Development of an in vitro diagnostic method to determine the genotypic sex of Xenopus laevis.

A genotypic sex determination assay provides accurate gender information of individuals with well-developed phenotypic characters as well as those with poorly developed or absent of phenotypic characters. Determination of genetic sex for Xenopus laevis can be used to validate the outcomes of Tier 2 amphibian assays, and is a requirement for conducting the larval amphibian growth and development assay (LAGDA), in the endocrine disruptor screening program (EDSP), test guidelines. The assay we developed uses a dual-labeled TaqMan probe-based real-time polymerase chain reaction (real-time PCR) method to determine the genotypic sex. The reliability of the assay was tested on 37 adult specimens of X. laevis collected from in-house cultures in Eurofins EAG Agroscience, Easton. The newly designed X. laevis-specific primer pair and probe targets the DM domain gene linked-chromosome W as a master female-determining gene. Accuracy of the molecular method was assessed by comparing with phenotypic sex, determined by necropsy and histological examination of gonads for all examined specimens. Genotypic sex assignments were strongly concordant with observed phenotypic sex, confirming that the 19 specimens were male and 18 were female. The results indicate that the TaqMan® assay could be practically used to determine the genetic sex of animals with poorly developed or no phenotypic sex characteristics with 100% precision. Therefore, the TaqMan® assay is confirmed as an efficient and feasible method, providing a diagnostic molecular sex determination approach to be used in the amphibian endocrine disrupting screening programs conducted by regulatory industries. The strength of an EDSP is dependent on a reliable method to determine genetic sex in order to identify reversals of phenotypic sex in animals exposed to endocrine active compounds.

Effects of bisphenol a on fecundity, egg hatchability, and juvenile growth of Marisa cornuarietis.

Recent work on the snail Marisa cornuarietis has claimed to show endocrine disruption in response to bisphenol A (BPA). The present experimental design was optimized to detect effects of BPA on fecundity, egg hatchability, and juvenile growth, with an emphasis on reproduction, since previous studies suggested this to be a sensitive endpoint. No differences in eggs/female/ month between unexposed snails and snails exposed to nominal concentrations of 0.1, 1, 25, and 640 microg BPA/L during six months of exposure were found. No effect of BPA on the percentage of eggs hatching successfully was found, as was no difference in time to hatching between the control and any BPA treatment. We observed a significant decrease in female growth and a marginal effect on female wet weight in the 640-microg/L treatment compared to the control and a significant increase in male growth rate and a marginal increase in male wet weight in the 1-microg/L treatment compared to the control. However, a much greater proportion of the variability in juvenile growth was explained by variation between pairs and between siblings from the same pair than by BPA treatment. We conclude that effects of BPA in the nominal exposure range 0.1 to 640 microg/L (measured range 0.062-696 microg/L) are unlikely to be of significance for field populations of this species. An additional adult fecundity trial at 22 degrees C (in contrast to all other experiments that were conducted at 25 degrees C) found no evidence to suggest that snails are more sensitive to BPA at the lower temperature, as has been previously claimed. The present results indicate that the sensitivity of M. cornuarietis to BPA is similar to that of other aquatic invertebrates for which data are available.

Does bisphenol A induce superfeminization in Marisa cornuarietis? Part I: intra- and inter-laboratory variability in test endpoints.

It has been claimed that bisphenol A (BPA) induces superfeminization in the freshwater gastropod, Marisa cornuarietis. To explore the reproducibility of prior work, here we present results from a three-laboratory study, the objectives of which were to determine the mean and variability in test endpoints (i.e., adult fecundity, egg hatchability, and juvenile growth) under baseline conditions and to identify the sources of variability. A major source of variability for all of the measured endpoints was due to differences within and among individuals. With few exceptions, variability among laboratories and among replicate tanks within laboratories contributed little to the observed variability in endpoints. The results highlight the importance of obtaining basic knowledge of husbandry requirements and baseline information on life-history traits of potential test species prior to designing toxicity test protocols. Understanding of the levels and sources of endpoint variability is essential so that statistically robust and ecologically relevant tests of chemicals can be conducted.

Does bisphenol A induce superfeminization in Marisa cornuarietis? Part II: toxicity test results and requirements for statistical power analyses.

This study presents results of the effects of bisphenol A (BPA) on adult egg production, egg hatchability, egg development rates and juvenile growth rates in the freshwater gastropod, Marisa cornuarietis. We observed no adult mortality, substantial inter-snail variability in reproductive output, and no effects of BPA on reproduction during 12 weeks of exposure to 0, 0.1, 1.0, 16, 160 or 640 microg/L BPA. We observed no effects of BPA on egg hatchability or timing of egg hatching. Juveniles showed good growth in the control and all treatments, and there were no significant effects of BPA on this endpoint. Our results do not support previous claims of enhanced reproduction in Marisa cornuarietis in response to exposure to BPA. Statistical power analysis indicated high levels of inter-snail variability in the measured endpoints and highlighted the need for sufficient replication when testing treatment effects on reproduction in M. cornuarietis with adequate power.

Effects of husbandry parameters on the life-history traits of the apple snail, Marisa cornuarietis: effects of temperature, photoperiod, and population density.

These experiments are part of a larger study designed to investigate the influence of husbandry parameters on the life history of the apple snail, Marisa cornuarietis. The overall objective of the program is to identify suitable husbandry conditions for maintaining multi-generation populations of this species in the laboratory for use in ecotoxicological testing. In this article, we focus on the effects of photoperiod, temperature, and population density on adult fecundity and juvenile growth. Increasing photoperiod from 12 to 16 h of light per day had no effect on adult fecundity or egg hatching and relatively minor effects on juvenile growth and development. Rearing snails at temperatures between 22 degrees C and 28 degrees C did not influence the rates of egg production or egg clutch size. However, the rates of growth and development (of eggs and juveniles) increased with increasing temperature in this range, and when temperatures were reduced to 22 degrees C egg-hatching success was impaired. Juvenile growth and development were more sensitive to rearing density than adult fecundity traits. On the basis of the present results, we conclude that rearing individuals of M. cornuarietis at a temperature of 25 degrees C, a photoperiod of 12L:12D, and a density of <0.8 snails L(-1) (with lower densities for juvenile snails) should provide favorable husbandry conditions for maintaining multi-generation populations of this species.

Effects of food type, feeding frequency, and temperature on juvenile survival and growth of Marisa cornuarietis (Mollusca: Gastropoda).

The present experiments are part of a larger study designed to investigate the influence of husbandry parameters on the life history of the ramshorn snail, Marisa cornuarietis, in order to identify suitable husbandry conditions for maintaining multi-generation populations in the laboratory for use in ecotoxicological testing. In this paper we focus on the effects of a combination of food types and feeding frequencies (i.e., the frequency with which the snails were offered food) on juvenile growth and survival at different temperatures. Offspring produced in the laboratory by wild specimens of M. cornuarietis, from Puerto Rico, were used to test the effects of three types of food (lettuce, alginate with fish food, alginate with snail mix) fed at three frequencies (given ad libitum on 4/4, 2/4, or 1/4 d) on juvenile survival and growth. The 4-d feeding regimens were repeated four times, giving a total of 16 d for the experiments. The experiments were conducted at two temperatures (22 degrees and 25 degrees C) under a 12 h light:12 h dark photoperiod. Juvenile growth rates increased with increasing feeding frequency for all food types. The most rapid growth rates occurred in the high-frequency lettuce treatments and the slowest growth rates in the low-frequency lettuce and alginate with snail mix treatments. Juvenile snails grew faster at 25 degrees than at 22 degrees C, and mortality was about twice as high at the lower temperature. Growth rates were used to provide a rough estimate of time to maturity, which was determined to take about twice as long at 22 degrees than at 25 degrees C. The results showed that lettuce is the best food if supplied in abundance, but effects on growth are very dependent on feeding frequency and temperature. We conclude that 25 degrees C is a more appropriate temperature for maintaining populations than 22 degrees C, that lettuce provides a suitable food source, and that food should be supplied continuously for husbandry and toxicity testing of populations of M. cornuarietis.

Development of freshwater water-quality criteria for perchlorate.

The anion perchlorate (ClO4-) is an oxidizing component commonly used in solid propellants for rockets and missiles; in explosives, flares, fireworks, chemical processes, and automobile air-bag inflators; and for other assorted uses. With recent advances in analytical detection capability, perchlorate has been found in a variety of ground and surface waters throughout the United States. Because perchlorate has been associated with thyroid problems in humans and may have similar effects on wildlife, it is desirable to develop a water-quality criterion to assist in identifying concentrations of perchlorate in water likely to pose an ecological health risk. In the present study, we compiled all available data regarding the effects of perchlorate to aquatic organisms, and we performed additional toxicity and bioconcentration tests as required by the U.S. Environmental Protection Agency (U.S. EPA) for the development of water-quality criteria for aquatic life. A criterion maximum concentration of 20 mg/L and a criterion continuous concentration of 9.3 mg/L were calculated based on the test results. Although these are not formal Clean Water Act Section 304(a) criteria, which must be published by the U.S. EPA, these criteria may be useful in the determination of remedial action levels for contaminated sites, for National Pollutant Discharge Elimination System permit limits, and other water-quality management practices.