Utilizing soil characteristics, tissue residues, invertebrate exposures and invertebrate community analyses to evaluate a lead-contaminated site: A shooting range case study Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy In the Graduate School of The Ohio State University By Sarah R. Bowman, M.S. Graduate Program in Evolution, Ecology, and Organismal Biology The Ohio State University 2015 Dissertation Committee: Roman Lanno, Advisor Nicholas Basta Susan Fisher Copyright by Sarah R. Bowman 2015 Abstract With over 4,000 military shooting ranges, and approximately 9,000 non-military shooting ranges within the United States, the Department of Defense and private shooting range owners are challenged with management of these sites. Ammunition used at shooting ranges is comprised mostly of lead (Pb). Shooting ranges result in high soil metal concentrations in small areas and present unique challenges for ecological risk assessment and management. Mean natural background soil Pb is about 32 mg/kg in the eastern United States, but organisms that live in soil or in close association with soil may be at risk from elevated levels of Pb at shooting ranges. Previous shooting range studies on the ecotoxicological impacts of Pb, with few exceptions, used total soil Pb levels as a measure of exposure. However, total soil Pb levels are often not well correlated with Pb toxicity or bioaccumulation. This is a result of differences in Pb bioavailability, or the amount of Pb taken up by an organism that causes a biological response, depending on soil physical/chemical characteristics and species-specific uptake, metabolism, and elimination mechanisms. Therefore, in order to better characterize exposure and risk at these sites, it is necessary to use site-specific measures of bioaccessibility, bioaccumulation, toxicity tests, and characterizations of biotic communities. The study sites for this research included a private shooting range (1,457–10,044 mg total Pb/kg soil) and an off-site reference area (Gallant Woods, Delaware County, OH; 6-11 mg total Pb/kg soil) located in central Ohio, USA. The shooting range ii consisted of three areas of interest: shotfall (where most Pb shot falls at the site), extracted (where Pb shot was removed by wet sieving in 2009), and on-site reference area. Elevated soil Pb levels at shooting ranges present a potential risk for wildlife living in the vicinity of ranges, with the direct ingestion of Pb shot and contaminated soil as the main pathways of exposure. Receptors such as earthworms live in the various soil horizons and are exposed to Pb through the soil pore water (dissolved Pb ions) and through direct ingestion and processing of organic matter. Lead can be incorporated into different tissue fractions within earthworms including the metal rich granule (MRG) and the non-metal rich granule (non-MRG) fractions. These fractions are important because the MRG fraction may be used as a Pb sequestration mechanism in earthworms, reducing the internal fraction of bioavailable Pb, and hence, toxicity. Another key component in ecological risk assessment includes the depuration (clearing of the gut) of organisms used for tissue analysis. Earthworms can be up to 30% soil by weight, therefore, it may be important to depurate organisms during bioaccumulation studies. However, during trophic interaction studies, it may be beneficial to use non-depurated earthworms since predators would consume food as-is. Standard earthworm reproduction and bioaccumulation assays were conducted with soils from the three areas from the shooting range and the off-site reference area. Earthworm reproduction (number of cocoons and juveniles) was reduced in the shooting range soils. Bioaccumulation varied for each treatment, but was higher for shooting range soils than off-site reference and negative control soils. Shooting range field-collected earthworm tissues had significantly higher Pb (range: 121–1,574 mg/kg) than worms iii collected at the off-site reference area (range: 2–4 mg/kg). Total Pb in depurated earthworms exposed to on-site reference soil and shotfall soil was lower than total Pb in non-depurated earthworms exposed to the same soils. A weak extraction (ammonium nitrate; ISO19730:2008(E)) of Pb also followed a similar trend. The ammonium nitrate extractable Pb is a measure of easily soluble Pb fractions of soil. This measure may be a better estimate of earthworm Pb exposure than total Pb because earthworms are exposed to Pb through pore water which would include the easily extractable Pb. Total earthworm Pb was highest when compared to the MRG and non-MRG fractions for all lab-exposed and field-collected earthworms, while MRG fractions contained more Pb (for each soil/treatment type) than the non-MRG tissue fractions. The results also indicate that the percentage of non-MRG Pb is smaller for field-collected earthworms than for lab- exposed earthworms. Ground beetles also live in close association with soil and feed on plant material or other insects. They can be used as biological indicators of ecosystem change since they are relatively easy to collect and identify with proper training. In order to better understand the community-level effects of Pb along a Pb gradient at the shooting range, the abundance and diversity of ground beetle communities were examined. Ground beetles (Carabidae) were collected using pitfall traps at the shooting range to determine if community structure (i.e., diversity and abundance) changes along a Pb gradient. Twenty-three genera were collected on the shooting range site from 2010–2012 and overall taxa richness and ground beetle abundance was highest in the extracted area. An indicator species analysis suggested that four taxa were indicators of the shotfall site, six taxa were indicators of the extracted site, and three taxa were indicators of the on-site iv reference area. The results suggest that habitat disturbance of soil wet screening and subsequent rye grass slice-seeding in the extracted area was more important in determining ground beetle abundance and diversity than soil Pb concentrations. Total Pb in depurated Harpalus ground beetles from the shooting range sites (5.8–12.4 mg/kg Pb) was higher than depurated Harpalus ground beetles from the off-site reference area (0– 1.3 mg/kg Pb). Average total Pb in depurated beetles from the extracted area (8.7 ± 1.9 mg/kg Pb) was lower than average total Pb in non-depurated beetles from the extracted area (31.4 ± 7.9 mg/kg Pb). Small mammals also live in close association with soil and depending on the species, consume plant material, insects, or other small mammals. Many small mammals also exhibit burrowing behavior. Small mammals may be exposed to Pb by direct ingestion of soil or contaminated food items. The meadow vole (Microtus pennsylvanicus), is one of the most common small mammals in the northern United States and is a receptor of interest for this study. Meadow voles feed mostly on monocot and dicot shoots, and to a lesser extent, seeds, roots, fungi, and insects. In mammals, Pb accumulates in the liver, kidney, and bone tissue. Meadow voles were collected using snap-traps at the shooting range and off-site reference area for tissue Pb analysis. Lead levels in kidney tissue of voles sampled from the shooting range (105 ± 17 mg/kg) exceeded published critical renal Pb values and were significantly higher than Pb levels in kidneys of voles sampled from the off-site reference area (0.34 ± 0.08 mg Pb/kg). Lead levels in liver tissue from voles at the shooting range (17 ± 2 mg/kg) were also significantly higher than samples from the reference site (0.15 ± 0.03 mg/kg). The percent of soil in the diet of meadow voles (4%) v at the site was estimated using element tracers. The Ohio State University in vitro gastrointestinal (OSU-IVG) method, a liquid extraction technique designed to mimic physiological conditions in the human stomach, was used to estimate the bioaccessible pool of Pb that may be dissolved in the stomach. The OSU-IVG extraction value was divided by the total Pb content of a soil to obtain the percent bioaccessible Pb in the soil. Values for shooting range soils (69–75%) were not different from positive control soil (83%), but were different than off-site reference (19%) and negative control (19%) soils. Literature values and field measurements were used to calculate hazard quotients for meadow voles at the site. Even with adjustments for bioaccessibility of Pb in soil, hazard quotients were greater than one. Based on laboratory and field-based analysis, it appears that receptors at the site (earthworms, beetles, and small mammals) may be under stress from elevated Pb concentrations in the soil. Future earthworm studies should focus on multi-generational effects of Pb on earthworm reproduction and bioaccumulation (total Pb, MRG Pb, and non-MRG Pb). Ground beetle laboratory toxicity studies should be conducted in the future to determine concentrations in the soil and tissue residues that cause acute and chronic endpoints. Future ground beetle community field studies should be conducted to monitor trends in taxa abundance, richness, and indicator species at other contaminated sites. Before ground beetles can be used as indicators of contaminated or reference sites, more data collection needs to occur. Future mammal studies should focus on pinpointing tissue concentrations that correlate with histological change endpoints. Population-level effects such as adaptation of shooting range mammals to high soil Pb concentrations should also be investigated. Bioaccessibility assays should be developed and validated for vi use with ecological receptors. Future studies at the site should consider higher level carnivores (e.g., raptors) to determine if impacts to lower trophic levels affect carnivores that rely on lower trophic levels for food. It is also recommended that similar studies be conducted at other shooting range sites that underwent similar management techniques (i.e., Pb shot removal) to see if similar trends exist in invertebrate and mammal data.