1 ml glass vial for analysis by GC-MS in SCAN-mode and SIM-mode, respectively.

Prochloraz

20 µg of propiconazol (internal standard) in acetone (1036 ng/µl) was added to the 10 ml sample, corresponding to an aqueous concentration of approximately 2.1 µg/ml. The aqueous samples were then extracted (shaken mechanically for 20 minutes) with 1 ml pentane. The pentane phase was transferred to 1 ml glass vials. 100 µl was transferred to another glass vial, evaporated to dryness, and redissolved in 100 µl of hexane. The hexane extract was subsequently analysed by capillary gas chromatography combined with mass spectrometry. The mass spectrometer was operated in the selective ion monitoring mode (GC-MS-SIM).

2.2.11 Data treatment and reporting

With the Probit analysis it was possible to obtain concentrations-response curves to determine the values of 96 h LC₅₀ and 96 h EC₅₀. The ratio between these two values gives the Teratogenic Index (TI).

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) was 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 D.A. et al. 1989).

Fodnoter

[1] 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