Embryotoxic effects of environmental chemicals: Tests with the South African clawed toad (Xenopus laevis)

ECOTOXICOLOGY

AND

ENVIRONMENTAL

SAFETY

13,324-338

(1987)

Embryotoxic Effects of Environmental Chemicals: Tests with the South African Clawed Toad (Xenopus laevis)’ KLAUS DUMPERT Battelle-Institut e. VI, Post Box 900 160, D-6000 Frankfurt am Main 90. Federal Republic of Germany Received March 21, 1986 In the course of the investigations reported below, it was shown that p-chloroaniline has a lethal effect on the embryos of.Xenopus laevisat a concentration of 100 ppm and is development inhibiting (teratogenic) at concentrations of 1 and 10 ppm, respectively. In the case of aniline, a significant development-inhibiting effect was observed at a concentration as low as I ppm. A toxic effect was caused by concentrations between 30 and 40 ppm during embryogenesis and by concentrations above 40 ppm during larval development. A very conspicuous finding was an inhibiting effect of 20 to 40 ppm aniline on pigmentation during embryogenesis and ofa concentration as low as 1 ppm on the body size ofthe young toads. In the case of potassium dichromate, it was possible to barely detect a weak development-inhibiting effect during embryogenesis but no development-retarding effect during larval development. Toxic effects of potassium dichromate occurred during embryogenesis at concentrations of 5 and 7.5 ppm and during the larval development at concentrations above 10 ppm. Sodium dodecylbenzenesulfonic acid at a concentration of 50 ppm was found to have such a strong embryolethal effect that 80% of the eggs showed no cell division at all and the remaining 20% developed to only the bicellular stage. A teratogenic effect of this substance was not observed. Phenol. too, was found to be toxic at a concentration of 50 ppm; in contrast to sodium dodecylbenzenesulfonic acid, however, it did not show any lethal effect on the embryos but it did on the tadpoles, mainly in the first stages of larval development. Lower concentrations ofphenol (5 and 10 ppm) had a nonsignificant inhibiting effect on the growth of the larvae. A teratogenic effect of phenol was not detected. 0 1987 Academic

Press. Inc.

1 INTRODUCTION

The German law for protection from hazardous substances took effect on January 1st 1982. This law provides that applications for the registration of new substances have to be accompanied by test certificates which permit decisions to be made on the possible hazardous effects of the substance concerned on humans or on the environment. If the substance for which an application for registration is being filed is marketed in the EC member states with an annual amount exceeding 100 t, additional proof of examination for carcinogenic, genotoxic, and embryotoxic properties has to be furnished (Umweltbundesamt, 1980). The test organism selected for exhibiting the embryotoxic (teratogenic and embryolethal) properties of environmental chemicals was the South African clawed toad (Yenopus Levis), which can be induced to spawn throughout the year and thus is always available for testing procedures (Dumpert, 1983; Dumpert and Zietz, 1984). ’ The investigations reported here were sponsored by the German Federal Environmental Agency (Umweltbundesamt) in Berlin.

0 147-65 13187 $3.00 Copyright 0 1987 by Academx Press, Inc. All rights of reproduction in any form reserved.

324

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The South African clawed toad appears to be particularly suitable as a test organism not only because of its reaction to three known teratogenic substances tested so far (Dumpert and Zietz, 1984) but also because performing the tests is much easier and involves much lower cost than tests with the usual test organisms (mainly rabbits). This last point is of greatest importance for a standard test required by law because it is the prerequisite for performing the test routinely under the “Law on Chemicals.” Another condition is that the test organism must react to the embryotoxic substances. Under these conditions, it is possible to use the embryotoxicity test with the clawed toad as a screening test. In the case of a positive result-in particular, for better transferability to humans-mammals can be used as the test organism. For further characterization of the reaction range and the sensitivity of the clawed toad to embryotoxic effects of environmental chemicals, the results of tests with a total of five test chemicals are reported. The following substances from a list of reference substances issued by the Umweltbundesamt were selected as test chemicals: p-chloroaniline, aniline, potassium dichromate, sodium dodecylbenzenesulfonic acid, and phenol. The selection criteria applied were solubility in water, resistance to hydrolysis, and either particularly large production volumes or a high hazard potential. p-Chloroaniline: Its solubility in water is 2.6 g/liter. This substance occurs in the environment, e.g., as a metabolite of pesticides (aromatic carbamates). The annual production ofp-chloroaniline in the EC amounts to 500 t. Aniline: Its solubility in water is 35 g/liter. The annual production in the Federal Republic of Germany alone is as high as 150.000 t. As to the possible carcinogenic effect of this substance, contradictory results exist: the teratogenic effect of substance remains to be investigated. Potassium dichromate: Its solubility in water is 150 g/liter. Production volumes are not known. Mutagenic effects are suspected. Sodium dodecylbenzenesulfonic acid: Its solubility in water is 400 to 1100 g/liter. A total amount of about 500,000 t/year is released into the environment worldwide via detergents. Phenol: Its solubility in water is 82 g/liter. This substance is produced worldwide in an amount of 3,000,OOO t/year as a technical raw product and disinfectant: of this amount, a.bout 75,000 t/year is released into the environment. 2 MATERIAL

AND

METHODS

The breeding stock comprised eight females and eight males of the South African clawed toad X. la&s, seven pairs of which had been imported from Cape Town, South Africa, as animals born in the wild state and captured: therefore, it was not possible to indicate the age of these animals. The 8th pair of animals originated from breeding a.t the Hubrecht Laboratory; at the start of the tests, the female was 4 years old and the male was 2 years old. The experiments were performed within a period of about 2 years. A comparison of the breeding pairs of different origin shows that the pairs imported from South Africa are markedly larger than the animals bred in the Netherlands. While the female from the Hubrecht Laboratory had a length of 10 cm and the male was 7 cm l.ong (length including head and trunk but excluding extremities), the ani-

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mals captured in the wild state reached average lengths of 12 cm and 9 cm, respectively. The breeding animals were kept in pairs in Macrolon basins of 26 X 42 cm floor space and 14 cm height, which were covered with stainless-wire grating. These basins in which the breeding animals were kept were continuously supplied with tap water from a 200-liter storage vessel. This storage vessel, in turn, was supplied from a second 200-liter vessel which was connected with the waterline network via a “Cellit” filter. A mechanical device ensured that the second vessel was always filled with fresh tap water. By strong ventilation, the free chlorine was expelled from the tap water, which had a constant temperature of 22°C in the second storage vessel. The flow was adjusted such that an amount of 10 liters was fed into each of the breeding basins per day, which corresponds to the volume of each of these basins. The breeding animals were induced to spawn by administering 2 X 400 IE Predalon (human chorionic gonadotropin) to the males and 1 X 1.O IE Predalon to the females in the form of injections into the dorsal lymph vessels. Subsequently, the breeding animals were placed into darkened spawning basins with a water temperature of 15°C which gradually assumed the room temperature of 22°C. Under these conditions, the females deposited between about 1800 and 3800 eggs. The fertilization rate was between 67 and 95%. Two pairs originating from South Africa, however. showed significantly lower spawning results and therefore were excluded. All the other breeding pairs were induced to spawn approximately once every 3 months. Spontaneous spawning of the breeding animals was not observed. The tap water in which the breeding animals were kept had a total hardness of 16 to 19 d H’, a carbonate hardness of 13 to 15 d H2, a chloride content of 25 to 40 mg/ liter, and a sulfate content of 35 to 75 mg/liter. The water for the tests with the eggs and the further development stages of Xenupzls was prepared from deionized water by addition of 0.35 g NaCl, 0.005 g KCl, 0.02 g MgC12, 0.01 g CaC12, and 0.02 g NaC03 per liter (modified according to Holtfreter, 193 1). The water was changed once a week. The test chemicals were added immediately after each change of water; no further chemicals were added in the interim. During the embryonal and larval development, the test and control basins were aerated by means of a diaphragm pump. The air was fed into the basins through Pasteur pipets at a rate such that uniformly about five air bubbles ascended per second. The breeding animals were fed twice a week with beef heart, to which “Cornicon” was added at intervals of 4 weeks. The tadpoles were fed on a mixture of 200 parts of finely-ground nettle leaves, 16 parts of Cornicon, 16 parts of pulverized egg white, 16 parts of brewer’s yeast, and 16 parts of yeast. The mixture was suspended in water and passed through a sieve of approximately 200-pm mesh size. The filtrate was further diluted with water and fed to the test animals daily in equal amounts which were increased with the development of the tadpoles. The room temperature was adjusted at 22 + 05°C. The darkened room was illuminated with white neon tubes which were switched on from 6.00 to 18.00 hr. In the course of the day on which the breeding animals had spawned (spawning always occurred early in the morning), the spawn was distributed to the test and control basins with two to five parallels each. The development was recorded daily. After completion ofthe embryonal development, the number of both test and control animals in the basins filled with 10 liters water was reduced to 10 animals.

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3 RESULTS 3. I Test:; with p-Chloroaniline The tests covering the examination ofp-chloroaniline for embryotoxic effects were carried out in 14 basins with 20 test animals in each of them; 2 of these basins contained identical test concentrations of the substance under investigation. The test concentrations used were 0.001, 0.01, 0.1, 1, 10, and 100 ppm. The water was changed once a week. After each change of water, the respective concentrations of the test chemicals were adjusted anew. The test animals were exposed to the test conditions in the egg stage at the beginning of the test. During the 90-day test period, no marked malformations or abnormalities in behavior of the animals in the test and the controls were observed. It should be noted, however, that all the animals in the test basins with the highest concentrations of the test chem.icals died within the first 3 weeks of the test period. In the other test basins, the death rate was highest (32%) in the basin with 0.001 ppm, and in the other test basins and in the control basins it ranged between 0 and 15%. The first froglet was observed in a control basin after 6 weeks: the development of all the other control and test animals-with the exception of those in the test basins with 1 and 10 ppm p-chloroaniline-was completed after 13 weeks at the latest. At that time, the test basins with 1 ppm p-chloroaniline still contained 3 and 1 tadpole(s) (stages 55-57). respectively, and the basins with 10 ppm p-chloroaniline still contained 15 and 12 tadpoles (stages 49-56), respectively. In relation to the original number of test animals and considering the death rate, this means that at the time when all the animals in the oth.er test basins developed into toads, the average proportions of test animals which had completed their larval development in the basins with 1 and 10 ppm pchloroaniline were approximately 85 and 20%, respectively. This means that p-chloroaniline at a concentration of 100 ppm has an embryolethal effect and at a concentration of 1 ppm is weakly development inhibiting and at a concentration of 10 ppm is marked.ly development inhibiting, and thus teratogenic. 3.2 Tests with Aniline In the first test the following concentrations were used: 1, 10, 100, 1000, and 10,000 ppm. Each of these concentrations was filled into three basins. Twenty tadpoles of stage 38 (Nieuwkoop and Faber, 1956) were placed into each of the basins. Immediately after the animals had been placed into the test basins, very drastic toxic effects were observed in the basins with 10,000 ppm aniline. The animals first swam hectically through the basin and then soon swam increasingly slower, and after about 30 set all were dead. In the test basin with 1000 ppm aniline it took about 2 days until all the test animals were dead. With the concentration of 100 ppm aniline, the test animals died within a period of 10 days, while in all the remaining basins no effects were observed. The test was discontinued after 2 weeks, after the acute toxic range for the tadpoles under investigation had been determined to be above concentrations of 100 ppm aniline. In the second test, the effect of aniline at concentrations of 10, 30, 50, 70, 80, 90, and 100 ppm was investigated. The test conditions corresponded to those in the first test. In this case also, tadpoles of stage 38 were placed into the test and control basins,

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FIG.1, Comparison of a test animal developed from stage 38 in a concentration of 70 ppm aniline (left) with a control animal (right).

the number of animals in each basin being reduced to 10 instead of 20 as in the first test. All the test animals in the basins with an aniline concentration of 90 and 100 ppm died in the course of 12 days. The test animals in the basins with 70 and 80 ppm were markedly retarded in their size development compared with the controls, and on the whole they were less pigmented (Fig. 1). Even at aniline concentrations below 70 ppm the death rate was extremely high, so that finally only tadpoles in aniline concentrations from 1 to 30 ppm developed into toads. The duration of development as a function of the aniline concentration is shown in Fig. 3. It was found that all the surviving controls developed into toads within 1 week (7th to 8th week from the start ofthe test), whereas the last surviving test animals had completed their larval development only after 16 weeks. Surprisingly, the larval development of the test animals in 1 ppm aniline took longer than that of the test animals kept in 10 ppm or even in 30 ppm. The proportions of surviving animals were 40% in the test basin with 10 ppm aniline, 50% in the basin with 1 ppm aniline, and 70% in the basin with 30 ppm aniline and in the control basins. The percentage of control animals which died in the course of the larval development thus was relatively high, i.e., 30% (3 out of 10 animals). Nevertheless, this second test shows both a development-inhibiting and an embryolethal effect of aniline. In the third test-unlike in the first two aniline tests-it was not the tadpoles but the eggs which were exposed to the various aniline concentrations. This was to examine how much more sensitive the embryos of the clawed toad were than the tadpoles. The following concentrations were tested in three parallels: 1, 10, 30, and 40 ppm. Each of the respective amounts of aniline was added to 20 liters water. About 200 eggs were placed into each of the test basins and into the control basins, where they were left until completion of their embryonal development. After that, the number of test animals in each basin was reduced to 20. After completion of the embryonal development, the test animals showed the first effects. The animals that had developed in concentrations from 20 to 40 ppm had a

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329

a

d FIG. 2. Examples of tadpoles whose embryonal development took place (a) in 20 ppm aniline. (b) in 30 ppm aniline, and (c) in 40 ppm aniline. in comparison with (d) a control animal of the same age.

markedly weaker pigmentation than the controls (Fig. 2) while no difference existed in the stage of development between test animals and controls. In the basins containing the highest aniline concentration (30 and 40 ppm), 5 -+ 5% and 15 -t 10% of the test animals, respectively, died at the embryonal stage: the death rate of the animals in the other test basins and in the control basins was 0%. During larval development, no conspicuous morphological or behavioral differences between the test animals and controls were observed, but there were marked

330

KLAUS DUMPERT loo %

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FIG. 3. Percentage of fully developed South African clawed toads as a function of development time and aniline concentration (mean values from three parallels); number of test animals per basin, 20.

differences in the duration of development. While all the control animals developed into toads in the 6th and 8th week, the development of the animals in 1 ppm aniline took 12 weeks at the most, in 10 ppm aniline 13 weeks, in 20 and 30 ppm aniline 14 weeks, and in 40 ppm aniline 15 weeks (Fig. 3). These differences from the controls were statistically significant both for the concentrations from 10 to 40 ppm (P = 0.0 1) and for the concentrations of 1 ppm (P = 0.05). In addition to a marked development-inhibiting effect, an embryolethal effect was observed. The survival rate of the controls was lOO%, in 10 ppm aniline it averaged 95%, in 1 and 30 ppm it was 80%, in 20 ppm it was 65%, and in 40 ppm it was on the average as low as 20%. A comparison of the fully developed toads from the control and test basins shows that the toads that had developed in a concentration as low as 1 ppm aniline are much smaller in size than the respective controls (Fig. 4). 3.3 Tests with Potassium Dichromate The tests with potassium dichromate were carried out in parallel to the tests with aniline. The first test was made with concentrations of 0.0 1,O. 1, 1, 10, and 100 ppm potassium dichromate, three basins being used for each concentration. Twenty tad-

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FIG. 4. Comparison of a froglet (Xenopus laevis) which had developed in a concentration of 1 ppm aniline (left) with a control animal of the same age (right).

poles of development stage 38 were placed into each of the test basins and into the control basins. One week after placing the tadpoles into the test basins, all the animals in the basins with 100 ppm potassium dichromate were dead, whereas no difference was observed between the other test animals and the controls. This test-as with the corresponding aniline test-was discontinued after 2 weeks. It had been found that a concentration of 100 ppm potassium dichromate has a strongly toxic effect and that the limit of toxicity and sublethal effects are to be expected in the range between 10 and 100 ppm. This range of concentration was examined more closely in the second test, which covered potassium dichromate at concentrations of 1,2.5,5,7.5, 10,25, and 50 ppm. The test conditions corresponded to those in the first test. In this case, too, tadpoles at development stage 38 were placed into the test and control basins, the numbers of animals being reduced to 10, instead of to 20 as in the first test. All the animals in the basins with 25 and 50 ppm potassium dichromate died in the course of 4 weeks from placing the tadpoles into the test basins. Among the effects of aniline that the test animals experienced at higher concentrations were retarded body size, compared to the control animals, and a weaker pigmentation; these effects were observed with potassium dichromate, but to a much lesser extent (Fig. 5). Only test animals kept in concentrations of potassium dichromate up to 10 ppm developed into toads. The development was fastest in the test basin with 1 ppm potassium dichromate, where all surviving test animals developed into toads between the 5th and 6th week; the development was slowest in the test basins with 2.5 ppm potassium dich.romate, where the first animals developed after 7 weeks and the last ones after 12 weeks. The development periods of all the other test and control animals were between these extremes. The survival rate of both test and control animals ranged between 50 and 90%, respectively; in this case, just as in the case of the development time, no concentration dependence of the effects was recognized. In the third test, the eggs instead of the tadpoles were exposed to the various test concentrations. The following concentrations, with three parallels, were used: 0.1, 1,

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FIG. 5. Comparison of two test animals (top) which had developed from stage 38 in a concentration of 25 ppm potassium dichromate with a control animal of the same age (bottom)

2.5, 5, and 7.5 ppm. About 200 eggs from fresh spawn were placed into each of the test and the control basins. Five days later, the numbers of test and control animals were reduced to 20 per basin. Whereas no difference was found to exist between the test animals kept in the concentrations of 0.1 and 1 ppm potassium dichromate and the control animals, the animals kept in the concentration of 2.5 ppm showed a markedly weaker pigmentation (Fig. 6). In the test basin with 5 ppm potassium dichromate, 15 + 5% of the embryos died, and in the basin with 7.5 ppm potassium dichromate 30 f 15%. In addition to this embryolethal effect, marked differences were observed between the animals kept in the various test basins. Whereas the controls and the test animals from the basins with 0.1 and 2.5 ppm potassium dichromate had all reached stage 41 and swam freely around in the water, the animals kept in 5 ppm were at stage 37 and the animals kept in 7.5 ppm were at stages 35 to 36 (Fig. 6). These differences in development, just as the increased death rate and the weaker pigmentation effected by potassium dichromate, show that the embryos of X. luevis react more sensitively to this test substance than do the larvae. In the further development of the test and control animals reduced to 20 per basin the differences in development and pigmentation were soon balanced, so that no further substance-related peculiarities existed, nor did a development-inhibiting effect that had become obvious under the influence of aniline (Fig. 3) occur in the test basins with potassium dichromate (Fig. 7). Only a weak toxic effect is indicated by the fact that the survival rate until completion of metamorphosis of all the test and control animals both in the control and the test basins containing 0.1, 1, and 2.5 ppm potassium dichromate was lOO%, but in the test basins with 5 ppm potassium dichromate it was 95 f 5% and in the basins with 7.5 ppm 90 + 10%. 3.4 Tests with Sodium Dodecylbenzenesulfonic

Acid

The following concentrations were used in three parallels for the tests with sodium dodecylbenzenesulfonic acid: 0.1, 1, 10, and 50 ppm. About 200 eggs were placed

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d FIG. 6. Tadpoles of Xenopus laevis after a development time of 5 days in (a) 2.5 ppm potassium dichromate, (b) 5 ppm, and(c) 7.5 ppm ofthe same substance; (d) control animal of the same age.

into each of the test and the control basins. As in all the other tests, aeration was effected by a diaphragm pump through Pasteur pipets at a rate of about five bubbles per second. The number of larvae was reduced to 20 per basin immediately after completion of the embryonal development. During embryonal development, no differences were observed between the test animals from the basins with 0.1, 1, 5, and 10 ppm and the control animals. Only

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100 %

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FIG.7. Percentage of fully developed South African clawed toads as a function of development time and potassium dichromate concentration (mean values from three parallels); number of test animals per basin, 20.

the concentration of 50 ppm sodium dodecylbenzenesulfonic acid was found to be so strongly embryotoxic that the eggs developed to the two-cell stage at the most. In the case of most of the eggs (ca. 80%) kept in a concentration of 50 ppm sodium dodecylbenzenesulfonic acid, no cell division occurred at all. The larval development of the surviving test animals showed no further peculiarities. The test and control animals completed metamorphosis after 6 to 9 weeks, without any effect of the test substances becoming obvious. This test proved that sodium dodecylbenzenesulfonic acid is embryolethal with toxicity limits between 10 and 50 ppm. A teratogenic effect of this substance on X. laevis was not recognized.

3.5 Tests with Phenol Phenol was tested at concentrations of 0.1, 1,5, 10, and 50 ppm. Each of the phenol concentrations was examined for its teratogenic and embryolethal effect in three parallel basins. Three basins without phenol were used as controls. To counteract the effect of the test substance being expelled from the water by the aeration of the basin, water of the same composition as that contained in the test basins and of the respective phenol concentration was continuously pumped into the basins from storage

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vessels. The amount of liquid which was thus pumped into each of the basins was 100 ml per day. The test animals contained in one of the two basins with 50 ppm phenol died within 6 days from completion of their embryonal development. The test animals kept in the second and the third basin with the same phenol concentration died within 3 weeks at the most. All the other test animals developed without any conspicuous differences from the controls. Exact measurements of the body lengths after a test period of 5 weeks showed that the test animals in the concentrations of 5 and 10 ppm phenol were on the average 8% smaller than the test animals kept in concentrations of 0.1 and I ppm phenol and the controls. This difference is, however, not statistically significant. 4 DISCUSSION The embryos and the larvae of amphibians have been used repeatedly for test purposes. Tests have so far been performed, in addition to X. Levis, with Ambystoma mexicanum (Johnsson, 1972), with A. opacum and A. maculatum (Hall and Swineford, 198 l), with Rana sphenocephala, R. sylvaticu, Bufo americana, and Acris crepitans (Hall and Swineford, 198 I), with M. ornata (Ghate and Mulherkar, 1980), with B. bufi (Cuoma and d’Angelo, 1977), with R. catesbeiana (Hall and Swineford, 198 1; Saber and Dumson, 1978), with R. temporaria (Rzehak et al., 1977), and particularly frequently with R. pipiens (Greenhouse, 1976a; Dial, 1976; Chang et al., 1974; Brown and Caston, 1962). The eggs for the tests with all the above species except for A. mexicanum and R. pipiens that could be bred in the laboratory had to be collected outdoors. Nevertheless, R. pipiens has some major drawbacks as a test organism compared with X. laevis (Greenhouse, 1976): (1) Xenopus is easily maintained in a disease-free condition, whereas ranid species are very often overcome by bacterial infections when kept in the laboratory. (2) Xenopus will thrive on commercially available trout food, whereas Rana must be kept in cold storage or fed live food. (3) Xenopus females will lay eggs several times per year for a period of several years in response to commercially available human chorionic gonadotropin, whereas each Rana female can be used only once and requires Rana hormone which must be prepared in the laboratory. (4) Xenopus embryos metamorphose to froglets in 6- 10 weeks, whereas R. pipiens embryos require 6- 12 months. The embryos and the larvae of X. Zaevis, which have frequently been used as test organisms, were found to be sensitive to environmental pollutants, in particular, heavy metals (Aleksandrowicz et al., 1975; Birge and Just, 1974; Browne and Dumont, 1979), fungicides, herbicides, and insecticides (Bancroft and Prahlad, 1972; Marchal-Segault and Ramade, 198 1; Anderson and Prahlad, 1976), as well as other organic compounds such as hydrocarbons (Green, 1954; Csaba et al., 1974), substituted amines (Davis et al., 198 1; Dumont et al., 1979) and hydrazines (Greenhouse, 1976a,b). These investigators differ substantially with respect to the breeding, spawning and test conditions. In the investigations by Dumont et al. (1979) and by Robinson et al. ( 1972), for example, the eggs were dejellied by means of mercaptoethanol; Greenhouse (1976a,b) dejellied the eggs with cysteine and papain; and the tests by Bancroft and Prahlad ( 1972) and by Prahlad et al. ( 1974) were made with eggs that had not been dejellied. According to the two last-mentioned authors, many sub-

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TABLE 1 Concentration (ppm)

Aquatic species p-Chloroanihne LCsO (96 h) Fathead minnow (Pimephalespromelas) LCsO (96 h) Rainbow trout (Salmo gairdneri) L& (96 h) Bluegill (Lepomis macrochirzu) LC50 (48 h) Zebra fish (Brachvdanio rerio) ECSO(24 h) Water flea (Daphnia magna)

12 14 2 46 0.06

Aniline LCsO (96 h) Rainbow trout (Salmo gairdneri) LCO Golden orfe (Leuciscus idus. m.) LC50 Water flea (Daphnia magna) E& (24 h) Water flea (Daphnia magna)

20-4 I 18-49 0.5 23

Sodium dodecylbenzenesulfonic acid LCsO (96 h) Bluegill (Lepimis macrochirus)

5.6

Phenol LC&, (48 h) Golden orfe (Leuciscus idus me/.) LCsO (48 h) Water flea (Dephnia magna) LCsO (24 h) Rotifera (Brachionus rubens)

25 12 600

stances are better taken up by embryos whose jelly and vitellin membrane are intact. The tests made by Robinson et al. (1972) and Knutson and Prahlad (1971) also showed that tritium is taken up to a higher extent by the embryos from nondejellied eggs. The author’s own investigations with dejellied eggs, however, suggest that the embryolethal effect of aniline is stronger on dejellied than on nondejellied eggs. In general, however? it is highly substance dependent whether the sensitivity of the embryos is increased or reduced by dejellying the eggs. A contribution to the standardization of a test with Xenopus larvae was made by Edmiston and Bantle ( 1982) who described a 96-hr flowthrough toxicity test with 3week-old Xenopus tadpoles performed with naphthalene. The toxicity criteria used by these authors were-apart from mortality-the depigmentation of the tadpoles and the reduction of their swimming activity. Among the substances used in the tests covered by the present paper, p-chloroaniline and aniline proved to be most effective. Concentrations of these substances as low as 1 ppm were sufficient to produce weak development-inhibiting effects; 10 ppm had a marked development-inhibiting effect. The limit values of lethality for both substances range between 10 and 100 ppm. Aniline, which was investigated more closely than p-chloroaniline, showed a lethal effect on larvae from 20 ppm. Potassium dichromate is less teratogenic than aniline, but more toxic. The test animals kept in concentrations between 5 and 7.5 ppm showed a slight retardation in embryonal development, which was caught up fast in the further development. Additional tera-

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togenic efFectsdid not occur in this case. The embryolethal effect of potassium dichromate, however, started at a concentration as low as 5 ppm; a concentration of 10 ppm had ,a toxic effect on tadpoles. Sodium dodecylbenzenesulfonic acid and phenol showed no teratogenic effects. Both substances had no toxic effects in a concentration of 10 ppm: at a concentration of 50 ppm the toxic effect was so drastic that no test animal survived. In the case of sodium dodecylbenzenesulfonic acid, this toxic effect influenced the fertilized eggs and impeded almost any further development; a phenol concentration of 50 ppm had a toxic effect only on the tadpoles but not on the embryos. CONCLUSIONS These results show that the embryos of Xenopus react more sensitively than the tadpoles to many, but not all, the tested substances. To evaluate the teratogenic and embryotoxic effects of test substances, it is therefore important to observe the whole development of the test animals under the influence of the test substances. To assess the sensitivity of the Xenopus test to the chemical under investigation, the results of tests concerning the toxicity of these substances to other aquatic animals (from Rippen, 1984) are compiled in Table 1. It was shown that in particular the teratogenic effects ofp-chloroaniline and aniline occur mostly at much lower concentrations than correspond to the L&, values of most of the test organisms. REFERENCES ALEKSANDROWICZ, J., DUBROWOLKI, J., AND LISIWIECZ, J. (1975). Effect of selenium on immunosuppressive and teratogenic properties of aflatoxin B, Rev. Exp. Oncol. 22,239-247. ANDERSON, R. J., AND PRAHLAD, K. V. (1976). The deleterious effects of fungicides and herbicides on Xenopus laevis embryos. Arch. Environ. Contam. Toxicd. 4,3 12-323. BANCROFT, R., AND PRAHLAD, K. V. (1972). Effect of ethylenebis (dithiocarbamic acid) disodium salt (nabam) and ethylenebis (dithiocarbamato) manganese (maneb) on Xenopus laevis development. Teratology 7, 143- 150. BIRGE, H. J., AND JUST, J. (1974). Sensitivity of Vertebrate Embryos to Heav.v Metals as a Criterion of Water Quality. OWR-R061-Washington, DC. BROWNE, C. L., AND DUMONT, J. N. (I 979). Toxicity of selenium to developing Xenopus laevis embryos. J. To.uicol. Environ. Health ~$699-709. BROWNE, C. L., AND DUMONT, J. N. (1980). Cytotoxic effects of sodium selenite on Xenopus laevis tadpoles. Arch. Environ. Contam. Toxicol. 9, 18. CHANG. L. W., REUHL, K. R., AND DUDLEY, A. W. (1974). Effects of methylmercury chloride on Rana pipiens tadpoles. Environ. Res. 8,82-9 I CUOMA, M. G., AND D’ANGELO, L. S. (I 977). Teratogenic effects of L-asparaginasis on anuran amphibians. Riv. Biol. IO, 297-302. DAVIS, K. R., SCHULTZ, T. W., AND DUMONT, J. N. (1981). Toxic and teratogenic effects of selected aromatic amines on embryos of the amphibian Xenopus laevis. Arch. Environ. Contam. Toxicol. 10, 371-391. DIAL, N. A. (1976). Methylmercury: Teratogenic and lethal effects in frog embryos. Teratology 13, 327-334.

DUMONT, J. N., SCHULTZ, T. W., AND JONES, R. D. (1979). Toxicity and teratogenicity of aromatic amines to Xenopus laevis. Bull. Environ. Contam. Toxicol. 22, 159- 166. DUMPERT, K. (1983). Der Krallenfrosch (Xenopus laevis) als Testorganismus ftir embryotoxische Wirkungen von Umweltchemikalien. Umweltbundesamt (Ed.), Chemikaliengesetz Heft 3, Priifung und Bewertung von Stoffen auf ihre Umweltgeftihrlichkeit. pp. I53- 157. Berlin.

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DUMPERT, K., AND ZIETZ, E. (1984). Platanna (Xenopus la&s) as a test organism for determining the embryotoxic effectsof environmental chemicals. Ecotoxicol. Environ. Saf 8,55-15. EDMISTON, E., AND BANTLE, J. A. (1982). Use of Xenopus luevis larvae in 96-hour, flow-through toxicity tests with naphthalene. Bull. Environ. Contam. Toxicol. 29,392-399. GHATE, H. V., AND MULHERKAR, L. (1980). Effect of sodium diethyldithiocarbamate on developing embryos of the frog Microhyla ornata. Indian J. Exp. Biol. 18,1040-1042. GREEN, E. V. (1954). Effects of hydrocarbon-protein conjugates on frog embryos. I. Arrest of development by conjugates of 9, lo-dimethyl- 1,2benzanthracene. Cancer Res. 14,59 1. GREENHOUSE,G. (1976a). The evaluation oftoxic effects ofchemicals in fresh water by using frog embryos and larvae. Environ. Poll&. l&303-3 15. GREENHOUSE, E. (1976b). Evaluation of the teratogenic effects of hydrazine, methylhydrazine, and dimethylhydrazine on embryos of Xenopus laevis, the South African clawed toad. Teratology 13, 167-178.

HALL, R. J., AND SWINEFORD,D. M. (198 1). Acute toxicities of toxaphene and aldrins to larvae of seven species of amphibians. Toxicol. Lett. 8,33 l-336. HOLTF~ETER, J. (193 I). Uber die Aufzucht isolierter Teile des Amphibienkeimes II. Ziichtung von Keimen und Keimteilen in Salzlosung. Arch. Entwicklungsmech. 124,404-466. JOHNSSON,B. G. (1972). Effects ofthalidomide on the embryonic development ofthe axolotl (Ambystoma mexicanurn).

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KNUTSON, T. L., AND PRAHLAD, K. V. (197 1). 3,5,3-Triiodo-L-thyronine uptake and development of metamorphic competence by Xenopus laevis embryonic tissues. J. Exp. Zool. 178,45-58. MARCHAL-SEGAULT, D., AND RAMADE. F. (198 I). The effects of lindane, an insecticide, on hatching and postembryonic development ofxenopus laevis (Daudin) anuran amphibian. Environ. Res. 24,250-258. NIEUWKOOP, P. D., AND FABER, J. (1956). Normal Table of Xenopus laevis (Daudin). North-Holland, Amsterdam. PRAHLAD, K. V., BANCROFT, R., AND HANZELY, L. (1974). Ultrastructural changes induced by the fungicide ethylenebis (dithiocarbonic acid) disodium salt (nabam) in Xenopus tissue during development. Cytobios 9, 12 1. RIPPEN, G. (1984). Handbuch der Umweltchemikalien. Ecomed, Landsberg/Lech. ROBINSON, D., PRAHLAD, K. V., AND HAMPEL, A. E. (1972). Amino acid uptake by Xenopus luevis embryos: Effect of triiodo+thyronine. Camp. Physiol. 43B, 749-754. RZEHAK, K., MARYANSKA-NADACHOWSKY, A., AND JORDAN, M. (1977). The effect of Karbotox 75. a carbaryl insecticide, upon the development of tadpoles of Rana temporaria and Xenopus laevis. Folia Biol. (Krakow) 25,39 l-399. SABER, P. A., AND DUMSON, W. A. (1978). Toxicity ofbog water to embryonic and larval anuran amphibians. J. Exp. Zool. 204,33-34. Umweltbundesamt (Ed.) (1980). Umweltchemikalien, Priifung und Bewertung von Stoffen auf ihre Umweltgefahrlichkeit im Sinne des neuen Chemikaliengesetzes, Berlin.