David B. Buchwalter

Aquatic insect ecophysiological traits reveal phylogenetically based differences in dissolved cadmium susceptibility

We used a phylogenetically based comparative approach to evaluate the potential for physiological studies to reveal patterns of diversity in traits related to susceptibility to an environmental stressor, the trace metal cadmium (Cd). Physiological traits related to Cd bioaccumulation, compartmentalization, and ultimately susceptibility were measured in 21 aquatic insect species representing the orders Ephemeroptera, Plecoptera, and Trichoptera. We mapped these experimentally derived physiological traits onto a phylogeny and quantified the tendency for related species to be similar (phylogenetic signal). All traits related to Cd bioaccumulation and susceptibility exhibited statistically significant phylogenetic signal, although the signal strength varied among traits. Conventional and phylogenetically based regression models were compared, revealing great variability within orders but consistent, strong differences among insect families. Uptake and elimination rate constants were positively correlated among species, but only when effects of body size and phylogeny were incorporated in the analysis. Together, uptake and elimination rates predicted dramatic Cd bioaccumulation differences among species that agreed with field-based measurements. We discovered a potential tradeoff between the ability to eliminate Cd and the ability to detoxify it across species, particularly mayflies. The best-fit regression models were driven by phylogenetic parameters (especially differences among families) rather than functional traits, suggesting that it may eventually be possible to predict a taxons physiological performance based on its phylogenetic position, provided adequate physiological information is available for close relatives. There appears to be great potential for evolutionary physiological approaches to augment our understanding of insect responses to environmental stressors in nature.

Differential exposure, duration, and sensitivity of unionoidean bivalve life stages to environmental contaminants

Abstract Freshwater mussels (superfamily Unionoidea) are in serious global decline and in urgent need of protection and conservation. The declines have been attributed to a wide array of human activities resulting in pollution and water-quality degradation, and habitat destruction and alteration. Linkages among poor water quality, pollutant sources, and mussel decline in rivers and streams have been associated with results of laboratory-based tests of specific pollutants. However, uncertainties remain about the relationship of laboratory data to actual contaminant exposure routes for various mussel species, life stages, and in the habitats occupied during these exposures. We evaluated the pathways of exposure to environmental pollutants for all 4 life stages (free glochidia, encysted glochidia, juveniles, adults) of unionoidean mussels and found that each life stage has both common and unique characteristics that contribute to observed differences in exposure and sensitivity. Free glochidia typically are exposed only briefly (e.g., seconds to days) through surface water, whereas adults sustain exposure over years to decades through surface water, pore water, sediment, and diet. Juveniles live largely burrowed in the sediment for the first 0 to 4 y of life. Thus, sediment, pore water, and diet are the predominant exposure routes for this life stage, but surface water also might contribute to exposure during certain periods and environmental conditions. The obligate parasitic stage (encysted glochidia stage) on a host fish might be exposed from surface water while partially encysted or from toxicants in host-fish tissue while fully encysted. Laboratory methods for testing for acute and chronic exposures in water have advanced, and toxicant-specific information has increased in recent years. However, additional research is needed to understand interactions of life history, habitat, and long-term exposure to contaminants through water, pore water, sediment, and diet so that the risks of environmental exposures can be properly assessed and managed.

Framework for traits‐based assessment in ecotoxicology

A key challenge in ecotoxicology is to assess the potential risks of chemicals to the wide range of species in the environment on the basis of laboratory toxicity data derived from a limited number of species. These species are then assumed to be suitable surrogates for a wider class of related taxa. For example, Daphnia spp. are used as the indicator species for freshwater aquatic invertebrates. Extrapolation from these datasets to natural communities poses a challenge because the extent to which test species are representative of their various taxonomic groups is often largely unknown, and different taxonomic groups and chemicals are variously represented in the available datasets. Moreover, it has been recognized that physiological and ecological factors can each be powerful determinants of vulnerability to chemical stress, thus differentially influencing toxicant effects at the population and community level. Recently it was proposed that detailed study of species traits might eventually permit better understanding, and thus prediction, of the potential for adverse effects of chemicals to a wider range of organisms than those amenable for study in the laboratory. This line of inquiry stems in part from the ecology literature, in which species traits are being used for improved understanding of how communities are constructed, as well as how communities might respond to, and recover from, disturbance (see other articles in this issue). In the present work, we develop a framework for the application of traits-based assessment. The framework is based on the population vulnerability conceptual model of Van Straalen in which vulnerability is determined by traits that can be grouped into 3 major categories, i.e., external exposure, intrinsic sensitivity, and population sustainability. Within each of these major categories, we evaluate specific traits as well as how they could contribute to the assessment of the potential effects of a toxicant on an organism. We then develop an example considering bioavailability to explore how traits could be used mechanistically to estimate vulnerability. A preliminary inventory of traits for use in ecotoxicology is included; this also identifies the availability of data to quantify those traits, in addition to an indication of the strength of linkage between the trait and the affected process. Finally, we propose a way forward for the further development of traits-based approaches in ecotoxicology.

Selenium bioaccumulation and maternal transfer in the mayfly Centroptilum triangulifer in a life-cycle, periphyton-biofilm trophic assay.

Selenium contamination in aquatic ecosystems provides management challenges because bioaccumulation in animals is largely a function of dietary exposure, whereas regulatory entities have traditionally focused on direct water to organism interactions. Selenium is known to be readily absorbed by primary producers and can potentially biomagnify in food webs and elicit adverse effects in higher trophic levels. However, selenium bioaccumulation in the invertebrate prey of many predatory animals is poorly understood. Here, we used 75Se (as selenite) as a radiotracer to characterize Se bioaccumulation into natural periphyton biofilms and subsequent dietary and maternal transfer in the mayfly, Centroptilum triangulifer, in a life-cycle assay. On average periphyton biofilms bioconcentrated selenium 1113 (+/-430)-fold following 7-9 days of exposure to a range of environmentally relevant dissolved concentrations (2.4-13.9 microg L(-1)). Mayflies grown to adulthood on these diets further biomagnified Se with trophic transfer factors averaging 22 (+/-0.4)-fold in postpartum maternal tissues. Adults then transferred 46.5 (+/-8.8)% of their body burdens to eggs with an observed reduction in fecundity for mayflies fed on diets greater than approximately 11 microg g(-1). These results suggest that at environmentally feasible dietary Se concentrations insects are potentially affected by Se exposure, and that the current presumption that insects are simply conduits of Se to higher trophic levels is inaccurate.

Saving freshwater from salts

Ion-specific standards are needed to protect biodiversity Many human activities—like agriculture and resource extraction—are increasing the total concentration of dissolved inorganic salts (i.e., salinity) in freshwaters. Increasing salinity can have adverse effects on human health (1); increase the costs of water treatment for human consumption; and damage infrastructure [e.g., amounting to

Cadmium exposure route affects antioxidant responses in the mayfly Centroptilum triangulifer.

700 million per year in the Border Rivers catchment, Australia (2)]. It can also reduce freshwater biodiversity (3); alter ecosystem functions (4); and affect economic well-being by altering ecosystem goods and services (e.g., fisheries collapse). Yet water-quality legislation and regulations that target salinity typically focus on drinking water and irrigation water, which does not automatically protect biodiversity. For example, specific electrical conductivities (a proxy for salinity) of 2 mS/cm can be acceptable for drinking and irrigation but could extirpate many freshwater insect species (3). We argue that salinity standards for specific ions and ion mixtures, not just for total salinity, should be developed and legally enforced to protect freshwater life and ecosystem services. We identify barriers to setting such standards and recommend management guidelines.

Use of reconstituted waters to evaluate effects of elevated major ions associated with mountaintop coal mining on freshwater invertebrates

Aquatic organisms accumulate metals directly from water and from their diets. Exposure to metals is known to generate oxidative stress in living organisms and this stress may be ameliorated via activation of antioxidant enzymes and non-enzymatic antioxidants. To determine if antioxidant physiology is dependent on Cd exposure route in the mayfly Centroptilum triangulifer, we exposed larvae to environmentally relevant concentrations of Cd from isolated dissolved or dietary exposure routes to achieve comparable tissue concentrations. Dissolved Cd had no effect on the antioxidant enzymes examined. However, dietary Cd significantly suppressed catalase and superoxide dismutase activities, and decreased concentrations of the reduced (active) form of glutathione in C. triangulifer larvae. These findings suggest that dietary Cd is potentially more toxic than aqueously derived Cd in this mayfly. We further examined the effect of dietary Cd tissue loading rates on antioxidant enzyme suppression and found that absolute tissue load appeared more important than loading rate. These results may help explain why insects are routinely unresponsive to dissolved metal exposures in the laboratory, yet highly responsive to metal pollution in nature.

Trophic transfer of Cd from natural periphyton to the grazing mayfly Centroptilum triangulifer in a life cycle test.

In previous laboratory chronic 7-d toxicity tests conducted with the cladoceran Ceriodaphnia dubia, surface waters collected from Appalachian sites impacted by coal mining have shown toxic effects associated with elevated total dissolved solids (TDS). The objective of the present study was to evaluate the effects of elevated major ions in chronic laboratory tests with C. dubia (7-d exposure), a unionid mussel (Lampsilis siliquoidea; 28-d exposure), an amphipod (Hyalella azteca; 28-d exposure), and a mayfly (Centroptilum triangulifer; 35-d exposure) in 3 reconstituted waters designed to be representative of 3 Appalachian sites impacted by coal mining. Two of the reconstituted waters had ionic compositions representative of alkaline mine drainage associated with mountaintop removal and valley fill-impacted streams (Winding Shoals and Boardtree, with elevated Mg, Ca, K, SO₄, HCO₃), and a third reconstituted water had an ionic composition representative of neutralized mine drainage (Upper Dempsey, with elevated Na, K, SO₄, and HCO₃). The waters with similar conductivities but, with different ionic compositions had different effects on the test organisms. The Winding Shoals and Boardtree reconstituted waters were consistently toxic to the mussel, the amphipod, and the mayfly. In contrast, the Upper Dempsey reconstituted water was toxic to the mussel, the amphipod, and the cladoceran but was not toxic to the mayfly. These results indicate that, although elevated TDS can be correlated with toxicity, the specific major ion composition of the water is important. Moreover, the choice of test organism is critical, particularly if a test species is to be used as a surrogate for a range of faunal groups.

Phase III Interlaboratory Study of Fetax, Part 2: Interlaboratory Validation of an Exogenous Metabolic Activation System for Frog Embryo Teratogenesis Assay-Xenopus (Fetax)

In streams, periphyton biofilms are important sinks for trace metals such as cadmium and are primary food sources of many invertebrate consumers. To study Cd trophic transfer, we produced differentially contaminated diets by exposing natural periphyton to environmentally relevant dissolved Cd ranging from 0 to 10 microg L(-1) for 6-7 days using a radiotracer approach. On average, periphyton grown during three different seasons bioconcentrated Cd similarly--approximately 1315 (+/-442) -fold above dissolved concentrations. However, mayfly larvae (Centroptilum triangulifer) raised on these differentially contaminated diets (first instar through adulthood) had significantly higher trophic transfer factors from periphyton grown in Aug and Nov 2008 (4.30 +/- 1.55) than from periphyton grown in Jan 2009 (0.85 +/- 0.21). This Cd bioaccumulation difference is only partially explained by apparent food quality and subsequent growth differences. Taken together, these results suggest that primary producers at the base of food webs drive metal bioaccumulation by invertebrate grazers.

Four reasons why traditional metal toxicity testing with aquatic insects is irrelevant.

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.