Incorporating Ground Beetle (Coleoptera: Carabidae) Assemblage Data and Earthworm Bioassays in the Ecological Risk Assessment of a Trap and Skeet Shooting Range

Home , Acupalpus, Adephaga, Agonum punctiforme, Amara familiaris, Amphasia, Amphasia interstitialis

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Joshua Lee Bryant

Graduate Program in Entomology

The Ohio State University

2010

Master's Examination Committee:

Roman Lanno “Advisor”

David Horn

Richard Bradley

Joshua Lee Bryant

2010

Abstract

Ecological risk assessment is an important tool for evaluating potentially hazardous contaminants on a site-specific basis. Few methods exist for evaluating population and community level effects of contaminants on terrestrial invertebrates, though such assessments may aid in site evaluation and subsequent decision-making with regard to site remediation. A particularly promising group of invertebrates for terrestrial site-based risk assessment is ground beetles (Carabidae) due to their high diversity in most terrestrial ecosystems. Ground beetles have also been shown to be important indicators of physical disturbances in their habitats. In the current study, we investigated the utility of ground beetles as indicators of elevated metal concentrations originating from lead-based shot in a trap and skeet shooting range. Concentration of the metals lead (Pb), arsenic

(As), and antimony (Sb) were 3668, 46, and 0.28mg/kg in the shotfall and 537, 22, and

0.15mg/kg in the reference site, respectively. Ground beetles were collected in 2008 and

2009 using pitfall traps placed in the shotfall region of the shooting range and an adjacent reference site. Overall, ground beetle species richness did not differ between sites.

Abundance of the most common species was either not significantly different between sites or they were more abundant in the reference site. A total of 45 species was collected in the two sites over both years. In addition to the ground beetle data, bioassays using the earthworm Eisenia andrei were used to determine bioavailability and sub-lethal effects of the metals in the shooting range. Bioavailability of Sb, As, and Pb was greater in the ii shotfall soil compared to the reference soil. There was no mortality in any of the test soils during the 28-day toxicity test. Earthworm cocoon production was reduced in shotfall soil relative to the soil from the reference area and Webster, a lab standard soil. Although earthworm data suggests that there are sub-lethal reproductive effects from exposure to shotfall associated metals, ground beetle diversity appeared to be robust to these environmental changes. Additional years of sampling may reveal additional trends in ground beetle distribution and abundance.

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Dedication

This document is dedicated to my family

Acknowledgments

I would like to thank my advisor, Dr. Roman P. Lanno, for his support and assistance in the development of this project. I also would like to thank my fellow lab mates, David Sovic, Shuo Yu, Meaghan Sutherland, and Chris Hurdzan, for their input and guidance during various parts of my project.

I would like to thank the following friends and colleagues for helping me either set up traps or collect samples; Erin Morris, Kaitlin Uppstrom, Allison Byrnes, and

George Keeney.

I would also like to thank my committee members David Horn and Richard

Bradley for their guidance and input in this project.

I wish to thank Foster Purrington for confirming my carabid identifications and teaching many tricks for discerning closely related species.

Vita

January 28, 1985...... Born, Chillicothe, Ohio, USA

2003 ...... Piketon High School

2007 ...... B.S. Biology, The Ohio State University

2008 to 2009 ...... Robert H. Edgerley Ecotoxicology Fellow

2007 to present ...... Graduate Teaching Associate, Department

of Entomology, The Ohio State University

Fields of Study

Major Field: Entomology

Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgments ...... v

Vita ...... vi

Fields of Study ...... vi

Table of Contents ...... vii

List of Tables ...... xi

List of Figures ...... xii

Chapter 1: Introduction and Literature Review ...... 1

1.1 Ground beetles ...... 1

1.2 Pitfall traps ...... 2

1.3 Biological Indicators ...... 3

1.4 Effects of heavy metals on arthropods ...... 5

1.5 Bioavailability ...... 11

1.6 Bioavailability and distribution of metals in shooting ranges ...... 13

1.7 Studies of soil arthropod communities in shooting ranges ...... 15

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1.8 Scope of the current study ...... 16

1.9 Hypotheses ...... 16

Chapter 2: Diversity of Ground Beetle assemblages of a shooting range and adjacent reference area ...... 18

2.1 Introduction ...... 18

2.2 Methods ...... 18

2.2.1Study site ...... 18

2.2.2 2008 Field Season ...... 20

2.2.3 2009 Field Season ...... 20

2.2.4 Sample processing and identification ...... 22

2.2.5 Data analysis ...... 23

2.3 Results ...... 24

2.3.1 2008 field season ...... 24

2.3.2 2009 field season ...... 24

2.3.3 Ground beetle activity over time ...... 26

2.3.4 Species abundance patterns, richness, and evenness ...... 28

2.3.5 Site preference ...... 30

2.2.6 Trap disturbance ...... 31

2.3 Discussion ...... 32

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Chapter 3: Bioavailability and sublethal effects of shotfall associated metals ...... 38 to Eisenia andrei ...... 38

3.1 Introduction ...... 38

3.2 Methods ...... 38

3.2.1 Preparation of soils ...... 38

3.2.2 Test organisms ...... 39

3.2.3 Bioaccumulation Assay...... 39

3.2.4 Reproduction Bioassay ...... 40

3.2.5 Bioaccumulation factor ...... 41

3.2.6 Analysis of metals in E. andrei tissues and test soils ...... 41

3.2.7 Data analysis ...... 42

3.3 Results ...... 43

3.3.1 Metal concentration in test soils ...... 43

3.3.2 Uptake of shotfall associated metals by E. andrei...... 43

3.3.3 Comparison of adult and hatchling As and Pb tissue concentrations...... 45

3.3.4 Bioaccumulation factor (BAF) ...... 46

3.3.5 Reproduction Bioassay ...... 47

3.4 Discussion ...... 48

Chapter 4: General Discussion and Conclusions ...... 51

References ...... 54

List of Tables

Table 1. Cumulative total catches of each species of ground beetles (Carabidae) in each site for both sampling years...... 25

Table 3. Total soil concentration (mg/Kg dry weight) (mean±SD) of Lead (Pb), Arsenic

(As), and Antimony (Sb) in three test soils. Reported p-values from one-way ANOVA, n=3 for each metal. Pairwise comparisons were made using Tukey’s method. Means in rows having different superscripts are significantly different (P<0.05)...... 43

Table 4. Tissue concentrations (mean±SD) of As, Sb, and Pb (mg/kg) accumulated in tissues of the earthworm E. andrei hatchlings. One way-ANOVA p-values reported multiple comparisons were made using Tukey’s test. Means in rows having different superscripts are significantly different (P<0.05) ...... 45

Table 5. Bioaccumulation factors BAF (mean±SD) based on 28-day dry weight tissue concentrations of adult E. andrei as a factor of the mean soil concentration of the test soils. n=3 for each site and age class...... 47

Table 6. Number of cocoons, juveniles, and juveniles/cocoon (mean±SD) produced by adult E. andrei exposed to Webster, reference, or shotfall soils for 28 days. Means in columns having different superscripts are significantly different (P<0.05)...... 47 xi

List of Figures

Figure 1. Topographic map showing the research site in grey along with the surrounding area. Credit: U.S. Geological Survey Department of the Interior/USGS ...... 19

Figure 2. An example of one of the traps used during the 2009 field season...... 21

Figure 3. Diagram of trap layout used in the 2009 field season. Each numbered square represents a set of 4 traps that are positioned ~10 m apart. Figure not to scale...... 22

Figure 4 . Seasonal activity of ground beetles (beetles/day*trap, mean (+/- SE)) in the shotfall and reference samples in 2008 (a) and 2009 (b). Means plotted against the midpoint of the day of year...... 27

Figure 5. Rank-abundance distributions of ground beetle assemblages collected by pitfall trapping in 2008 (a) and 2009 (b)...... 29

Figure 6: Metal concentrations (mg/kg, dry weight) (Pb -a; As - b) in E. andrei over a 28- day exposure period...... 44

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Chapter 1: Introduction and Literature Review

1.1 Ground beetles

The beetle family Carabidae, ground beetles, is a highly diverse group, widely distributed throughout the world. Carabidae is currently recognized as the 3rd most diverse beetle family based on the number of known species (Triplehorn et al., 2005). As of 1993, there were 2,502 species of ground beetles known to occur in the United States, excluding the Hawaiian and Alaskan faunas, of which 456 were recorded from Ohio

(Bousquet and Larochelle, 1993).

The Ohio ground beetle species occupy a diverse array of niches and include both habitat generalists and specialists. Many ground beetles demonstrate a high affinity for specific habitats such as woodland, arboreal, riparian, or prairie habitats where others overlap many habitat types. (Lindroth, 1969; Silverman et al., 2008; Larsen et. al., 2003;

Lovei and Sunderland, 1996). Ground beetles are well represented in studies of biodiversity and habitat specificity as they tend to change in distribution across habitat types or disturbance gradients (Ranio and Niemelä, 2003).

Many studies have revealed the importance of ground beetles as predators in agricultural ecosystems especially in organic agriculture (Kromp, 1999; Thiele, 1977).

The majority of the species in this family are considered generalist predators; however,

1 there are several genera that occasionally feed on plant material or specialize on plant and seed feeding during all or part of their life cycle (Lövei and Sunderland, 1996; Thiele,

1977). Within the predatory guild of this taxon, there are also many groups of species that specialize on specific prey; for example, members of the genus Brachinus have been identified as parasitoids and carabids in the tribe Cychrini and the genus Dicaelus are morphologically well adapted slug and snail feeding specialists (Arnett et al., 2000; Lövei and Sunderland, 1996; Thiele, 1977).

The distribution of ground beetles in the landscape is determined by a number of biotic and abiotic factors including food availability, habitat type, temperature, humidity, light, soil texture, and even soil pH (Thiele 1977). Many of these factors can influence one another. For example, environmental quality or habitat type may influence food availability and this alteration in food availability would be expected to modify the abundance and distribution of habitat specialists. Due to the wide-ranging habits and numerous species, this group is a good candidate for studies on the impact of disturbances on terrestrial habitats.

1.2 Pitfall traps

Pitfall traps are one of the most common methods used for sampling ground beetle communities (Lövei and Sunderland, 1996). Pitfall trapping is often seen as an inadequate method of sampling as it tends to give biased estimates of abundance. It is important to note that pitfall trapping is a method of measuring activity and not absolute abundance (Thiele, 1997). The factors that influence trap yields for pitfalls include the 2 size of the organism, activity level, and agility. Large bodied organisms tend to be over represented in pitfall samples compared to smaller individuals largely due to their greater mobility (Thiele, 1977). Another issue is the patchy, stochastic nature of species distribution in the landscape. Although other sampling methods exist for determining absolute abundance, they tend to be much more labor and time intensive.

One issue associated with studies of community structure using measures of activity is the question of which species are resident and which may be transient or incidental captures. This may be more important when many species are represented by few individuals or even by a single specimen. It is difficult to discern whether a species is a resident of the community or just happens to be trapped while passing through the sampled habitat. One aspect of using pitfall traps for studies of ground beetles is the influence of habitat edges and ecotones on the movement of ground beetles. Silverman et al (2008) demonstrated that ground beetle activity was higher in an ecotone than in the forest interior or in the open habitat nearby.

1.3 Biological Indicators

Biological indicators are organisms that are used in environmental monitoring and environmental risk assessment to gage the impact of a disturbance on an ecosystem.

Much of the work in the area of ecological risk assessment has focused on aquatic ecosystems where agricultural runoff, sewage treatment plant and industrial effluent, and other human inputs often reduce the quality of water and thus the ability for sensitive species to thrive there. For freshwater streams, synthesis of an abundance of data has led 3 to the development of risk assessment tools that utilize the diversity of aquatic benthic invertebrates, fishes, and mollusks to develop ratings and indices to quantify adverse effects. The development of Indices of Biotic Integrity (IBI) for aquatic systems has relied on comparisons of potentially impacted streams to reference streams with similar characteristics not receiving chemical inputs (Southerland et al., 2007).

In the terrestrial environment the use of invertebrates for environmental monitoring and risk assessment has not been utilized to its full potential due to a lack of toxicological data (Van Straalen et al., 1993). When analytical techniques are used in conjunction with ecological data, decision making concerning potential environmental risk comes from a more holistic viewpoint, taking into consideration both actual concentrations of contaminants and observable effects of the interaction of the contaminants and environment on ecological receptors (Van Straalen et al., 1993).

McGeoch (1998) identified many criteria that are important in the selection of a group of terrestrial species or individual species as environmental or biodiversity indicators. Some of the relevant criteria for the selection of an indicator taxon include the temporal and spatial sampling scale, the ability of the organisms to respond predictably to an environmental change, abundance, diversity, ease of sampling, cost of sampling, available taxonomic literature on group, and ease of identification (McGeoch, 1998;

Ranio and Niemelä, 2003; Andreasen et al, 2001).

Ground beetles have been used as biological indicators in studies on the effects of habitat fragmentation, urbanization, forest and land management (Larsen et al., 2003;

Villa-costillo and Wagner, 2002), climate change, and biodiversity (Ranio and Niemelä,

2003). In their review of ground beetles as indicators, Ranio and Niemelä (2003) found that response of ground beetle communities did not always numerically predict the responses of other communities; however, the Shannon and Simpson indices for ground beetles often correlate positively with the indicies of other taxa. Based upon their survey of the literature, ground beetles species richness may not change in response to disturbance; however changes in species abundance patterns such as a decrease abundance of specialist species and an increase in generalists is often observed in response to environmental disturbance.

1.4 Effects of heavy metals on arthropods

The toxicity of metals to arthropods has long been recognized as evidenced by their use as first-generation insecticides. Lead arsenate, inorganic sulfur, and other metal- based insecticides were commonly used until DDT, botanicals, and organophosphate insecticides became widely available. The use of metals as insecticides was reduced in part because they were recognized to be general poisons, persisted in the environment, and tended to be harmful to all organisms (Casida et al., 1998). The relationship of increased concentrations of certain metals and increased mortality of pest invertebrates was of interest to early developers of insecticides.

Early studies on the accumulation of metals in arthropods demonstrated that heavy metals tended to accumulate to a higher degree with increasing trophic level (Price et al., 1974). Futher studies revealed that species within the same trophic category varied

5 markedly in their tissue concentrations of metals (Morgan et al., 1986). In this study, detritivores had the highest level of most metals including Pb and cadmium (Cd).

The effects of heavy metal exposure on arthropods vary depending on the species and the form of metal and exposure concentration. One study demonstrating the effects of metal exposure on growth and development was performed by Gintenreiter (et. al.,

1993a). Their study of Lymantria dispar, Gypsy moth, reared on a metal-contaminated artificial diet demonstrated many morphometric differences in metal-exposed organisms compared to those reared on an uncontaminated artificial diet. Head capsule width, larval weight, and pupal weight all decreased with increasing exposure concentration of Pb, Cd, copper (Cu), and zinc (Zn). Exposure to metals also increased the number of larval instars needed to reach maturity. In some cases as many as ten instars were observed in comparison to the normal number of six and larval mortality was high in supernumerary instars. Some metal treatments yielded 100% mortality including nominal dietary concentrations of 500 µg/g Pb, 1,250 µg/g Cu, 2,500 and 12,500 µg/g Zn (Gintenreiter et al., 1993b). In a similar study by the same authors assessing the bioaccumulation of metals, it was found that four metals had differing concentration factors with Pb concentrating to less than 0.4 (0, 4, and 20 µg/g exposure concentrations), Zn 0.7 to 3.5

(0, 100, and 500µg/g), Cu 2 to 5 (0, 10, and 50µg/g), and Cd was concentrated to a level

2 to 4 times greater than the dietary exposure concentraiton (0, 2, or 10µg/g). Modes of elimination included passing metals in the feces, especially the meconium for Cu, and loss during molting in the head capsule and exuviae. They also noted a significant relationship between the exposure concentration of the adult and the metal concentration 6 of the newly hatched offspring indicating vertical transfer of metals (Gintenreiter et. al.,

1993b).

In the grasshopper Tetrix tenuicornis, genetic irregularities were observed in individuals collected in a Zn-Pb mining area with elevated levels of Zn, Pb, Cu, and Cd.

The germ cells of 45.8 percent of females were observed to have anomalous numbers of chromosomes, compared to literature values of five to ten percent reported for most populations. The abnormalities included abnormally high numbers of tetraploid and octoploid oocytes, possibly due to disruption of the cytoskeleton during cell division, as well as to direct effects on DNA resulting in aberrant chromosomes (Warchałowska-

Śliwa et al., 2005).

Heikens et al. (2001) in their survey of the literature found trends of metal bioaccumulation in terrestrial invertebrates. This literature study examined four metals

(Pb, Cd, Cu, Zn) and all except Zn showed a positive correlation between whole-body and total soil metal concentration. Of the three metals where soil concentration was correlated with whole body concentrations, a trend was noted for most taxa, Pb had the greatest slope indicating that it generally concentrated to a higher level relative to the soil concentration followed by, Cd, then Cu. The reduced assimilation of Cu and Zn is likely due physiologically to regulation of uptake due to their role as essential trace metals.

Exposure of invertebrates to elevated metal levels had varied effects. Some arthropods show alterations in life history trajectory in response to exposure and other species have no visible symptoms. Many organisms have mechanisms for dealing with exposure to moderate levels of metals by reducing assimilation, eliminating excess metals

7 during molting, or possibly transferring excess metals into ova. Bioaccumulation of metals also varies across taxa, but a common theme is that essential metals tend to be regulated to a constant concentration with changing environmental concentrations while the bioaccumulation of non-essential metals is correlated with environmental concentrations.

There have been few investigations of the effects of metals on ground beetles. The topics of investigation include bioaccumulation of metals, developmental effects of dietary metal, energetics, diversity, and cross generational effects. The largest amount of work in this group has been done in the area of metal bioaccumulation.

Of the studies investigating bioaccumulation, many found little evidence of bioaccumulation of metals in ground beetles (Morgan et al., 1986); however there appear to be species’ specific differences in respect to bioaccumulation, in particular for non- essential metals (Puchart et al., 2007; Jelaska et al., 2007). Ground beetles inhabiting a metal contaminated site in Croatia demonstrated relatively constant concentrations of some metals (Mn, Cu, Zn, and Fe) but concentrations of Pb and Cd appear to be related to the concentration of metals in the soil. Another study at an industrial site in Russia found that ground beetles were also accumulators of Cd and Pb when compared to other taxa collected in the same study (Van Straalen et. al., 2001).

The direct effects of metals on ground beetle communities are not easy to address but ground beetles are often included in larger biodiversity studies because of their high species richness and abundance. Read et al. (1998) studied the effects of a metal-polluted woodland in England on multiple invertebrate taxa. Their investigation found no

8 significant correlations between the number of taxa and level of metal contamination, except in two taxa, Coleoptera larvae and Hymenoptera, where abundance correlated positively with metal concentration. When the data were analyzed using the Shannon index, an inverse relationship with metal concentration was a notable trend in all groups except mollusks. With respect to ground beetles, Read et al. (1998) found many more species preferred sites with lower levels of metals than the polluted sites and noted that the species preferring the polluted sites tended to be larger-bodied.

Another study on the effects of metals at a mining site on ground beetle diversity did not find any effect, possibly due to the low bioavailability of metals at the site (Lock et al., 2001). The sampled sites may have also been different microhabitats and thus effects of metals on ground beetles were masked by differences in other influential environmental parameters (Lock et al., 2001)

The effects of metals on the larval development of ground beetles is important in understanding how populations and communities may respond to elevated levels of metals in the environment. Larvae of Pterostichus oblongopunctatus fed a Cd, Zn, or Cd and Zn enriched diet had a shorter median survival time and lower mass. Larvae of adult beetles collected from contaminated and reference sites did not differ in their responses to metal exposure indicating that there is not a heritable mechanism of metal tolerance in the studied populations (Mozdzer et al., 2003). Larval exposure to Cd and Zn fed fly pupae was negatively correlated with several mophometric measurements in adults of Poecilus cupreus. Beetles fed contaminated fly pupae were also smaller than those fed uncontaminated fly pupae as measured by calorimetery (Maryanski et al., 2002). Another

9 investigation of the cross generational effects of mixed metal exposure found that the fecundity of females was positively correlated with increased metal concentraion (r =

0.316, p = 0.017), but the hatchability of the eggs was negatively correlated (rS= –0.423, p= 0.001); the regressions used the ln[Zn] (Zn ranged from 150 to 10,000) as an index of metal concentration as all of the metals were correlated. The authors also noted a significant negative relationship (Linear Mixed-effect Models, p<0.001 for females p =

0.007 for males) between the exposure histories of the field collected generation and the body mass of the adults of the F1 generation (Lagisz and Laskowski, 2008).

The metabolism and excretion of Hg, methyl-Hg, Cd, and Zn from Pterostichus niger was studied by Lindqvist et al. (1995). After a single dietary exposure to contaminated food; the tissue concentrations were measured and autoradiograms were used to examine the distribution of the metals in the tissue after 5 and 15 days. The metals responded differently in both excretion patterns as well as distribution within the organism. Cd and inorganic Hg, which were primarily retained in the midgut epithelial cells, were eliminated more quickly than Zn and methyl-Hg, which were more evenly distributed throughout tissues of the organism.

Stone et al. (2001) investigated the response of Pterostichus oblongopunctatus collected at five sites across a gradient of mixed metal (Pb, Cd, and Zn) pollution to additional physiological stresses. For this study they determined median survival times for food deprivation and insecticide exposure. The beetles originating from the most contaminated sites demonstrated significantly reduced median survival times to both stressors indicating that exposure to environmental metals increased susceptibility to

10 additional physiological pressures. A study of the activity of detoxification enzymes non- specific carboxylesterase and glutathione S-transferase in Pterostichus oblongopunctatus in the same locality indicated that they did not respond in a predictable way to elevated metals. Interestingly, males did not show elevated levels of enzyme activity where enzyme activity in females was elevated in some cases (Stone et al., 2002).

The effects of metals on carabids have been measured for many different metals and several endpoints have been assessed. Some ground beetle species appear to accumulate metals more readily than others, indicating that species specific differences are important to ground beetle metal metabolism. Retention time, distribution, and elimination of metals from the body varied depending on the form of the metal. Larval exposure to increased concentrations of metals often resulted in reduced body size and in some cases significant mortality in immature beetles. Metals showed a mixed effect on fecundity and hatchability in one species; however consequences of metal exposure were noted in the unexposed offspring of previously exposed adults. In diversity studies more ground beetle species showed an affinity for clean sites more often than for contaminated sites.

1.5 Bioavailability

The concept of bioavailability is very important in many fields dealing with the absorption of an element or compound by an ecological receptor or human. This concept has application to medicine and pharmacology as well as environmental risk assessment. 11

The term bioavailability or bioavailable fraction describes the amount of substance that is actually or potentially bioaccessible to be taken up and assimilated by the exposed organism. Rarely is a substance absorbed in toto as there is some that cannot be take up.

The fraction that is absorbed can be influenced by the physiology of the exposed organism as well as the chemical state of the substances and various environmental factors that alter bioavailability. For example, bioavailability of some ingested substances such as metals and ionizable organic compounds is greatly influenced by the pH of the digestive system.

There are many methods in the literature for estimating bioavailability and the effects of elevated tissue concentrations of metals on organisms. These methods include both chemical and biological means of determining bioavailability. One commonly used method is to use laboratory bioassays with standard test organisms to examine bioaccumulation, metabolism, excretion, and toxicity (acute or sublethal) of various substances. Common test organisms used in laboratory soil bioassays include land snails,

Collembola, and earthworms. Other surrogate methods such as extraction with many types of solvents and solutions also aim to estimate bioavailability of metals in the environment through chemical means (Harmsen, 2007; Lanno et al., 2004)

There are a number of studies that have worked toward developing and standardizing protocols for using earthworms in toxicity testing with soils (Environment

Canada, 1997). Some tests have developed artificial test soils for calculating EC50 and

LC50 values of single contaminants (Spurgeon and Hopkin, 1995; Environment Canada,

2004). Earthworms have been used to investigate the uptake and elimination kinetics of

12 metal in artificial lab soils as well as field-collected contaminated soils. In a review of bioavailability of chemicals in soil to earthworms by Lanno et al. (2004), many different methods of estimating chemical bioavailability to earthworms were discussed; however it was noted that actual chemical residues in earthworm tissue may be the best measure of bioavailability since this measurement integrates all of the factors that may alter bioavailability. The use of non-biological measures of bioavailability (e.g., soil extracts) must be correlated to either some effect in earthworms or chemical bioaccumulation to be useful.

1.6 Bioavailability and distribution of metals in shooting ranges

Lead shot in shooting ranges has been of concern as a potential hazard to organisms living within the area of distribution. The concern for toxicity of Pb shot in shooting ranges has primarily been associated with the ingestion of whole shot by waterfowl in contaminated wetlands or as grit by passerine birds (e.g., mourning dove).

The toxicity of Pb-based shot to waterfowl had led to the prohibition of its use in waterfowl hunting. In feeding trials, Mallard ducks (Anas platyrhynchos) were demonstrated to have 90% mortality in 30 days when fed only eight Pb shot pellets in comparison to zero mortality when fed the same number of steel-based shot (Brewer et al., 2003). The high acute toxicity of Pb shot to waterfowl leads to the question of how terrestrial organisms metabolize and respond to these metals released from Pb shot in soils.

Shooting ranges present an ideal situation for studying metal metabolism by terrestrial organisms as the distribution of metals is in a relatively well defined area with an artificially elevated concentration of metals that would otherwise be less abundant in the surrounding environment. The distribution of intact shot follows a predictable distribution along the axis of shot with the highest concentrations from 30-80 m with a lower concentration peak around 180 m with only minor amounts of shot travelling more than 220 m from the origin (Craig et al., 2002). This distribution will likely differ some between shooting ranges depending on the distances of the targets, changes in topography, and site history. Because of the gradient of shot concentration, it is possible to sample in areas of high and low impact and gauge these effects.

In soils, as Pb shot weathers, it can form different species of Pb, some of which have been shown to be water soluble. The solubility of weathered Pb is dependent on many environmental factors, the most important being pH. Overall, the soluble concentration of Pb increases with the concentration of total Pb as determined by the toxicity characteristic leaching procedure (Cao et al., 2003). The mineralogy of Pb speciation products on the crust of pellets is primarily in the form hydrocerussite

(Pb3(CO3)2(OH)2), cerussite (PbCO3), and anglesite (PbSO4), with other forms such as massicot (PbO), and hydroxypyromorphite [(Pb10(PO4)6(OH)2] being present depending on mineralogy of the surrounding soil (Cao et al., 2003a; Cao et al., 2003b; Lin et al.,

1995).

The total concentration of Pb in the soil is highest on the surface but the presence of an organic material may contribute to migration of Pb into subsoils through the

14 formation of soluble organic metal complexes. Subsoils with a high Cation Exchange

Capacity (CEC) tend to retain and concentrate the soluble Pb in the subsoils (Cao et al.,

2003a; Cao et al., 2003b). In addition to pH, CEC, and organic matter, many factors can influence bioavailability and speciation of metals from Pb-based shot in soils.

In addition to Pb, other trace elements are also present as alloy components in Pb shot. The trace elements may include antimony [Sb], arsenic [As], nickel [Ni], Cu, bismuth [Bi], titanium [Ti], and mercury [Hg]. Most of these metals are only present as small components of shot, but some like Sb, can be present at 5% by weight and contribute to the bioavailable fraction of metals in the soil (Johnson et al., 2005).

The well defined local area of elevated metals, along with soluble metal species, contributes to the value of shooting ranges as study sites for the effects of metals on arthropod communities. Often, laboratory studies on the effects of metals on arthropods consider a single species or few species and do not take into account the dynamics that exist within a community. Studying the arthropod communities in a shooting range allows one to investigate the interaction between potential influences of metals as well as competitive interactions of constituent species.

1.7 Studies of soil arthropod communities in shooting ranges

Few studies have surveyed the arthropod communities of shooting ranges. These studies tend to investigate the composition of these communities with broad taxonomic scope. Migliorini et al. (2003), attempting to investigate the effects of metals on the shooting range to arthropods, identified 17 taxa at the order level. This study did not 15 identify any differences in abundance of taxa in the study site, although it was found that

Symphyla were nearly absent in the shotfall samples and more abundant in the uncontaminated sites. Diplura and Protura were found in greater abundance in the contaminated sites when compared to the uncontaminated sites. Similarly, Rantalainen et al., (2006) did not find any difference in microarthropod diversity (Acari: Prostigmata,

Astigmata, Mesostigmata, Orbatida; Hexapoda: Colembola) in their shooting range investigation. As previously discussed, the abundance of individuals within a broad taxon may not change greatly due to a disturbance; however, with greater taxonomic resolution it is possible to elucidate the responses of individual species and diversity patterns.

1.8 Scope of the current study

The objectives of the current study are to:

1) Assess species richness and diversity patterns of carabid beetles inhabiting a

central Ohio trap and skeet shooting range and a corresponding reference site, and

2) Examine the toxicity of soils collected from Pb shot-contaminated areas to

earthworms in laboratory tests

1.9 Hypotheses

If the elevated metals in shooting ranges are toxic to carabids, then the assemblage of Carabidae species will differ in composition relative to the assemblage without elevated shotfall associated metals. 16

If the metals associated with Pb-based shot are bioavailable, then we predict that earthworms will accumulate them in their tissues. Additionally we predict that acute and sub-lethal toxic effects of metals will be greatest in the soil with the highest bioavailable metal.

Chapter 2: Diversity of Ground Beetle Assemblages of a Shooting Range and Adjacent Reference Area

2.1 Introduction

Metals in the environment in elevated concentrations can be hazardous to ecological receptors. Shooting ranges present an unique area of study due to the elevated levels of Pb and other minor constituent metals being present in a relatively discrete area.

The objectives of this study aimed to determine whether there were any detectable differences in the assemblages of ground beetles inhabiting the primary shotfall region of a trap and skeet shooting range compared to an adjacent reference site. Understanding how ground beetles respond to shotfall is necessary to determine if ground beetles may be a useful indicator taxon for use in sites with elevated metals of anthropogenic origin.

2.2 Methods

2.2.1Study site

The study site is a privately owned trap and skeet shooting range with a shotfall area of approximately 4 hectares. The site is located in central Ohio, but the exact locality as well as the name have been excluded due to the request of the range owner. The 18 property has a history of use by various clubs starting in 1948. The sight was developed into the current shooting facility in 2004. The topography of the range is relatively flat and maintained by occasional mowing. The ground cover includes a diverse assemblage of woody and herbaceous forbs, grasses, and immature trees. The mowed site is bordered to the north by a fallow agricultrual field. The west perimeter has a narrow forested fence row seperating the study site from a hay field. The land use of the surrounding area is primarily a mix of agricultural and residential (Figure 1).

200 m

Figure 1. Topographic map showing the research site in grey along with the surrounding area. Credit: U.S. Geological Survey Department of the Interior/USGS

2.2.2 2008 Field Season

Insects were sampled using unbaited pitfall traps. Pitfall traps consisted 946-ml plastic deli containers placed so that the rim the cup was even with the soil surface.

Approximately 100ml of propylene glycol based antifreeze, Splash© brand RV and marine antifreeze (SuperClean brands Inc, Saint Paul, MN), was used a killing agent and trap preservative. The opening of the trap was covered with a rain guard, which consisted of an 0.2 x 0.2 m square of plywood stilted with nails on each corner. A total of 20 traps was placed in 2 transects with 10 traps approximately 10 m apart. One transect was placed in the shotfall area, the area with the heaviest deposition of Pb, and the other was placed in the reference area, a near area outside the primary shotfall region. Transects were positioned in order to optimize the site similarity between the reference and shotfall sampling areas, while reducing the effect of edges and ecotones.

2.2.3 2009 Field Season

In 2009, arthopods were sampled using a modified pitfall trap array making use of lead-ins constructed of landscape edging. Edging was cut into lengths of 0.5 m and 4 units were placed at approximately 90º from the adjacent lead-in. The trap and rain guard were located at the center of the lead-ins (Figure 2). Traps were placed in clusters of 4 traps with 3 groupings in each sampling area (Figure 3). Traps within a cluster were placed approximately 10m apart in a square arrangement. Trap clusters were placed a

20 distance of equal to or greater than 25 m apart. Propylene glycol based RV and Marine

Splash© brand antifreeze was used as a preserving agent in the pitfall traps.

Figure 2. An example of one of the traps used during the 2009 field season.

Figure 3. Diagram of trap layout used in the 2009 field season. Each numbered square represents a set of 4 traps that are positioned ~10 m apart. Figure not to scale.

2.2.4 Sample processing and identification

Samples were collected from the field on average every 9.13±0.67 days in 2008 and 11.5±1.28 days in 2009. In 2008, traps were installed on May 19th and the last samples were collected on July 31st, and in 2009 traps were installed on May 11th and

22 the last samples were collected on August 10th. Samples were transferred into labeled

946-ml zip seal bags and the preserving fluid was refreshed. The samples were maintained at 4ºC until they were processed. Processing first required the samples to be rinsed through a sieve (~0.5 mm mesh size) with distilled water to remove the used propylene glycol residue. The rinsed sample was then transferred to a pan for sorting. All

Carabidae were removed and transferred to 50-ml medical centrifuge tubes containing

70% ethanol. The sample was further examined under a dissecting microscope to ensure all carabid specimens had been removed. The remaining sample was transferred into additional centrifuge tubes and retained in 70% ethanol. The ground beetles were pinned, labeled, and sorted to morphologically similarly species groups and identified. A subsample of the tentatively identified and unidentified beetles was confirmed and served as voucher specimens for the collection. Voucher specimens will be retained in the ecotoxicology lab of Dr. Roman Lanno at the Ohio State University.

2.2.5 Data analysis

Species rank-abundance distributions was created for each habitat for each year.

The total cumulative samples over the season were used. Species were first ranked by most abundant and converted to proportion of the total site sample. A two-sample

Kolmogorov-Smirnov test was used to compare the shape of the rank abundance distributions between sites for each year (Magurran, 2007). Due to a change in experimental design between 2008 and 2009 we did not compare the rank-abundance distributions between sampling years. A two-sample T-test assuming unequal variances 23 was used to compare beetle numbers trapped in the shotfall and reference site in 2008 and

2009. Site preference was assessed by a Mann-Whitney U test for two independent samples. Each year was run independently. Analysis was run on the species that were represented by a minimum 10 individuals over the entire sampling season.

2.3 Results

2.3.1 2008 field season

The 2008 sampling season yielded a total of 244 ground beetle specimens belonging to 22 different species. 15 species of ground beetle were represented in the samples collected in the shotfall with 68 individuals. In comparison, 13 species were identified from the reference site samples out of 176 specimens. Seven species comprised

89% of the pooled sample and were represented by five or more individuals and the remaining 15 species were represented by three or fewer specimens.

2.3.2 2009 field season

A total of 573 carabids belonging to 44 species was collected during the 2009 field season. There were 36 species represented in the samples from the shotfall and 35 species represented in the reference samples. There were significantly more beetles collected in the reference traps than in the shotfall traps (Two-sample T-test, unequal variance, T=-3.27, df=15.53, p<0.005)

Table 1. Cumulative total catches of each species of ground beetles (Carabidae) in each site for both sampling years.

Species of Carabidae caught by pitfall trap during 2008 and 2009 Shotfall Reference Shotfall Reference Species 2008 2008 2009 2009 Total Acupalpus pauperculus Dejean 0 0 0 1 1 Agonum cupripenne (Say) 3 7 2 5 17 Agonum nutans (Say) 0 19 7 24 50 Agonum punctiforme (Say) 0 0 2 1 3 Amara aenea (DeGeer) 1 0 1 1 3 Amara familiaris (Duftschmid) 0 0 6 6 12 Amara impuncticollis (Say) 0 0 0 6 6 Amphasia interstitialis (Say) 0 0 1 0 1 Anisodactylus harrisii LeConte 0 2 3 0 5 Anisodacylus ovularis (Casey) 0 3 15 11 29 Anisodacylus rusticus (Say) 1 0 1 0 2 Badister notatus Haldeman 0 0 3 0 3 Bembidion affine Say 0 0 0 1 1 Bradycellus nigriceps Leconte 0 0 1 3 4 Chlaenius pusillus Say 1 0 0 1 2 Chlaenius tricolor Dejean 2 3 6 52 63 Cicindella punctulata Oliver 0 0 1 0 1 Cicindella sexguttata Fabricius 0 0 1 2 3 Clivina bipustulata (Fabricius) 0 0 3 4 7 Colliuris pensylvanica (Linné) 0 0 2 2 4 Cyclotrachelus sodalist (Leconte) 0 0 3 2 5 Dicaelus elongatus Bonelli 0 2 6 0 8 Diplocheila obtuse (LeConte) 0 0 1 5 6 Harpalus caliginosus (Fabricius) 0 0 0 4 4 Harpalus compar Leconte 1 0 16 25 42 Harpalus faunus Say 0 0 1 1 2 Harpalus herbivagus Say 0 0 3 0 3 Harpalus pensylvanicus (DeGeer) 3 0 30 59 92 Notiophilus semistriatus Say 0 0 5 2 7 Ophonus puncticeps Stephens 0 0 19 4 23 Patrobus longicornis (Say) 0 0 4 1 5 Poecilus chalcites (Say) 1 44 5 45 95 Poecilus lucublandus (Say) 35 24 10 13 82 Pterostichus atratrus (Newman) 0 1 1 2 4 Pterostichus commutabilis (Motschulsky) 2 1 3 2 8 Pterostichus femoralis (Kirby) 1 1 1 0 3 Pterostichus melanarius (Illiger) 0 0 0 1 1 Pterostichus permundus (Say) 1 0 0 2 3 Scaratini sp. 0 0 0 1 1 Scarites quadriceps Chaudoir 14 66 25 76 181 Scarites subteraneus Fabricius 0 1 2 4 7 Selenophorus opalius (LeConte) 1 0 0 0 1 Stenolophus ochropezus (Say) 0 3 1 5 9 Stenolophus rotundatus LeConte 0 0 2 2 4 Trichotichus fulgens (Csiki) 1 0 4 0 5 Sum 68 177 197 376 818

2.3.3 Ground beetle activity over time

In both study years ground beetles were trapped between May and August. Here we compared the activity patterns between sampling years and between the reference and shotfall sites. Beetle numbers were converted to 퐵푒푒푡푙푒푠 to correct for differences in length 퐷푎푦 of sampling interval. The mean number of beetles per day was plotted as a function of the day of the year (Figure 4).

(a) 1 Shotfall 2008 0.9 Reference 2008

0.8

SE) - 0.7 0.6 0.5 0.4 0.3 0.2

Beetles/trap*day mean(+/ Beetles/trap*day 0.1 0 134 144 154 164 174 184 194 204 214 Sample date mid point (day of year)

(b) 1 0.9 Shotfall 2009 Reference 2009

0.8

SE) - 0.7 0.6 0.5 0.4 0.3 0.2

Beetles/trap*day mean(+/ Beetles/trap*day 0.1 0 134 144 154 164 174 184 194 204 214 Sample date mid point (day of year)

Figure 4 . Seasonal activity of ground beetles (beetles/day*trap, mean (+/- SE)) in the shotfall and reference samples in 2008 (a) and 2009 (b). Means plotted against the midpoint of the day of year.

2.3.4 Species abundance patterns, richness, and evenness

The total ground beetle assemblage of the shooting range and adjacent reference area yielded a total of 45 species over both sampling seasons. Absolute species richness, the total number of species collected in each sampling site, was 15 in the 2008 shotfall sample and 13 in the reference site. In contrast, the shotfall yielded 36 species and the reference had 35 species in 2009.

A comparison of rank-abundance distributions between the reference and shotfall area in 2008 did not yield a significant result at the α=0.5 level (Two-sample

Kolmogorov-Smirnov test, Critical D0.05(13,15) =0.515, Observed D= 0.140) indicating that patterns of species dominance do not differ between the two sites (Figure 5A). In

2009, there was also no significant difference between the shotfall and reference site

(Figure 5B) (Two-sample Kolmogorov-Smirnov test, Critical D0.05(35,36)=0.32,

Observed D=0.049).

(a) 60% Shotfall 2008 50% Reference 2008

40%

30%

20% Proportion %) (of Proportion sample 10%

0% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Species Rank

(b) 25% Shotfall 2009 20% Reference 2009

15%

10%

5% Proportion sample of %) ( Proportion 0% 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 Species rank

Figure 5. Rank-abundance distributions of ground beetle assemblages collected by pitfall trapping in 2008 (a) and 2009 (b).

2.3.5 Site preference

Some species of ground beetles showed consistent differences in site preference both years; Scarites quadriceps, Poecilus chalcites, and Agonum nutans all differed significantly in their distribution and were found most abundantly in the reference site

(Mann-Whitney U, p-value<0.05) (Table 2). Ophonus puncticeps and Chlaenius tricolor showed a preference for the reference site in 2009 as well but were not collected in enough numbers in 2008 to make a comparison across both years (Mann-Whitney U, p- value<0.05). A number of species showed no site preference these included; Harpalus pennsylvanicus, Harpalus compar, Anisodactylus ovularis, Poecilus lucublandus, Amara familiarus, and Agonum cuprepenne (Mann-Whitney U, p-value>0.05).

Table 2. Results of Mann-Whitney U comparison of Pitfall trap samples from 2008 (a) and 2009 (b) Species compared included only those that were represented by 10 or more individuals over the entire sampling season. a Site Species Mann-Whitney U Wilcoxon W P-value Preference Scarites quadraceps 5.000 60.000 0.001 Reference Poecilus lucublandus 41.500 96.500 0.509 None Poecilus chalcites 16.500 71.500 0.004 Reference Agonum nutans 20.000 75.000 0.005 Reference Agonum cuprepenne 39.500 94.500 0.326 None

b Site Species Mann-Whitney U Wilcoxon W P-value Preference Scarites quadraceps 10.5 88.5 0.001 Reference Harpalus pensylvanicus 42 120 0.081 None Chlaenius tricolor 25 103 0.004 Reference Poecilus chalcites 30.5 108.5 0.012 Reference Harpalus compar 53.5 131.5 0.269 None Agonum nutans 34 112 0.018 Reference Anisodacylus ovularis 58 136 0.397 None Poecilus lucublandus 63.5 141.5 0.558 None Ophonus puncticeps 36 114 0.022 Shotfall Amara familiaris 69 147 0.844 None

2.2.6 Trap disturbance

Trap disturbance led to a loss of 0% and 2.5% of samples in the shotfall and reference sites respectively in 2008. In 2009, 7.29% and 6.25% of samples were lost due to disturbance in the shotfall and reference sites.

2.3 Discussion

The most common species either showed no preference or a preference toward the reference site. These results were consistent across both sampling years. One interesting observation was that the two Poecilus species behaved differently in respect to site preference. Over both sampling years, Poecilus chalcites was more often encountered in samples from the reference site than in the shotfall samples, whereas Poecilus lucublandus showed no difference in its site preference. Read et al. (1998) also found that species within the same genus did not always respond consistently to polluted and unpolluted sites. The distribution of Ophonus puncticeps as well as Harpalus species is possibly influenced by the distribution of plant species such as Daucus carota and other umbelliferous plants as their larvae are known to feed largely on the seeds of these plants

(Theile, 1977). Analysis of feeding guild (herbivorous, omnivorous, carnivorous) and distribution may be useful as the metals in this site, Pb in particular, do not generally have high accumulation or toxicity to plants, whereas populations of soil invertebrates that serve as a primary food source for omnivorous and carnivorous species may be reduced due to toxicity from metal exposure or alteration of food availability.

Rank-abundance distributions visually represent patterns of species dominance therefore reflect evenness of the species in the sample. The pattern is also intrinsically related to the sampling method (i.e absolute vs. relative). The Kolmogorov-Smirnov two- sample tests which can be used to compare the shape of two continuous distributions are

32 a conservative estimate of similarity (Magurran, 2007). In our study, no significant difference was observed between the reference and shotfall in regard to the rank abundance distributions. One benefit of comparing rank abundance distributions is that it does not compare named species directly so it is useful in comparing habitats with high β diversity given a standard method of sampling was used.

The trapping methods were modified between the 2008 and 2009 field season in an effort to increase the number of species and specimens collected per trap/day. The addition of lead-ins appeared to increase the diversity of ground beetles represented in the traps; however, this could not be statistically compared as both methods were not implemented concurrently to take into account the effect of year to year variation due to abiotic environmental factors.

Trap disturbance which led to a loss of sample was a source of potential lost data or lost sampling time. During some sampling intervals traps were disturbed by rain leaving traps less than ideally nested in the ground. In these situations the sample was generally intact and processed as such. The primary source of lost samples were suspected disturbance by animals and mowing. Mowing appeared to be a larger disturbance source with the trapping array used in 2009 as the addition of the landscape edging lead-ins created a larger footprint for each trap. In only a few cases, traps were completely pushed from the ground by the raising water table during rainfall.

Overall more ground beetle specimens were collected in the traps placed in the reference site. This trend was largely consistent across both sampling seasons (Figure 4).

Although the reference traps yielded more specimens the absolute species richness of the 33 sites were not different. The observed difference in activity could be a result of the elevated metals in the soil or due to other differences in habitat that could not be controlled in the field such as fluctuations in soil moisture, differences in the soil structure, or distribution of preferred prey or food sources. A majority of the species encountered are ecological generalists, often common in disturbed habitats such as agricultural fields (Larsen et al, 2003).

One issue that was encountered was that for most species only one or two specimens were collected which limits the ability to do formal inference on them. There are tools that have been developed to deal with these types of data including indices that investigate similarity, diversity, richness, or evenness of species in a habitat, but these are only valid with large sample sizes >1000 individuals (Magurran 2004).

Another important different between the two sampling years was the increase in activity and new species toward the end of the 2009 field season. Two of the most abundant species Harpalus pensylvanicus and Harpalus compar only were collected in the last two sampling intervals in 2009 and constituted a substantial portion of the overall sample in 2009. The presence of new species toward the end of the second sampling season is likely related to the bimodal peaks in activity of ground beetles, which general exhibit highest activity in the Spring and Autumn (Lövei and Sunderand, 1996; Thiele,

1977). When studying community composition, it is advisable to sample over the entire activity period of the organisms of interest. In the case of the current study, we were unfortunately restricted to the early season sampling and were unable to collect those species which peak in activity during the late summer and autumn. This was a result of

34 working on a privately owned facility where the traps had to be removed to allow for site maintenance.

Changing the trapping regime between the 2008 and 2009 field seasons reduced our ability to make comparisons between years. However we can comment on differences and similarities in relative abundances as well as species abundance distributions.

Additionally, the use of landscape edging lead-ins did appear have a positive effect on the number of species collected, although this could be due to a number of factors between the two sampling years. One down side to the use of lead-ins is that disturbance by human activity was higher as the footprint of the trap was larger and more easily disturbed during mowing which happened to 4 traps on one sampling date. Although trap disturbance was high, the samples were intact but lost samples did account between 0 and

6.5% of samples within a site over the entire sampling season. Loss of samples due to flooding was a concern during execution of this project; however, flooding resulted in few lost samples. The use of rain guards appeared to be sufficient to prevent direct filling of the reservoirs. On one occasion, rain did elevate the traps from their set position due to water seeping into the space under the container.

Seasonal variations in weather patterns may also be a challenge for pitfall traps, although with the current investigation trap disturbance was not a major issue. Since reference and shotfall sties were in close proximity the weather patterns influencing the traps generally had the same effect on both sites. Sampling of multiple sites separated by large distances, thus under different weather regimes, may lead to asymmetrical

35 disturbances among them making it more difficult to account for the effect of disturbances due to the year effect.

Many biotic and abiotic factors may influence the spatial distribution of species in the landscape (Thiele, 1977). As previously mentioned, the selection of the reference site was such that differences other than in metal chemistry between sites would be minimized; however, it is not certain which factors other than metal distribution are influencing the diversity and distribution of ground beetle species.

Pitfall traps have a tendency be size biased. This is due to the fact that large carabids are generally more mobile; thus large bodied species are over represented relative to their absolute densities, whereas smaller species may be under estimated. One factor influencing this is the slight physical barrier that exists between the soil and the aperture of the trap. Small organisms may also be deterred by minute incongruence between the level of the trap aperture and soil that do not present a barrier to large carabids.

Dispersal ability of ground beetles varies from species to species; some have a proclivity to fly, whereas others are incapable of flight or within the same species they may have both macropterous and micropterous forms (Lindroth 1969). In this case it is possible that more mobile species may have moved between sampling sites. Some studies indicate that site fidelity is quite high for some species of ground beetles and thus some likely do not move large linear distances. Large species in general, have greater speed up to 250 m in one month but generally just a few meters in a 24-h period are reported for large species. Smaller species tend to have lower dispersal and range from a minimum of

0.05 to maximum of 10 m per day (Thiele, 1977). Some species are capable fliers and have been caught on ships up to 30 km off the nearest coast (Thiele, 1977). Best et al.

(1982) investigated the dispersal ability, dispersion, and density of H. pensylvanicus, S. quadriceps, and P. chalcites, three of the most numerous species in the present study. It was concluded that S. quadriceps, P. chalcities and H. pensylvanicus moved on average

12.2, 8.5, and 10.2 m/day respectively; however, the recapture rates of P. chalcites and S. quadriceps were much greater than H. pensylvanicus indicating that it likely dispersed more quickly. Some of the P. chalcities and S. quadriceps were caught and released as many as 4 times indicating relatively high site fidelity compared to H. pensylvanicus.

Because ground beetles differ in their dispersal ability and site fidelity, for some species it is likely that movement between sites was a significant factor influencing our ability to detect differences in the assemblages.

Ground beetle assemblages appear to be robust to the presence of elevated shotfall associated metals. Species richness and rank-abundance distributions did not differ greatly between sites. Overall the number of specimens collected in the shotfall was lower than the reference in both sampling years. Scarites quadriceps, C. tricolor, P. chalcities, and A. nutans, were most active in the reference site relative to the shotfall thus they may be sensitive to the influences of shotfall associated metals. Additional investigation is required to determine what key factors are influencing this distribution.

Chapter 3: Bioavailability and Sublethal Effects of Shotfall Associated Metals to Eisenia andrei

3.1 Introduction

Metals associated with Pb-based shot are initially inert; however, through transformation processes, the metals are transformed into bioavailable forms. The first objective of the study was to assess the bioaccumulation potential of shotfall associated metals. Our second aim was to assess the potential of sub-lethal effects of these with regard to the reproductive endpoints of cocoon production and number of juveniles produced. This study is necessary to determine if metals in this trap and skeet shooting range have a potential to move from the abiotic to the biotic components in the ecosystem.

3.2 Methods

3.2.1 Preparation of soils

Bioassays were conducted with soils collected from the shotfall and reference areas and a standard laboratory reference soil, Webster loam. The field soils were a composite sample consisting of 10 subsamples collected at random from each locality.

Each subsample was taken from the top 15 cm of the soil after the vegetation and thatch were removed. The soil was roughly broken up, homogenized, and the approximately 38

2000 g of each soil was removed for use in the experiments. The soil was further prepared by removing all large rocks, plastics, and debris. The soils were maintained at 4º

C until the beginning of the bioassay. Unused soils were stored at 4°C. 1200 g of each dry test soil was hydrated to 50% of the water holding capacity for use in the bioassay as follows: Webster (24%), shotfall (36%), and reference (38%).

3.2.2 Test organisms

Bioaccumulation tests and reproduction bioassays were conducted with laboratory-reared Eisenia andrei. The master culture has been maintained at The Ohio

State University for five years cultured in a bed of separated dairy solids (SDS) and provided with supplemental food (cooked rolled oats).

3.2.3 Bioaccumulation Assay

The bioaccumulation potential of metals associated with the shotfall area was assessed using E. andrei following a standard protocol protocol (Environment Canada

2004). Ten worms were placed on the surface of the soil (200 g dry weight) in each test chamber (glass mason jars; 500 ml; Ball, Muncie, IN), and test chambers were sealed with a perforated metal lid (one hole, ~2.0 mm, to allow gas exchange) and screw collar.

During the tests, all the test chambers were maintained under continuous fluorescent lighting at 20±2ºC. Worms were fed with 25 g of SDS once a week, and excess food was removed weekly to maintain the total volume of soil. Worms were removed for metal

39 analysis on day 0, 7, 14, and 28 with day 0 worms taken from worms depurated prior to conducting the tests. Three worms were removed from each replicate on sampling days and pooled to ensure enough tissue for analysis. The worms were depurated for 24 hours on moistened filter paper to empty the remove gastrointestinal tract prior to rinsing with distilled water and freezing. Samples were frozen and maintained at -70ºC until they were analyzed.

3.2.4 Reproduction Bioassay

Reproduction was assessed using a similar protocol (Environment Canada 2004).

For each replicate, ten adult E. andrei with well developed clitella were used. Ten worms were placed on the surface of the soil (200 g dry weight) in each test chamber (glass mason jars; 500 ml; Ball, Muncie, IN), and test chambers were sealed with a perforated metal lid (one hole, ~2.0 mm, to allow gas exchange) and screw collar. During the tests, all the test chambers were maintained under continuous fluorescent lighting at 20±2ºC.

Worms were fed with 25 g of SDS once a week, and excess food was removed weekly to maintain the total volume of soil. The adult worms were removed after 28 days in the test chamber and depurated on damp filter paper for 24 hours to remove gastrointestinal contents. Following depuration, the worms were frozen at -70ºC pending metals analysis.

Soil was removed from each test chamber to a flat pan to allow for counting of cocoons.

Cocoons were counted for each of the test chambers, the test chambers reassembled, and cocoons were replaced into the test chambers. After an additional 28 days, E. andrei

40 hatchlings were counted, depurated for 24 hours, and frozen at -70ºC until they were analyzed for metal content.

3.2.5 Bioaccumulation factor

The 28-day bioaccumulation factor (BAF) was calculated for each replicate for

As and Pb. The BAFs were compared across test soils for each metal using one-way

ANOVA. The BAF was calculated based on the following equation (Equation 1).

BAF = 푚푒푡푎푙 푤표푟푚 푚푒푡푎푙 푠표푖푙

Equation 1. In this equation, 푚푒푡푎푙 푤표푟푚 represents the total concentration of metal in the worm in mg/kg dry weight and 푚푒푡푎푙 푠표푖푙 is the concentration of metal in the soil in mg/kg dry weight.

3.2.6 Analysis of metals in E. andrei tissues and test soils

Individual earthworms samples were dried in an oven at 105ºC for 2 hours, and weighed to obtain dry mass. Each adult earthworm sample consisted of 3 worms pooled together to ensure adequate tissue for analysis. In the analysis of hatchlings, all individuals recovered were pooled. The sample was mixed with 10 ml of 25% (v/v) concentrated trace metal-grade HNO3 (Fisher Scientific, Pittsburgh, PA), and digested in a closed Teflon bottle in a microwave oven (Ethos 320; Milestone Inc., Monroe, CT) at

180ºC for 10 min. After cooling at ambient temperature, the solution was diluted to 50 ml with deionized water in a volumetric flask. Concentrations of Pb, As, and Sb in the

41 digests were determined with inductively coupled plasma mass spectroscopy (ICP-MS;

Elan 6000; Perkin Elmer Sciex., Woodbridge, ON Canada). Metal concentrations were reported as mg/kg based on dry weight.

Total soil metal concentrations were measured in the soils used for the bioassays.

First, 0.5 g of test soil was oven-dried at 105ºC for 2 h, weighed, mixed with 10 ml of concentrated trace metal-grade HNO3 (Fisher Scientific, Pittsburgh, PA), and digested in a closed Teflon bottle in a microwave oven (Ethos 320; Milestone Inc., Monroe, CT) at

180ºC for 10 min. After cooling at ambient temperature, the solution was diluted to 50 ml with deionized water, and then any residual soil was removed by filtration (0.22 µm).

Concentrations of Pb, As, and Sb in the digests were determined with ICP-MS (Elan

6000; Perkin Elmer Sciex., Woodbridge, ON Canada), and reported based upon the dry weight of soil.

3.2.7 Data analysis

Soil metal concentrations and earthworm reproduction parameters were compared using one-way ANOVA (α = 0.05). A linear model was fitted to the bioaccumulation data over the exposure. Tissue concentrations and BAFs of metals between adults and juveniles were compared using two-way ANOVA (α = 0.05) with the age and exposure soil as main factors. All post-hoc tests performed used Tukeys test with a family-wise error rate of (α = 0.05).

3.3 Results

3.3.1 Metal concentration in test soils

Arsenic, Pb, and Sb concentrations differed significantly among all three test soils

(Table 3). Lead, As, and Sb, which are all associated with Pb shot, followed an expected pattern of increase with the Webster soil having the lowest concentration of each metal followed by successive increases in the reference and shotfall soils.

Metal Soil (mg/kg) Webster Reference Shotfall P-value Pb a18.0±1.0 b537.3±18.0 c3668.3±38.3 <0.001 As a9.18±0.85 b21.67±4.04 c45.67±2.52 <0.001 Sb a0.02±.005 b0.15±.04 c0.28±.005 <0.001

Table 3. Total soil concentration (mg/Kg dry weight) (mean±SD) of Lead (Pb), Arsenic (As), and Antimony (Sb) in three test soils. Reported p-values from one-way ANOVA, n=3 for each metal. Pairwise comparisons were made using Tukey’s method. Means in rows having different superscripts are significantly different (P<0.05).

3.3.2 Uptake of shotfall associated metals by E. andrei

Arsenic and Pb concentrations were significantly different (p<0.001) across all test soils after 28 days of exposure. The linear model yielded R2=0.76, R2=0.98 and

R2=0.87 for the shotfall, reference, and Webster soils, respectively (Figure 6a).

Accumulation of As yeiled a linear fit of R2=0.98, R2=0.97, and R2=0.96 for the shotfall, reference, and Webster soils respectively (Figure 6b).

Lead Bioacculation a 350 Webster R² = 0.76 300 Reference 250 Shotfall 200

150

100

Pb mg/kg weight mg/kg dry Pb R² = 0.98 50

0 0 7 14 21 R² = 0.87 28 Exposure time (Days)

Arsenic Bioacculation b 18 R² = 0.98 16 Webster 14 Reference 12 Shotfall 10 8 6 R² = 0.97

As mg/kg weight dry mg/kg As 4 2 0 0 7 14 21 R² = 0.96 28 Exposure time (Days)

Figure 6: Metal concentrations (mg/kg, dry weight) (Pb -a; As - b) in E. andrei over a 28- day exposure period.

3.3.3 Comparison of adult and hatchling As and Pb tissue concentrations.

Tissue concentrations of Pb in juveniles and adults were compared by two-way

ANOVA. The effects of both main factors, soil type (F(2, 12) = 5964.92, p<0.001)and age (F(1, 12) = 120.43, p<0.001) were significant, suggesting that adult E. andrei accumulated significantly higher concentrations of Pb than juveniles and that Pb bioaccumulation was affected by soil type. The interaction term was also significant (F(2,

12) = 9.86, p=0.003) suggesting that adults and juveniles respond differently to Pb uptake with regard to soil treatment.

Tissue concentrations of As in juveniles and adults were also compared by two- way ANOVA. The effects of both main factors, soil type (F(2, 12) = 3797.54, p<0.001) and age (F(1, 12) = 411.65, p<0.001) were significant, indicating that adult E. andrei accumulated significantly greater amounts of As than juveniles and that As bioaccumulation was affected by soil type. The interaction term was also significant (F(2,

12) = 13.87, p=0.001) suggesting that adults and juveniles respond differently to As uptake with regard to soil treatment.

Metal Shotfall reference Webster p-value Sb a0.62 ± 0.07 b0.2633 ± 0.03 c0.0443 ± 0.002 <0.001 As a9.11 ± 0.70 b1.58 ± 0.04 c0.6567 ± 0.04 <0.001 Pb a126.67 ± 4.51 b17.667 ± 1.53 c0.5800 ± 0.02 <0.001

3.3.4 Bioaccumulation factor (BAF)

Assimilation of metal into the tissue of E. andrei varied with the concentration of metal in the soil. In general, as the concentration of soil metals increased a larger portion of metals relative to the soil concentration were accumulated by the worms (Table 5).

Adults and juveniles E. andrei differed significantly with respect to the observed BAFs, adults accumulated significantly more metals than juveniles over the exposure period. A

Two-way ANOVA revealed significant effects of both main factors, soil type (F(2, 12) =

442.08, p<0.001) and age (F(1, 12) = 225, p<0.001) were significant, indicating that adult

E. andrei on average had significantly higher As BAFs than juveniles and that the test soil also significantly influenced BAF values for As. The interaction term was also significant (F(2, 12) = 37.92, p=0.001) suggesting that adults and juveniles respond differently in their bioaccumulation of As with regard to test soil.

A two-way ANOVA with age and soil type as main factors for Pb BAF again revealed significant effects of both main factors, soil type (F(2, 12) = 39.62, p<0.001) and age (F(1, 12) = 217, p<0.001) were significant, indicating that adult E. andrei on average had significantly higher Pb BAFs than juveniles and that the test soil also significantly influenced BAF values for Pb. The interaction term was also significant

(F(2, 12) = 28.21, p=0.001) suggesting that adults and juveniles respond differently to in their bioaccumulation of Pb with regard to test soil.

Metal As Pb Soil Adult Juvenile Adult Juvenile Webster 0.107±0.004 0.072±0.004 0.042±0.006 0.032±0.001 Reference 0.139±0.002 0.073±0.002 0.050±0.002 0.033±0.003 Shotfall 0.337±0.015 0.200±0.015 0.070±0.001 0.035±0.001

3.3.5 Reproduction Bioassay

The mean number of cocoons and juveniles produced decreased in worms exposed to shotfall soils (p=0.003 for cocoons, p=0.004 for juveniles) relative to reproduction in reference and Webster soils (Table 6). The mean number of cocoons and juveniles did not differ between reference and Webster soils. The number of juveniles recovered per cocoon did not differ significantly across soil treatments (one-way

ANOVA, p=0.122).

Soil Cocoons Juveniles Juveniles/Cocoon Webster a 53.3 ± 4.9 a 99.7 ± 3.2 a1.88±0.22 Reference a 43.3 ± 6.1 a 82.7 ± 23.8 a 1.89±0.30 Shotfall b27.7 ± 5.1 b 36.3 ± 7.6 a 1.35±0.38

3.4 Discussion

The most significant result from our study is that reproduction was reduced in the shotfall soil relative to the other two test soils. The observed reduction in reproductive output was likely a consequence of an altered energy budget. Spurgeon and Hopkin

(1996) found that the congeneric earthworm, Eisenia fetida, exhibited a reduced rate of growth and maturation, lower reproductive output assessed by the rate of cocoon production, and higher mortality when exposed to soils with mixed metal contaminants particularly high Pb and Zn. In this case it is hypothesized that the energy required for metal detoxification and sequestration reduced the amount of energy allocated for growth, maturation, and reproductive processes. This is most certainly the case with the shotfall soils in our experiment. Although the number of cocoons produced differed across treatments, the number of juveniles produced from each cocoon did not differ significantly between treatments (p=0.122). This result suggests that toxicity of elevated metals to developing embryos and developing juveniles was negligible over our exposure period.

Since the shooting range soils exceeded published soil invertebrate ecological soil screening levels (Eco-SSL) for Pb, the sublethal reproductive effects observed in our study are not unexpected. The concentration of Pb in the composite sample from the shotfall is more than double the invertebrate Eco-SSL for Pb (1,700 mg/kg dry weight).

The mammalian and avian Pb Eco-SSLs were also exceeded in the shotfall as well as reference soils (USEPA, 2005a). An invertebrate Eco-SSL has not been derived for As; however, the level of As in the shotfall was approximately equal to the Eco-SSLs for 48 mammalian and avian ground insectivores of 46 and 43 mg/kg dw, respectively (USEPA,

2005b). The level of Sb was below the invertebrate Eco-SSL and within the normal background range for all test soils (USEPA, 2005c). The Eco-SSLs, which are used as a screening value, provide a conservative estimate of contaminant concentrations that could adversely affect wildlife.

The 28-day tissue concentrations observed for Pb are less than the values observed by Langdon et al. (2005) in their 28-day bioassay utilizing Pb(NO3)2 spiked soils of similar concentrations (406 mg/kg dw in soil with nominal concentrations of

3000 mg/kg Pb and 637 mg/kg in soil with nominal concentration of 4000 mg/g dw). The reduced bioavailability compared to the soils in the aforementioned study suggests that the metal species that exist in the soil have lower availability and soil modifying factors at the site are likely further altering the bioavailability.

The differences in the 28-day BAF across test soils showed some interesting trends. Arsenic BAFs were very similar between worms exposed to the Webster and reference soils; however, three-fold greater BAF was observed in the shotfall. For Pb the

BAFs had the general trend of increasing with increasing total soil Pb concentration. The observed differences are likely due to a number of factors, but one likely contributing factor is the differences in the bioavailable or bioaccessible fraction of metals relative to the total concentration of Pb in the soil. Bioavailability may be altered by the interaction of soil physical and chemical properties with the Pb species in the soil. Another potential contributing factor is that at higher concentrations, mechanisms the worms use to maintain Pb homeostasis become less effective. In general, Langdon et al. (2005)

49 reported an opposite trend in BAF. In soils contaminated with a single metal species,

Pb(NO3)2, the Bioconcentration factor decreased as the soil concentration increased. One important note is that tissue concentrations of metals did not appear to be approaching steady state during the test period in our study, thus the BAF derived from steady state values may have been more appropriate to compare across the test soils.

Arsenic and Pb concentrations in juvenile E. andrei were significantly lower

(P<0.001) than As and Pb concentrations in adult worms likely due in part to the variability of actual exposure time. The juveniles were not synchronized with respect to age in the treatments, with the hatching of cocoons spread over the 28-day period following the removal of adult worms. An alternative explanation is that organisms of different age classes may also differ in physiological responses, and thus the uptake and elimination rates of As and Pb may be different than for adult earthworms.

Chapter 4: General Discussion and Conclusions

One important observation is that mortality was not observed in the adult worms during either of the bioassays. Sub-lethal effects of the metals do seem to be an issue with respect to the reproductive endpoints of cocoon and juvenile production. The reproduction differences between the shotfall and reference soils are not likely due to the test matrix, as most of the soil properties should be relatively similar given the proximity of the two sites. A few possible explanations of lower reproductive output in the shotfall test chambers is that metal detoxification is an energetically expensive process and therefore the amount of energy that can be allocated to reproduction is reduced as an increase of energy is demanded for detoxification mechanisms.

Because we found that laboratory exposed earthworms showed some sub-lethal effects of metal exposure and readily accumulated metals, we attempted to assess metal body burdens of field collect worms to see how our 28-day bioaccumulation values relate to earthworms exposed to the soils over the duration of their lives. Although earthworms were collected abundantly in the pitfall samples, they were degraded quickly and not of good enough quality to accurately and confidently analyze the metal content. Attempts to hand collect or use mustard solution to acquire earthworms at the field site were unfortunately not overwhelmingly successful and only a few worms were collected by

51 these methods. Metal analysis of field-collected earthworms was therefore not performed due to low numbers and sporadic distribution of collected individuals.

Earthworms constitute a food source for predatory carabid beetles, thus the evaluation of bioaccumulation potential using E. andrei also elucidates one potential route of dietary Pb exposure to this feeding guild (Forsthye 1982). In contrast to earthworms, most plants do not accumulate Pb to very high levels except in plant roots

(Labare et al, 2004; Manninen and Tanskanen 1993). The dietary exposure concentration of Pb to granivourous and phytophagous ground beetles such as individuals belonging to the genera Harpalus, Anisodactylus, and Amara are likely much lower than in Scarites,

Poecilus, Chlaenius, and Agonum where a large portion of the diet is other soil invertebrates (Forsthye 1982; White et al, 2007). Future investigations of ground beetles in this study site should consider the role of feeding guild in the distribution of and activity of different species.

There have been few studies that investigate the effects of shooting range metals on arthropods (Migliorini et al. 2004; Rantalainen et al. 2006). Our present study is the first that addresses the potential effects on assemblages at the species level of resolution.

Overall, it appeared that ground beetle species richness and rank abundance distribution was not impacted by elevated shotfall associated metals; however, the activity of many of the common species was lower in the shotfall samples compared to the reference samples.

In future, continued sampling using the lead-in method that was employed the second year would help identify consistent trends in ground beetle activity. Furthermore,

52 it would be useful to identify an additional more distant reference site as the level of both

Pb and As were above reported background levels in the area we designated as reference

(USEPA 2005) A 56-day test to asses bioaccumulation potential may further elucidate uptake trends as we did not observe great deviations from linearity in the 28 day bioassay. The evaluation of other methods for sampling earthworm populations would be helpful in the future to determine actual Pb and As concentrations in earthworms exposed over their lifetimes.

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