Occurrence and Characterization of Steroid Growth Promoters Associated with Particulate Matter Originating from Beef Cattle Feedyards.

Studies of steroid growth promoters from beef cattle feedyards have previously focused on effluent or surface runoff as the primary route of transport from animal feeding operations. There is potential for steroid transport via fugitive airborne particulate matter (PM) from cattle feedyards; therefore, the objective of this study was to characterize the occurrence and concentration of steroid growth promoters in PM from feedyards. Air sampling was conducted at commercial feedyards (n = 5) across the Southern Great Plains from 2010 to 2012. Total suspended particulates (TSP), PM10, and PM2.5 were collected for particle size analysis and steroid growth promoter analysis. Particle size distributions were generated from TSP samples only, while steroid analysis was conducted on extracts of PM samples using liquid chromatography mass spectrometry. Of seven targeted steroids, 17α-estradiol and estrone were the most commonly detected, identified in over 94% of samples at median concentrations of 20.6 and 10.8 ng/g, respectively. Melengestrol acetate and 17α-trenbolone were detected in 31% and 39% of all PM samples at median concentrations of 1.3 and 1.9 ng/g, respectively. Results demonstrate PM is a viable route of steroid transportation and may be a significant contributor to environmental steroid hormone loading from cattle feedyards.

Establishment, characterization, and toxicological application of loggerhead sea turtle (Caretta caretta) primary skin fibroblast cell cultures.

Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 µM following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 µM benzo[a]pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells.

Embryotoxicity of Corexit 9500 in mallard ducks (Anas platyrhynchos).

Embryotoxicity of the oil dispersant Corexit 9500 was examined using fertilized mallard duck eggs. Corexit 9500 was topically applied below the air cell to eggs in volumes ranging from 0 to 100 µL on day 3 of incubation. The highest incidence of mortality occurred at developmental stage 4, one day post-Corexit 9500 application. Hatching success was significantly decreased among eggs treated with ≥ 20 µL of Corexit 9500 as compared to controls (P ≤ 0.047). No egg treated with ≥ 40 µL successfully hatched. The application volume resulting in 50% mortality (corrected for control survival) was determined to be 15.5 µL. Developmental stage at embryo death was also significantly decreased compared to controls in eggs exposed to 40 µL (P = 0.0042) and above.

Exposure of Foraging Bees (Hymenoptera) to Neonicotinoids in the U.S. Southern High Plains.

Exposure to pesticides is a major threat to insect pollinators, potentially leading to negative effects that could compromise pollination services and biodiversity. The objectives of this study were to quantify neonicotinoid concentrations among different bee genera and to examine differences attributable to body size and surrounding land use. During the period of cotton planting (May-June), 282 wild bees were collected from habitat patches associated with cropland, grassland, and urban land cover and analyzed for three neonicotinoids (thiamethoxam, clothianidin, and imidacloprid). Twenty bees among eight genera contained one or more of the neonicotinoid compounds and detections occurred in all landscape types, yet with the most detections occurring in cropland-associated habitats. Apis Linnaeus (Hymenoptera: Apidae), Melissodes Latreille (Apidae), Perdita Smith (Andrenidae), and Lasioglossum Curtis (Halictidae) had multiple individuals with neonicotinoid detections. Two of the largest bees (Apis and Melissodes) had the greatest number of detections within genera, yet the relatively small-bodied genus Perdita had the three highest neonicotinoid concentrations reported. The number of detections within a genus and average generic body mass showed a marginally significant trend towards larger bees having a greater frequency of neonicotinoid detections. Overall, the relatively low percentage of detections across taxa suggests practices aimed at conserving grassland remnants in intensified agricultural regions could assist in mitigating exposure of wild bees to agrochemicals, while differences in bee traits and resource use could in part drive exposure. Further work is needed to address variable agrochemical exposures among pollinators, to support strategies for conservation and habitat restoration in affected landscapes.

Persistence of elevated concentrations of PM, affiliated pharmaceuticals, and tetracycline resistance genes downwind of feedyards.

Beef cattle feedyards have been identified as sources of large amounts of particulate matter (PM) which may transport affiliated chemicals including steroids, beta agonists, and antibiotics from feedyards into the environment. This study is the first to examine persistence of PM-affiliated pharmaceuticals downwind of feedyards using multiple downwind samples collected at increasing distances from feedyard boundaries (n = 5). Concentrations of antibiotics and ractopamine per gram of PM remained consistent at all downwind locations (out to 4.8 km) whereas concentrations per m3 air decreased significantly at distances between 0.1 and 0.7 km downwind, corresponding to significant decreases in mass of PM. Monensin was present in the highest concentrations of any measured pharmaceutical, with concentrations of 37 µg/g PM (376 ng/m3) air in samples collected within 0.1 km downwind of feedyards. Total copy count of tetracycline resistance genes (tetW, tetQ, tetO, tetM, tetL, and tetB) were also significantly increased in samples collected within 0.1 km downwind of feedyards (106 copies) as compared to samples collected upwind (103 copies) and farther downwind (104 copies) of feedyard boundaries. These results suggest that transport of pharmaceutical-laden PM into the terrestrial environment is occurring primarily via PM deposition within 0.7 km of the feedyard, while aerial transport persists over longer distances (>4.8 km).

Ractopamine in particulate matter emitted from beef cattle feedyards and playa wetlands in the Central Plains.

Beef cattle in the United States are routinely administered ractopamine, a ß-adrenergic receptor agonist, to enhance growth. The present study is the first to quantify ractopamine in feedyard-emitted particulate matter and playa wetlands near feedyards. Ractopamine was present in 92% of particulate matter samples, 16% of playa sediment samples, and 3% of playa water samples, at maximum concentrations of 4.7 µg/g, 5.2 ng/g (dry wt), and 271 ng/L, respectively. These data suggest that aerial transmission and deposition of particulate matter is a transport mechanism for ractopamine between feedyards and aquatic systems in the region. Environ Toxicol Chem 2018;37:970-974. © 2017 SETAC.

Antibiotics, bacteria, and antibiotic resistance genes: aerial transport from cattle feed yards via particulate matter.

BACKGROUND: Emergence and spread of antibiotic resistance has become a global health threat and is often linked with overuse and misuse of clinical and veterinary chemotherapeutic agents. Modern industrial-scale animal feeding operations rely extensively on veterinary pharmaceuticals, including antibiotics, to augment animal growth. Following excretion, antibiotics are transported through the environment via runoff, leaching, and land application of manure; however, airborne transport from feed yards has not been characterized. OBJECTIVES: The goal of this study was to determine the extent to which antibiotics, antibiotic resistance genes (ARG), and ruminant-associated microbes are aerially dispersed via particulate matter (PM) derived from large-scale beef cattle feed yards. METHODS: PM was collected downwind and upwind of 10 beef cattle feed yards. After extraction from PM, five veterinary antibiotics were quantified via high-performance liquid chromatography with tandem mass spectrometry, ARG were quantified via targeted quantitative polymerase chain reaction, and microbial community diversity was analyzed via 16S rRNA amplification and sequencing. RESULTS: Airborne PM derived from feed yards facilitated dispersal of several veterinary antibiotics, as well as microbial communities containing ARG. Concentrations of several antibiotics in airborne PM immediately downwind of feed yards ranged from 0.5 to 4.6 µg/g of PM. Microbial communities of PM collected downwind of feed yards were enriched with ruminant-associated taxa and were distinct when compared to upwind PM assemblages. Furthermore, genes encoding resistance to tetracycline antibiotics were significantly more abundant in PM collected downwind of feed yards as compared to upwind. CONCLUSIONS: Wind-dispersed PM from feed yards harbors antibiotics, bacteria, and ARGs.

Phthalate ester leachates in aquatic mesocosms: implications for ecotoxicity studies of endocrine disrupting compounds.

Aquatic mesocosm experiments have become common throughout the fields of ecology and ecotoxicology. Mesocosm containers are often composed of plastic materials as these are lighter and cheaper than steel cattle tanks. Plastics may contain phthalate esters which may leach from containers, potentially confounding experiments via toxicity or endocrine disruption. The objective of this experiment was to determine the extent to which plastic containers (="tanks") used for mesocosms leach phthalates, and if there are significant differences between tank types and phthalate profiles. We investigated four tank types: fiberglass, polyethylene, poly-vinyl chloride, and polyethylene tanks containing an established aquatic community. We measured six common phthalate esters in water samples collected from each tank every 2 weeks for 8 weeks. There was a significant effect of tank type, time, and time x type interactions on phthalate ester concentrations. Di-(2-ethylhexyl) phthalate was the predominant congener measured in all samples. Fiberglass tanks had greater concentrations of dimethyl phthalate compared to other tank types (more than 600x larger concentration), but no other differences in phthalate profiles among tank types were evident. In addition, tanks with established communities had much higher concentrations of most phthalates at the 6 and 8 week timepoints. Our study confirms that mesocosm tanks of different materials leach phthalates starting immediately after they are filled and continuing for at least 8 weeks, but do so at different rates. Future ecotoxicity experiments should consider the potentially confounding effects of phthalates that may leach from tanks used in experimental mesocosms.

Canine toys and training devices as sources of exposure to phthalates and bisphenol A: quantitation of chemicals in leachate and in vitro screening for endocrine activity.

Chewing and mouthing behaviors exhibited by pet dogs are likely to lead to oral exposures to a variety of environmental chemicals. Products intended for chewing and mouthing uses include toys and training devices that are often made of plastics. The goal of the current study was to determine if a subset of phthalates and bisphenol A (BPA), endocrine disrupting chemicals commonly found in plastics, leach out of dog toys and training devices (bumpers) into synthetic canine saliva. In vitro assays were used to screen leachates for endocrine activity. Bumper leachates were dominated by di-2-ethylhexyl phthalate (DEHP) and BPA, with concentrations reaching low µg mL(-1) following short immersions in synthetic saliva. Simulated chewing of bumpers during immersion in synthetic saliva increased concentrations of phthalates and BPA as compared to new bumpers, while outdoor storage had variable effects on concentrations (increased DEHP; decreased BPA). Toys leached substantially lower concentrations of phthalates and BPA, with the exception of one toy which leached considerable amounts of diethyl phthalate. In vitro assays indicated anti-androgenic activity of bumper leachates, and estrogenic activity of both bumper and toy leachates. These results confirm that toys and training devices are potential sources of exposure to endocrine disrupting chemicals in pet dogs.

Effects of 17α-trenbolone and melengestrol acetate on Xenopus laevis growth, development, and survival.

The synthetic growth-promoting hormones trenbolone and melengestrol acetate have been detected in the environment near beef cattle feedlots and are reportedly transported via wind-borne particulate matter. Therefore, movement of synthetic hormones from beef cattle feedlots to water bodies via particulate matter is possible. Our objective was to evaluate potential effects of 17α-trenbolone (17α-TB), melengestrol acetate (MGA), and combinations of both on growth, development, and survival of Xenopus laevis larvae. On post-hatch day 2 (stage 33/34), X. laevis larvae were exposed to three nominal concentrations of 17α-TB (10, 100, and 500 ng/L), MGA (1, 10, and 100 ng/L), a combination of both (1/10, 10/100, and 100/500 ng/L MGA/17α-TB), frog embryo teratogenesis assay-Xenopus medium, or a solvent control. Significant increases in all X. laevis growth metrics were observed among larvae in the 1 ng/L MGA + 10 ng/L 17α-TB and 10 ng/L MGA + 100 ng/L 17α-TB treatments. Stage of development was increased among larvae in the 1 ng/L MGA + 10 ng/L 17α-TB treatment group and significantly decreased among those in the 500 ng/L 17α-TB treatment. Total body mass and snout-vent length of X. laevis larvae were significantly reduced in the 100 ng/L MGA and 100 ng/L MGA + 500 ng/L 17α-TB treatment groups. Larvae exposed to 500 ng/L 17α-TB had decreased total body mass, snout-vent length, and total length. In general, growth measurements decreased with increasing concentration of MGA, 17α-TB, or a combination of both. Survival among all treatments was not significantly different from controls. Amphibians exposed to MGA and 17α-TB in the environment may experience alterations in growth and development.

Embryotoxicity of mixtures of weathered crude oil collected from the Gulf of Mexico and Corexit 9500 in mallard ducks (Anas platyrhynchos).

Dispersants are applied to marine crude oil spills to enhance microbial degradation and reduce impacts of crude oils on ecosystems. In summer 2010, the dispersant Corexit 9500 was applied to crude oil in the Gulf of Mexico. The co-occurrence of the Deepwater Horizon oil spill with nesting efforts of birds in the Gulf region may have resulted in exposure of adult birds, and subsequently bird eggs, to combinations of crude oil and Corexit 9500. The objective of this study was to examine the embryotoxicity of 50:1 and 10:1 mixtures of weathered crude oil collected from the Gulf of Mexico and Corexit 9500 applied to mallard duck eggs. Combinations of weathered crude oil and Corexit 9500 were applied to eggshells of mallard ducks via paintbrush in varying masses ranging from 0.1 to 59.9 mg and 0.1 to 44.9 mg for 50:1 and 10:1 mixtures, respectively. Conservatively derived median lethal applications for 50:1 and 10:1 mixtures of weathered crude oil and Corexit 9500 were 21.3±4.9 mg/egg (321.8 µg/g egg) and 33.1±11.8 mg/egg (517.0 µg/g egg), respectively. Spleen mass of hatchlings exposed to the 50:1 mixture was the only physiological measure significantly different from controls of both mixtures. Results indicated that decreasing ratios of dispersant relative to weathered crude oil decreased toxicity to mallard embryos. In comparison to treatments of eggs with weathered crude oil alone, toxicity increased when the oil to dispersant ratio was 50:1, but decreased with the mixture that contained more dispersant (10:1).

Embryotoxicity of weathered crude oil from the Gulf of Mexico in mallard ducks (Anas platyrhynchos).

Weathered crude oil in the Gulf of Mexico can result from oil spills such as the Deepwater Horizon incident that occurred on April 20, 2010 or from natural seeps. Adult waterbirds of the Gulf Coast region may become exposed to weathered crude oil while foraging, wading, or resting, and residues can then be transferred to nests, eggs, and hatchlings. Although the toxicity of many types of crude oil to avian embryos has been thoroughly studied, the effects of weathered crude oil on developing avian embryos are not well characterized. The objective of the present study was to examine embryotoxicity of weathered crude oil collected from the Gulf of Mexico in June 2010 using mallard ducks (Anas platyrhynchos) as a model species. Weathered crude oil was applied to fertilized mallard duck eggs by paintbrush in masses ranging from 0.1 to 99.9 mg on day 3 of incubation. Mortality occurred as early as day 7 and the conservatively derived median lethal application of weathered crude oil was 30.8 mg/egg (0.5 mg/g egg) or 30.7 µl/egg (0.5 µl/g egg). Body mass, liver and spleen mass, crown-rump and bill lengths, and frequency of deformities were not significantly different among hatchlings from oiled and control eggs. In comparison to published reports of fresh crude oil embryotoxicity, weathered crude oil was considerably less toxic. We conclude that avian toxicity varies according to the degree of crude oil weathering and the stage of embryonic development at the time of exposure. Results indicate bird eggs exposed to weathered crude oil from the Gulf of Mexico during summer 2010 may have had reduced hatching success.