Developmental energetics in the oviparous corn snake, Pantherophis guttatus, confirms a conservative evolutionary pattern in snakes.

Eggs of oviparous reptiles are ideal models for studying evolutionary patterns of embryonic metabolism since they allow tracking of energy allocation during development. Analyzing oxygen consumption of whole eggs throughout development indicates three patterns among reptiles. Embryos initially grow and consume oxygen exponentially, but oxygen consumption slows, or drops before hatching in some species. Turtles, crocodilians, and most lizards follow curves with initial exponential increases followed by declines, whereas embryonic snakes that have been studied exhibit a consistently exponential pattern. This study measured oxygen consumption of corn snake, Pantherophis guttatus, embryos to determine if this species also exhibits an exponential increase in oxygen consumption. Individual eggs, sampled weekly from oviposition to hatching, were placed in respirometry chambers for 24-h during which oxygen consumption was recorded. Embryos were staged and carcasses and yolk were weighed separately. Results indicate steady inclines in oxygen consumption during early stages of development, with a rapid increase prior to hatching. The findings support the hypothesis that embryonic oxygen consumption of snakes differs from most other non-avian reptiles. Total energy required for development was determined based on calorimetry of initial yolk compared to hatchlings and residual yolk and by integration of the area under the curve plotting oxygen consumption versus age of embryos. The cost of development estimates based on these two methods were 6.4 and 10.0 kJ, respectively. Our results emphasize the unique physiological aspects of snake embryogenesis and illustrate how the study of physiological characteristics can contribute to the broader understanding of reptilian evolution.

Behavioral preference for microclimate conditions across elevation in Plethodon montanus.

The ability to behaviorally regulate body conditions is critical for ectotherms, particularly in the face of global climate change when seeking stable refugia in a changing environment could facilitate survival. This is especially important for montane species that are limited to high elevations. In the Northern Gray-cheeked salamander (Plethodon montanus), studies have demonstrated that population demographics improve at higher elevations and physiological constraints may prevent them from moving into lower-elevation habitats. However, little is known about the species' ability to utilize microhabitats and behaviorally regulate by selecting preferable microclimates. Here, we used continuous position-sensing gradient chambers to examine the behavioral preference for temperature and relative humidity (RH) in P. montanus to better understand their microhabitat use and behavioral thermoregulation across an elevation gradient. We investigated the seasonal variation in both thermal and RH preference of P. montanus collected from different elevations. Our results suggest that most recently experienced environmental temperatures influence thermal preference in animals at high elevations but not those at lower elevations. Salamanders preferred the highest available RH conditions regardless of environmental conditions or elevation. Data on shuttling behavior (movement across the behavior arena) from the experiments suggest that while salamanders shuttled a similar number of times in both types of trials, they spent significantly less time exploring when exposed to the RH gradient compared to the thermal gradient. Together these results suggest that while thermal preference is influenced by acclimation, preference for moisture conditions is less elastic.

Predation cues influence metabolic rate and sensitivity to other chemical stressors in fathead minnows (Pimephales promelas) and Daphnia pulex.

The response of aquatic species to contaminants is often context dependent as illustrated by the influence that predation cues can have on the toxicity of some chemicals. We sought to gain additional insight into this interaction by examining how predation cues (alarm cue and fish kairomone) influence metabolic rate and the acute toxicity of sodium chloride and cadmium to fathead minnow larvae (Pimephales promelas) and sodium chloride to Daphnia pulex neonates. Consistent with a "flight or fight" response, the metabolic rate of fish larvae was elevated in the presence of alarm cue and growth of the minnows was also significantly reduced when exposed to alarm cue. The average 48-h LC50 for fathead minnows exposed to sodium chloride was significantly lower in the presence of alarm cue and kairomone combined as compared to tests with the salt alone. Analysis of the dose and survival response indicated alarm cue increased sensitivity of the fish to mid-range salt concentrations in particular. These results suggest an energetic cost of exposure to predation cues that resulted in enhanced toxicity of NaCl. Exposure to kairomone alone had no significant effect on salt toxicity to the minnows, which could be related to a lack of previous exposure to that cue. The acute toxicity of cadmium to the fish larvae was also not affected by the presence of predation cues which could be due to a metal-induced sensory system dysfunction or reduced bioavailability of the metal due to organic exudates from the predation cues. In contrast to the fathead minnow results, the metabolic rate of D. pulex and toxicity of NaCl to the daphnids were reduced in the presence of certain predator kairomones. This suggests an anti-predator response that enhanced tolerance to the salt. This study illustrates that the effect of predation cues on toxicity of aquatic contaminants can vary significantly based on the prey species, type of cue, and chemical stressor.

Can site-specific heuristic toxicity models predict the toxicity of produced water?

An empirically derived model of major ion toxicity was combined with other toxicity assessments to account for the observed toxicity in field-collected produced water and produced water contaminated groundwater. The accuracy and precision of the ion toxicity model, calculated using model deviation ratios (MDR) and simple linear regressions, was determined for fathead minnows, Ceriodaphnia dubia, and Daphnia magna. Model accuracy for produced water fell within a factor of two for all three organisms. The precision, or variability explained by the model, was 47.9%, 56.1%, and 0.00% for fathead minnows, C. dubia, and D. magna, respectively. Incorporating other measured potential toxicants improved predictive precision for fathead minnows to 67.0% using ion toxicity and pH and to 30.9% for D. magna using ion toxicity, pH, and total ammonia. The observed toxicity to Daphnia pulex was also evaluated using D. magna model predictions and other measured parameters, but no consistent relationship was found. Dissimilar results were found for produced water contaminated groundwaters with model predictions for D. magna falling within a factor of two of and explaining 53.8% of the observed variability in D. pulex responses. These results indicate that predicted major ion toxicity, combined with other measured parameters, can accurately and precisely account for observed responses in test organisms to field-collected samples.

Occurrence of organic wastewater and other contaminants in cave streams in northeastern Oklahoma and northwestern Arkansas.

The prevalence of organic wastewater compounds in surface waters of the United States has been reported in a number of recent studies. In karstic areas, surface contaminants might be transported to groundwater and, ultimately, cave ecosystems, where they might impact resident biota. In this study, polar organic chemical integrative samplers (POCISs) and semipermeable membrane devices (SPMDs) were deployed in six caves and two surface-water sites located within the Ozark Plateau of northeastern Oklahoma and northwestern Arkansas in order to detect potential chemical contaminants in these systems. All caves sampled were known to contain populations of the threatened Ozark cavefish (Amblyopsis rosae). The surface-water site in Oklahoma was downstream from the outfall of a municipal wastewater treatment plant and a previous study indicated a hydrologic link between this stream and one of the caves. A total of 83 chemicals were detected in the POCIS and SPMD extracts from the surface-water and cave sites. Of these, 55 chemicals were detected in the caves. Regardless of the sampler used, more compounds were detected in the Oklahoma surface-water site than in the Arkansas site or the caves. The organic wastewater chemicals with the greatest mass measured in the sampler extracts included sterols (cholesterol and beta-sitosterol), plasticizers [diethylhexylphthalate and tris(2-butoxyethyl) phosphate], the herbicide bromacil, and the fragrance indole. Sampler extracts from most of the cave sites did not contain many wastewater contaminants, although extracts from samplers in the Oklahoma surface-water site and the cave hydrologically linked to it had similar levels of diethylhexyphthalate and common detections of carbamazapine, sulfamethoxazole, benzophenone, N-diethyl-3-methylbenzamide (DEET), and octophenol monoethoxylate. Further evaluation of this system is warranted due to potential ongoing transport of wastewater-associated chemicals into the cave. Halogenated organics found in caves and surface-water sites included brominated flame retardants, organochlorine pesticides (chlordane and nonachlor), and polychlorinated biphenyls. The placement of samplers in the caves (near the cave mouth compared to farther in the system) might have influenced the number of halogenated organics detected due to possible aerial transport of residues. Guano from cave-dwelling bats also might have been a source of some of these chlorinated organics. Seven-day survival and growth bioassays with fathead minnows (Pimephales promelas) exposed to samples of cave water indicated initial toxicity in water from two of the caves, but these effects were transient, with no toxicity observed in follow-up tests.

Toxicity of copper, lead, and zinc mixtures to Ceriodaphnia dubia and Daphnia carinata.

Acute and chronic bioassays were conducted to determine the effects of copper, lead, and zinc mixtures on Ceriodaphnia dubia and Daphnia carinata. Copper, lead, and zinc combined at up to 5.2, 4.5, and 51.8 microg/L, respectively, did not cause significant mortality during acute exposures, although mixtures of 10.6, 9, and 101.1 microg/L and higher resulted in 65-100% mortality. Binary combinations of Cu+Zn (1.3+13.0 microg/L) and Cu+Pb (1.3+1.1 microg/L) and ternary combinations of Cu+Pb+Zn (1.3+1.1+13.0 microg/L) had a significant effect on reproduction of C. dubia. Toxic units and associated confidence intervals were calculated to characterize the nature of metal interactions. In most cases, and based on confidence intervals encompassing a value of 1, most of the metal interactions would be classified as additive. However, a more than additive effect was indicated by the acute tests for both species exposed to Cu+Pb, for D. carinata exposed to Cu+Zn, and for C. dubia exposed to all three metals.

Tadpole size, cholinesterase activity, and swim speed in four frog species after exposure to sub-lethal concentrations of chlorpyrifos.

While physiological biomarkers exist to verify exposure of amphibians in natural populations to agricultural chemicals, the ecological relevance of changes in these parameters is often difficult to determine. We compare the relationship between tadpole cholinesterase (ChE; a common enzymatic biomarker of exposure to OP pesticides) and measures of size and swim speed in four native North American species of anurans (Hyla chrysoscelis, Rana sphenocephala, Acris crepitans, and Gastrophryne olivacea). We used four environmentally realistic levels (1, 10, 100 and 200microg/l) of a commonly used organophosphate pesticide (OP), chlorpyrifos, and examined tadpole response at the conclusion of 4 days of exposure. We further examined if the presence or absence of pond sediment influenced tadpole responses, and, in two species, we determined how a 12-day exposure influenced responses. We found species-specific differences in response to the pesticide, with H. chrsysoscelis and G. olivacea being most sensitive; however, the levels of inhibition of ChE activity were generally not sufficient to exert an effect on swim speed as we measured it. Generally, tadpole mass was reduced 20-35% in the highest concentration after 4 days of exposure. We found the presence of sediment to influence these responses, although the effects were not consistent among species. Given these differences in ecologically relevant responses, we recommend exercising caution when making generalizations across different anuran amphibian taxa regarding responses to pesticide exposure.

Intra- and interlaboratory variability in acute toxicity tests with glochidia and juveniles of freshwater mussels (Unionidae).

The present study evaluated the performance and variability in acute toxicity tests with glochidia and newly transformed juvenile mussels using the standard methods outlined in American Society for Testing and Materials (ASTM). Multiple 48-h toxicity tests with glochidia and 96-h tests with juvenile mussels were conducted within a single laboratory and among five laboratories. All tests met the test acceptability requirements (e.g., >or=90% control survival). Intralaboratory tests were conducted over two consecutive mussel-spawning seasons with mucket (Actinonaias ligamentina) or fatmucket (Lampsilis siliquoidea) using copper, ammonia, or chlorine as a toxicant. For the glochidia of both species, the variability of intralaboratory median effective concentrations (EC50s) for the three toxicants, expressed as the coefficient of variation (CV), ranged from 14 to 27% in 24-h exposures and from 13 to 36% in 48-h exposures. The intralaboratory CV of copper EC50s for juvenile fatmucket was 24% in 48-h exposures and 13% in 96-h exposures. Interlaboratory tests were conducted with fatmucket glochidia and juveniles by five laboratories using copper as a toxicant. The interlaboratory CV of copper EC50s for glochidia was 13% in 24-h exposures and 24% in 48-h exposures, and the interlaboratory CV for juveniles was 22% in 48-h exposures and 42% in 96-h exposures. The high completion success and the overall low variability in test results indicate that the test methods have acceptable precision and can be performed routinely.

Cholinesterase activity and behavior in chlorpyrifos-exposed Rana sphenocephala tadpoles.

Recent studies have found a correlation between organophosphate (OP) pesticide exposure and declines in amphibian populations. We evaluated the hypothesis that this relationship is driven by behavioral changes in developing larvae. Specifically, we examined how exposure to a common OP pesticide, chlorpyrifos, influenced cholinesterase (ChE) activity, mass, and swim speed in Rana sphenocephala tadpoles. We also determined how the presence of natural pond sediments in exposure chambers influenced response to the pesticide and how mass and survival were affected when tadpoles were exposed to an invertebrate (odonate) predator in addition to the pesticide. Mass and swim speed were measured after 4- and 12-d laboratory exposures to 1, 10, 100, and 200 microg/L of chlorpyrifos in test chambers that either did or did not contain pond sediments. These same parameters also were examined in mesocosms dosed with 200 microg/L of chlorpyrifos to evaluate responses under more environmentally realistic conditions. The effect of the invertebrate predators on survival and/or growth of tadpoles was evaluated in the mesocosm study and in separate laboratory experiments. In laboratory tests, no pesticide-induced mortality was observed; however, tadpole ChE activity in the two highest concentrations was significantly lowered, with a longer exposure duration further decreasing activity (maximum inhibition, 43%). Mass also was lower at higher concentrations, but this effect was not enhanced with longer duration of exposure. Reductions in ChE activity of tadpoles exposed in mesocosms were similar to those observed in laboratory experiments for the first 4 d. Tadpole swim speed and survival in the presence of a predator were not affected, with the latter largely resulting from pesticide-induced predator mortality.

The influence of salinity on metal uptake and effects in the midge Chironomus maddeni.

The influence of different porewater salinities (up to 12 g/L) on the toxicity and bioaccumulation of copper, zinc and lead from metal-spiked sediments was assessed using the midge, Chironomus maddeni. Survival of the larvae was significantly reduced at a porewater salinity of 12 g/L, but no effects were observed at 4 or 8 g/L. Both growth and survival of C. maddeni were reduced after exposure to salt/metal spiked sediments as compared to those exposed to sediments spiked with metals or salt alone. Increased salinity resulted in increased bioaccumulation of copper and zinc, but decreased bioaccumulation of lead. The observed patterns of bioaccumulation were not entirely explained by the modelled free ion activities of the metals, indicating that factors such as osmotic stress, consumption of metal-contaminated sediments or metal interactions may have been important as well. These results highlight the need to consider the influence of existing or potential salinization when undertaking hazard assessments of freshwater systems impacted by contaminants such as trace metals.

Cholinesterase inhibition and impacts on behavior of the Asian clam, Corbicula fluminea, after exposure to an organophosphate insecticide.

This study assessed the effects of exposure to an insecticide formulation containing the organophosphate, chlorpyrifos on cholinesterase activity, siphoning and burrowing ability in the Asian clam, Corbicula fluminea. Clams were exposed to concentrations of the pesticide ranging from 0.05 to 50 mg/L (as chlorpyrifos) in a series of 96-h static bioassays. Those organisms exposed to pesticide concentrations at or above 3.13 mg/L avoided exposure through valve closure during the 96-h test period, with no resultant effects on cholinesterase activity or behavior. Similarly, no effect was observed at low-test concentrations ranging from 0.05 to 0.1 mg/L, even though clams actively siphoned in these treatments. Asian clams exposed to the mid-range of concentrations, 0.5-1.0 mg/L, experienced a significant reduction in cholinesterase activity and a reduced capacity to burrow into the substrate. While these data indicate that cholinesterase activity in C. fluminea could be used as a biomarker of organophosphorous pesticide exposure, valve closure can clearly influence exposure profiles and biomarker response.

Assessing the application of an additive model to estimate toxicity of a complex effluent.

A number of industries monitor levels of chemicals in their effluent, but few have undertaken prolonged biological monitoring of this wastewater. The focus of the present study was to determine whether past chemical data for effluent from a lead smelter could be used to estimate its past toxicity. Since the interactive effects of metals in effluents are often assumed to be additive, it was hypothesized that an additive model, 100/[sigma(metal concentration in effluent/EC50 for individual metal)], could be used to generate an EC50 from chemical data (where EC50 is the concentration of test material that affects 50% of the test organisms). To test the approach, a larval development toxicity test with the marine polychaete, Galeolaria caespitosa, was used to test 26 separate samples of effluent from a lead smelter, generating empirical EC50 values. EC50 values for each individual metal in the effluent were also generated using the larval development toxicity test. The concentrations of trace metals in each effluent sample were determined and, using the additive model, EC50 values were calculated. For the majority of effluent samples tested, the additive model underestimated toxicity, suggesting the presence of additional unidentified contaminants in the effluent samples. Additionally, a nonlinear rather than linear regression curve was found to best describe the relationship between the model and empirically derived EC50 values. This relationship was then used to estimate past trends in toxicity of the smelter effluent. Forty-eight percent of the variability in measured toxicity was explained by the model, with the model underestimating toxicity in the majority of samples.

Toxicity evaluation of a commercial bioremediation agent mixed with crude oil.

The toxicity and efficacy of a bacteria-based commercial bioremediation agent (CBA) was assessed through bioassays with juvenile inland silverside minnows, Menidia beryllina, and flask studies of oil degradation. Addition of the CBA to weathered Alaska, USA North Slope crude oil (ANS-521) prior to testing increased toxicity of the water-soluble fraction (WSF) of the oil in both chronic (growth) and acute (mortality) toxicity tests. Time-course toxicity assessment of the water-soluble fraction of the CBA/oil combination indicated increases in effect after 7 to 14 d of mixing that coincided with elevated concentrations of both alkanes and aromatics in the WSF. Under controlled laboratory conditions, the CBA significantly enhanced degradation of the oil compared with a treatment with nutrients alone. The alkane fraction was degraded by nearly 100% over a 42-d period while the aromatic fraction was decreased by 70%. While toxicity testing is not currently required to list bioremediation agents on the product schedule of the National Oil and Hazardous Substances Pollution Contingency Plan, the potential interaction between bioremediation agents and oil should be further investigated and listing requirements reassessed as necessary. Recommendations for future investigations of this issue include characterization of temporal trends in toxicity of CBA and oil mixtures, use of multiple test methods (battery testing) when evaluating toxicity, comparative evaluations of indigenous versus product-derived microorganisms in efficacy studies, and the use of a comparable oil between studies to facilitate comparison of efficacy and toxicity data for different products.

Genetic diversity and metal tolerance of two marine species: a comparison between populations from contaminated and reference sites.

Long-term contamination of the marine environment surrounding a lead smelter offered a unique opportunity to examine how pollutants might have acted to alter genetic characteristics of populations of organisms in the receiving system. This study used random amplified polymorphic DNA analysis to compare the genetic diversity of populations of the prawn, Leander intermedius, and the isopod, Platynympha longicaudata from the smelter discharge site with reference populations. The genetic diversity of the prawn population from the smelter discharge site (Port Pirie) was lower than that found in one reference population, and not significantly different from the other two reference populations. Genetic diversity of the Port Pirie population of isopods was found to be significantly lower than that of all reference populations. Prawns and isopods were also exposed to metal mixtures in the laboratory in a similar ratio to that found in seston near the smelter effluent discharge site. Both pre-exposed and reference populations of prawns tolerated elevated levels of metals and exhibited no significant difference in response. This contrasted with the isopods, with the pre-exposed isopod population showing greater tolerance to elevated metal levels compared with the reference population. These results highlight the need to include a number of reference populations for comparative purposes in genetic diversity studies, and the need to assess the influence of pollution on the genetic diversity of more than one species if genetic diversity analyses are to be used to gauge remediation success.