In situ effects of pesticides on amphibians in the Sierra Nevada.

For more than 20 years, conservationists have agreed that amphibian populations around the world are declining. Results obtained through laboratory or mesocosm studies and measurement of contaminant concentrations in areas experiencing declines have supported a role of contaminants in these declines. The current study examines the effects of contaminant exposure to amphibians in situ in areas actually experiencing declines. Early larval Pseudacris regilla were translocated among Lassen Volcanic, Yosemite and Sequoia National Parks, California, USA and caged in wetlands in 2001 and 2002 until metamorphosis. Twenty contaminants were identified in tadpoles with an average of 1.3-5.9 (maximum = 10) contaminants per animal. Sequoia National Park, which had the greatest variety and concentrations of contaminants in 2001, also had tadpoles that experienced the greatest mortality, slowest developmental rates and lowest cholinesterase activities. Yosemite and Sequoia tadpoles and metamorphs had greater genotoxicity than those in Lassen during 2001, as determined by flow cytometry. In 2001 tadpoles at Yosemite had a significantly higher rate of malformations, characterized as hemimelia (shortened femurs), than those at the other two parks but no significant differences were observed in 2002. Fewer differences in contaminant types and concentrations existed among parks during 2002 compared to 2001. In 2002 Sequoia tadpoles had higher mortality and slower developmental rates but there was no difference among parks in cholinesterase activities. Although concentrations of most contaminants were below known lethal concentrations, simultaneous exposure to multiple chemicals and other stressors may have resulted in lethal and sublethal effects.

Joint toxicity of chlorpyrifos and endosulfan to Pacific treefrog (Pseudacris regilla) tadpoles.

Recent ecotoxicology studies have focused on the potential interaction of pesticides and the effects these interactions may have on aquatic ecosystems. We examined the combined effects of two insecticides, endosulfan and chlorpyrifos, that have been previously examined individually on survival, growth, and development of Pacific treefrog (Pseudacris regilla) tadpoles. Historically, both pesticides have been heavily used in the Central Valley of California, been identified in downwind montane areas, and are highly toxic to amphibian larvae. Tadpoles were exposed to chlorpyrifos and endosulfan individually and in combination from Gosner stage 25 through metamorphosis to evaluate the individual effects and the interaction between these insecticides. Chlorpyrifos alone did not affect survival or body size after 30 days, even at concentrations greater than the previously reported LC50. Survival and body size decreased with increasing endosulfan concentrations. The interactive effects of the insecticides depended on concentration and exposure duration. In combination, 137 µg/L chlorpyrifos inhibited the negative effects of endosulfan on growth and survival. The presence of both insecticides in combination facilitated the development of axial malformations. In the presence of endosulfan with either 266 or 394 µg/L chlorpyrifos, malformations occurred in 33 and 87 % of tadpoles, respectively. Our results indicate that organophosphorus and organochlorine pesticides with different modes of action can result in varying interactions depending on their concentrations and on the end points being assessed. Further examination of contaminant impacts on natural aquatic systems should continue to focus on the effects of multiple contaminants and their potential for unpredictable, nonadditive interactions.

Correlation between heavy metals and turtle abundance in ponds near the Paducah Gaseous Diffusion Plant, Kentucky, USA.

Reptiles are declining globally, and environmental contamination has been suggested as a contributing factor; however, few studies have investigated the relationship between contamination and reptile populations. We performed a mark-recapture study at ponds near the Paducah Gaseous Diffusion Plant (PGDP), Kentucky, to determine if heavy metals had an impact on turtle populations. We measured concentrations of cadmium, chromium, copper, lead, and mercury in red-eared slider turtle (Trachemys scripta elegans) tissues and pond sediment and determined the correlation between metal concentrations and red-eared slider density. Metal concentrations measured in the current study were low, and turtle density was not significantly correlated with metal concentrations in tissues or sediment. However, we observed a trend of decreasing turtle density in ponds that had greater metal concentrations. Sex ratio and proportion of juveniles were significantly different among ponds, but it is unclear if these differences are related to contamination associated with the PGDP.

Accumulation of polychlorinated biphenyls (PCBs) and evaluation of hematological and immunological effects of PCB exposure on turtles.

Concentrations of total polychlorinated biphenyls (PCBs), Aroclor 1260, and 26 congeners were measured in liver, fat, and eggs of red-eared slider turtles (Trachemys scripta elegans) collected from ponds near or on the Paducah Gaseous Diffusion Plant (PGDP), Kentucky, USA. Concentrations of total PCBs (wet mass) ranged from 0.002 to 0.480 mg/kg, 0.028 to 0.839 mg/kg, and 0.001 to 0.011 mg/kg in liver, fat, and eggs, respectively. Concentrations of Arochlor 1260 did not exceed 0.430, 0.419, and 0.007 mg/kg in liver, fat, and eggs, respectively. Exposure to PCBs in red-eared sliders collected from the PGDP is characterized by low concentrations of moderately chlorinated mono-ortho and di-ortho congeners (PCB 153, 180, and 118). Although PCB concentrations measured in the current study were low, chronic exposure to PCBs may have altered hematology and immunity of the turtles examined. Total white blood cell count and number of heterophils were negatively correlated with concentrations of total PCBs and Arochlor 1260, respectively. However, disease and other contaminants in the study area may influence the results. Because little is known regarding the influence of PCBs on hematology and immune function in turtles, additional study is needed to better evaluate results observed in the current study.

Metal accumulation and evaluation of effects in a freshwater turtle.

A variety of contaminants have been detected in aquatic and terrestrial environments around the Paducah Gaseous Diffusion Plant (PGDP), Kentucky. The presence of these contaminants at the PGDP may pose a risk to biota, yet little is known about the bioaccumulation of contaminants and associated effects in wildlife, especially in aquatic turtles. The current study was initiated to evaluate: (1) the accumulation of heavy metals (Cd, Cr, Cu, Pb, and Hg) in aquatic ecosystems associated with the PGDP using red-eared slider turtle (Trachemys scripta elegans) as biomonitors; (2) maternal transfer of heavy metals; and (3) potential hematological and immunological effects resulting from metal accumulation. A total of 26 turtles were collected from 7 ponds located south, adjacent, and north of the PGDP. Liver Cu concentrations were significantly different among ponds and Cu concentrations in eggs were positively correlated with female Cu concentrations in kidney. The concentrations of heavy metals measured in turtle tissues and eggs were low and, based on previous studies of reptiles and established avian threshold levels of heavy metals, did not appear to have adverse effects on aquatic turtles inhabiting ponds near the PGDP. However, total white blood cell counts, heterophil to lymphocyte ratio, and phytohemagglutinin stimulation index were correlated with metal concentrations. Because other factors may affect the hematological and immunological indices, further investigation is needed to determine if these effects are associated with metal exposure, other contaminants, or disease.

Toxicity of coal-tar pavement sealants and ultraviolet radiation to Ambystoma Maculatum.

Polycyclic aromatic hydrocarbons (PAHs) can affect amphibians in lethal and many sublethal ways. There are many natural and anthropogenic sources of PAHs in aquatic environments. One potentially significant source is run off from surfaces of parking lots and roads that are protected with coal tar sealants. Coal tar is 50% or more PAH by wet weight and is used in emulsions to treat these surfaces. Break down of sealants can result in contamination of nearby waters. The toxicity of PAHs can be greatly altered by simultaneous exposure to ultraviolet radiation. This study exposes larvae of the spotted salamander (Ambystoma maculatum) to determine if coal tar sealant can have negative effects on aquatic amphibians and if coal tar toxicity is influenced by ultraviolet radiation. Spotted salamanders were exposed to 0, 60, 280 and 1500 mg coal tar sealant/kg sediment for 28 days. Half of the animals were exposed to conventional fluorescent lighting only and half were exposed to fluorescent lighting plus ultraviolet radiation. No significant mortality occurred during the experiment. Exposure to sealants resulted in slower rates of growth, and diminished ability to swim in a dose-dependent fashion. Exposure to ultraviolet radiation affected the frequencies of leukocytes and increased the incidence of micronucleated erythrocytes. There was an interactive effect of sealant and radiation on swimming behavior. We conclude that coal-tar sealant and ultraviolet radiation increased sublethal effects in salamanders, and may be a risk to salamanders under field conditions.

Mercury concentrations in wetlands associated with coal-fired power plants.

There have been contradictory reports of the relative proportion of mercury from coal-fired power plants that deposits locally. Our objective was to determine any local effect of coal-fired power plants on total mercury concentrations in wetland sediment and tadpole samples. Four power plants and 45 wetlands were selected for study. Total mercury concentrations were determined in 75 sediment samples (range: 8-82 ng/g dry weight) and 100 bullfrog (Lithobates catesbeiana) and green frog (Lithobates clamitans) tadpoles (range: 5-318 ng/g wet weight). Tadpole and sediment total mercury did not significantly vary by power plant or distance from the plant. Only one power plant had a significantly greater concentration of total mercury in sediment downwind compared to upwind wetlands. A similar (but non-significant) trend was found for tadpole total mercury surrounding the same plant. Tadpole total mercury was negatively correlated with both tadpole weight and total length. Tadpole and sediment total mercury concentrations were not significantly correlated with one another. The results of the current study suggest that coal-fired power plants are not significantly affecting mercury concentrations in surrounding wetlands.

Toxicity of two insecticides to California, USA, anurans and its relevance to declining amphibian populations.

Contaminants have been associated with population declines of several amphibian species in California (USA). Pesticides from the Central Valley of California are transported by winds into the Sierra Nevada Mountains and precipitate into wet meadows where amphibians breed. The present study examined the chronic toxicity of two of the insecticides most commonly used in the Central Valley and found in the mountains, chlorpyrifos and endosulfan, to larval Pacific treefrogs (Pseudacris regilla) and foothill yellow-legged frogs (Rana boylii) and discusses the implications of this toxicity to declining amphibian populations. Larvae were exposed to the pesticides from Gosner stages 25 to 26 through metamorphosis. The estimated median lethal concentration (LC50) for chlorpyrifos was 365 microg/L in P. regilla and 66.5 microg/L for R. boylii. Time to metamorphosis increased with concentration of chlorpyrifos in both species, and cholinesterase activity declined with exposure concentration in metamorphs of both species at Gosner stages 42 to 46. For endosulfan, the estimated LC50 was 15.6 microg/L for P. regilla and 0.55 microg/L for R. boylii. All R. boylii exposed to concentrations of greater than 0.8 microg/L died before they entered metamorphosis. Pseudacris regilla remains relatively abundant and is broadly distributed throughout California. In contrast, R. boylii is among the species experiencing severe population declines. The present study adds to the increasing evidence that pesticides are very harmful to amphibians living in areas that are miles from sources of pesticide application.

Toxicity of glyphosate as Glypro and LI700 to red-eared slider (trachemys scripta elegans) embryos and early hatchlings.

More than 8.2 billion ha of cropland, gardens, and forests are treated with the herbicide glyphosate each year. Whereas the toxicity of glyphosate and associated adjuvants has been measured in other vertebrates, few, if any, studies have looked at their effects in reptiles. In some instances, management of turtle habitat requires control of successional stages through application of herbicides. Adults and juvenile turtles may be exposed directly, whereas embryos may contact the chemicals through the soil. In the present study, we exposed eggs of red-eared sliders (Trachemys scripta elegans) to single applications of herbicide ranging from 0 to 11,206 ppm wet weight of glyphosate in Glypro and 0 to 678 ppm of the surfactant, LI700. Hatching success at the highest concentration was significantly lower (73%) than in other treatments (80-100%). At hatch, turtles at the highest concentration weighed less than those at other concentrations. During a 14-d holding period, we observed dose-response relationships in the ability of hatchlings to right themselves when turned on their backs. At the end of the holding period, hatchlings at the highest dose level were still lighter, and somatic indices were lower, than those in other treatments. Genetic damage, as measured by flow cytometry, increased with treatment concentration except for the highest dose. We conclude that because of the high concentrations needed to produce effects and the protection offered by several centimeters of soil or sediment, glyphosate with LI700 poses low levels of risk to red-eared slider embryos under normal field operations with regards to the endpoints measured in the present study. Carelessness in handling glyphosate or failure to follow label directions may produce adverse effects. There also is a risk that the health of turtle embryos may be affected in ways not measured in the present study.

Interactive effects of climate change with nutrients, mercury, and freshwater acidification on key taxa in the North Atlantic Landscape Conservation Cooperative region.

The North Atlantic Landscape Conservation Cooperative LCC (NA LCC) is a public-private partnership that provides information to support conservation decisions that may be affected by global climate change (GCC) and other threats. The NA LCC region extends from southeast Virginia to the Canadian Maritime Provinces. Within this region, the US National Climate Assessment documented increases in air temperature, total precipitation, frequency of heavy precipitation events, and rising sea level, and predicted more drastic changes. Here, we synthesize literature on the effects of GCC interacting with selected contaminant, nutrient, and environmental processes to adversely affect natural resources within this region. Using a case study approach, we focused on 3 stressors with sufficient NA LCC region-specific information for an informed discussion. We describe GCC interactions with a contaminant (Hg) and 2 complex environmental phenomena-freshwater acidification and eutrophication. We also prepared taxa case studies on GCC- and GCC-contaminant/nutrient/process effects on amphibians and freshwater mussels. Several avian species of high conservation concern have blood Hg concentrations that have been associated with reduced nesting success. Freshwater acidification has adversely affected terrestrial and aquatic ecosystems in the Adirondacks and other areas of the region that are slowly recovering due to decreased emissions of N and sulfur oxides. Eutrophication in many estuaries within the region is projected to increase from greater storm runoff and less denitrification in riparian wetlands. Estuarine hypoxia may be exacerbated by increased stratification. Elevated water temperature favors algal species that produce harmful algal blooms (HABs). In several of the region's estuaries, HABs have been associated with bird die-offs. In the NA LCC region, amphibian populations appear to be declining. Some species may be adversely affected by GCC through higher temperatures and more frequent droughts. GCC may affect freshwater mussel populations via altered stream temperatures and increased sediment loading during heavy storms. Freshwater mussels are sensitive to un-ionized ammonia that more toxic at higher temperatures. We recommend studying the interactive effects of GCC on generation and bioavailability of methylmercury and how GCC-driven shifts in bird species distributions will affect avian exposure to methylmercury. Research is needed on how decreases in acid deposition concurrent with GCC will alter the structure and function of sensitive watersheds and surface waters. Studies are needed to determine how GCC will affect HABs and avian disease, and how more severe and extensive hypoxia will affect fish and shellfish populations. Regarding amphibians, we suggest research on 1) thermal tolerance and moisture requirements of species of concern, 2) effects of multiple stressors (temperature, desiccation, contaminants, nutrients), and 3) approaches to mitigate impacts of increased temperature and seasonal drought. We recommend studies to assess which mussel species and populations are vulnerable and which are resilient to rising stream temperatures, hydrological shifts, and ionic pollutants, all of which are influenced by GCC.

Temporal and spatial variation of atmospherically deposited organic contaminants at high elevation in Yosemite National Park, California, USA.

Contaminants used at low elevation, such as pesticides on crops, can be transported tens of kilometers and deposited in adjacent mountains in many parts of the world. Atmospherically deposited organic contaminants in the Sierra Nevada Mountains of California, USA, have exceeded some thresholds of concern, but the spatial and temporal distributions of contaminants in the mountains are not well known. The authors sampled shallow-water sediment and tadpoles (Pseudacris sierra) for pesticides, polycyclic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls in four high-elevation sites in Yosemite National Park in the central Sierra Nevada twice during the summers of 2006, 2007, and 2008. Both historic- and current-use pesticides showed a striking pattern of lower concentrations in both sediment and tadpoles in Yosemite than was observed previously in Sequoia-Kings Canyon National Parks in the southern Sierra Nevada. By contrast, PAH concentrations in sediment were generally greater in Yosemite than in Sequoia-Kings Canyon. The authors suggest that pesticide concentrations tend to be greater in Sequoia-Kings Canyon because of a longer air flow path over agricultural lands for this park along with greater pesticide use near this park. Concentrations for DDT-related compounds in some sediment samples exceeded guidelines or critical thresholds in both parks. A general pattern of difference between Yosemite and Sequoia-Kings Canyon was not evident for total tadpole cholinesterase activity, an indicator of harmful exposure to organophosphorus and carbamate pesticides. Variability of chemical concentrations among sites, between sampling periods within each year, and among years, contributed significantly to total variation, although the relative contributions differed between sediment and tadpoles.

Pesticide distributions and population declines of California, USA, alpine frogs, Rana muscosa and Rana sierrae.

Atmospherically deposited pesticides from the intensively cultivated Central Valley of California, USA, have been implicated as a cause for population declines of several amphibian species, with the strongest evidence for the frogs Rana muscosa and Rana sierrae at high elevation in the Sierra Nevada mountains. Previous studies on these species have relied on correlations between frog population status and either a metric for amount of upwind pesticide use or limited measurements of pesticide concentrations in the field. The present study tested the hypothesis that pesticide concentrations are negatively correlated with frog population status (i.e., fraction of suitable water bodies occupied within 2 km of a site) by measuring pesticide concentrations in multiple media twice at 28 sites at high elevation in the southern Sierra Nevada. Media represented were air, sediment, and Pseudacris sierra tadpoles. Total cholinesterase (ChE), which has been used as an indicator for organophosphorus and carbamate pesticide exposure, was also measured in P. sierra tadpoles. Results do not support the pesticide-site occupancy hypothesis. Among 46 pesticide compounds analyzed, nine were detected with ≥ 30% frequency, representing both historically and currently used pesticides. In stepwise regressions with a chemical metric and linear distance from the Central Valley as predictor variables, no negative association was found between frog population status and the concentration of any pesticide or tadpole ChE activity level. By contrast, frog population status showed a strong positive relationship with linear distance from the Valley, a pattern that is consistent with a general west-to-east spread across central California of the amphibian disease chytridiomycosis observed by other researchers.

Toxicity of coal-tar and asphalt sealants to eastern newts, Notophthalmus viridescens.

Between 1970 and 2000 the concentration of total polycyclic aromatic hydrocarbons (TPAH) in several lakes across the country increased whereas those of other persistent organic pollutants (POPs) tended to remain stable or declined. Urbanized watersheds experienced greater rises in TPAH concentration compared to non-urban lakes. Sources for urban PAHs include industrial wastes, vehicular exhausts and oil leaks and sealants from pavement surfaces. Both coal-tar and asphalt sealants are used to protect surfaces but runoff from surfaces coated with coal-tar can have mean concentrations of 3500 mg TPAHs kg(-1), much higher than runoff from asphalt-sealed or cement surfaces. Unaltered parent compounds of PAHs can have many lethal and sublethal toxic effects, but oxidation and UV radiation can alter the toxicity of these compounds, sometimes creating degradates that are many times more toxic than parent compounds. The purposes of this study were to determine if coal-tar sealants can be toxic to adult eastern newts (Notophthalmus viridescens) and to compare the toxicity of coal-tar sealant to that of asphalt sealant. Newts were exposed to sediments containing dried sealants ranging from 0 mg kg(-1) to 1500 mg kg(-1) under simultaneous exposure to UV radiation and visible light to determine concentration/response relationships. No significant mortality occurred with any treatment. Significant effects due to sealants included decreased righting ability and diminished liver enzyme activities. Coal-tar sealant was more effective in inducing these changes than was asphalt sealant.

Alteration of larval development and metamorphosis by nitrate and perchlorate in southern leopard frogs (Rana sphenocephala).

Amphibians are sensitive to a great variety of agrochemicals. Nitrate compounds are commonly applied as fertilizers, whereas perchlorate salts occur as by-products of the military industry and in some nitrate fertilizers. Both compounds are highly soluble and can easily diffuse between ground and surface water, thus potentially affecting amphibians. Nitrate reduces embryonic and larval survival and development, whereas perchlorate has a well-known goitrogenic effect and inhibits metamorphosis. We present the results of an experiment that assessed the combined effects of these two chemicals on the development and metamorphosis of southern leopard frogs (Rana sphenocephala) larvae. Individual frogs were exposed in a block design throughout their larval stages until they metamorphosed. Nitrate at 100 mg/l increased larval mortality and reduced the growth of developing tadpoles. Perchlorate at 15 mg/l was not lethal to larvae, but inhibited metamorphosis, resulting in high mortality during metamorphosis compared with control animals. At 30 mg/l of perchlorate, larvae experienced increased mortality. Together, the chemicals exhibited additive effects on developmental rate and survival. When nitrate and perchlorate occur simultaneously in the environment, they can have greater effects on an amphibian population than either chemical alone.

Effects of lead-contaminated sediment on Rana sphenocephala tadpoles.

We exposed larval southern leopard frogs (Rana sphenocephala) to lead-contaminated sediments to determine the lethal and sublethal effects of this metal. Tadpoles were laboratory-raised from early free-swimming stage through metamorphosis at lead concentrations of 45, 75, 180, 540, 2360, 3940, 5520, and 7580 mg/kg dry weight in sediment. Corresponding pore water lead concentrations were 123, 227, 589, 1833, 8121, 13,579, 19,038, and 24,427 microg/L. Tadpoles exposed to lead concentrations in sediment of 3940 mg/kg or higher died within 2 to 5 days of exposure. At lower concentrations, mortality through metamorphosis ranged from 3.5% at 45 mg/kg lead to 37% at 2360 mg/kg lead in sediment. The LC50 value for lead in sediment was 3728 mg/kg (95% CI = 1315 to 72,847 mg/kg), which corresponded to 12,539 microg/L lead in pore water (95% CI = 4000 to 35,200 microg/L). Early growth and development were depressed at 2,360 mg/kg lead in sediment (8100 microg/L in pore water) but differences were not evident by the time of metamorphosis. The most obvious effect of lead was its pronounced influence on skeletal development. Whereas tadpoles at 45 mg/kg lead in sediment did not display permanent abnormalities, skeletal malformations increased in frequency and severity at all higher lead concentrations. By 2360 mg/kg, 100% of surviving metamorphs displayed severe spinal problems, reduced femur and humerus lengths, deformed digits, and other bone malformations. Lead concentrations in tissues correlated positively with sediment and pore water concentrations.

Contaminant exposure and effects in red-winged blackbirds inhabiting stormwater retention ponds.

Stormwater wetlands are created to retain water from storms and snow melt to reduce sediment, nutrient, and contaminant pollution of natural waterways in metropolitan areas. However, they are often a source of attractive habitat to wetland-associated wildlife. In this study of 12 stormwater wetlands and a larger, older reference site, elevated concentrations of zinc and copper were found in sediments and carcasses of 8-day-old red-winged blackbird (Agelaius phoeniceus) nestlings inhabiting stormwater sites. Although nesting success in the stormwater wetlands was comparable to national averages, sediment zinc concentrations correlated with clutch size, hatching success, fledgling success, and Mayfield nest success, suggesting that the nestlings may have been stressed and impaired by elevated zinc. This stress may have been direct on the nestlings or indirect through effects on the availability of food organisms.