Developmental toxicity of p,p'-dichlorodiphenyltrichloroethane, 2,4,6-trinitrotoluene, their metabolites, and benzo[a]pyrene in Xenopus laevis embryos

Since 1995, high incidences of deformed frogs have been documented in Kitakyushu, Japan. In this area, relatively high concentrations of DDT, trinitrotoluene (TNT), their metabolites (p,p'-dichlorodiphenyldichloroethylene [DDE], p,p'-dichlorodiphenyldichloroethane [DDD], 2-amino-4,6-dinitrotoluene [2ADNT], and 4-amino-2,6-dinitrotoluene [4ADNT]), and benzo[a]pyrene [BaP]) have been identified from field samples. I used a standardized assay with Xenopus laevis embryos (frog embryo teratogenesis assay--Xenopus, FETAX) to examine the developmental toxicity of these compounds. Both DDE and BaP were considered nearly nontoxic in embryonic development because they induced low (< 10%) mortality and malformation incidence even at the highest concentrations tested (DDE, 393 microM; BaP, 13.2 microM). The DDD (96-h median lethal concentration [LC50] = 44.1 microM, 96-h median effective concentration [EC50] for malformation = 14.9 microM) was more lethal and teratogenic than its parent compound, DDT (96-h LC50 = 101 microM, 96-h EC50 = 41.5 microM). Predominant symptoms observed were axial malformations (DDT and DDD) and irregular gut coiling (DDT). However, DDT and DDD should not act as major lethal or teratogenic toxicants in the aquatic environment within a short-term exposure via water because their 96-h LC50 and 96-h EC50 values were extremely high, considering their low solubility in water. The TNT (96-h LC50 = 16.7 microM) was more lethal than 2ADNT (96-h LC50 = 166 microM) or 4ADNT (96-h LC50 = 115 microM). Although 4ADNT (96-h EC50 = 85.8 microM) induced various tadpole malformations, it was a weak teratogen compared with TNT (96-h EC50 = 9.78 microM) and 2ADNT (96-h EC50 = 16.9 microM). The most typical malformations observed were axial malformations, eye abnormalities (TNT), edema, and irregular gut coiling (2ADNT and 4ADNT). The 96-h LC50 and 96-h EC50 values of TNT, 2ADNT, and 4ADNT were lower than their saturated concentrations in water. Therefore, these nitroaromatic compounds may show lethal or teratogenic effects on aquatic animals if their habitats are severely contaminated with TNT.     

Joint actions of carboxylic acid binary mixtures on Xenopus embryo development: Comparison of joint actions for malformation types

The joint action for malformations induced by 18 binary mixtures of four carboxylic acids was determined, using Xenopus embryos. The acids were selected to evaluate whether joint actions for mixtures changed when the data were analyzed by specific malformations or malformation syndromes as opposed to joint actions determined when any type of malformation was included in the analysis. Each of six binary combinations of the acids were tested three or four times in 96-h staticrenewal tests, starting with late-blastula stage embryos. Toxic unit analysis indicated that all but five of 56 possible analyses showed concentration addition joint actions. Concentration addition was observed for all instances in which microcephaly was included as a malformation (with or without other malformations being included) and for all possible mixtures where only eye defects were evaluated. In five instances, evaluating only abnormal gut coiling, the joint action was response addition, including all three mixtures for the valproic acid and pentanoic acid combination. The other 29 analyses which included abnormal gut coiling showed concentration addition. The results indicate that, for chemicals that are structurally similar and induce the same types of defects in frog embryos, there is no significant change in the joint action when the data is analyzed by specific malformations or syndromes rather than by including all malformations together. Abnormal gut coiling, a malformation induced by most toxic chemicals, appears to be less reliable for joint action determination, especially if it is the primary manifestation of abnormal development.     

Initial evaluation of developmental malformation as an end point in mixture toxicity hazard assessment for aquatic vertebrates

The joint toxic action of three binary mixtures was determined for the embryo malformation endpoint of the aquatic FETAX (frog embryo teratogenesis assay: Xenopus) test system. Osteolathyrogenic compounds and short-chain carboxylic acids, representing separate, distinct modes of action for induction of malformation, were selected for testing in 96-hr, static-renewal tests. Three mixtures were tested for each combination, with each combination being tested on three separate occasions. Using toxic unit analysis, the combination of osteolathyrogens and the combination of carboxylic acids produced strictly additive (concentration addition) rates of malformation, while the combination of an osteolathyrogen and a carboxylic acid was less-than-additive (response addition) for induction of malformation. Therefore, developmental malformation may have value as an endpoint in mixture toxicity hazard assessment.     

Stage-specific malformations and phenotypic changes induced in embryos of amphibian (Xenopus tropicalis) by triphenyltin

Tributyltin (TBT) and triphenyltin (TPT) can induce unique malformations in embryos of amphibian (Xenopus tropicalis). We further exposed X. tropicalis embryos to TPT during different stages. Severe malformations were observed in the embryos after exposure to 5–10 μgSn/L TPT during S32–40 and S40–46. Comparatively, the embryos showed slight or moderate malformations after exposure during S10–25, S25–32 or S46–47. The most characteristic malformations were narrow or no fins, followed by enlarged proctodaeums and skin hypopigmentation. The developed fins were still affected after TPT exposure, but the inhibited fins could not recover even after the removal of TPT exposure. In TPT treatment groups, the proctodaeums of embryos externally expanded, and the pigment cells of embryos were poor, small and dot like. Our results suggested that TPT-induced teratogenicity was highly stage-specific. The sensitive window of fin defects indicated that TPT affected the formation rather than induction or expansion of fins.     

Response of larval Xenopus laevis to atrazine: assessment of growth,metamorphosis, and gonadal and laryngeal morphology

Larval Xenopus laevis were exposed to one of four concentrations of atrazine (0, 1, 10, or 25 microg/L, 11 replicate tanks per treatment, 60-65 larvae per replicate) dissolved in an artificial pond water (frog embryo teratogenesis assay- Xenopus [FETAX]) medium beginning 48 h after hatching until the completion of metamorphosis. Separate groups of larvae (six replicate tanks per treatment, 60-65 larvae per replicate) were exposed to estradiol (100 microg/L), dihydrotestosterone (100 microg/L), or ethanol vehicle control dissolved in FETAX medium. None of the treatments affected posthatch mortality, larval growth, or metamorphosis. There were no treatment effects on sex ratios except for estradiol, which produced a greater percentage of female offspring. Exposure to either estradiol or 25 microg atrazine/L increased the incidence of intersex animals based on assessment of gonadal morphology. Atrazine did not reduce the size of the laryngeal dilator muscle, a sexually dimorphic muscle in this species. We conclude that environmentally relevant concentrations of atrazine do not influence metamorphosis or sex ratios and do not inhibit sexually dimorphic larynx growth in X. laevis. The incidence of atrazine-induced intersex animals was small (<5%) and occurred only at the greatest concentration of atrazine tested, a concentration that is rarely observed in surface waters in the United States.     

两栖类动物在环境毒理学研究中的应用

两栖类动物(amphibian)在食物链中具有水陆两栖的独特地位,其生活周期比较复杂,幼体生长速度快,卵、鳃和皮肤具有渗透性,污染物能在其体内富集.这些特性使其成为监测环境污染的前哨物种,在环境毒理学研究中发挥着越来越重要的作用,该文以非洲爪蟾(Xenopus laevis)和牛蛙(R.catesbeiana)为例综述了其在环境毒理学(急性毒性试验、蛙胚胎致畸试验、变态试验、微核试验)及环境内分泌干扰物检测等研究中的应用.     

Application of frog embryo teratogenesis assay-Xenopus to ecological risk assessment

An expert workshop recently was convened to consider the frog embryo teratogenesis assay-Xenopus (FETAX) as a screening method for identifying the potential developmental toxicity of single chemicals and chemical mixtures. One recommendation from the workshop was that, in order to determine the utility of FETAX for ecological risk assessments, additional consideration of how the assay is conducted is necessary. In addition, a comparative evaluation would be useful of FETAX endpoints (i.e., survival, malformations, growth) versus each other, endpoints from aquatic toxicity tests using more commonly tested species of cladocerans and fish, and tests with other amphibian species. This review provides an evaluation and critique of the current FETAX protocol from two perspectives: Practical considerations relative to conducting the test and sensitivity of the assay (and associated endpoints) compared to tests with other species. Several aspects of the current standard protocol, including test temperature, diet, loading rates, and chemical exposure options, need to be modified to ensure that the assay is robust technically. Evaluation of FETAX data from the open literature indicates that growth is the most sensitive endpoint in the assay, followed by malformations and then survival; unfortunately, the growth endpoint often is not considered or reported in the assay. Comparison of FETAX data with acute toxicity data from tests with other amphibians or traditional aquatic test species indicates FETAX is relatively insensitive. This suggests that environmental risk assessments using acute hazard data from tests with traditional aquatic test species usually would be more protective of native amphibian species than risk assessments that use hazard data from FETAX.     

Effects of low pH,metals, and water hardness on larval amphibians

The interactions between pH, metals, and water hardness in determining the suitability of vernal ponds for amphibian reproduction were investigated through a fully factorial laboratory microcosm experiment. Naturally-occurring metals (Al, Cu, Fe, Pb, Zn) at concentrations analogous to those observed in prior field studies had variable effects on acute (7 day) and chronic (28 day) exposure survival for both the Jefferson salamander, Ambystoma jeffersonianum, and the wood frog, Rana sylvatica. Acute exposure to Al and Cu significantly reduced wood frog survival; increased water hardness significantly increased acute exposure wood frog survival. Acute exposure mortality of the Jefferson salamander was significantly higher in the Al and Cu treatments; in toxic metal treatments (Al and Cu), survival was higher at the low pH level. Chronic exposure of wood frogs to Al and Cu, higher pH level, and lower hardness levels greatly reduced survival. Chronic exposure of Jefferson salamander larvae to Al and Cu significantly reduced survival. Chronic Jefferson salamander survival was significantly greater in the higher pH treatments in the presence of non-toxic metals, however, survival was significantly decreased in the higher pH treatments in the presence of Al and Cu. There were no statistically significant effects of metals, pH, or hardness on wet mass of the pre-metamorphic larvae.The four conditions tested (pH, hardness, presence of metals, and amphibian species) are important determinants of toxicity observed in temporary pond amphibian breeding sites. Clearly, there is no single water chemistry parameter that can explain the toxicity of temporary ponds to the embryos and larvae of terrestrial amphibians that use the ponds for breeding.     

Waterfowl and the bacteriological quality of amenity ponds

This study investigated the impact of waterfowl on the bacteriological quality of village ponds in East Yorkshire, north-east England. Water and sediment samples were collected from ponds with and without resident ducks and geese; faecal indicator and potentially pathogenic bacteria were assayed by membrane filtration and by selective enrichment. Escherichia coli, faecal streptococci and, to a degree, Clostridium perfringens were more abundant in ponds with waterfowl; Salmonella was isolated in June-August from the sediment of a pond with waterfowl. The results suggested that the bacteriological quality of village ponds might be adversely affected by waterfowl. All water samples from ponds with waterfowl had faecal indicators at higher concentrations than EU requirements for bathing waters. Although these ponds are not bathing waters we suggest skin contact and accidental ingestion of water should be avoided.     

Selenium teratogenesis in natural populations of aquatic birds in Central California

The frequency and types of malformations are described that were encountered during the spring of 1983 in a natural population of aquatic birds exposed to agricultural drainwater ponds and food items containing high concentrations of selenium in central California. A total of 347 nests of aquatic birds containing 1,681 eggs was selected for study at Kesterson Reservoir located in the Kesterson National Wildlife Refuge (NWR), Merced County, California. Embryos collected during incubation or from eggs that failed to hatch were examined to determine the age at death and presence of malformations. Embryonic death was generally high; approximately 17–60% of the nests of different species contained at least one dead embryo. The incidence of malformed embryos was also high; approximately 22–65% of the nests where at least two embryos were examined contained abnormal embryos. American coots (Fulica americana) and black-necked stilts (Himantopus mexicanus) experienced the highest incidence of malformed embryos. For all species, the average percentage of eggs containing dead or live abnormal embryos was 16.1 whereas the average percentage containing live abnormal embryos was 10.7. Multiple gross malformations of the eyes, brain, and feet were often present. Brain defects included hydrocephaly and exencephaly. Eye defects included both unilateral and bilateral anophthalmia and microphthalmia. Eye and foot defects with ectrodactyly and swollen joints were the most common in coots. Beak defects also occurred frequently and most often included incomplete development of the lower beak of ducks (Anas spp.) and stilts. Wing and leg defects were most prevalent in stilts and ducks, with ectromelia and amelia most prevalent in stilts. Other malformations occurring at lower frequencies included enlarged hearts with thin ventricular walls, liver hypopiasia, and gastroschisis. Based upon simultaneous examination of a control population of aquatic birds of the same species and published studies, the incidences of embryonic mortality and deformities were 9–30 times greater than expected. The role of the form of selenium responsible for teratogenesis in laboratory studies is discussed.     

Risk of malformations associated with residential proximity to hazardous waste sites in Washington State

Hazardous waste sites often contain substances harmful to fetal development. Using linked birth-hospital discharge and hazardous sites data for Washington State, we evaluated the association between malformation occurrence and maternal residential proximity to hazardous waste sites. Cases (N=63,006) were infants born 1987-2001 with malformations. Controls (N=315,030) were randomly selected infants without malformations born during these years. Distance between maternal residence and nearest hazardous waste site was measured using geographic information systems (GIS) software. Odds ratio (OR) estimates of the relative risk of malformation at varying distances were calculated. Relative to living >5 miles from a site, living < or = 5 miles was associated with increased risk of any malformations in offspring (for >2- < or = 5 miles: OR 1.15: 95% Confidence Interval (CI): 1.10, 1.21; for >1- < or = 2 miles: OR 1.26, 95% CI: 1.20, 1.32; for >0.5- < or = 1 miles: OR 1.28, 95% CI: 1.22, 1.35; for < or = 0.5 miles: OR 1.33, 95% CI: 1.27, 1.40.) Risk estimates varied by urban vs. rural maternal residence and by specific malformation type. Hazardous waste sites are often located within populated areas. Thus, the possibility of increased malformation occurrence among those in close proximity deserves closer scrutiny.     

Investigation of the relationship between s-methoprene and deformities in anurans

Evidence that amphibian deformities are on the rise has prompted a number of researchers to question a variety of natural and man-made substances, including chemicals such as s-methoprene (trademark Altosid), an insect growth regulator applied to water bodies for mosquito control. Despite conclusions reached by the U.S. Environmental Protection Agency and a considerable body of scientific evidence demonstrating that it is unlikely that s-methoprene or any of its degradation products are causing the high rates of deformities found in Minnesota and other states, concerns persist about potential negative impacts of s-methoprene on nontarget species, specifically anurans. Water analyses in field and laboratory conditions and a comparison of reported Altosid use with reported frog deformities in Minnesota demonstrate that a connection between frog deformities and Altosid use is unlikely. These results indicate that factors other than s-methoprene and its degradation products are contributing to the recent outbreak of frog deformities.     

Impact of a Commercial Peat Moss Operation on Water Quality and Biota in a Small Tributary of the Richibucto River, Kent County, New Brunswick, Canada

The St-Charles Plain (Kent County, New Brunswick, Canada) commercial peat moss operation has been ongoing since 1983. To process the peat, a dry extraction method is used that requires extensive drainage of the peat bog. The water is directed toward sedimentation ponds, where it drains into a small brook, which feeds into a river affected by tidal salt water. Water discharge from the bog contains large amounts of peat particles that deposit in the surrounding watershed. As a result, the pH of the freshwater sites that receive the drainage water from the commercial operation, is fairly acidic (pH 3.9–4.7). Water samples from or near the peat moss operation have a higher concentration of total phosphorous and total organic carbon. The peat particles contain relatively high levels of total mercury, as reflected by analysis of peat sediments. However, the water samples contained low levels of dissolved mercury. Indigenous samples of biota—namely, sand shrimps (Crangon septemspinosa) and mummichogs (Fundulus heteroclitus)—did not contain mercury levels higher in the impacted sites than in the reference sites. Introduced blue mussels (Mytilus edulis) did not accumulate significant amounts of mercury during a 62-day exposure in the study area. Overall, the data suggest that although relatively large amounts of mercury-containing peat particles are discharged into the ecosystem, bioaccumulation of mercury in the biota does not occur. Received: 12 June 2001/Accepted: 10 December 2001     

Toxicity of Guthion® and Guthion® 2S to Xenopus laevis embryos

The development of Xenopus laevis (African clawed frog) embryos exposed to the pesticide Guthion^® (technical grade) and Guthion^® 2S (commercial formulation) was evaluated in modified Frog Embryo Teratogenesis Assay—Xenopus (FETAX) tests. The embryos were exposed to five or six increasing concentrations of pesticide in 10- and 100-ml exposure volumes of test solution for 96 h. Embryos exposed in 10-ml volumes of Guthion exhibited increased mortality, increased deformation, and decreased size as compared to those exposed in 100-ml volumes. LC50s for embryos exposed in the 10-ml Guthion tests ranged from 6.1 to 6.3 mg/L as compared to 10.6 to 11.9 mg/L for those in the 100-ml tests. The percentage of deformities at 3 mg/L Guthion in test survivors in 10-ml tests ranged from 73 to 89%, while in the 100-ml tests less than 2% were deformed at the same concentration. Mean control embryo lengths at test completion were 8.2 and 10.6 mm, respectively, for 10- and 100-ml tests. The LC50 for embryos in 100 ml Guthion 2S was 1.6 mg/L active ingredient, indicating a much greater toxicity of the commercial formulation. NOAEL (No Observed Adverse Effect Level) values for Guthion and Guthion 2S ranged from 0.48 to 7.96 mg/L, depending upon basis (length, deformity, mortality) and pesticide formulation, and were many times greater than the existing water quality criterion of 0.01 g/L.     

Assessment of environmental stressors potentially responsible for malformations in North American anuran amphibians

Several species of anuran amphibians from different regions across North America have recently exhibited an increased occurrence of malformations, predominantly of the hindlimb. Research concerning the potential causes of these malformations has focused extensively on three stressors: chemical contaminants, ultraviolet (UV) radiation, and parasitic trematodes. In this overview of recent work with each of these stressors, we assess their plausibility as contributors to the malformations observed in field-collected amphibians. There is as yet little evidence that chemical contaminants are responsible for the limb malformations. This includes chemicals, such as the pesticide methoprene, that could affect retinoid-signaling pathways that are critical to limb development. Exposure to UV radiation also seems to be an unlikely explanation for hindlimb malformations in amphibians. Although solar UV can cause hindlimb deficiencies in amphibians, a probabilistic assessment based on empirical dose-response and exposure data indicates that UV exposures sufficient to induce limb defects would be uncommon in most wetlands. Results of controlled studies conducted with some affected species and field-monitoring work suggest infection by digenetic trematodes as a promising explanation for the malformations observed in anurans collected from many field sites. Controlled experimentation with additional species and monitoring across a broader range of affected sites are required to assess fully the role of trematodes in relation to other stressors in causing limb malformations. If trematode infestations are indeed related to the recent increases in malformed amphibians, then the question remains as to what alterations in the environment might be causing changes in the distribution and abundance of the parasites.     

Rapid dissipation of glyphosate in small forest ponds

Glyphosate was applied to the water surface of four small boreal forest ponds and sixin situ microcosms at a rate of 0.89 Kg a.i./ha. Water samples collected over a period of up to 255 days were analyzed for glyphosate and its primary metabolite aminomethylphosphonic acid (AMPA). Glyphosate dissipated rapidly from all ponds with first order half-lives ranging from 1.5 to 3.5 days. The slowest dissipation rate occurred in the pond with the most calcareous water and sediments. Glyphosate remained at or above the treatment concentration in microcosms containing only water but decreased rapidly in the presence of sediments. AMPA levels in ponds and microcosms were consistently low. Concentrations on microcosm wall samples were temporally variable, probably a result of adsorption to periphytic biofilms. Glyphosate in the sediments of treated microcosms generally increased with time during the period of observation. These results confirm that glyphosate dissipates rapidly from the surfaces waters of lentic systems, and suggest that sediment adsorption or biodegradation were the major means of glyphosate loss from the water column.     

Effects on sea urchin fertilization and embryogenesis of water and sediment from two rivers in Campania,Italy

Sea urchin embryos and sperm were utilized for evaluating the toxicity of water and sediment from two rivers, the Sarno (S) River and the Volturno (V) River, in the Campania region, Italy. The effects on developing embryos were evaluated by scoring developmental defects, whereas sperm exposure was tested for the effects on fertilization and offspring quality. Ten sampling sites from the rivers (S.1–S.4 and V.1–V.6) were monitored for water and sediment quality. Water sampling was carried out biweekly for a year (1988–1989); the samples were tested at dilutions 10^–4 to 10^–2 in natural seawater. Sediment tests were carried out on solid phase samples (collected in 1989 to 1992), at concentrations ranging from 2 to 10 mg/ml (dry wt) in seawater. The tests conducted on water samples mostly led to nonsignificant results in either embryo-or spermiotoxicity, possibly due to sharp changes in pollutant levels in the water column. Unlike water, sediment samples displayed clear-cut results both on embryogenesis and on fertilization success. The grain size of sediment failed to reveal any relationship with toxicity, which could only be referred to the presence of toxic contaminants (Melluso et al., 1993). The most polluted sediment samples displayed a dramatic embryo-toxicity, up to approximately 87% developmental arrest in embryos reared in 2 mg/ml of sediment from site S.1 (affected by leather tanning effluent). These results were independent of storage conditions of sediment samples (i.e., at +4°C or –20°C). A decrease in fertilization success was also induced by sediment samples from polluted sites (not by a relatively unpolluted reference sample). Interestingly, sediment spermiotoxicity appeared to be related to storage temperature in samples from two agricultural sites (S.3 and V.6), in that freezing caused a drop in sample spermiotoxicity compared to refrigeration; e.g., a sample from site V.6 stored at +4°C decreased fertilization rate (FR) to 9% versus a control value of 86%, whereas the same sample induced but a minor change following storage at –20°C (FR=68%).In general, a better reliability was observed for sediment bioassays than for water bioassays, thus suggesting that future biomonitoring studies should mainly focus on sediment quality. The use of solid phase sediment in toxicity testing by sea urchin embryos and sperm is warranted by the present results.     

锌对镉所致胚胎毒性的保护作用研究

实验用Ｗｉｓｔａｒ大鼠，分为正常组、低锌组、低锌染镉组、正常染镉组和染镉高锌保护组，分别饲养，其中三组在孕８和１０天注射镉（２．０ｍｇ／ｋｇ）。妊娠第２０天处死动物，取血，并剖腹检查孕鼠内脏和胎鼠畸形情况。研究发现：与正常对照组相比，单纯缺锌即可引起胎鼠的吸收率增高和畸形的发生；而染镉（２．０ｍｇ／ｋｇ）也可引起胎鼠的吸收胎和迟死胎显著增加，并可引起皮下水肿、卷尾等外观畸形。在孕期缺锌的同时，给予孕鼠２．０ｍｇ／ｋｇ体重的镉，孕鼠在整个孕期体重呈负增长，胚胎不能形成；如给予孕鼠高锌饲养（２２７ｍｇ／ｋｇ），胎鼠可得到较好的保护，其吸收胎、死胎率明显下降，存活率显著上升，且胎鼠体重、身长也与对照组相差无几。提示孕鼠缺锌可引发胎儿畸形，缺锌染镉加重胚胎毒作用，而高锌膳食能拮抗镉所造成的胚胎毒作用。     

Toxicity and Pathogenicity Testing of the Insect Pest Control Fungus Metarhizium anisopliae

Renewed interest in the use of Metarhizium anisopliae and its toxins for insect control prompted the following safety assessment. A neutral extract (methylene chloride, pH 7.2), derived from M. anisopliae cultures, was evaluated for toxicity and mutagenicity using aquatic animal bioassays and the Ames test. The average LC₅₀ of the neutral extract obtained in static, acute 96-h tests conducted with ≤24-h-old Mysidopsis bahia was 2.41 mg L⁻¹. By partially purifying destruxins from the neutral extract, it was shown that destruxins alone were not responsible for the observed toxicity in mysids. The neutral extract was fetotoxic to developing grass shrimp, Palaemonetes pugio, and frog, Xenopus laevis, embryos; the LC₅₀ values were 52 and 32 mg L⁻¹, respectively. Eye spot abnormalities were observed in shrimp and frog embryos exposed to the neutral extract. In extract-exposed frog embryos, moderate to severe cranial, facial, and gut malformations were also observed. The neutral extract was toxic to juvenile mosquito fish, Gambusia affinis, at an LC₅₀ value of 141 mg L⁻¹. Adult female G. affinis surviving a 24-h exposure to 200 μg ml⁻¹ of the neutral extract produced healthy broods. After 3 months, no mortalities or adverse effects were observed in adult G. affinis fed a diet partially composed of a freeze-dried M. anisopliae culture. The neutral extract did not show mutagenicity in the Ames test using strains TA98 and TA100 with and without metabolic activation by rat liver S9. Significant (p  ≤ 0.05) mortalities were obtained when embryos of grass shrimp and inland silverside fish, Menidia beryllina, were exposed to the same lot of M. anisopliae conidiospores. Exposure of frog embryos to M. anisopliae conidiospores did not cause significant (p > 0.05) mortalities or malformations. Received: 31 October 1997/Accepted: 5 February 1998     

Potential ecotoxic effects of polychlorinated biphenyls on Xenopus laevis

We examined potential ecotoxic effects of polychlorinated biphenyl (PCB)3, PCB5, Aroclor 1254, and Aroclor 1242 on Xenopus laevis. Tadpoles were exposed to PCBs from stage 46/47 (system of Nieuwkoop and Faber) to the completion of metamorphosis. We demonstrated, to our knowledge for the first time, forelimb malformations caused by PCBs (malformation rate, > 70%). The malformed forelimbs were fixed in the adduction-backward rotation position and could not move. Therefore, malformed male frogs were destined to have no offspring, because they could not grasp the females with their forelimbs to mate. Alcian blue-alizarin red double-staining indicated that the forelimb malformation resulted from the shoulder abnormality. Compared with the normal shoulder joint, the proximal humerus with the humerus inter-rotated 90 degrees in the abnormal shoulder joint. Moreover, testes from more than a third of male frogs with exposed to PCBs exhibited feminization to different degrees at gross morphology and histology, with fewer or abnormal spermatogonia and oocytes. Gonadal abnormalities would lead directly to reproductive dysfunction and population decline. These results suggest that PCBs have potentially ecotoxic effects on amphibian populations. We infer that PCBs could play roles in amphibian malformations and population declines, at least at sites that are polluted heavily with PCBs.