The symbiosis between developing Spotted Salamanders (Ambystoma maculatum) and the unicellular alga known as Oophila amblystomatis appears to be mutualistic, with involved parties trading apparent benefits within the salamander egg capsule, but the spatiotemporal ecology of the interaction has yet to be thoroughly explored. Using newly developed primers for quantitative polymerase chain reaction (PCR), we sampled 149 salamander egg masses of various ages from six breeding ponds in northeast Alabama. We documented the presence of algal DNA within egg capsules throughout the developmental process. Regression via linear mixed model estimation showed a positive relationship between the age of egg masses and the probability of algal DNA detection and a negative trend when comparing egg-mass age and qPCR cycle quantification value. These trends indicate an increase in both the probability that a given egg will contain algal DNA and the amount of algal DNA an egg contains, if present, as age increases. We found no effects of pond, site within pond, or year of sampling in either case.

Arsenic (As) and chromium (Cr) are two contaminants that are detected in aquatic and terrestrial habitats. Using the spotted salamander, Ambystoma maculatum, to assess impacts from these contaminants may be advantageous as adults live and breed in such environments. Adult amphibians typically exhibit elevated tissue concentrations of contaminants present in their environment, while larval stages were found to exhibit increased sensitivity to pollutants. From January through March of 2015, during the spring breeding season, 5 adults and approximately 32 egg masses were collected from a local breeding site. Field levels of As and Cr ranged from 5.99 to 8.88 mu g/L and 1.45 to 2 mu g/L, respectively, while mean adult As tissue concentrations were 56.74 mu g/g dry weight for heart, 0.92 mu g/g for liver, and 1.21 mu g/g for tail tissue. Mean tissue concentrations for Cr were 87.64 mu g/g for heart, 1.47 mu g/g for liver, and 6.92 mu g/g for tail. Developing larvae that were collected from the field and exposed in a lab setting for 12d to 0.2 or 20mg/L of either As or Cr displayed little DNA damage attributed to As, but marked damage due to exposure to 20mg/L Cr when assessed using the comet assay. Exposure to a mixture of either 0.25:0.1 or 25:10mg/L As and Cr resulted in significant DNA damage at the lower concentration of 0.25:0.1mg/L. As adult spotted salamanders were found to possess high concentrations of these contaminants in cardiac tissue, and larvae were shown to be susceptible to DNA damage from increased exposures, assessing impacts and potential declines of amphibian populations exposed to As and Cr is needed.

Potential developmental toxicities of three different cigarette butt leachates were evaluated using the frog embryo teratogenesis assay–Xenopus (FETAX). Xenopus laevis embryos were exposed to regular cigarette butt (RCB), menthol (MCB) and electronic (ECB) in concentrations ranging from 0 to 4 butts/l for RCB and MCB and 0–10 butts/l for ECB. The embryos were from stage 8 to 11 and were exposed for a 96-h period in static renewal test conditions. Median lethal concentration (LC50), malformation (EC50), non-observed adverse effect concentration (NOAEC), and lowest observed adverse effect concentration (LOAEC) were calculated. Results from these studies suggest that each tested leachate is teratogenic for X. laevis embryos. The lowest LC50 was determined for ECB exposure at 17.9 cigarette butts/L. The LC50 value was the highest with RCB and MCB having LC50s of approximately 1 cigarette butt/L. There were notable EC50 differences with RCB having the highest and ECB the lowest. The NOAEC and LOAEC levels for RCB and MCB were below 1 cigarette butt/L for both mortality and malformations; over 8 butts/L for ECB mortality and over 4 butts/L for malformations. From these results, we conclude that RCB leachate is the most toxic compound, while MCB leachate has the higher teratogenicity. ECB leachate has the lowest toxic and teratogenic effects on embryos but there were still noticeable effects. The results confirmed that the FETAX assay can be useful in an integrated biological hazard assessment for the preliminary screening for ecological risks of cigarette butts, and electronic cigarettes, in aquatic environment.

Anthropogenic activity has contributed to elevated environmental concentrations of arsenic (As) and chromium (Cr). The spotted salamander, Ambystoma maculatum, may be useful for identifying developmental effects produced by exposure to these contaminants as adults breed and larvae develop in water that may contain As or Cr. Three sample sets among 700 developing larvae were exposed to a range of As, Cr, or 2.5:1 mixture of As:Cr concentrations, respectively. From these 700 larvae, samples containing approximately 24 larvae showed different patterns of whole-body As and Cr from individual and mixture exposure. Whole-body As concentrations were 20.27 and 45.4 µg/g dry weight for larvae exposed to 20 mg/L As and 25:10 mg/L As:Cr, respectively, while whole-body Cr concentrations were 24.8 and 22 µg/g dry weight for larvae exposed to 20 mg/L Cr and 25:10 As:Cr, respectively. Observed malformations included edema, tail kinking, facial deformities, and abnormal bending. Twelve-day lethal concentrations for As and Cr in Ambystoma maculatum larvae were 261.17 mg/L and 71.93 mg/L, respectively, while 12-d effective concentrations to induce malformations were 158.82 and 26.05 mg/L, giving teratogenic indices of 1.64 and 2.76 for individual metal exposure. Exposure to a mixture of As and Cr resulted in a response addition and yielded lower lethal and effective concentration values with a teratogenic index of 2.78, indicating that these contaminants are developmentally toxic at lower concentrations when exposed as a mixture. Data demonstrate that As and Cr affect development of amphibian larvae, and that Ambystoma maculatum may be a useful indicator of environmental toxicity for these metals.

Phthalates, compounds used to add flexibility to plastics, are ubiquitous in the environment. In particular, the diethyl (DEP), di-n-propyl (DnPP), and di-n-butyl (DBP) phthalates were found to exert detrimental effects in both mammalian and non-mammalian studies, with toxic effects varying according to alkyl chain length. Embryos of Xenopus laevis, the African clawed frog, have been used to assess toxicity and teratogenicity of several compounds and serves as a model for assessing adverse and teratogenic effects of ortho-phthalate esters. The purpose of this study was to develop a model for comparison of developmentally toxic effects of ortho-phthalate esters using Xenopus embryos. In this study developing Xenopus laevis embryos were exposed to increasing concentrations of DEP, DnPP, and DBP using the 96-h Frog Embryo Teratogenesis Assay-Xenopus (FETAX), with 96-h lethal concentrations, effective concentrations to induce malformations, teratogenic indices, and concentrations to inhibit growth determined. DEP, DnPP, and DBP showed enhanced toxicity with increasing ester length. Developing Xenopus laevis exposed to DEP, DnPP, and DBP showed similar malformations that also occurred at lower concentrations with increasing alkyl chain length. Teratogenic risk did not change markedly with alkyl chain length, with data showing only DBP to be teratogenic.

The pungent natural compound allyl isothiocyanate isolated from the seeds of Cruciferous (Brassica) plants such as mustard is reported to exhibit numerous beneficial health-promoting antimicrobial, antifungal, anticarcinogenic, cardioprotective, and neuroprotective properties. Because it is also reported to damage DNA and is toxic to aquatic organisms, the objective of the present study was to determine whether it possesses teratogenic properties. The frog embryo teratogenesis assay-Xenopus (FETAX) was used to determine the following measures of developmental toxicity of the allyl isothiocyanate: (a) 96-h LC50, defined as the median concentration causing 50% embryo lethality; (b) 96-h EC50, defined as the median concentration causing 50% malformations of the surviving embryos; and (c) teratogenic malformation index (TI), equal to 96-h LC50/96-h EC50. The quantitative results and the photographs of embryos before and after exposure suggest that allyl isothiocyanate seems to exhibit moderate teratogenic properties. The results also indicate differences in the toxicity of allyl isothiocyanate toward exposed embryos observed in the present study compared to reported adverse effects of allyl isothiocyanate in fish, rodents, and humans. The significance of the results for food safety and possible approaches to protect against adverse effects of allyl isothiocyanate are discussed.

The embryo toxicities of two food-processing-induced toxic compounds, acrylamide and furan, with and without added L-cysteine were examined individually and in mixtures using the frog embryo teratogenesis assay-Xenopus (FETAX). The following measures of developmental toxicity were used: (a) 96 h LC50, the median concentration causing 50% embryo lethality; (b) 96 h EC50, the median concentration causing 50% malformations of the surviving embryos; and (c) teratogenic index (96 h LC50/96 h EC50), an estimate of teratogenic risk. Calculations of toxic units (TU) were used to assess possible antagonism, synergism, or response addition of several mixtures. The evaluated compounds demonstrated counterintuitive effects. Furan had lower than expected toxicity in Xenopus embryos and, unlike acrylamide, does not seem to be teratogenic. However, the short duration of the tests may not show the full effects of furan if it is truly primarily genotoxic and carcinogenic. L-Cysteine showed unexpected properties in the delay of hatching of the embryos. The results from the interaction studies between combination of two or three components (acrylamide plus L-cysteine; furan plus L-cysteine; acrylamide plus furan; acrylamide plus furan and L-cysteine) show that furan and acrylamide seem to have less than response addition at 1:1 toxic unit ratio in lethality. Acrylamide and L-cysteine show severe antagonism even at low 19 acrylamide/1 L-cysteine TU ratios. Data from the mixture of acrylamide, furan, and L-cysteine show a slight antagonism, less than would have been expected from binary mixture exposures. Bioalkylation mechanisms and their prevention are discussed. There is a need to study the toxicological properties of mixtures of acrylamide and furan concurrently formed in heat-processed food.

Schwarzauer-Rockett, K., Al-Hamdani, S. H., Rayburn, J. R. and Mwebi, N. O. 2013. Utilization of kudzu as a lead phytoremediator and the impact of lead on selected physiological responses. Can. J. Plant Sci. 93: 951–959. This study was carried out to evaluate kudzu [Pueraria montana var. lobata (Willd.)] in lead phytoremediation. The impact of lead (PbNO3) concentrations of 100 and 200 mg L−1on plant growth, photosynthetic pigments, photosynthesis, and stomatal conductance were evaluated. Additionally, concentrations of phenolic compounds and anthocyanin were determined. The lead was dissolved in 0.142 g L−1ethylenediaminetetraacetic acid (EDTA) and an EDTA control treatment was added to evaluate the impact of EDTA on the above selected plant responses. Root accumulation of lead was significantly higher than in the shoot. Kudzu growth in response to the presence of lead and EDTA in the Hoagland's solution was similar to that of the EDTA control, except for plants growing at 200 mg L−1, which showed significantly lower root dry weight. Total phenolic compounds increased with the presence of EDTA and lead in the Hoagland's solution. Photosynthetic rate, stomatal conductance, chlorophyll a and chlorophyll b, carotenoids, and anthocyanin were not different (P≤0.05) among the treatments, with the exception that carotenoids were significantly higher in plants growing in the presence of EDTA without lead compared with 200 mg L−1lead concentration. Kudzu accumulated most of the lead in the root and therefore can be considered as a rhizofiltrator. As kudzu was able to accumulate 1.02% (wt/wt) of lead it can be considered a hyper-accumulator.

The two tarantula species used in this study were Grammostola rosea, found in Chile, and Haplopelma lividum, found in Africa. As tarantulas become more common in the pet trade, questions about the effects and components of their venom have arisen. Many types of venom are known to contain toxins with pharmacological action. However, the exact mode of action of tarantula venom is unknown. To address the hypothesis that the two tarantula species would have different teratogenicity based on their different evolutionary histories, venom was collected from tarantulas housed in similar conditions. The venom was stored at −20°C until used. A 96-hr frog embryo assay with 20 embryos was performed to determine the LC50 (lethal), EC50 (malformation), and growth effects. Venom concentrations ranged from 0-0.2% (v/v). Mortality, malformation, and length were analyzed using Tox Tools and SYSTAT software. The Grammostola rosea venom had a 96-hr LC50 of 0.101% (v/v) and an EC50 of 0.0963% (v/v). It also resulted in a consistent spinal malformation and reduction of embryo growth. The Haplopelma lividum venom had a 96-hr LC50 of 0.0713% (v/v) and an EC50 >> 0.075% v/v.

Introducing students to the process of scientific inquiry is a major goal of high school and college labs. Environmental toxins are of great concern and public interest. Modifications of a vertebrate developmental toxicity assay using the frog Xenopus laevis can support student-initiated toxicology experiments that are relevant to humans. Teams of students formulate hypotheses, perform experiments, analyze data, and present their results. By performing experiments to investigate the toxicity of household chemicals, pharmaceuticals, or agricultural chemicals, students will gain an appreciation of the environmental effects of improper disposal of common chemicals and industrial or agricultural run-off.

Hellbenders (Cryptobranchus alleganiensis) historically occurred in large numbers in the eastern United States, but they have undergone a considerable decline due to habitat modification and its subsequent effects on stream quality. To evaluate the Hellbender's current status in Alabama, we verified several recent anecdotal sightings of Hellbenders, and conducted 355 person hours of surveys and 31 trap nights in most historical collection localities and at several additional sites that we considered suitable for this species. We failed to find any Hellbenders during our survey. If Hellbenders still exist in Alabama, they represent relict populations that occur in very low population densities, and likely will be extirpated from the state in the near future. The apparent decline and possible extirpation of Hellbenders in Alabama is probably related to degradation of habitat and water quality resulting from large scale impoundments and land use patterns. As such, land use and water utilization practices will likely persist, and we recommend that conservation efforts intended for this species in Alabama be focused on other species, or on other areas of this species' range where conservation success is more feasible.

Dietary acrylamide is largely derived from heat-induced reactions between the amino group of the free amino acid asparagine and carbonyl groups of glucose and fructose during heat processing (baking, frying) of plant-derived foods such as potato fries and cereals. After consumption, acrylamide is absorbed into the circulation and is then distributed to various organs, where it can react with DNA, neurons, hemoglobin, and essential enzymes. In the present study, we explored the potential of L-cysteine (CySH), N-acetyl-L-cysteine (NAC), reduced glutathione (GSH), and the amino acid glycine (Gly) to protect frog embryos against acrylamide-induced developmental toxicity in the frog embryo teratogenesis assay - Xenopus (FETAX). To test the antiteratogenic potential, based on concentration-response study ranging from 0.07 to 4.22 mM acrylamide in FETAX solution (pH 8.1), we selected concentrations of acrylamide that induced 100% malformations and mortality. At the end of 96 h, we counted survivors and malformed embryos and measured embryo length. The data show that CySH, NAC, and GSH protected the embryos against acrylamide induced malformations and mortality to different degrees. CySH and GSH protected the embryos against both malformations and mortality, whereas NAC protected only against mortality. Gly had no protective effect. Possible mechanisms of the protective effects and the dietary significance of the results of this and related studies for food safety and human health are discussed.

Pseudoephedrine and caffeine are found in many over-the-counter drugs including decongestants and weight loss chemicals. Both are proven central nervous system stimulants, and they may potentially interact on human health. Frog Embryo Teratogenesis Assay-Xenopus (FETAX) was used to determine the developmental toxicity of pseudoephedrine and caffeine mixtures and to determine if synergism or antagonism occurs between the two on developmental toxicity. FETAX is a 96-hour developmental toxicity assay that screens for direct acting teratogens. Both have both been evaluated for developmental toxicity in FETAX but not as mixtures. The 96-hr LC50, 96-hr EC50, MCIG, and TI were determined for pseudoephedrine and caffeine in various mixtures. We tested five binary mixturesofthe two chemicals. The mixtures were basedonthe toxic unitsofeach chemical; where one toxic unit was equal to the 96-hr LC50. The toxic unit mixtures were pseudoephedrine to caffeine at toxic unit ratios of 0:1, 1:0, 3:1, 1:1, and 1:3 based on published literature. Toxic units were plotted on an isobole graph to determine if synergism, concentration response or antagonism occurred. Mixtures acted in a concentration additive interaction on the developmental toxicity. Malformations in mixtures were similar to those seen in caffeine with the exception of the mixture containing the most pseudoephedrine.

Grass shrimp are abundant, ecologically important inhabitants of estuarine ecosystems that have been used extensively in laboratory experiments. However, optimal laboratory feeding conditions have not been determined. We used a laboratory experiment to determine the effects of food availability on survival, growth and reproduction in adult Palaemonetes pugio Holthuis and hatching success of their offspring, Fifteen groups of 50 shrimp were fed 1 of 5 rations of TetraMin (R) flake food, a standard laboratory diet, for 12 wk; supplementary a-mounts of newly hatched Artemia sp. nauplii were also given twice weekly. Adult survival, female reproductive condition, and embryo hatching success were determined every 2 wk. Growth was determined from measurements at the beginning and end of the study, Limited food availability caused severe mortality and reduced growth of female shrimp, whereas male shrimp were not affected, Reproduction, as measured by percent gravid females and individual clutch size, was not affected by food availability. However, estimated population-level embryo production was reduced indirectly for the lower rations through mortality and smaller size of females. Hatching success was low in all treatments, Further studies axe needed to determine optimal laboratory feeding conditions for grass shrimp so they may be better utilized as predictors of responses in the field.

Embryos of estuarine grass shrimp Palaemonetes pugio have demonstrated sensitivity to various solvents and petroleum products, indicating utility for evaluating estuarine contamination. Testing was performed to establish concentration‐response curves for methylene chloride, copper chloride, and 6‐aminonicotinamide, three known teratogenic chemicals. Two exposure periods were used, 4 d and 12 d, and both periods extended through hatching. The average 4‐d LC50 values for methylene chloride, copper chloride, and 6‐aminonicotinamide were 0.071% v/v, 1.82 mg/L, and 0.21 mg/ml, respectively. The average 12‐d LC50 values for methylene chloride, copper chloride, and 6‐aminonicotinamide were 0.031% v/v, 1.44 mg/L, and 0.057 mg/ml, respectively. Eye malformations were observed with embryos exposed to concentrations greater than 3 mg/L copper chloride or greater than 0.07% v/v methylene chloride. Very few abnormalities were observed in embryos exposed to 6‐aminonicotinamide. Abnormal larval development was found with exposure to copper chloride at concentrations greater than 1 mg/L. The sensitivity and low variability found here further supports the development of these relatively simple methods using grass shrimp embryos. Establishment of sublethal developmental endpoints warrants further investigation because of their potential correspondence to mechanisms of toxic action.

As part of an effort to improve the safety of plant foods, a need exists to define the relative toxicities of structurally different glycoalkaloids and metabolites which may be present in Solanum plant species such as potatoes, tomatoes and eggplants. The objectives of this study were to determine the relative toxicities and the modes of action of the eggplant (Solanum melongena) glycoalkaloids solamargine and solasonine in Xenopus laevis frog embryos, using membrane potential and embryo growth and teratogenicity assays. In the cell membrane assays, adverse effects on embryos were evaluated by measuring membrane potentials using an electrochromic dye, di-4-ANEPPS, as a fluorescence probe for the integrity of the membranes. In the embryo growth and teratogenesis assays, the survival of the embryos and organ malformations was used as an index of embryo toxicity. The relative potencies of glycoalkaloids are similar for frog embryo effects (survival and teratogenicities) and for membrane effects (membrane potential). Experiments with solasonine at pH 6 and 8 suggest that the unprotonated form of the glycoalkaloids appears to be involved in the membrane effects. The nature of the carbohydrate side-chains of the steroidal glycosides governs relative potencies. The possible significance of the findings to food safety and plant physiology and possible application of the membrane assays to bacterial toxins are discussed.

Interlaboratory validation of an exogenous metabolic activation system (MAS) developed for the alternative, short-term developmental toxicity bioassay, Frog Embryo Teratogenesis Assay - Xenopus (FETAX) was performed with cyclophosphamide and caffeine. Seven study groups within six separate laboratories participated in the study in which three definitive concentration-response experiments were performed with and without the MAS in a side-by-side format for each chemical. Since both chemicals had been previously tested in FETAX, the test concentrations were provided to each laboratory prior to testing. Interlaboratory coefficient of variation (CV) values for unactivated cyclophosphamide (no MAS) were 15%, 15%, 29%, and 25% for the 96-hr LC50, 96-hr EC50 (malformation). Minimum Concentration to Inhibit Growth (MCIG), and Teratogenic Index (TI) values, respectively. Addition of the MAS increased the CV values of each endpoint at least 3.9-fold. Interlaboratory CV values for unactivated caffeine were 31%, 18%, 31%, and 46% for the 96-hr LC50, 96-hr EC50 (malformation), MCIG, and TI values, respectively. Addition of the MAS decreased the CV values of each respective endpoint by at least 1.6-fold. Results indicated that bioactivated toxicants may be prone to greater variability in response amongst laboratories than compounds, which are detoxified. Even though more variability was noted with activated cyclophosphamide, results were within interlaboratory variation expected for other aquatic-based bioassays. Thus, results from these studies warrant the continued use and further refinement of FETAX for alternative developmental toxicity assessment.

Embryos of the grass shrimp (Palaemonetes pugio) have shown sensitivity to the water soluble fraction of number 2 fuel oil. To determine the possible use of carrier solvents in grass shrimp bioassays, detailed concentration-response experiments for ethanol (EtOH), dimethylsulfoxide (DMSO), and acetone were performed and LC50 values were obtained using two test methods. The 4-d assay included development prior to the time of hatch through the time of hatch, a critical life stage of these embryos. The 12-d assay included development from the tissue cap stage embryos (late gastrula) through two days post-hatch. The average 4-d LC50s for EtOH, DMSO, and acetone were 12.07, 22.57, and 6.78 g/L, whereas the average 12-d LC50s were 3.63, 12.33, and 6.94 g/L, respectively. The coefficient of variation for each test was less than 25.2%. Based on concentration-response curves, the maximum allowable limit of EtOH, DMSO, and acetone to be used as a carrier in the grass shrimp embryo toxicity studies should be <1, <6, and <4 g/L, respectively.