ABSTRACT

ACCUMULATION OF ENVIRONMENTAL AND DIETARY HEAVY METALS BY THE WOLF MILVINA (ARANEAE, LYCOSIDAE)

by Lucas Christopher Erickson

Invertebrates provide a key link in moving toxic heavy metals from the environment into the vertebrate food web. Studying the uptake and accumulation of metals in invertebrates can help explain bioaccumulation of metals further up the food web. In this study, I decoupled dietary and substrate exposure to heavy metals and quantified accumulation of cadmium, copper, lead, and zinc in the epigeic Pardosa milvina. I also looked at how foraging and lifespan might change in response to heavy metal exposure. I found that P. milvina absorbs and accumulates all four of these heavy metals through its cuticle. However, only metals that also are micronutrients (e.g. zinc) were accumulated from dietary consumption; non-nutritive metals were excreted without accumulation. Female mortality was not affected by heavy metal exposure, while male mortality was negatively affected by substrate exposure to metals but not by dietary exposure. As P. milvina is a common prey item for a variety of vertebrate and invertebrate species, it likely provides a vector for heavy metals to move from the soils into the vertebrate food web.

ACCUMULATION OF ENVIRONMENTAL AND DIETARY HEAVY METALS BY THE WOLF SPIDER PARDOSA MILVINA (ARANEAE, LYCOSIDAE)

A Thesis

Submitted to the

Faculty of Miami University

in partial fulfillment of

the requirements for the degree of

Master of Science

by

Lucas Christopher Erickson

Miami University

Oxford, Ohio

2018

Advisor: Dr. Ann Rypstra

Reader: Dr. Al Cady

Reader: Dr. Melany Fisk

©2018 Lucas Christopher Erickson

This Thesis titled

ACCUMULATION OF ENVIRONMENTAL AND DIETARY HEAVY METALS BY THE WOLF SPIDER PARDOSA MILVINA (ARANEAE, LYCOSIDAE)

by

Lucas Christopher Erickson

has been approved for publication by

The College of Arts and Science

and

Department of Biology

______Dr. Ann Rypstra

______Dr. Al Cady

______Dr. Melany Fisk

Table of Contents List of Tables ...... iv List of Figures ...... v Dedication ...... vi Acknowledgements ...... vii Introduction ...... 1 Methods ...... 5 Results ...... 10 Discussion...... 12 Future Studies ...... 16 Literature Cited ...... 17 Tables/Figures ...... 21 Appendix ...... 40

iii

List of Tables Table 1 ...... 25 Table 2 ...... 26 Table 3 ...... 27 Table 4 ...... 28 Table 5 ...... 29 Table 6 ...... 30 Table 7 ...... 31 Table 8 ...... 32

iv

List of Figures Figure 1 ...... 33 Figure 2 ...... 34 Figure 3 ...... 35 Figure 4 ...... 36 Figure 5 ...... 37 Figure 6 ...... 38 Figure 7 ...... 39 Figure 8 ...... 40 Figure 9 ...... 41 Figure 10 ...... 42 Figure 11 ...... 43

v

Dedication

For my parents. Thanks for everything.

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Acknowledgments

There are quite a few people I need to thank for their help and support in getting through graduate school. First and foremost is my advisor Ann Rypstra; without her guidance, none of this work would have been possible. In addition, I would like to thank my committee members Al Cady and Melany Fisk for their insight and feedback throughout my work. Thank you to Mary Gardiner and James Harwood for supplying me with the heavy metal contaminated soil. Thanks as well to John Morton for his help with ICP-MS, especially in working with small amounts of biomass. Thanks to everyone in the Rypstra lab for their help and support (both scientific and emotional) during my time at Miami. An especially heartfelt thank you goes out to Amber Dailey, who has provided constant motivation and support, whether she knew it or not. And finally, thanks to the undergraduate professors that nurtured my spark of scientific curiosity: Bob Verb, Jay Mager, Leslie Riley, Stephen Kolomyjec, and Terry Keiser. Without them, I wouldn’t have applied to graduate school in the first place.

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Introduction Environmental contamination with heavy metals is a pressing concern for both human and environmental health (UNEP 2013). The risks posed by heavy metals have increased in recent decades as human use of heavy metals and their release into the environment has increased (Tchounwou et al. 2012). Anthropogenic activities from industrialization such as burning of fossil fuels and mining are primary drivers of heavy metal deposition into the environment (Alloway 2012). As growing populations in developing countries are increasingly industrialized, the impact of heavy metals in the environment is expected to increase (Nagajyoti et al. 2010). Heavy metals can have negative ecological effects at both individual and population levels (Alloway 2012). Individual exposure to heavy metals may impair development, damage neurological function, cause systemic damage, or otherwise interfere with physiological processes (Tchounwou et al. 2012). Heavy metals may also have non-lethal effects that reduce population viability. Exposure of fish to non-lethal concentrations of heavy metals impaired swimming ability (Eissa et al. 2010), while increased mercury exposure has been tied to reduced nesting rates in bird populations (Heath and Frederick 2005). Heavy metal uptake and accumulation has been widely studied in vertebrates, but has received relatively little attention in soil (Gall et al. 2015). Furthermore, the majority of studies have focused on detritivores and herbivores, ignoring how heavy metals may move through the invertebrate food web into predators (Gall et al. 2015). Field studies have shown that heavy metals accumulate in invertebrate predators, but the factors impacting uptake and accumulation are unknown (Heikens et al. 2001, Gall et al. 2015). Laboratory studies have shown the potential for transfer of heavy metals through predation in invertebrates, but results can vary based on the metals and system under consideration (Gall et al. 2015). Cadmium and zinc have been shown to accumulate in ladybugs through consumption of aphids (Green et al. 2010). In contrast, Cheruiyot et al. (2013) found that soldier bugs did not accumulate nickel from consuming nickel contaminated caterpillars. In addition to consumption, invertebrates can also directly absorb heavy metals through their cuticle (Rabitsch 1995). These two mechanisms of exposure (dietary and environmental) may contribute differentially to both heavy metal

1 accumulation and consequences in invertebrates (Maelfait and Hendrickx 1998; Heikens et al. 2001). (Araneae) provide an excellent opportunity to study the movement of heavy metals in an invertebrate predator. Dietary uptake of heavy metals is known to occur since Peterson et al. (2003) found that nickel could accumulate in spiders from consuming nickel-contaminated grasshoppers. Additionally, spiders have been shown to directly absorb heavy metals through their cuticle (Rabitsch 1995; Heikens et al. 2001), and heavy metal accumulation seems driven by microhabitat selection: ground dwelling spiders tend to accumulate more than web spiders (Larsen et al. 1994, Marc 1999). This disparity may be driven by dietary uptake as ground dwelling spiders would consume more herbivores and detritivores that accumulate metals (Larsen et al. 1994) and also produce fewer enzymes that allow for sequestration and excretion of heavy metals relative to web building spiders (Wilzcek and Babczynska 2000). Environmental exposure may also explain some of this disparity. While ground dwelling spiders receive continuous exposure (and thus potential for cuticular absorption) to soil contaminants (Maelfait and Hendrickx, 1998), web dwelling spiders are at greatest risk for direct absorption when the web itself is contaminated with heavy metals (Clausen 1986, 1989). Wolf spiders (Araneae: Lycosidae) in particular provide a good model for heavy metal biomagnification in terrestrial invertebrate food webs (Maelfait and Hendrickx 1998). Lycosid spiders are ground dwelling, leading to potentially strong absorption and accumulation of heavy metals through cuticle contact with substrate contaminants (Rabitsch 1995; Heikens et al. 2001). In addition, wolf spiders are generalist invertebrate predators, allowing a broad study of predator-prey heavy metal transfer (Wilczek et al. 2004). Wolf spiders are known accumulators of heavy metals (Jung and Lee 2012), but the pathways and mechanisms are not fully understood (Gall et al. 2015). Field studies showed that lycosid spiders also exhibit sexual dimorphism, which results in sexual differences in heavy metal accumulation (Wilczek 2007). Females are thought to take up more dietary heavy metals than males due to ingestion of more prey, but it is unclear why males accumulate more heavy metals (Wilczek 2007). Pardosa milvina (Hentz 1844) is a typical wolf spider species found throughout the United States from the eastern coast to the Rocky Mountains (Kaston 1972). This species 2 displays sexual dimorphism, with females being larger, more aggressive, and more voracious than males (Walker and Rypstra 2002), which may result in differential sexual effects on heavy metal uptake and accumulation. Since P. milvina is known to respond to chemical and tactile cues in the environment (Persons et al. 2001; Evans et al. 2010), it may be possible for P. milvina to detect heavy metal contamination in the environment and/or prey and alter foraging behavior to minimize exposure (e.g. by reducing feeding). In this study, I documented heavy metal accumulation in P. milvina, specifically assessing uptake and accumulation of cadmium, copper, lead, and zinc, as these metals are commonly studied in soil invertebrates (Jung and Lee 2012). Copper and zinc are both micronutrients, so P. milvina may be able to regulate their dietary uptake and excretion (but not necessarily environmental uptake). In contrast, cadmium and lead are not nutritious, so digestive regulation may be lacking in those metals (Ardestani et al. 2014). By attempting to uncouple uptake from substrate exposure versus prey consumption, I pulled apart the drivers of heavy metal accumulation in a predatory invertebrate. Adult P. milvina were exposed to heavy metals in their substrate, diet, or both and the accumulation of heavy metals was compared under each set of conditions and by sex. As P. milvina exhibits behavioral sexual dimorphism, I predicted that behavioral responses as well as the resulting heavy metal uptake would differ between the sexes. I predicted that contamination in the soil substrate and contamination of prey species with heavy metals would each contribute differently to the overall accumulation of heavy metals in the predator. Specifically, I predicted that copper and zinc accumulation would be driven by environmental contamination alone, while cadmium and lead accumulation is driven by both environmental and dietary accumulation with an interactive effect. Additionally, I compared how both routes of exposure affected foraging behavior over a period of six weeks to determine if P. milvina could detect and respond to the presence of heavy metals, predicting that both male and female spiders would reduce feeding in response to heavy metal contamination in their prey, but that females would see a greater reduction in feeding. I also compared survivorship among the treatment groups and predicted that contamination of either soil or prey with heavy metals would reduce the spider’s lifespan, and combining routes of exposure would have an even greater effect on mortality. For all

3 monitored outcomes, I predicted that the strength of the effects would increase as spiders spent more time being exposed to heavy metals (Figure A1).

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Methods Care All Pardosa milvina used in this study were captured in agricultural fields at Miami University’s Ecology Research Center in southwest Ohio (39°31'53" N, 84°43'22” W). Spiders were kept in translucent plastic cylinders 6cm in diameter and 7cm tall enclosed with a lid filled to a depth of 2-3cm with either a control substrate (1:1 potting soil/peat moss mixture) or known contaminated soil (see below). The soil was moistened on a weekly basis with 1 mL distilled water and any water that was not absorbed by the substrate after two minutes was removed. The containers were kept in a climate controlled room at 25° C, 60% humidity, and 13:11 hours light/dark cycle. For the duration of the experiment, spiders were fed Sinella curviseta (Collembola: Entomobryidae) () and Gryllodes sigillatus (Insecta: Gryllidae) (banded crickets) once weekly (see feeding schedule below). When a spider produced an egg sac, it was removed and frozen. At the onset of the study, I measured each spider’s carapace width and abdomen width with a digital micrometer attached to a microscope. These measurements were used to estimate body condition according to Jakob (1996) to verify there were no differences among treatment groups prior to the study. I estimated body condition in the spiders by regressing the natural log of body mass on the natural log of carapace width and comparing the residuals (Jakob et al. 1996). I then compared the body conditions among treatment groups to ensure that differences in consumption of prey and/or mortality among treatment groups was not due to any previous differences between the treatment groups. Contaminated Soil Contaminated soil came from vacant residential lots in Cleveland, Ohio and were obtained via coring (3.5cm in diameter) to a depth of 10-12cm. Coring sites were 1, 6, or 11 meters away from the road. A total of twelve cores were taken at each site; these were combined to make a composite sample. This soil had elevated levels of heavy metals including cadmium, lead, and zinc and were used as part of a larger study examining heavy metal contamination in urban settings and the potential associated risks (Sharma et al. 2015). Soil pH was previously found to be fairly neutral, averaging 6.97 and was predominantly composed of sand (69.4%) and silt (21.14%) with a small amount of clay

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(9.47%) (Sharma et al. 2015). I thoroughly mixed the soil samples from all of the lots together and autoclaved them before using them as a substrate for spider habitats. Experimental Procedures In order to uncouple diet and soil substrate as routes of heavy metal uptake, I maintained spiders according to one of four possible treatment conditions for six weeks. The four treatments were: uncontaminated substrate and uncontaminated food; uncontaminated substrate and contaminated food; contaminated substrate and uncontaminated food; and contaminated substrate and contaminated food. I kept twenty- one adult male and female Pardosa milvina under each treatment condition (total forty-two spiders) under each set of conditions and 168 spiders in total. At the conclusion of the six- week period, any spiders left alive were euthanized by freezing and then stored in the freezer; spiders that died during the study were frozen within three days of dying. Contaminated Food I fed the spiders one of two types of S. curviseta for the duration of the experiment, classified as either “contaminated” or “uncontaminated.” Uncontaminated springtails came from existing laboratory cultures raised and maintained on the standard laboratory soil/peat substrate. Three weeks before the study began, stock S. curviseta were cultured on contaminated soil to allow the Collembola populations time to grow (Gist et al. 1974) and accumulate heavy metals via absorption through the cuticle, as springtails tend to avoid eating food contaminated with heavy metals and are efficient at excreting consumed metals (Fountain and Hopkins 2001). The S. curviseta raised on the contaminated substrate were the contaminated food source for the spiders in this study. I maintained Collembola cultures in translucent cylindrical containers approximately 8cm in diameter and 5cm in height. The containers had 2-3cm of soil added, followed by enough distilled water to saturate the substrate. A piece of potato ~1cm3 in size was sprinkled with yeast and added as a food source. Finally, 15-20 adult Collembola were added. Initially, the adult Collembola came from laboratory stocks, but subsequent contaminated Collembola cultures were established with Collembola from contaminated cultures with high populations. Contaminated cultures were given a minimum of three weeks to develop before being used in the experiment.

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Feeding Schedule I fed spiders once per week for the duration of the study. For each feeding, I placed thirty S. curviseta into a glass feeding bowl. I then added a spider and covered the container with a loose-fitting lid. After sixty minutes feeding time it was it from the feeding dish and the remaining springtails were counted to determine consumption. I then gave the spider a 3mm cricket to ensure enough caloric intake in the event that the spider did not fully satiate itself on Collembola. I checked the spiders after 24 hours to verify the cricket was consumed. Heavy Metal Analysis I used inductively coupled plasma mass spectroscopy (ICP-MS) to analyze heavy metal concentrations in substrates, spiders, and contaminated food in accordance with Thomas (2001). Fifteen samples were analyzed of both laboratory and Cleveland substrate after the spiders had been on them for six weeks. I also analyzed fifteen samples of each substrate after they had been used to raise S. curviseta. I dried soil samples for 48 hours at 45 degrees Celsius, weighed them, and then ashed the soil in a muffle furnace at 350° C for eight hours followed by 550° C for twelve hours to remove any organic matter. I coarsely ground the ashes with a mortar and pestle and weighed out approximately 100mg of the ash for each sample and dissolved it for 36 hours in 1mL of concentrated nitric acid set on a hot plate. I then mixed solution with 50mL distilled water before analyzing it in an ICP mass spectrometer to measure concentrations of cadmium, copper, lead and zinc in the soils. For chemical analysis, I combined egg sacs from female spiders with the parent spider to account for any metals that may have been offloaded into egg sacs. Some spiders did not have enough mass for individual analysis; when that occurred, spiders were combined with others of the same treatment, sex, and survival (number of weeks). I also combined Collembola into samples of at least 10mg each (approximately 150 individuals). I took fifteen samples from the uncontaminated Collembola population and fifteen from the contaminated population. For each sample, I weighed the invertebrates, dried them for 48 hours at 45 degrees Celsius, and then weighed them again. Dried samples were digested in concentrated nitric acid on a hotplate for 36 hours and then removed them from the heat

7 and left to sit until the acid had evaporated entirely (approximately four days). Samples were then brought up in 3-28mL of weak nitric acid based on the dry sample mass and this solution was analyzed in an ICP mass spectrometer to measure concentrations of cadmium, copper, lead and zinc in the organisms. Statistics I used one-way ANOVA to test if concentrations of cadmium, copper, lead, and zinc were different between contaminated and uncontaminated soils. Similarly, I used one-way ANOVA to determine if the contaminated cadmium, copper, lead and zinc concentrations differed between contaminated and uncontaminated springtails. To compare heavy metal accumulation within the spiders, I first used one-way ANOVA to check for sexual differences in heavy metal accumulation. Because the sexes accumulated metals differently, I separated them for further analysis. For each metal, I conducted a three-factor ANOVA with soil type, food type, and lifespan as factors to compare how environmental conditions relate to metal accumulation and test for effects of exposure duration. Spiders were divided into two lifespan groups: spiders that died within the first three weeks (short lived) and those that survived until the end of the treatment (long lived). In this ANOVA, lifespan was used as a proxy for duration of exposure. Finally, for each heavy metal I used two-factor ANOVAs to test for interactions among sex, soil type, food type, and lifespan. I used a right-censored proportional hazard analysis to compare survivorship between males and females across all treatments. Because of large differences between the sexes I tested the hypothesis that soil and prey contamination would affect survival separately for males and for females. I used the proportional hazard test to examine the effects of soil type, prey type and their interaction on survival of females separate from their effects on the survival of males. I compared the average weekly consumption of Collembola between males and females using ANOVA. As above the large differences in prey capture between the sexes caused me to run separate analyses on males and females. I used a two-way ANOVA to example the effects of soil type, prey type and any interaction on average weekly consumption of prey for each sex.

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To examine whether the response to prey or soil contamination changed over time, I conducted a MANOVA with repeated measures on the number of prey consumed per week. I analyzed the data from spiders that lived through week 4 because by week 5 I had less than 25% of the males left in the experiment. I first compared males and females and then tested the response of males and females to our treatments over time in separate MANOVAs.

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Results Body condition There was no initial difference in body condition between treatments or sexes among spiders (Table A1). Heavy Metal Analysis Soil analysis revealed that concentrations of lead and zinc were significantly higher in the contaminated Cleveland soil relative to the uncontaminated soil (Table 1). Lead was almost twenty times more concentrated in the contaminated soil, and zinc was about twice as concentrated. There was no difference in soil concentrations of either cadmium or copper between the two soils (Table 1). Collembola on contaminated soils accumulated significantly more cadmium, copper, and lead than did Collembola on uncontaminated soils (Table 2). There was no difference in zinc accumulation for Collembola between the substrates (Table 2). These results are in contrast to the relative heavy metal contamination in the two substrates. Copper and zinc concentrations were both significantly higher in male spiders than in female spiders (Figure 1a, Table 3). Cadmium and lead concentrations were not different between the sexes (Figure 1b, Table 3). For female P. milvina, all four metals had a higher concentration in spiders that lived on the contaminated soil (Table 4, Figure 2). However, only copper and zinc accumulation increased in spiders that consumed contaminated prey and female spiders showed no differences in cadmium or lead accumulation from contaminated prey (Table 4, Figure 3). There were no differences in accumulation for any metal based on lifespan alone in females (Table 4, Figure 4). Cadmium, copper, and zinc all exhibited a similar trend whereby short-lived females on contaminated soil accumulated the most metal (Table 4). Additionally, copper accumulation showed an interactive effect between environmental and dietary exposure (Table 4). Male P. milvina heavy metal accumulation followed trends similar to those in females. Living on contaminated soil resulted in greater accumulation of all heavy metals except for copper (Table 5, Figure 2), and consuming contaminated prey resulted in greater accumulation of only copper and zinc (Table 5, Figure 3). However, cadmium, copper, and lead all had significantly lower concentrations in the male spiders that survived to the end

10 of the study compared to the male spiders that died within the first three weeks (Table 5, Figure 4). Similar to females, cadmium and copper accumulation was greatest in the short- lived spiders that were exposed to contamination in the soil (Table 5). Male P. milvina also showed an interactive effect of contamination when exposed to both dietary and environmental heavy metals (Table 5). Lifespan Overall, females survived significantly longer than males: 61% of the females made it to the end of the experiment whereas only 20% of the males were alive in the sixth week of the experimental period (Table 6, Figure 5). There was no effect of the treatments on female survivorship but soil contamination significantly reduced male survival (Figure 6; Table 6). There were no effects of prey type on male or female survival (Figures 6 and 7; Table 6). Foraging Behavior The overall average weekly Collembola consumption was significantly higher for females than it was for males (Figure 8; Table 7). Males ate four to six Collembola every week, while females ate seven to nine (Figure 8). Female Collembola consumption was affected by soil type and there was a nearly significant interaction between soil type and prey type (Figure 9a, Table 6). Spiders that were held on contaminated soil and fed contaminated Collembola ate significantly fewer prey on average than those held on laboratory soil and fed contaminated prey, consuming on average two fewer Collembola than the other treatment groups (Figure 9b, Table 6). There was no impact of our treatments on the average number of Collembola that males consumed during the experiment (Figure 9b, table 6). Females ate fewer prey when they were on contaminated soil and while prey consumption decreased over time, the change was not related to our treatments (Table 6, Figure 10). For males, there were no overall effects, but the treatments affected their consumption differently over time. Specifically, males held on contaminated soil reduced their consumption over time but the consumption of contaminated prey increased as the experiment progressed (Table 6, Figure 11).

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Discussion Heavy metal accumulation in P. milvina was primarily driven by soil contamination in this study. Prey contamination also played a significant role in accumulation, but only for certain metals. There were few sexual differences in metal accumulation, but when present they showed similar trends as dietary accumulation of metals. While spiders that died younger had significantly higher concentrations of all heavy metals, environmental exposure to heavy metals resulted in significantly increased mortality only in males. Females reduced foraging while on heavy metal contaminated soil, while males exhibited varied changes in foraging over time depending on treatment conditions. All metals examined in this experiment showed accumulation through soil contact in Pardosa milvina, likely by cuticular absorption, which agreed with my initial predictions. This is surprising given that the contaminated soil from Cleveland only showed elevated concentrations of lead and zinc. This suggests that other properties of the soils are responsible for the availability of metals for uptake rather than concentration (Ramirez et al. 2011). One difference was that the amount of organic matter was much greater in the laboratory soil (Table 2a). Soil organic matter can complex with metals and reduce their bioavailability, so this may explain why cadmium and copper were more readily absorbed from the Cleveland soil (Brady and Weil, 2008). It is important to note that, overall, this study likely underestimates the total environmental uptake of heavy metals by spiders from the atmosphere. Deposition of metals directly on spiders has been suggested as a factor in accumulation, but could not be simulated in this study (Clausen 1984). An additional route of heavy metal exposure is consumption from non-prey sources such as contaminated drinking water. Metals consumed in water would be potentially more bioavailable than metals consumed in prey, as they could be free ions or simpler compounds, rather than being complexed to organic matter. As a result, an increased soil organic matter content would decrease the concentration of heavy metals in the soil water solution. This route of exposure was not isolated in this study so its impacts here are unknown; A more rigorous follow up study should investigate that avenue further.

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In this study, dietary accumulation of metals occurred only for the micronutrients copper and zinc and not for cadmium or lead, which is the opposite of my initial predictions. I predicted females would accumulate more metals from consuming more prey. However, while females consumed more prey, males showed higher levels of dietary accumulation. This difference suggests that when heavy metals with no apparent nutritive purpose are consumed, they are efficiently excreted by the spider. It is interesting that the accumulation of essential metals (zinc, copper) does not seem to be internally regulated. While they are needed in small amounts, at high concentrations they can provoke lethal or sub-lethal effects (Zmudzki and Laskowski 2012). However, given that there were no dietary effects on mortality in this study, it is likely that these metals did not reach harmful concentrations. An intriguing possibility is that micronutrients are stored as a reserve. Pardosa milvina may alter its foraging based on nutrition content of prey (Schmidt et al. 2012), so it would be interesting to test whether micronutrients incluence prey selection. It is important to note that while females accumulated lower concentrations of heavy metals than males, but this did not seem to impact mortality. Sexually dimorphic physiology has been found in other species of spiders, where males and females process heavy metals with different chemical pathways (Wilczek et al. 2008). My findings suggest that P. milvina also has sexual dimorphism in physiological pathways for coping with heavy metal ingestion/absorption and that females are more efficient at excreting heavy metals. Perhaps the most surprising find in the heavy metal food chain was zinc accumulation. The concentration of zinc was much greater in the contaminated soil. Strangely, the Collembola placed on this soil did not accumulate significantly more zinc, but the spiders that ate heavy-metal contaminated food did tend to accumulate more zinc. One plausible explanation is that zinc acts as a catalyst for enzyme activity (Bertini and Luchinat 1994). As a result, spiders may have required additional zinc in order to activate enzymatic pathways to eliminate heavy metals or prevent and repair damage caused by heavy metals (Bertini and Luchinat 1994). Despite both sexes accumulating metals, increased mortality was only evident for males that were exposed to environmental metals. Dietary metals did not affect male mortality in this study, and female mortality was not significantly affected across treatment groups. This finding is similar to work on a related spider, Pardosa astrigera, which also 13 suffered no lethal effects from heavy metal exposure (Jung et al. 2005). However, in the study by Jung et al. (2005), the spiders were only exposed to metals for a portion of their lifespan. Pardosa astrigera juveniles exposed to heavy metals showed delayed development and reduced weight gain in the short term, so longer term lethal effects remain a possibility (Jung et al. 2005). Similarly, my spiders were only exposed to heavy metals as adults at the end of their life span. As a result, age-related (or stress-related) mortality may have masked lethality from heavy metals. Nevertheless, the relatively low lethality found here supports the idea that P. milvina could act as a vector for heavy metal movement up the food chain. These spiders could accumulate relatively high concentrations of metals before being consumed by predators. At least one example is already known in the eastern U.S., where songbirds along the Shenandoah River in Virginia were found to accumulate high levels of methylmercury and approximately 75% of the mercury they accumulated was traced back to spider consumption, despite spiders only contributing to approximately 20% of the birds’ diet (Cristol et al., 2008) Lifespan and metal concentration also showed a surprising relationship. Male spiders that survived longer had lower levels of cadmium and lead. This is perhaps unexpected given the limited lethal effects from heavy metal exposure. This suggests that there may be a great deal of individual variability in susceptibility to heavy metal accumulation and toxicity. Additionally, males that accumulated more cadmium and lead may have, as a result, been more susceptible to other causes of death. Not exclusive with that is the possibility that there is a delay between when heavy metal exposure first occurs and when physiological defense (such as producing costly enzymes) takes effect (Wilczek et al. 2004). In this case, spiders that died early may have died before excretion of absorbed metals could occur. Only females adjusted foraging habits based on metal contamination. In contrast to my predictions, they reduced prey consumption when environmental contamination was present, rather than when the prey itself was contaminated. As heavy metal contamination is presumably novel to these spiders, it possible that this is a response to a change in environmental conditions rather than a response specific to heavy metals. Males exhibited an interesting response over time. Males on contaminated soil decreased prey consumption over time, while males eating contaminated prey increased prey consumption. These 14 contrasting responses by males suggest that dietary and environmental exposure routes can elicit different behavioral responses and bears further investigation. The different behavioral responses between males and females may relate to ecological differences. Females may not be able to reduce feeding as they need energy to invest in eggs, while males can be more plastic in their feeding behavior without the burden of parental care (Walker and Rypstra 2002). In summary, P. milvina accumulates heavy metals via direct absorption through the cuticle. Consumption of heavy metals can result in accumulation of micronutrients, while non-nutritive metals are excreted. This accumulation has limited effects on mortality, allowing the spiders to pass metals up the food web. Males of this species accumulate higher levels of heavy metals than females, which is consistent with field studies on other epigeal spiders. Foraging responses to heavy metals were mixed in this study, as it remains unclear if or how P. milvina alters foraging behavior to account for the presence of potentially toxic heavy metals.

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Future Studies • How do other kinds of prey influence the accumulation of heavy metals in P. milvina? • Given a choice, would P. milvina selectively choose a substrate with lower concentrations of heavy metals? Is there a gradient of preference or a threshold? • Does P. milvina factor heavy metal contamination into prey selection? • Does method of uptake affect what chemical form a heavy metal is stored in? How does that impact bioavailability further up the food web? • What impacts, if any, does age of the spider have on heavy metal uptake? • Do females offload heavy metals into eggs or egg sacs? • What are the sublethal effects of heavy metal exposure? Do they vary based on route of uptake?

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Literature Cited Alloway, B. J. (2012). Environmental pollution #22: Heavy metals in soils: Trace metals and metalloids in soils and their bioavailability. Dordrecht: Springer. Ardestani, M. M., van Straalen, N. M., and van Gestel, C. A. M. (2014). Uptake and elimination kinetics of metals in soil invertebrates: A review. Environmental Pollution, 193, 277- 295. Biddinger, G.R., and Gloss, S.P. (1984). The importance of trophic transfer in the bioaccumulation of chemical contaminants in aquatic ecosystems. Residue Reviews, 91, 103-145. Brady, N. C. and Weil, R. R. (2008). The Nature and Properties of Soils: Fourteenth edition. New York, NY: Pearson Bertini, I., and Luchinat, C. (1994). The reaction pathways of zinc enzymes and related biological catalysys. Bioinorganic Chemistry. California: University Science Books Press. Cheruiyot, D. J., Boyd, R. S., Coudron, T. A., and Cobine, P. (2013). Biotransfer, bioaccumulation and effects of herbivore dietary Co, Cu, Ni and Zn on growth and development of the insect predator Podisus maculiventris (Say) (Hemiptera: Pentatomidae). Journal of Chemical Ecology, 39, 764–772. Clausen, I. H. S. (1984). Lead (Pb) in spiders: A possible measure of atmospheric Pb pollution. Environemtnal Pollution Series B, Chemical and Physical, 8 (3), 217-230. Clausen, I. H. S. (1986). The use of spiders (Araneae) as ecological indicators. Bulletin of the British Arachnological Society, 7 (3), 83–86. Clausen, I. H. S. (1989). On the dynamics of cadmium and lead in Steatoda bipunctata (Araneae). In: Bohac, J., Ruzicka (Eds.), Proceedings of the 5th International Conference of Bioindicatores Deteriorisationis Regionis, 315–318. Cristol, D. A., Brasso, R. L., Condon, A. M., Fovargue, R. E., Friedman, S. L., Hallinger, K. K., … and White, A. E. (2008). The movement of aquatic mercury through terrestrial food webs. Science, 320, 335.

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Eissa, B. L., Ossana, N. A., Ferrari, L., and Salibian, A. (2010). Quantitative behavioral parameters as toxicity biomarkers: Fish response to waterborne cadmium. Archives of Environmental Contamination and Toxicology, 58, 1032-1039. Evans, S. C., Griesinger, L. M., and Rypstra, A. L. (2010). Effects of a glyphosate-based herbicide on mate location in the wolf spider Pardosa milvina. 95th ESA Annual Meeting, Pittsburgh, August 1-6. Fountain, M. T. and Hopkins, S. P. (2001). Continuous monitoring of Folsomia candida (Insecta: Collembola) in a metal exposure test. Ecotoxicology and Environmental Safety, 48, 275-286. Gall, J. E., Boyd, R. S., and Rajakaruna, N. (2015). Transfer of heavy metals through terrestrial food webs: a review. Environmental Monitoring and Assessment, 187-201. Gist, C. S., Crossley, Jr. D. A., and Merchant, V. A. (1974). An analysis of life tables for Sinella curviseta (Collembola). Environmental Entomology, 3 (5), 840-844. Green, I. D., Diaz, A., & Tibbett, M. (2010). Factors affecting the concentration in seven- spotted ladybirds (Coccinella septempunctata L.) of Cd and Zn transferred through the food chain. Environmental Pollution, 158, 135–141. Heath, J. A. and Frederick, P. C. (2005). Relationships among mercury concentrations, hormones, and nesting efforts of white ibises (Eudocimus albus) in the Florida Everglades. The Auk, 122 (1), 255-267. Heikens, A., Peijnenburg, W. J. G. M., and Hendriks, A. J. (2001). Bioaccumulation of heavy metals in terrestrial invertebrates. Environmental Pollution, 113, 385-393. Jakob, E. M., Marshall, S. D., and Uetz, G. W. (1996). Estimating fitness: A comparison of body indices. Oikos, 77 (1), 61-67. Jung, C. S., Lee, S. B., Jung, M. P., Lee, J.H., Lee, S., and Lee, S. H. (2005). Accumulated heavy metal content in wolf spider, Pardosa astrigera (Araneae: Lycosidae) as a bioindicator of exposure. Journal of Asia-Pacific Entomology, 8(2), 185-192. Jung, M. P. and Lee, J. H. (2012). Bioaccumulation of heavy metals in the wolf spider, Pardosa astrigera L. Koch (Araneae: Lycosidae). Environmental Monitoring and Assesment, 184 (3), 1773-1779. Kaston, B. J. (1972). How to Know the Spiders. McGraw-Hill: New York City.

18

Larsen, K.J., Brewer, S. R., and Taylor, D. H. (1994). Differential accumulation of heavy- metlas by web spiders and ground spiders in an old field. Environmental Toxicology and Chemistry, 13(3), 503-508. Maelfait, J. P. and Hendrickx, F. (1998). Spiders as bio-indicators of anthropogenic stress in natural and semi-natural habitats in Flanders (Belgium): some recent developments. Proceedings of the 17th European Colloquium of Arachnology, 1997. Marc, P., Canard, A., and Ysnel, F. (1999). Spiders (Araneae) useful for pest limitation and bioindication. Agriculture Ecosystems & Environment, 74, 229-273. Morgan, A. J., Morris, B., James, N., Morgan, J. E., and Leyshon, K. (1986). Heavy metals in terrestrial macroinvertebrates: Species differences within and between trophic levels. Chemistry and Ecology, 2(4), 319-334. Nagajyoti, P. C., Lee, K. D., and Sreekanth, T. V. M. (2010). Heavy metals, occurrence, and plants: a review. Environmental Chemistry Letters, 8(3), 199-216. Persons, M. H., and Rypstra, A. L. (2001). Wolf spiders show graded antipredator behavior in the presence of chemical cues from different predators. Journal of Chemical Ecology, 27(12), 2493-2504. Peterson, L. R., Trivett, V., Baker, A. J.M., Aguiar, C., & Pollard, A. J. (2003). Spread of metals through an invertebrate food chain as influenced by a plant that hyperaccumulates nickel. Chemoecology, 13, 103–108. R Core Team (2016). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R- project.org/. Rabitsch, W. B. (1995). Metal accumulation in arthropods near a lead/zinc smelter in Arnoldstein, Austria. III. Arachnida. Environmental Pollution, 90 (2), 249–257. Ramirez, M. G., McCallum, J.E.B., Landry, J. M., Vallin, V. A., Fukui, S. A., … and Sy, C. L. (2011). Relationships between physiological characteristics and trace metal body burdens of banded garden spiders Argiope trifasciata (Araneae, Araneidae). Ecotoxicology and Environmental Safety, 74(4), 1081-1088. Schmidt, J. M., Sebastian, P., Wilder, S. M., and Rypstra, A. L. (2012). The nutritional content of prey affects the foraging of a generalist arthropod predator. PLoS ONE, 7 (11).

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Sharma, K., Basta, N. T., and Grewal, P.S. (2015). Soil heavy metal contamination in residential neighborhoods in post-industrial cities and its potential human exposure risk. Urban Ecosystems, 18 (1), 115-132. Tchounwou, P. B., Yedjou, C.G., Patlolla, A. K., and Sutton, D. J. (2012). Heavy metals toxicity and the environment. Molecular, Clinical and Environmental Toxicology: Experientia Supplementum, 101, 133-164. Thomas, R. (2001). A beginner’s guide to ICP-MS. Spectroscopy, 17 (2), 42-48. UNEP (2013). Global Mercury Assessment 2013: Sources, Emissions, Releases and Environmental Transport. UNEP Chemicals Branch, Geneva, Switzerland Van den Brink, N., Lammertsma, D., Dimmers, W., Boerwinkel, M., and van der Hout, A. (2010). Effects of soil properties on food web accumulation of heavy metals to the wood mouse (Apodemus sylvaticus). Environmental Pollution, 158, 245-251. Van Straalen, N. M., Butovsky, R. O., Pokarzhevskii, A. D., Zaitsev, A. S., and Verhoef, S. C. (2001). Metal concentration and invertebrates in the vicinity of a metallurgical factory near Tula (Russia). Pedobiologia, 45, (451-466). Walker, S. E. and Rypstra, A. L. (2002). Sexual dimorphism in trophic morphology and feeding behavior of wolf spiders (Aranea: Lycosidae) as a result of differences in reproductive roles. Canadian Journal of Zoology, 80, 679-688. Wilczek, G., & Babczynska, A. (2000). Heavy metals in the gonads and hepatopancreas of spiders (Araneae) from variously polluted areas. Ekologia, 3, 283–292. Wilczek, G., Babczyńska, A, Augustyniak, M., and Migula, P. (2004). Relations between metals (Zn, Pb, Cd and Cu) and glutathione-dependent detoxifying enzymes in spiders from a heavy metal pollution gradient. Environmental Pollution, 132 (3), 453-461. Wilczek, G., Babczynska, A., Wilczek, P., Dolezycha, B., Migula, P., and Mlynska, H. (2007). Cellular stress reactions assessed by sex and species in spiders from areas variously polluted with heavy metals. Ecotoxicology and Environmental Safety, 71(1), 127-137. Zmudzki, S. and Laskowski, R. (2012). Biodiversity and structure of spider communities along a metal pollution gradient. Ecotoxicology, 21(5), 1523-1532.

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Table 1. Mean heavy metal concentration (ppm) (± SD) in the uncontaminated laboratory soil and contaminated Cleveland soil. The concentration of each metal was compared between contaminated and uncontaminated soils in separate ANOVAs. (n = 30 for each type of soil) Metal Uncontaminated Contaminated Soil df Test p Soil statistic (F) Cadmium 0.579 ± 0.061 0.660 ± 0.041 1 1.22 0.273 Copper 35.3 ± 3.13 32.1 ± 1.77 1 0.773 0.383 Lead 12.4 ± 0.954 240 ± 11.9 1 363 <0.0001 Zinc 125 ± 10.6 265 ± 14.8 1 59.2 <0.0001

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Table 2. Mean concentration (ppm) (± SD) of selected heavy metals in the Sinella curviseta used as food for Pardosa milvina. Uncontaminated Collembola were reared on standard laboratory soil, while the contaminated Collembola were raised on the contaminated soil. Metal accumulations were compared between the two groups using ANOVA. (n = 15 Collembola cultures for each treatment. Each culture contained ~150 Collembola) Metal Uncontaminated Contaminated df Test statistic p Collembola Collembola (F) Cadmium 0.298 ± 0.014 0.509 ± 0.023 1 63.7 <0.0001 Copper 60.7 ± 3.86 91.9 ± 3.59 1 34.9 <0.0001 Lead 0.177 ± 0.045 17.3 ± 5.18 1 10.9 0.00263 Zinc 116 ± 2.96 121 ± 3.58 1 1.14 0.295

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Table 3. Mean concentration (ppm) (± SD) of heavy metals in Pardosa milvina separated by sex. Concentrations were compared between the sexes using one-way ANOVA. In all cases where there is a significant difference, the concentration was higher in males (see figures 1a and 1b). (n = 84 for each sex) Metal Females Males df F p Cadmium 5.64 ± 0.47 6.40 ± 1 1.04 0.309 0.572 Copper 182 ± 10.2 277 ± 19.1 1 19.29 <0.0001 Lead 35.1 ± 7.46 32.5 ± 4.72 1 0.087 0.768 Zinc 575 ± 32.5 7340 ± 35.4 1 11.69 <0.0001

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Table 4. Three-factor ANOVA table for how soil type, prey type, and lifespan affected accumulation of heavy metals in female Pardosa milvina (n=21 per treatment). Model and factor tested df F p Cadmium Accumulation Soil 1 46.36 <0.0001 Prey 1 3.108 0.082 Lifespan 1 2.659 0.107 Soil * Prey 1 1.072 0.304 Soil * Lifespan 1 7.012 0.0099 Prey * Lifespan 1 2.169 0.146 Soil * Lifespan * Prey 1 8.750 0.00415

Copper Accumulation Soil 1 64.22 <0.0001 Prey 1 14.09 <0.0001 Lifespan 1 1.846 0.178 Soil * Prey 1 9.183 0.003 Soil * Lifespan 1 13.53 <0.0001 Prey * Lifespan 1 1.658 0.202 Soil * Lifespan * Prey 1 14.40 <0.001

Lead Accumulation Soil 1 26.84 <0.0001 Prey 1 0.971 0.328 Lifespan 1 0.214 0.645 Soil * Prey 1 1.156 0.286 Soil * Lifespan 1 0.133 0.716 Prey * Lifespan 1 4.606 0.0351 Soil * Lifespan * Prey 1 2.941 0.0901

Zinc Accumulation Soil 1 63.535 <0.0001 Prey 1 9.725 0.0026 Lifespan 1 2.362 0.129 Soil * Prey 1 3.114 0.0817 Soil * Lifespan 1 13.38 0.0047 Prey * Lifespan 1 2.016 0.160 Soil * Lifespan * Prey 1 11.929 0.0009

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Table 5. Three-factor ANOVA table for how soil type, prey type, and lifespan affected accumulation of heavy metals in male Pardosa milvina (n=21 per treatment). Model and factor tested df F p Cadmium Accumulation Soil 1 15.17 0.0002 Prey 1 2.506 0.118 Lifespan 1 5.484 0.0219 Soil * Prey 1 1.616 0.208 Soil * Lifespan 1 4.521 0.0369 Prey * Lifespan 1 1.799 0.1838 Soil * Lifespan * Prey 1 0.173 0.679

Copper Accumulation Soil 1 1.939 0.169 Prey 1 27.24 <0.0001 Lifespan 1 7.435 0.0080 Soil * Prey 1 12.64 0.0007 Soil * Lifespan 1 11.137 0.0013 Prey * Lifespan 1 6.319 0.014 Soil * Lifespan * Prey 1 13.26 0.0005

Lead Accumulation Soil 1 134.29 <0.0001 Prey 1 0.727 0.397 Lifespan 1 23.801 <0.001 Soil * Prey 1 1.710 0.195 Soil * Lifespan 1 25.655 <0.001 Prey * Lifespan 1 0.642 0.426 Soil * Lifespan * Prey 1 1.265 0.264

Zinc Accumulation Soil 1 11.633 0.0010 Prey 1 6.574 0.0124 Lifespan 1 0.515 0.4751 Soil * Prey 1 10.881 0.00149 Soil * Lifespan 1 2.651 0.10765 Prey * Lifespan 1 0.020 0.887 Soil * Lifespan * Prey 1 6.995 0.0010

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Table 6. The effects of soil and prey treatments on survival and foraging in Pardosa milvina with the sexes analyzed separately. Survival was analyzed using the proportional hazards model, average consumption was analyzed with ANOVA and the actual prey consumption was analyzed using MANOVA with repeated measures (n=21 per sex per treatment).

Model and factor tested df Test statistic p Female survival Soil 1 2=2.54 0.1106 Prey 1 2=0.01 0.9303 Soil * prey 1 2=0.77 0.3817

Male survival Soil 1 2=4.43 0.0353 Prey 1 2=1.80 0.1797 Soil * prey 1 2=0.18 0.6740

Average Weekly consumption of females Full model 3 F=3.55 0.0181 Soil 1 T=2.32 0.0230 Prey 1 T=1.17 0.2452 Soil * prey 1 T=1.95 0.0551

Average Weekly consumption of males Full Model 3 F=0.80 0.4958 Soil 1 T=1.19 0.2391 Prey 1 T=1.00 0.3203 Soil * prey 1 T=0.04 0.9651

Female foraging from weeks 1-4 (repeated measures) Between subjects Soil 1 F=4.50 0.0381 Prey 1 F=1.38 0.2453 Soil * prey 1 F=0.27 0.6049

Across time Time 3 F=3.52 0.0208 Soil 3 F=0.66 0.5807 Prey 3 F=0.73 0.5404 Soil * prey 3 F=0.73 0.5394

Male foraging from weeks 1-4 (repeated measures) Between subjects

Soil 3 F=1.78 0.1915 Prey 3 F=0.01 0.9243 Soil * prey 3 F=0.80 0.3773

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Across time Time 3 F=0.52 0.6724 Soil 3 F=0.52 0.1886 Prey 3 F=3.27 0.0339 Soil * prey 3 F=4.41 0.0106

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Table 7. Tests for overall differences in survival and foraging of male and female Pardosa milvina over six weeks (all treatments combined) (n=84 per sex).

Model df Test p statistic Survival (right censored) 1 2= 24.00 <0.0001 Average prey consumption/week – ANOVA 1 F=61.83 <0.0001

Prey consumption over four weeks - MANOVA Sex differences 1 F=42.62 <0.0001 Time effects 3 F=1.62 0.1847 Sex * Time 3 F=1.42 0.2364

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a) 1200.00

1000.00

800.00

600.00

400.00 Concentration(ppm)

200.00

0.00 Copper Zinc Metal

Female Male b) 45.00 40.00 35.00 30.00 25.00 20.00 15.00

Concentration(ppm) 10.00 5.00 0.00 Cadmium Lead Metal

Female Male

Figure 1. A) Mean (± SEM) copper and zinc concentrations (ppm) in male and female Pardosa mivina. B) Mean (± SEM) cadmium and lead concentrations (ppm) in male and female spiders. Summary statistics are in table 3 (n=84 per sex).

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*^ * 10 350 9 )

8 300 ppm

7 ( 250 6 200 5 4 150 3 copper 100 2 1 Female 50 Female 0 0

Male Male

Concentration Concentrationcadmium (ppm)

Soil type Soil type a) b) 90 *^ 80 *^ 70 60 50 40 30 20 10 Female 0

Male Concentrationlead (ppm)

Soil type c) d) 1000 800 600 400 200 0 Female

Male Concentrationzinc (ppm)

Soil type

Figure 2. Comparisons of mean (± SEM) (a) cadmium, (b) copper, (c) lead, and (d) zinc measured in Pardosa milvina on different soil types. Asterisks indicate significant differences between females within a treatment, while carats indicate significant differences between males within a treatment. Summary statistics can be found in tables 4 and 5 (n=42 per sex per treatment).

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10 *^ 8 500 6 400 4 300 2 200 100 0 Female (ppm) 0 Female Male (ppm)

Male

Concentration cadmium Concentrationcadmium Concentrationcopper

Food type Food type a) b) 60 50 *^ 40 1000 30 800 600 20 400 10 Female 200 0 0 Male Female

Concentrationlead (ppm) Male Concentrationzinc (ppm)

Food type Food type c) d) Figure 3. Comparisons of mean (± SEM) (a) cadmium, (b) copper, (c) lead, and (d) zinc measured in Pardosa milvina receiving either contaminated or uncontaminated Sinella curviseta. Asterisks indicate significant differences between females within that treatment, while carats indicate significant differences between males within that treatment. Summary statistics can be found in tables 4 and 5 (n=42 per sex per treatment).

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10 400 8 300 6 4 200 Female

(ppm) Female (ppm) 2 Male 100 Male 0 Concentrationcopper 0 Concentrationcadmium Short Long lived lived Short lived Long lived Lifespan Lifespan a) b) 70 ^ 1000 60 50 800 40 600 30 20 Female 400 Female 10 Male 200 Male 0

Short Long 0 Concentrationzinc (ppm) Concentrationlead (ppm) lived lived Short lived Long lived Lifespan Lifespan c) d) Figure 4. Comparisons of mean (± SEM) (a) cadmium, (b) copper, (c) lead, and (d) zinc measured in Pardosa milvina that died within the first three weeks (short lived) compared to those that did not. Asterisks indicate significant differences between females within that treatment, while carats indicate significant differences between males within that treatment. Summary statistics can be found in tables 4 and 5 (n=84 per sex).

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Figure 5. Kaplan-Meier survivorship curve for Pardosa milvina of each sex. Duration of the experiment is presented along the x-axis while the y-axis shows the proportion of the group still alive. Overall, females lived significantly longer than males (n=168, df = 1, χ2 = 26.5, p < 0.001).

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Figure 6. Survivorship curves for female Pardosa milvina of each treatment group. Duration of the experiment is presented along the x-axis while the y-axis shows the proportion of the group still alive. Neither soil nor food treatment significantly impacted survivorship (n=84).

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Figure 7. Survivorship curves for male Pardosa milvina of each treatment group. Duration of the experiment is presented along the x-axis while the y-axis shows the proportion of the group still alive. Soil contamination negatively affected male survivorship; there was no effect of food contamination on male survivorship (n=84).

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12

10

8

6 week

4

2

Average Average Collembolans consumerd per 0 Females Sex Males Figure 8. Mean (± SEM) weekly Sinella curviseta consumption for Pardosa milvina of each sex. One-way ANOVA showed that females ate significantly more than males each week (n=168, df = 1, F =61.83, p < 0.001).

36 a)

b) Figure 9. Mean (± SEM) weekly Sinella curviseta consumption broken up by treatment group for a) female and b) male Pardosa milvina. The treatments are: UU, uncontaminated soil and Collembola; UC, uncontaminated soil and Collembola contaminated with heavy metals; CU, heavy metal contaminated soil and uncontaminated Collembola; and CC, contaminated soil and contaminated Collembola. The treatment had a marginally non- significant effect on Collembola consumption in females, and no effect in males (n=168, see table 6).

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18 16 14 12 10 8

6 Prey Prey Consumed 4 2 0 Uncontaminated Soil Contaminated Soil

Uncontaminated Prey Contaminated Prey a) 18 16 14 12 10 8

6 Prey Prey Consumed 4 2 0 Uncontaminated Soil Contaminated Soil

Uncontaminated Prey Contaminated Prey b) Figure 10. Mean (± SEM) Sinella curviseta consumption for female Pardosa milvina at week 2 (a) and week 4 (b) (n=84). Summary statistics are in table six.

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10 9 8 7 6 5 4

Prey Consumed Prey 3 2 1 0 Uncontaminated Soil Contaminated Soil Uncontaminated Prey Contaminated Prey a)

10 9 8 7 6 5 4

Prey Prey Consumed 3 2 1 0 Uncontaminated Soil Contaminated Soil Uncontaminated Prey Contaminated Prey b) Figure 11. Mean (± SEM) Collembola consumption for male Pardosa milvina at week 2 (a) and week 4 (b) (n=84). Summary statistics are in table six.

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Appendix Table A1. Mean (± SD) starting conditions of Pardosa milvina within each treatment group. Body condition was calculated as by regressing ln(body mass) on ln(carapace width) and comparing each population to the whole with a t-test. In all cases, p > 0.95 (n=168). Treatment Group Sex Carapace Width Mass Body (mm) (mg) Condition Index Negative Control Males 1.88 ± 0.22 12.13 ± 3.09 4.7e-18 ± 0.11 Females 2.06 ± 0.18 19.12 ± 3.18 -5.8e-18 ± 0.15 Contaminated Soil Males 1.88 ± 0.17 12.59 ± 3.10 -5.2e-18 ± 0.09 Females 2.20 ± 0.14 21.57 ± 4.15 5.7e-18 ± 0.16 Contaminated Food Males 1.84 ± 0.13 11.89 ± 2.48 6.9e-18 ± 0.09 Females 2.12 ± 0.15 19.36 ± 4.40 6.12e-19 ± 0.11 Contaminated Soil Males 1.90 ± 0.16 11.92 ± 2.36 1.31e-17 ± 0.11 and Food Females 2.11 ± 0.21 17.96 ± 4.23 1.27e-17 ± 0.18

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Table A2. Comparison of Mean (± SD) soil organic matter (SOM) between laboratory and Cleveland soils. SOM was measured by ashing soil samples in a muffle furnace overnight and recording the mass lost (n = 30 for each soil type). Data was compared with a two- sample t-test. The laboratory soil was found to have significantly greater organic matter (t = -14.903, df = 59, p < 0.001). Soil Type Loss on Ignition (%) Laboratory (uncontaminated) 57.87 ± 18.21 Cleveland (contaminated) 6.56 ± 3.58

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Figure A1. Diagram representation of the experimental set up and predicted outcomes for each treatment group.

Adult Pardosa milvina

Uncontaminated Contaminated soil soil

Uncontaminate Contaminated Uncontaminate Contaminated d food food d food food

Predictions

Baseline No Zn, Cu Zn, Cu, Cd, Pb Zn, Cu, Cd, Pb group accumulation accumulation accumulation No Cd, Pb from soil from soil accumulation accumulation No change in Cd, Pb of metals from food foraging accumulation Normal Decreased Increased from food foraging foraging mortality Highest Lowest Increased overall mortality mortality accumulation Decreased foraging Increased mortality

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