The Effect of Arsenic Trioxide on the Grey Flesh Fly Sarcophaga bullata (Diptera: Sarcophagidae)

by

Nina Dacko B.S.

A Thesis

In

ENVIRONMENTAL TOXICOLOGY

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

Approved

Steven M. Presley PhD Committee Chair

Stephen B. Cox PhD

George P. Cobb PhD

Peggy Miller Dean of the Graduate School

May, 2011

Copyright 2011, Nina Dacko Texas Tech University, Nina Dacko, May 2011

ACKNOWLEDGMENTS

First and foremost, I would like to thank my advisor, Dr. Steven Presley, for he has been utterly helpful in thesis guidance and funding as well as with side work and professional connections. May I one day, follow in your footsteps as a medical entomologist. I look up to you as a scientist, a friend, and as a father figure.

I would like to thank committee member, Dr. George Cobb who gave me more than adequate advice about chemical analysis as well as suggestions for statistical analysis and suggested contacts for advice and knowledge pertaining to my research. I must admit being intimidated by your intelligence, but you were always easy to understand. Also thank you for your numerous suggestions in thesis writing.

I would like to thank committee member Dr. Stephen Cox who suggested statistical analyses, explained why these analyses correspond to presented research questions and most of all helped me to recognize why these analyses were superior to others in similar research. I also give credit to Stephen for helping me in logical thesis and defense organization skills.

I would like to thank the entire vector-borne zoonoses lab (VZL): specifically Anna Hoffarth, Juliet Kinyua, James Walls and Anna Gibson for their help, watching my fly colonies while I was away and Anna Gibson for teaching me numerous tasks in the laboratory- dissection of rabbits and oral dosing- for my thesis along with PCR, RNA isolation, colorimetric protein assays and other things for work experience. Others at TIEHH include Richie Ericson- for statistics suggestions, and Dawn Slekis- again for fly care.

Thanks to Ben Wozniak and Applied Speciation in Spokane, WA for performing arsenic speciation analyses as well as giving me suggestions for my own chemical

ii Texas Tech University, Nina Dacko, May 2011 analyses. You’ve been most helpful and a lot of results were dependent on the information you have provided me at no charge!

Dr. Galen Austin- thank you for insectary maintenance, such as fixing doors and setting up temperature monitors and for lending me your digital caliper. I would also like to thank Mitchell Burt for helping me build fly cages and constantly helping with heavy carboys.

Thanks to Amber Matthews and Mark Goza for rabbit housing and maintenance and euthanasia facilities.

Thanks to Dr. Marjolaine Giroux for identification of Sarcophaga bullata, to Dr. Lois Held for supplying fly standards in chemical analysis and James Cockendolpher for fly identification knowledge.

Thanks to professors and colleagues who frequently answered questions when asked including- Dr. Greg Mayer, Dr. Todd Anderson and Dr. Emily Notch. Thanks to Dr. Jaclyn Cañas for use of the metal analysis laboratory and Brad Thornhill and Marshall Pattee for help with the ICP-MS. Thanks to Shibin Li and Shuanying Yu for help and advice with digestions.

Thanks to my professors in the plant and soil science department and at the forensic science institute.

Thanks to Michael Lee Goff for experimental suggestions and inspiration.

Thanks to Dr. RJ Kendall and TIEHH for facilities. Thanks Faculty and Staff- Matthew Young for being curious!

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Thanks friends and family and Rob Bendkowski for driving me to the insectary in snow storms and sitting in the stinky insectary while I worked.

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TABLE OF CONTENTS ACKNOWLEDGMENTS ...... ii

ABSTRACT ...... vii

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

I. INTRODUCION ...... 1 Arsenic History in Industry, Crime, Warfare, Environment and Medicine ...... 1 Postmortem Interval, Forensic Entomology and Entomotoxicology ...... 3 Life History of Sarcophaga bullata ...... 5 Past Research in the Field of Entomotoxicology ...... 7 Case Studies ...... 7 Laboratory Studies ...... 10 Gaps in Research ...... 16 Objectives, Aims and Hypotheses ...... 17 Literature Cited ...... 18

II. MATERIALS AND METHODS ...... 23 Rabbits ...... 23 Chronic Exposure ...... 23 Acute Exposure ...... 24 Flies ...... 24 Collection ...... 24 Colony Establishment ...... 25 Larval exposure and measurements ...... 25 Pupae and adult maintenance, reproductive output and generation two ...... 25 Chemical Analyses ...... 26 Arsenic speciation and analyses ...... 26 Digestion ...... 26 Total arsenic analyses ...... 27 Statistycal analyses ...... 27 Fly growth rate and development trends ...... 27 Fly life paramters and arsenic concentrations in tissues...... 28 Literature Cited ...... 28

III. RESULTS AND DISCUSSION ...... 29 Observations ...... 29 Fly growth rate and development trends ...... 31 Emergence and metamorphic trends ...... 43 Mortality ...... 45 Arsenic in tissues ...... 47

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Reproductive Output ...... 53 Literature Cited ...... 54

III. CONCLUSIONS ...... 55 Literature Cited ...... 56

A. ARSENIC MSDS AND DIGESTION METHOD...... 57 B. RAW DATA ...... 74

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ABSTRACT Larvae of Sarcophaga bullata (Diptera: Sarcophagidae), a necrophagous insect commonly utilized in the field of entomotoxicology, were reared on rabbit tissues of rabbits that were previously exposed to arsenic trioxide (As2O3) by different methods. We observed the effects of arsenic (As) and As metabolites in acute versus chronic exposure in rabbit tissues on growth rate (mean larval length), metamorphosis, mortality and reproductive output of S. bullata. The New Zealand white rabbit was utilized as a vehicle to create natural concentrations of As and As metabolites in liver tissue and to serve as food media for S. bullata. Acutely exposed rabbits (2 per group) received three different doses of As2O3 corresponding to the dosages of half the median lethal dose or 0.5 MLD (10.1 mg/kg body weight (BW)), median lethal dose or MLD (20.2 mg/kg BW) and twice the median lethal dose or 2 MLD (40.4 mg/kg BW) and one control rabbit received dosing vehicle only. Chronically exposed rabbits received a dosage of 1.5 mg/kg BW daily for 35 days and one rabbit received dosing vehicle only. Rabbits were subsequently euthanized either eight hours post-exposure (acute) or on day 36 (chronic). The liver of these rabbits were removed and half was used as food media for 100 S. bullata larvae per experimental rabbit liver. The remaining half of the rabbit liver was used as a food media for 100 offspring of the prior experimental fly generation. Ten of these larvae were sampled and measured every eight hours until the onset of larval migration, for both generations of flies. Larval and pupal mortality was recorded, as well as the number of offspring, per dose group. ANOVA revealed a significant increase in mean larval body length in MLD as compared to the chronic dose group. Metamorphic time of S. bullata was positively correlated to the concentration of total As in exposed rabbit liver and mortality was negatively correlated to the concentration of total As in exposed rabbit liver. The reproductive output of S. bullata was not correlated to the concentration of total As in rabbit liver tissue.

Key Words: Diptera; necrophagous; entomotoxicology; arsenic; acute; chronic

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LIST OF TABLES 3.1 Regression Analysis Statistics for Metamorphosis...... 45 3.2 Pearson’s Chi Square for Mortality ...... 46 3.3 Regression Analysis Statistics for Mortality ...... 47 3.4 Arsenic in Rabbit Liver ...... 49 3.5 Arsenic in Insect Tissues ...... 49 3.6 Regression Analysis Statistics for As in Tissues ...... 52 3.7 Regression Analysis Statistics for Reproductive Output ...... 53 B.1 Rabbit Liver Weights ...... 72 B.2 Experimental Rabbit Doses ...... 72 B.3 Larval Length (Generation 1) ...... 73 B.4 Larval Length (Generation 2) ...... 79 B.5 Time in Pupae (Generation 1) ...... 84 B.6 Time in Pupae (Generation 2) ...... 84 B.7 Mortality (Generation 1) ...... 85 B.8 Mortality (Generation 2) ...... 85 B.9 Reproductive Output (Generation 1) ...... 86 B.10 Reproductive Output (Generation 2) ...... 86

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LIST OF FIGURES 1.1 Sketch of Sarcophaga bullata life stages ...... 6 3.1 Puparation differences observed between groups ...... 30 3.2 Mean Length over time generation 1 ...... 31 3.3 Mean larval length at 8 hours for generation 1 ...... 32 3.4 Mean larval length at 16 hours for generation 1 ...... 32 3.5 Mean larval length at 24 hours for generation 1 ...... 33 3.6 Mean larval length at 32 hours for generation 1 ...... 33 3.7 Mean larval length at 40 hours for generation 1 ...... 34 3.8 Mean larval length at 48 hours for generation 1 ...... 34 3.9 Mean larval length at 56 hours for generation 1 ...... 35 3.10 Mean larval length at 64 hours for generation 1 ...... 35 3.11 Mean larval length at 72 hours for generation 1 ...... 36 3.12 Mean larval length at 80 hours for generation 1 ...... 36 3.13 Mean larval length over time generation 2 ...... 37 3.14 Mean larval length at 8 hours for generation 2 ...... 37 3.15 Mean larval length at 16 hours for generation 2 ...... 38 3.16 Mean larval length at 24 hours for generation 2 ...... 38 3.17 Mean larval length at 32 hours for generation 2 ...... 39 3.18 Mean larval length at 40 hours for generation 2 ...... 39 3.19 Mean larval length at 48 hours for generation 2 ...... 40 3.20 Mean larval length at 56 hours for generation 2 ...... 40 3.21 Mean larval length at 64 hours for generation 2 ...... 41 3.22 Mean larval length at 72 hours for generation 2 ...... 41 3.23 Mean larval length at 80 hours for generation 2 ...... 42 3.24 Emergence trends generation 1 ...... 43 3.25 Emergence trends generation 2 ...... 44 3.26 Regression analysis for metamorphic time versus As concentrations in experimental rabbit livers ...... 44 3.27 Regression analysis for % mortality versus As concentrations in experimental rabbit livers ...... 46

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3.28 Regression analysis for % mortality versus As concentrations in experimental rabbit livers minus 2 x MLD ...... 47 3.29 Regression analysis for As in liver and insect tissues ...... 51 3.30 Regression analysis for reproductive output versus As concentrations in experimental rabbit livers ...... 53

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CHAPTER I

INTRODUCTION

Arsenic History in Industry, Crime, Warfare, Environment, and Medicine

Arsenic (As) is a metalloid present in the Earth’s crust and is toxic to many forms of life including plants, animals and fungi. Arsenic’s toxic properties were known as early as 2000 B.C. (Zaman and Pardini, 1995). In ancient Greece, arsenikon was synonymous with masculine (Vahidnia et al., 2007). The discovery of As is mainly accredited to Albertus Magnus, a German alchemist in the early 13th century. As an element, As appears on the periodic chart as number 33 with a molecular weight of 74.9216 atomic mass units (amu). The most common mineral forms of As exist as orpiment (As2S3) and realgar (As2S2), which were used as pigments for their brilliant gold and red colors, respectively. Additionally, copper acetoarsnite produced the pigment known as Paris Green also called Emerald Green or Scheele’s Green. These pigments were commonly used by painters for portraits, used to dye cloths and wallpapers, and unfortunately, food. Paris Green was also thought to be a toxic component of absinthe (Doyle, 2009). Arsenic is referred to as the “king of poisons” or the “poison of kings” and is possibly the most influential toxic compound used in history (Nriagu, 2002). During the early Bronze Age, As, as well as other metals and minerals, was used as an alloy to strengthen copper, and there is evidence that was linked to the first signs of occupational disease (Harper, 1988). It was thought that the Greek god of fire and technology, Hephaestus, had deformities linked to exposure to the fumes of As or lead (Nriagu, 1983). The application of As to produce silvery surfaces of mirrors and aid in embalming has also been noted in ancient China and Egypt (Nriagu, 2002). Presently, workers in the copper smelting industry may still be exposed to arsenic trioxide

(As2O3) during the copper smelting process (Beck et al., 2002).

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Many forms of As have been used for medicinal purposes. Ancient cultures, from Greek and Assyrian to Indian and Chinese have used As as a cure all. In the ancient Indian text, the Rasa, it “cures phlegm, poison, excess air and fear from ghosts, stops menstrual discharge, is soothing, increases appetite and cures leprosy”. Similarly, in Chinese texts it is noted to be “antifebrile, prophylactic, emetic, expectorant, deobstruent, arthritic, antihelmintic, antidotal and escharotics” in works by Pen Ts’au

(Nriagu, 2002). As2O3 is used in medical therapy today as a cancer therapeutic for patients with acute promyelocytic leukemia (Miller et al., 2002). Arsenic salts are readily available to the general public in insecticides, such as ant baits, which may be used as a homicidal or suicidal poison. It may also be accidentally ingested by children (Park and Currier, 1991). One of the most popular reports involving As poisoning is that of the death of Napoleon Bonaparte. Theories on his death range from being poisoned by the British army, to eating marzipan that was dyed with copper acetoarsenite, to the chronic poisoning from the Paris Green wallpaper in his bedroom around the time of his death (Doyle, 2009). In the Middle Ages, different forms of As were commonly used by women to kill unwanted husbands or lovers, as popularized in John Kesselring’s play, Arsenic and Old Lace. The ease of dissolving As in drink and blending into food of unknowing victims earned As2O3 the synonym “inheritance powder”. In the 1600s, Toffana of Palermo and Naples sold murderous oil, Aqua della Toffana, which effects were similar to inheritance powder (Nriagu, 2002). It became so popular as a homicidal poison that the United Kingdom outlawed the sale of As, except to pharmacist or doctor’s prescription through the Arsenic Act in 1851 (Doyle, 2009). As well as being a homicidal poison, As in gaseous form was used as one of the first chemical warfare agents. In ancient China, As sulfides were used to make “holy smokes” (bombs) and As containing oils and waxes in “death lamps” (Nriagu, 2002). In the 1920’s, chlorovinyl dichloroarsine gas also known as lewisite, was developed for use in warfare. (ATSDR, 2009). In the late 1940s, the Jewish organization, the Nakam, painted 2,000 loaves of bread with As which were delivered to camps housing

2 Texas Tech University, Nina Dacko, May, 2011 members of the Gestapo (secret police) and the SS or Shutzstaffel (military force) of Nazi Germany during the end of World War II. Although no one died, over two thousand people fell ill. (Walters, 2009) There are two major routes in which humans can be exposed to As, including ingestion and inhalation (ATSDR, 2000). Ingestion of As may occur unintentionally (through contaminated drinking water or contaminated food, or unmarked pesticide bottles), or intentionally (homicide, or in cases of attempted suicide). Inhalation occurs most commonly in industry from the smelting of metal. The adsorption capacity for inhaled As is not known exactly, but is estimated to range from 60 to 90% (ATSDR, 2000). Approximately 80 % of ingested As is absorbed from both human and other vertebrate gastrointestinal tracts. After absorption, As is then distributed primarily to the liver, kidney, lung, spleen, aorta and skin. Arsenic exits the body through excretion in urine at rates as high as 80% in 61 hours following oral exposure (ATSDR, 2000). In humans, inorganic As is methylated through the s-adenosylmethionine (SAM) process of phase one metabolism to monomethylarsonic acid (MMA), dimethylarsonic acid (DMA) and less commonly, trimethylatedarsine oxide (TMAO) (Lin et al., 2005). Methylation of As was thought to be a detoxification mechanism; however, this issue is under debate (Carter et al, 2003). Methylated As can be more readily excreted, but some reports suggest methylated As in trivalent form has comparable or greater potency than inorganic As. Trivalent MMA has been shown to be highly cytotoxic to human and cultured rat cells, and in many cases more cytotoxic than inorganic As or pentavalent methylated As (Vega et al., 2001); therefore, methylation may be a toxification rather than a detoxification (Beck et al., 2002).

The Postmortem Interval, Forensic Entomology and Entomotoxicology

The post mortem interval (PMI) may be described as the time period between death and the discovery of a corpse (Byrd and Castner, 2001). Oftentimes, entomological evidence is utilized to determine PMI. This can be helpful in medico-

3 Texas Tech University, Nina Dacko, May, 2011 criminal cases to determine the time that a murder may have occurred (Byrd and Butler, 1998). Necrophagous insects are typically the first organisms to arrive at a corpse, and colonize the corpse in a predictable sequence (Anderson, 1998). This, as well as interactions in and between species of arthropods, is known as faunal succession. Knowing the normal faunal succession pattern can be helpful in the determination of the PMI. If one can properly identify present insect species, know when those insects typically arrive to a corpse and determine the age of those insects, then an accurate PMI can be determined. While accounting for proper biological parameters, PMI can be estimated within hours of the actual time of death. It should be noted here that the age of these insects, commonly in the form of larvae, is determined by length. Many different extrinsic cofactors can affect the faunal succession of necrophagous insects, such as weather (humidity, temperature, precipitation etc.), location (forest, field, urban, rural etc.) and season. Considering these cofactors, the PMI can be more accurately determined. Toxicology is known as the study of poisons and the study of the way exogenous chemicals affect normal homeostatic bodily functions. Within forensic science, toxicological analyses are used to determine what drugs (if any) may have influenced a person involved in a crime, and often used as evidence in the courtroom. Entomotoxicology can be described as the application of toxicological analyses of carrion-feeding insects and other materials, such as frass, to identify the presence of toxicants in tissues when bodily fluids or tissues are not able to be used. Necrophagous insects have been successfully used in medico-criminal investigations to detect toxicants (Introna et al., 2001). Entomotoxicology also investigates the effects of substances on arthropod development in order to assist PMI estimates (Bourel et al., 2001).

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Life History of Sarcophaga bullata

Sarcophaga bullata Parker (Diptera: Sarcophagidae) is commonly found in and around the Texas Panhandle and throughout North America. This dipteran lives in diverse habitats, from arid to humid, which suggests that it is readily adaptive and can be easily reared in an insectary. Sarcophaga bullata is (order: Diptera, suborder: Cyclorrapha, family: Sarcophagidae, genus: Sarcophaga, subgenus: Neobellieria and species bullata) is commonly called the “grey flesh fly”. Flesh flies breed in carrion, excrement and other decaying organic matter (Al-Misned, 2002). This behavior explains the fly’s importance in the determination of the PMI in legal investigations (Giroux and Wheeler, 2009). As with all dipterans, S. bullata is holometabolous and has three main life stages; larvae or maggot, pupae and imago or adult. There are three larval instars which are identified by size. In the order of Diptera, the outer shell of the pupae is known as the puparium (Castner, 2001)

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Figure 1.1. Sketch of the various life stages of a sarcophagid fly. A- imago; B- freshly larviposited larvae; C- larva; D- pupa; E- metamorphic insect. Source: http://www.life.illinois.edu/entomology/illustrations/gifs/sarcophagid.gif

Flies in the family of Sarcophagidae commonly larviposit as opposed to oviposit (Girard et al., 1979). Eggs take a number of days to develop inside the female’s abdomen and once they hatch are ready for larviposition. These larvae, along with egg casings, are deposited by an ovipositor located at the end of the female’s abdomen. Larviposition is usually diurnal (Pape et al., 2002), but there is evidence that they may larviposit at night (Singh and Bharti, 2008). Larval age can be determined by length; however, many other parameters also influence developmental rate such as temperature, (Byrd and Butler, 1998) relative humidity, and competition (Al-Misned, 2002). Once larvae approach maximum length (at the end of the third instar), they enter the prepupal stage. This stage is noted by the migration away from the food source or “wandering” stage. In a natural environment, maggots burrow into the soil and become dormant. The insect is now entering the metamorphic stage.

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Upon entering the metamorphic stage, the larvae’s outer exoskeleton begins to harden and overall body length is reduced. Pupae first appear cream in color and gradually darken from reddish to dark brown within several hours. Pupal age is correlated with pupal color. It is in this stage that most S. bullata enter diapause. Sarcophaga bullata pupae can withstand temperatures up to -20⁰C for short periods. The production of glycogen phosphorylase enables temperature tolerance and is stimulated by the combination of cool temperature, hormones and photoperiod (Chen et al., 1991). During metamorphosis larvae undergo a series of changes such as growing legs, wings and compound eyes and sexual maturation. After these metamorphoses, the fly enters its final molt called emergence or eclosion. Mandubunyi (1986) noted that Sarcophaga haemorrhoidalis emerges within five hours of sunrise. During emergence, an organ called the ptilinium, fills and empties with fluid causing the puparium to burst open. The adult fly is then able to emerge and remains stationary while the soft exoskeleton indurates. This process is usually complete within a few hours (Castner, 2001). After the exoskeleton hardens, basic life functions, such as consumption of food and water, and excretion occur. Adult flies typically consume nectar and protein, including decaying organic matter. Mating occurs shortly after emergence and may last for several hours. Upon fertilization, the female will find decaying organic matter on which to larviposit, and thus completes the life cycle.

Past Research in the Field of Entomotoxicology

Case Studies

The field of entomotoxicology arose from the combination of ecological toxicology and entomology. The first use of insects to detect toxicants in a legal case is by Pekka Nuorteva in 1977. Nuorteva determined the geographic origin of an unidentified body that had been transported to the Finnish countryside. He discovered

7 Texas Tech University, Nina Dacko, May, 2011 low mercury concentrations within the maggots feeding on the body. This mercury content correlated with the town of Turku which had virtually no mercury pollution. It was later determined that the body fit the description of a missing student in the town. The use of these insects helped police focus their investigation in the correct city (Goff and Lord, 2001). The first case study demonstrating the use of insects in toxicological analyses was by Beyer and others (1980). They reported that Cochliomyia macellaria (Diptera: Calliphoridae) maggots served as reliable surrogates for the toxicological analysis of phenobarbital. At the time of the autopsy, maggots were only intended to assist with the estimation of the PMI. However, since no suitable tissues were available for toxicological analysis as a result of advanced decomposition, maggots were utilized. Kintz and others (1990a) further supported the idea of the use of maggots for toxicological analysis. The barbiturates triazolam, oxazepam, alimemazine, clomipramine and phenobarbital, were present in calliphorid larvae feeding on a cadaver that had been decaying for 67 days. Larvae contained much less barbiturates than the liver of the victim which, even though was semi-decayed, was still able to be analyzed using high performance liquid chromatography (HPLC). Kintz and others (1990b) assisted in a case in which a chronic heroin user was found dead nine days post-overdose. Along with taking tissues from various organs, calliphorid larvae were also analyzed. Both morphine and phenobarbital were detected in all tissues. This study further validated the use of flies as surrogates for human tissue samples when a corpse is badly decayed. Gunatilake and Goff (1989) assisted in a case in the Hawaiian Islands in which a man, who had a history of suicide attempts, was found dead in his parents’ crawlspace next to an eight ounce bottle of malathion (organophosphate). The malathion was detected from body fat and larvae feeding on the body. Based on larval development, a PMI of five days was estimated assuming normal ambient conditions. Insects were also observed feeding in the mouth and nasal passages with high concentrations of malathion. Noticeably, common necrophagous insect taxa normally found in similar

8 Texas Tech University, Nina Dacko, May, 2011 areas in Hawaii, were missing from the body. It was thought that malathion must have affected normal insect succession in the corpse. Calliphorid larvae tested positive for opiates when blood opiate concentration of a corpse was greater than 180 µg/kg. Chemical analyses were performed on larvae that fed on 40 liver samples from cadavers that had tested positive for morphine. (Introna et al., 1990). If the opiate concentration in blood was less than 180 µg/kg, there were no detectable concentrations of opiates in larvae. All analyses were conducted via radioimmunoassay (RIA) and verified with gas chromatography/mass spectrometry (GC/MS). This was the first paper to report a relationship between the quantifiable concentrations of chemicals in a cadaver and those in fly larvae. Nolte and others (1992) reported a case where skeletonized remains of a chronic intravenous cocaine addict were discovered in a forest three months after he was reported missing. His girlfriend explained that he injected himself with cocaine, became agitated and walked away, although no drug paraphernalia was found on or near his corpse. No bodily fluids were available for analysis; however, muscle samples and intact Calliphora vicina (Diptera: Calliphoridae) larvae and puparia were present and used for toxicological analysis. Cocaine was discovered in muscle tissue (via GC/MS), but could not be quantified due to the presence of decomposition products. Cocaine was quantified in larval and puparia samples; however, whether or not these larvae bioaccumulate or eliminate cocaine was not determined at the time of the experiment. Kintz and others (1994) reported the corpse of a heroin abuser found in his kitchen. The man had been dead for 10 days and femoral blood, bile and larvae were used for toxicological analysis. Both metabolites, morphine and codeine, were quantified in the human tissues and in larvae. Levine and others (2000) reported a case where a bottle of secobarbital was found near a skeletonized corpse. Because the body was in an advanced state of decomposition, maggot samples were utilized for toxicological analysis by gas

9 Texas Tech University, Nina Dacko, May, 2011 chromatography. Again, maggots served as a reliable surrogate to corpse tissue samples, as both tested positive for secobarbital. The use of insects as surrogates for corpse tissues during toxicological analyses when the corpse is badly decayed has been clearly demonstrated. It has also been observed that these necrophilic insects have chemicals present in their systems that may affect growth rate and development.

Laboratory Studies

Because chemicals may affect an insect’s growth and development, laboratory experiments aimed at determining what these effects are and how they may change the determination of a PMI have been conducted. As mentioned earlier, entomotoxicology is a hybrid field of ecological toxicology and entomology. The first half of this section deals with entomotoxicological experiments from an ecological and toxicological perspective. Pickett and Paterson (1963) discovered exposure of four species of adult flies to sub-lethal concentrations of arsenates, greatly reduced egg production One of the species studied was necrophagous, Musca domestica (Diptera: Muscidae) or the common housefly. This study was intended to test applicable efficacy of As, but the study’s results are also valuable for entomotoxicological purposes. An early ecotoxicological laboratory experiment is reported by Nuorteva and Nuorteva (1982). They fed Creophilus maxillosus (Coleoptera: Staphylinidae), a larviporous carrion beetle, on calliphorid larvae that fed on mercury-exposed fish tissues. They found that mercury/methylmercury bioaccumulated in the beetle. After three months, beetles exposed to mercury-laden larvae showed signs of methylmercury poisoning, including slowed movement, paralysis and death. Akori and Suzuki (1984) reported cadmium was retained in Sarcophaga peregrine larvae, however most cadmium was excreted immediately after emergence. Little if any cadmium was discovered in adult flies. Sarcophaga peregrina larvae were fed

10 Texas Tech University, Nina Dacko, May, 2011 homogenized porcine liver laced with cadmium as food media and then subsequent tissues, such as larvae, mucus from larvae, puparia, excreta from adults, and adults were analyzed for cadmium. The highest concentrations of cadmium were found in excreta immediately following emergence (50%), and a small amount remained in larval mucous and puparia. These data suggest that adult S. peregrina avoid the toxic effects of cadmium through the excretion process. Moe and others (2001) fed Lucilia sericata (Diptera: Calliphoridae) a cadmium- laden diet at different life stages to observe the effects on development. They exposed insects in the all life stages; larval, pupal or adult stage separately; or no cadmium in any stage. They determined mean pupal and adult masses were higher in cadmium- exposed populations than in control populations. However, higher mortality decreased the weight per group when compared to controls. There was also a lower survival rate among pupae of the cadmium-exposed population. Cadmium-exposed populations remained in all development stages 2-8 days longer than those of the control group. Cadmium exposure in the larval stage reduced adult longevity. Reproduction was reduced as a result of cadmium exposure at any stage. Cadmium accumulation was greater in exposed adults when compared to larvae that deposited cadmium in their puparia. This paper suggests that the insect will take longer to develop if it must synthesize metallothioneins in the process of detoxification, because less energy will be available for growth and development (Moe et al., 2001). Sarica and others (2004) reported that blowflies effectively bioaccumulate methylmercury (meHg) as larvae, and therefore predators of these larvae may be at greater risk for meHg poisoning. Ferhat and others (2010) found that the larvae of Calliphora vicina could tolerate certain essential metal salts, such as iron (Fe), copper (Cu) and zinc (Zn), but had significantly higher mortality and extended growth periods when exposed to cadmium (Cd), mercury (Hg), lead (Pb), nickel (Ni), and chromium (Cr). After Gunatilake and Goff assisted in the Hawaiian malathion suicide case, Goff and colleagues performed several laboratory experiments involving drugs and their

11 Texas Tech University, Nina Dacko, May, 2011 affect on growth rate, fecundity and mortality of sarcophagid flies. They suggest that these effects should be accounted for when using entomological evidence in the determination of PMI. All the experiments were similar in that three different doses were used including half median lethal dose (0.5 MLD), median lethal dose (MLD) and twice median lethal dose (2 MLD). Rabbits were used as the vehicle to expose insects to drugs. Most drugs were administered intravenously or by cardiac heart puncture. Rabbits were euthanized, their livers removed, and flesh flies were allowed to develop on contaminated rabbit livers. Larvae were then analyzed for drugs, and the chemical effects on growth and development were recorded. The first was their experiments utilized cocaine (Goff et al., 1989). Significant differences (P< 0.05) in the length of maggots were observed after 36-78 hours. The amount of cocaine was positively correlated with larval growth. Pupation occurred earliest in the 2 MLD beginning at 126 hours after larviposition. This was followed by the MLD at 138 hours, 0.5 MLD at 144 hours, and control group at 150 hours. All flies produced viable offspring. Goff and others (1991) reported significant differences in growth rate were observed between18 and 96 hours. As with cocaine, larvae exposed to more heavily heroin-laden livers reached their maximum size sooner than the control group. Similarly, the duration of the pupal stage was significantly greater among the heroin exposed flies. All flies produced viable offspring. Rates of development were significantly different after 30 hours when sarcophagids were fed methamphetamine-treated rabbit liver. The MLD and 2 MLD larvae reached maximum length 12 hours prior to 0.5 MLD and control group. Pupation occurred earliest in the MLD group, followed by the 0.5 MLD, and lastly, the 2 MLD and control group. Pupal weights were greatest in the control group, followed by 2 MLD, and less in the 0.5 MLD and MLD groups. Pupal stage duration was also significantly less for larvae exposed to the methamphetamine-laden tissues. Pupal mortality rates were approximately equal in the 2 MLD and control group (only 5%) and higher in the other two groups. Flies first emerged in the 2 MLD treatment

12 Texas Tech University, Nina Dacko, May, 2011 group (290 hours post-exposure), followed by 0.5 MLD treatment group (294 hours post-exposure), MLD treatment group (295 hours post-exposure) and control treatment groups (338 hours post-exposure). The MLD group larviposited dead larvae or unhatched eggs. The offspring of the 2 MLD treatment group failed to produce viable offspring (Goff et al., 1992). No significant differences in growth rate were observed when sarcophagids fed on liver from amytriptilene-exposed rabbits, but there were significant differences in larval duration. Larval duration was greatest in the 2 MLD treatment group and less time as doses decreased with shortest in the control group. Mortality ranged from 5.5% in the control group to 40.5-57.7% in dosed groups. Pupae in the MLD and 2 MLD treatment groups weighed more than 0.5 MLD and control treatment groups. The 0.5 MLD and control treatment groups produced viable offspring, the 2 MLD treatment groups only produced 50% viable larvae, and the MLD group failed to produce any larvae (Goff et al., 1993). Goff and others, (1994) determined no significant differences in larval length between groups when exposed to phencyclidine (PCP). Larval duration was the greatest in the control group and larval mortality was positively correlated with phencyclidine concentrations. Pupal duration was greatest in the MLD and shortest in the control group. Pupal mortality was greatest for the control and MLD treatment groups. Goff and others (1997), conducted experiments using 3,4 methylenedioxymethamphetamine (MDMA). The MDMA metabolite was only detected in puparia, and was absent in the rabbit tissues. These findings suggest that metabolism must have occurred in the larvae. Between 24 and 114 hours post exposure, larvae from the control and 2 MLD treatment groups developed more rapidly than the other treatment groups. Larval and pupal mortalities were lowest in the 2 MLD, followed by the MLD group and greatest in control group. The total period required for development from larviposition to adult was significantly shorter for the 2 MLD treatment group due to the shortened larval development period.

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Wilson and others (1993) reported a failed attempt to detect parcetamol in Calliphora vicina. It was later suggested by Sadler and others (1995) that “A drug will be detected in the larvae when its rate of absorption exceeds the rate of elimination…”. This may explain the absence of paracetamol in the previous report. Sadler and colleagues collected drug-laden tissues from three suicide cases and fed them to C. vicina. These drugs included the antidepressants/muscle relaxers amitriptyline, temazepam, trazodone and trimipramine. They reported detectable concentrations of all drugs in the maggots, with a decline in the amount of drugs detected after eight days associated with puparation. Similar uptake was observed after transferring larvae from drug-laden tissue to drug-free tissue. These drugs could be detected because their rate of elimination was not as rapid as parcetamol. Musvasva and others (2001) reported no differences in survivorship of the flesh fly Sarcophaga tibialis exposed to sodium methohexital (metabolic depressant) or hydrocortisone (metabolic stimulant). They reported both hydrocortisone and sodium methohexital retarded development, but metamorphosis occurred more quickly. There was no significant difference in larval and pupal growth rate combined. Their experiment included a small population size and larval length change was simply observed instead of quantified. Carvalho and others (2001) reported significantly higher larval weight in diazepam exposed populations of Chrysomya albiceps (Diptera: Calliphoridae) and Chrysopya putoria. The rate of pupation and emergence time was also accelerated for both species of flies exposed to the diazepam laden liver. Roeterdink and others (2004) tested larvae for lead (Pb), barium (Ba), and antimony (Sb) (common in gunshot residue) after shooting pieces of beef food medium with a revolver. There were two sections of beef that were shot and one that was not to serve as a control. Larvae were fed on shot beef, and then transferred to clean beef at 12, 24, 36, 48 and 60 hours post-feeding. Concentrations of all tested metals in transferred larvae significantly decreased, and were undetectable at12 and 24

14 Texas Tech University, Nina Dacko, May, 2011 hours, but lead and barium were present in transferred larvae at 36, 48 and 60 hours post-feeding. Kaneshrajah and Turner (2004) reported Calliphora vicina growth rate is significantly dependant on what food substrate insects were fed on. Larvae fed tissues other than liver had a significantly greater growth rate than those that were fed liver. Later. O’Brien and Turner (2004) reported paracetamol sped up the growth rate of C. vicina by up to twelve hours and was not dependant on the amount of paracetamol used. Tabor and others (2005) studied the effects of ethanol on faunal succession and insect developmental rate. They tested differences in faunal succession in the field, and tested growth and development rate changes in the laboratory. Pigs were dosed, euthanized, and a section of muscle was placed either in the field or the laboratory. Larval development was delayed by up to 11.9 hours. Kharbouche and others (2008) reported a 21 hour error in determination of the PMI when feeding codeine laced tissues to Lucilia sericata. Three codeine doses (i.e., therapeutic, toxic and potentially lethal) were added to swine liver homogenates, and fed to L sericata. Samples of larvae were collected every 12 hours (from 24 to 96 hours) and stored at -20⁰C. Pupae and imagoes were also sampled, weighed and stored at -20⁰C. All larvae from codeine-treated groups were positive for codeine, and the control group was negative. The quantity of codeine in larvae was positively correlated with the quantity of codeine detected in liver. Codeine levels were higher in liver than in larvae. Codeine metabolites, norcodeine and morphine were also found in the higher dosed larvae. These findings suggested that these metabolites may have different effects on larval development Oliveira and others (2009) demonstrated the use of Chrysomya megacephala larvae for chemical analysis and growth rate effects of butylscopolamine bromide, an acetylcholine antagonist drug used for calming muscle spasms. They found all larvae reared on decaying organic matter (bovine and fish) dosed with rat 0.25 MLD, .05

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MLD, MLD and 2 MLD were affected negatively. Larvae that fed on the drug-laden media showed shorter body lengths when compared to the control at the same stage. Yan-Wei and others (2010) evaluated the effects of malathion on the succession and development of the green bottle fly (Chrysomya megacephala). They dosed three domestic rabbits with malathion corresponding to 0.5 lethal dose (0.5 LD), LD, 4 LD. The rabbits were euthanized and placed 20 meters apart in an open field. Carcasses were inspected every 30 minutes until the occurrence of oviposition. Carcasses were then examined every twelve hours. All rabbit carcasses attracted blowflies within five minutes. Different species of calliphorids dominated the malathion-dosed carcasses after four days. There were no significant differences in the number of species of coleopterans found on each carcass; however there were fewer coleopteran adults (notably in 4 LD). This implies that some species of necrophagous insects are more malathion tolerant than others. Larvae were larger and took longer to develop on treated carcasses, which suggests an increase in acetylcholine. Malathion had a dose- dependent effect on larvae, but factors like temperature, environment, dosage and mode of exposure may have also influenced toxicity. These laboratory studies affirm the likelihood that different chemicals, doses of chemicals, and mode of exposure differentially affect multiple life stages of necrophagous insects. Each species of insect may be affected differently, and these effects may also be dependent on which organs or part of a corpse these insects are feeding. All of these factors should be considered when using entomological evidence in the determination of the PMI.

Gaps in Research

Arsenic trioxide was chosen for this experiment because there is a lack of published entomotoxicological reports that directly pertain to classically used homicidal/suicidal poisons, and little information pertaining to the influences of As combined with metabolites on necrophagous insects. The majority of past research

16 Texas Tech University, Nina Dacko, May, 2011 pertains to pharmaceuticals and illicit drugs. There is a lack of reported research on the growth and development of necrophagous insects fed upon acute exposure’s accumulation of As, necrophagous insects fed upon chronic exposure’s accumulation of As and the differences between these two types of exposure. It is unknown if Sarcophaga bullata possess the ability to methylate As as do humans. There is little known about metabolic differences between species of necrophagous insects, and how each species may exemplify different effects from the same chemical. There have been few entomological experiments performed in the field setting. This is likely because there is no control over environmental parameters that may influence insect growth and development, and would therefore be confounding factors to accurately assessing the effects. Because reports of field experimentation are lacking, there is limited information differences in faunal succession differences, differences in the Shannon index (diversity), differences in insect competition, differences in insect susceptibility, and differences in the mode that chemicals affect all of these dynamic processes of necrophagous insects. Chemical influences may also serve as synergists/antagonists to these other factors.

Objectives, Aims and Hypotheses

The primary objective of this project was to observe and quantify the responses of Sarcophaga bullata larvae when developing on liver tissues from rabbits that were previously exposed to As2O3. There are three specific aims including: 1. Determine the difference in larval development of Sarcophaga bullata when grown on livers of rabbits that received acute or chronic exposures to As, 2. Determine the influence of dose on subsequent larval growth rate, 3. Determine the influence of As dose on metamorphosis, mortality and reproductive output. Efforts were focused on four hypotheses, including: 1. Fly larvae developing on a chronically dosed rabbit will grow faster than those developing on liver from rabbits acutely dosed rabbit liver as a result of lesser concentrations of inorganic As

17 Texas Tech University, Nina Dacko, May, 2011 and As metabolites in the livers, 2. Fly larvae will spend a greater amount of time in metamorphosis relative to the concentration of As in rabbit liver, 3. Fly larval mortality will be greater dependant on the greater concentration of As in rabbit liver, 4. There will be less reproductive output for populations of adult flies exposed to As in the larval stage.

Literature Cited

Agency for Toxic Substances and Disease Registry (ATSDR), 2000. Division of Toxicology and Medicine. Arsenic Toxicity. http://www.atsdr.cdc.gov/csem/arsenic/ Agency for Toxic Substance and Disease Registry (ATSDR), 2009. Division of Toxicology and Medicine. Arsenic Toxicity: Case Studies in Environmental Medicine. http://www.atsdr.cdc.gov/csem/arsenic/docs/arsenic.pdf Al-Misned, F. A. M. 2002. Effects of larval population density of the whole life cycle of flesh fly Wohlfahrtia nubia (Weidmann) (Diptera:Sarcophagidae). Saudi J. Bio. Sci. 9:140-48. Anderson, G. S. 1998. Wildlife forensic entomology in the investigation of illegally killed black bears. Proceedings in A. Acoc. Forensic Sci. 4:45. Aoki Y., and K. T. Suzuki. 1984. Excretion of Cadmium and change in the relative ration of iso-cadmium-binding proteins during metamorphosis of fleshfly (Sarcophaga peregrine). Comp. Biochem. Physiol. 78:315-17. Azcue, J. M., J. O. Nriagu. 1994. Arsenic: Historical Perspectives. In J. O. Nriagu (eds.) Arsenic in the Environment, Part 1:Cycling and Characterization. John Wiley and Sons, Inc p 1- 17. Beck, B. D., T. M. Slayton, C. H. Farr, D. W. Sved, E. A. Creclius, and J. F. Holson. 2002. Systematic uptake of inhaled arsenic in rabbits. Human and Exper. Tox., 20:205-15. Beyer, J. C., W. F. Enos, and M. Stajic. 1980. Drug identification through analysis of maggots, J. Forensic Sci. 25:411-12. Bourel, B., G. Tournel, V. Hedouin, M. Deveaux, M. L. Goff and D. Gosset. 2001. Morphine extraction in necrophagous insects remains for determining ante- mortem opiate intoxication. Forensic Sci. Inter. 120:127-31. Byrd, J. H. and J. F. Butler. 1998. Effects of temperature on Sarcophaga Haemorrhoidalis (Diptera: Sarcophagidae) development. J. Med. Entomol. 35:694-98. Byrd, J. H. and J. L. Castner. 2001. Insects of Forensic Importance. In J. H. Byrd and J. L. Castner (eds.) Forensic Entomology: The Utility of Arthropods in Legal Investigations. CRC Press, Boca Raton, FL p 43-80.

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Carter, D. E., H. V. Aposhian and A. J. Gandolfi. 2003. The metabolism of inorganic arsenic oxides, gallium arsenide, and arsine: a toxicochemical review. Toxicol. App. Pharmaco. 193:309-34. Carvalho, L. M. L., A. X. Linhares and J. R. Trigo. 2001. In J. H. Byrd and J. L Castner (eds.) Determination of drug level and the effect of diazepam on the growth of necrophagous flies of forensic importance in southeastern Brazil. Forensic Sci. Inter. 120: 140-44. Castner, J. L. 2001. General Entomology and Arthropod Biology. In J. H. Byrd and J. L. Castner (eds.) Forensic Entomology: The Utility of Arthropods in Legal Investigations. CRC Press, Boca Raton, FL p 17-42. Charles, J. A. 1980. The Coming Age of Iron. In T. A. Wertime and J. D. Muhly (eds.) New Haven, CT:Yale University Press 580p. Chen, C., D. L. Denlinger and R. E. Lee. 1991. Seasonal variation in generation time, diapauses and cold hardiness in a central Ohio population of the flesh fly Sarcophaga bullata. Ecol. Entomol. 16:155-62. Doyle, D. 2009. Notoriety to respectability: a short history of arsenic prior to its present day use in hematology. Brit. J. Haem. 145:309-17. Ferhat, A., K. A. Yavuz and F. Kose. 2010. Effects of some toxic heavy metals on larval growth rates of Calliphora vicina (Diptera: Calliophoridae) and estimation of PMI. Fresenius Environ. Bulletin 19:1064-73. Girard, J. E., F. J. Germino, J. P. Burdis, R. A. Vita and M. P. Garrity. 1979. Pheromone of the male flesh fly Sarcophaga bullata. J Chem Ecol 5:125-30. Giroux, M., T. A. Wheeler. 2009. Systematics and phylogeny of the subgenus Sarcophaga (Neobellieria) (Diptera:Sarcophagidae). Ann. Entomol. Soc. Am. 102:567-87. Goff, M. L., A. I. Omori, and J. R. Goodbrod. 1989. Effects of cocaine in tissues on the development rate of Boettcherisca peregrine (Diptera: Sarcophagidae). J. Med. Entomol. 26:91-93. Goff, M. L., W. A. Brown, K. A. Hewadikaram, and A. I. Omori. 1991. Effects of heroin in decomposing tissues on the development rate of Boettcherisca peregrine (Diptera, Sarcophagidae) and implications of this effect on estimation of postmortem intervals using arthropod development patterns. J. Forensic Sci. 36:537-42. Goff, M. L., W. A. Wayne, and A. I. Omoti. 1992. Preliminary observations of the effects of methamphetamine in decomposing tissues on the development rate of Parasarcophaga ruficornis (Diptera: Sarcophagidae) and the implications of this effect on the estimations of postmortem intervals. J. of Forensic Sci. 37:867-72. Goff, M. L., W. A. Brown, A. I. Omori, D. A. LaPointe. 1993. Preliminary observations of the effects of amitriptyline in decomposing tissues on the development of Parasarcophaga ruficornis (Diptera: Sarcophagidae) and implications of this effect to the estimation of postmortem interval. J. of Forensic Sci. 38:316-22.

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Goff, M. L., W. A. Brown, A. I. Omori, and D. A. LaPointe. 1994. Preliminary Observations of the effects of phencyclidine in decomposing tissues on the development of Parasarcophaga ruficornis (Diptera: Sarcophagidae). J. Forensic Sci. 39:123-28. Goff, M. L., M. L. Miller, J. D. Paulson, W. D. Lord, E. Richards and A. I. Omori. 1997. Effects of 3,4-Methylenedioxymethamphetamine in decomposing tissues on the development of Parasarcophaga ruficornis (Diptera: Sarcophagidae) and detection of the drug postmortem blood, liver tissue larvae and puparia. J. Forensic Sci. 42:276-80. Goff M. L and Lord D. L. 2001. Entomotoxicology: Insects as Toxicological Indicators and the Impact of Drugs and Toxins on Insect Development. In J. H. Byrd and J. L Castner (eds.) Forensic Entomology: The Utility of Arthropods in Legal Investigations. CRC Press, Boca Raton, FL p331-41. Gunatilake, K., and M. L. Goff. 1989. Detection of Organophosphate Poisoning in a Putrefying Body by Analyzing Arthropod Larvae. J. Forensic Sci. 34:714-16. Harper, M. 1988. Occupational health aspects of arsenic extractive industry in Britain (1868-1925). British J. Indust. Med. 45:602-05. Introna, F. C. L. Dico, Y. H Caplan and J. E. Smialek. 1990. Opiate analysis in cadaveric blowfly larvae as an indicator of narcotic intoxication. J. Forensic Sci. 35:118-22. Introna, F., C. P. Campobasso, M. L. Goff. 2001. Entomotoxicology. Forensic Sci. Inter. 120:42-47. Kaneshrajah, G., and B. Turner. 2004. Calliphora vicina larvae grow at different rates on different body tissues. Int. J. Legal Med. 118:242-44. Kharbouche H., M. Augsburger, D. Cherix. F. Sporkert, C. Giroud, C. Wyss, C. Champod and P. Mangin. 2008. Codeine accumulation and elimination in larvae, pupae, and imago of the blowfly Lucilia sericata and effects on its development. Int. J. Legal Med. 122:205-11. Kintz, P., B. Godelar, A. Tracqui, P. Mangin, A. A. Lugnier and A. J. Chaumont. 1990a. Fly Larvae: A New Toxicicological Method of Investigation in Forensic Medicine. J. Forensic Sci. 35:204-07. Kintz, P., A. Tracqui and P. Mangin. 1990b. Toxicology and fly larvae on a putrefied cadaver. J. Forensic Sci. 30:243-46. Kintz, P., A. Tracqui and P. Mangin. 1994. Analysis of opiates in fly larvae sampled on a putrefied cadaver. J. Forensic Sci. 34:95-7. Levine, B., M. Golle and J. E. Smialek. 2000. An unusual drug death involving maggots. Amer. J. Forensic Med. Path. 21:59-61. Lin, C., M. Wu, Y. Hsueh, S. S. Sun, and A. Cheng. 2005 Tissue distribution of arsenic species in rabbits after single and multiple parental administration of arsenic trioxide: tissue accumulation and the reversibility after washout are tissue-selective. Cancer Chemother. Pharm. 55:170-78. Mandubunyi, L. C. 1986. Laboratory life history parameters of the red-tailed flesh fly, Sarcophaga haemorrhoidalis (Fallen) (Diptera:Sarcophagidae). Insect Sci. Applic. 7:617-21.

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Miller, W. H., H. M. Schipper, J. S. Lee, J. Singer, and S. Waxman. 2002. Mechanisms of action of arsenic trioxide. Cancer Research. 62:3893-3903. Moe, S. J., N. C. Stenseth and R. H. Smith. 2001. Effects of a toxicant on population growth rates: sublethal and delayed responses in blowfly populations. Funct. Ecol. 15:712-21. Musvasva, E., K. A. Williams, W. J. Muller and M. H Villet. 2001, Preliminary observations of the effects of hydrocortisone and sodium methohexital on development of Sarcophaga (Curranea) tibialis Macquart (Diptera: Sarcophagidae), and implications for estimation post mortem interval. Forensic Sci. Inter. 120:37-41. Nriagu, J. O. 1983. Lead and Lead Poisoning in Antiquity. John Wiley and Sons. New York, NY 437p. Nriagu, J. O. 2002. Arsenic Poisoning Through the Ages. W. T. Frakenberger Jr (eds.) Environmental Chemistry of Arsenic. Marcel Dekker Inc., New York, NY p1- 27. Nolte, K. B., R. D. Pinder and W. D. Lord. 1992. Insect larvae used to detect cocaine poisoning in a decomposed body. J. Forensic Sci. 37(4) 1179-85. Nuorteva, P. and S. Nuorteva. 1982. The fate of mercury in Sarcophagous flies and in insects eating them. Ambio. 11: 34-37. O’Brien, C. and B. Turner. 2004. Impact of paracetamol on Calliphora vicina larval development. Int. J. Legal Med. 118:188-89. Oliveira, H. G., G. Gomes, J. J. Morlin, C. J. Von Zuben, and S. X. Linhares. 2009. The effect of Buscopan ® on the development of the blow fly Chrysomya megacephala (F.) (Diptera: Calliphridae). J. Forensic Sci. 54:202-06. Pape, T., D. Dechmann and M. J. Vonhof. 2002. A new species of Sarcofahrtiopsis Hall (Diptera : Sarcophagidae) living in roosts of Spix's disk-winged bat Thyroptera tricolor Spix (Chiroptera) in Costa Rica. J. Nat. Hist. 36:991-98. Park, M. J. and M. Currier. 1991. Arsenic Exposure in Mississippi: A review of cases. S. Med. J. 84:461-64. Pickert, A. D., and N. A. Patterson. 1963. Arsenates: Effect on fecundity in some dipteral. Science. 140:493-94. Roeterdink, E. M., I. R. Dadour and R. J. Watling. 2004. Extraction of gunshot residues from the larvae of the forensically important blowfly Calliphora dubia (Macquart) (Diptera: Calliphoridae). Int. J. Legal Med. 118:63-70. Sadler, D. W., C. Fluke, F. Court and D. J. Pounder. 1995. Drug accumulation and elimination in Calliphora vicina larvae. Forensic Sci. Inter. 71:191-97. Sarica J., M. Amyot, J. Bey and L. Hare. 2005. Fate of mercury accumulated by blowflies feeding on fish carcasses. Environ Tox. Chem. 24:526-29. Singh, D. and M. Bharti. 2008. Some notes on nocturnal larviposition by two species of Sarcophaga (Diptera: Sarcophagidae) Forensic J. Inter. 177:e19-e20. Tabor, K. L., R. D. Fell, C. C. Brewster, K. Pelzer and G. S. Behonick. 2005. Effects of antemortem ingestion of ethanol on insect succestional patterns and development of Phormia regina (Diptera: Calliphoridae). J. Med. Entomol. 42:481-89.

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Vahidnia, A., G. B. van der Voet and F. A. de Wolf. 2007. Arsenic neurotoxicity; A review. Toxicology. 26:823-32. Vega, L., M. Styblo, R. Patterson, W. Cullen, C. Wang, and D. Germolec. 2001. Differential effects of trivalent and pentavalent arsenicals on cell proliferation and cytokine secretion in normal human epidermal derotinocytes. Toxicol. Appl. Pharmocol. 172:225-32. Walters, G. 2009. Hunting Evil: The Nazi War Criminals Who Escaped and the Quest to Bring Them to Justice. Broadway books, Crown Publishing Groups. Random House Inc. New York, NY 518p. Yan-Wei, S. L. Wiao-Shan, W. Hai-Yang and A. Run-Jie. 2010. Effects of Malathion on the insect succession and the development of Chrysomya megacephala (Diptera: Calliphoridae) in the field and implications for estimating postmortem interval. J. Forensic Sci. 31:46-51. Zaman, K., and R. S. Pardini. 1995. An insect model for assessing arsenic toxicity: Arsenic elevated glutathione content in the Musa domestica and Trichoplusia ni. Bull. Environ. Contam. Toxicol. 55:845-52. Zaman, K., R. S. MacGill, J. E. Johnson, S. Ahmad, and R. S. Pardini. 1995. An insect model for assisting oxidative stress related to arsenic toxicity. Arc. Insect Biochem. Physiol. 29:199-209.

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CHAPTER II

MATERIALS AND METHODS

Rabbits

Rabbits were used as the vehicle to provide physiologically natural concentrations of inorganic arsenic (As) and As metabolites in rabbit liver tissues used to feed flesh flies. Rabbits were dosed through chronic and acute exposure methods. Theoretically, these different exposure methods would result in the accumulation of different concentrations and As and its metabolites, which should have different effects on fly growth and development, mortality, metamorphosis, and reproductive output. The New Zealand white rabbit was chosen for this experiment because they metabolize As similarly to humans (Beck et al., 2002) and their livers are large enough to be used as fly feeding media.

Chronic Exposure

New Zealand white rabbits (strain 052) were obtained from Charles River Laboratory International Incorporated (http://www.criver.com/en- US/Pages/home.aspx) and maintained according to standard Texas Tech University Animal Care and Use Committee (ACUC) protocol. Three rabbits were designated as the chronic treatment group. A solution of 10% agarose, 20% sucrose to 70% distilled water was used as the vehicle for orally dosing using a plastic syringe. Two rabbits were dosed daily with As at 1.5 mg/kg (Lin et al., 2005) based on the average weight of the three rabbits (ranging from 2.67 – 2.71 kg). The control rabbit received only the dosing vehicle. After a period of 35 days, rabbits were euthanized in a CO2 chamber. Livers, kidneys and spleens were removed from the rabbits, transported to the lab on ice, frozen and stored at -80°C until used as fly feeding media. Approximately 3 mL of blood was collected using heart puncture for plasma analysis

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Acute Exposure

Rabbits were acquired and maintained similar to those in the chronic study. Dosing vehicle remained the same as the chronic study. Seven rabbits were included in the acute exposure group, and included three different dosages. Dosages were equivalent to half of the median lethal dose for the New Zealand white rabbit (0.5 MLD). or 10.1 mg/kg body weight (BW); a median lethal dose (MLD) or 20.2 mg/kg BW; two times the median lethal dose (2 MLD) or 40.4 mg/kg to achieve a low medium and high concentration of As and metabolites in rabbit liver. Two rabbits received each of the designated doses to account for metabolic differences between rabbits within each group. One rabbit served as a control, receiving dosing vehicle only. Rabbits within each group were dosed based upon individual body weights and according to the median lethal dose (LD50) listed in the material safety and data sheet (MSDS) (Acros Organics, 2003) and greater than the minimum lethal dose of 15 mg/kg (Carter et al., 2003). After dosing, rabbits were allowed eight hours to metabolize As, euthanized, organs and tissues were harvested and stored similarly to the livers from rabbits used in the chronic dose group.

Flies

Collection

On the 28th of August 2009 at 1800 hours, two 454g sections of fresh beef liver were placed in an open field approximately 11 km southwest of Lubbock, TX. Sections of liver were placed on a thin sheet of plastic inside wire Tomahawk ® live traps (Tomahawk Live Trap Company, Tomahawk, Wisconsin) so as not to be disturbed by local wildlife and pets. Liver sections were observed, kept moist with distilled water, and allowed to accumulated local fly specimens for 66 hours.

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Colony Establishment

Following accumulation of local fly species on liver sections, and larviposition by female flies, maggot-laden liver was relocated to the insectary. Insectary conditions were as follows: Temperature- 26-30⁰C, 75-85%RH and 12:12 photoperiod (Byrd, 1995). Upon observed larval migration, liver and accumulated maggots were placed upon a bed of vermiculite to facilitate pupation. Once pupation was completed, sarcophagid pupae were separated from other species based on size. Flies received a 1:1 diet of sucrose and powdered milk, and water ad libitum. Flies were maintained for three generations for acclimation to laboratory conditions, and to obtain a sufficient number of larvae to support experimental colonies.

Larval exposure and measurement

Once the third generation flies were ready to larviposit, three sections of beef liver (454 g) were placed into cages (one per cage). Flies were allowed to larviposit for two hours to ensure a sufficient number of larvae for experimental groups with limited variation in larval age. One-hundred larvae were placed on half of previously dosed rabbit liver sections. Each section of liver was weighed, then placed in labeled 226.796 g plastic containers on a bed of vermiculite, and placed on shelves in the insectary. Sampling of larvae was accomplished by dipping a metal spatula into the mass of maggots within each container. Maggots were measured every eight hours until the onset of migration away from the food source with a BMI® digital caliper.

Pupae and adult maintenance, reproductive output and generation two

Migrating larvae were placed into vermiculite to facilitate pupation. Four days after pupation, pupae were removed from the vermiculite and placed in 226.796 g plastic containers. Containers of pupae were placed in wire screen cages (30.48 cm x

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30.48 cm x 30.48 cm) until emergence. When adult female flies began larviposition, ten separate pieces of beef liver were placed in each cage. Flies were allowed to larviposit for two hours, after which each container of beef liver was removed. One- hundred larvae were placed onto the remaining halves of experimental rabbit liver. Larvae remaining on beef liver were placed in -80°C freezer after one day of growth, then larvae were counted by hand. Larvae deposited on wicks, food media or other decaying media were recorded and added into total reproductive output data.

Chemical Analyses

Arsenic speciation analyses

One to two gram samples of experimental rabbit livers, and one sample of maggots directly exposed to 50 mg/kg As2O3/pureed liver, were sent to Applied Speciation and Consulting in Spokane, Washington. Arsenic speciation included arsenate (As (V)), arsenite (As (III)), monomethylarsinic acid (MMA) and dimethylarsinic acid (DMA) and additional species of As, performed using ion chromatography- inductively coupled (argon) plasma- dynamic reaction cell- mass spectrometry (IC-ICP-DRC-MS).

Digestion

Total As concentrations were quantified in puparia and adult flies from fly generation 2.. Samples were rinsed with filtered water (>18 M Ω) Barnstead NANOpure infinity system (Dubuque, IA, USA) to remove surface contaminants prior to nitric acid/peroxide digestion. The digestion method used slight modification of EPA method 3050B (as suggested by Applied Speciation and Consulting) to be optimal for Inductively Coupled Plasma- Mass Spectrometry (ICP-MS) As analysis. Briefly, these modifications included multiple sample sizes ranging from 0.1 - 0.2g, and thus included different dilution factors.

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Total arsenic analyses

Digested samples were taken to the Civil Engineering Building at Texas Tech University and analyzed using the Perkin Elmer SCIEX model Elan PRC-e ICP-DRC- MS. Indium (In) at 10 ppb was used as an internal standard. Indium is ideal to use to measure As oxide (AsO) in the dynamic reaction cell (DRC) mode because the atomic masses of In and AsO are similar. Arsenic was measured in both regular (As) and DRC (AsO) modes to avoid chloride-argon interference. A curve of 0.1 to 100 ppb was used for calibration with As PerkinElmer Pure As Calibration Standard, Matrix:

2% HNO3. After calibration, a blank sample (consisting of a 2% HNO3 solution) and a 10 ppb As spike sample were analyzed to confirmation calibration. A 10 ppb spike was also analyzed every tenth sample to confirm proper calibration. All As output was performed with the Perkin Elmer default program.

Statistical Analyses

Fly growth rate and development trends

A mixed effects linear model was used to test for differences in growth rate. Dose groups were considered fixed effects, while rabbits within each dose group were considered a random (i. e. nested) effect. An initial mixed effects linear model was used to determine if there were effects over all time periods. Each eight hour interval was then analyzed separately. Tukey’s Honestly Significant Difference test was used for post-hoc comparisons.

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Fly life trends and arsenic concentrations in tissues

Pearson’s Chi square analysis was used to determine if the percent mortality was dependent on dose group. A linear regression was used to model the relationship between percent mortality and total As concentration in rabbit livers, the metamorphic time and the total concentration of As in rabbit livers, reproductive output, and the total As concentration in rabbit livers. Lastly, linear regression was used to model the relationship between the concentrations of total As in rabbit liver, puparia and imago tissues collected from the second generation of experimental fly colonies. All statistical analyses and linear regression graphs were performed/created using statistical program R (version 2.8.0). All other graphs and tables were constructed using Microsoft Office, Excel, 2007.

Literature Cited

Acros Organics, 2003. Material Safety and Data Sheet MSDS for Arsenic (III) Oxide. 5p. Beck, B. D., T. M. Slayton, C. H. Farr, D. W. Sved, E. A. Creclius, and J. F. Holson. 2002. Systematic uptake of inhaled arsenic in rabbits. Human Exper. Tox., 20:205-15. Byrd, J. A. 2001. Laboratory Rearing of Forensic Insects. In J. H. Byrd and J. L Castner (eds.) Forensic Entomology: The Utility of Arthropods in Legal Investigations. CRC Press, Boca Raton, FL p121-42. Environmental Protection Agency (EPA), 1996. Method 3050 B: Acid digestion of sediment, sledges and soils. http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3050b.pdf Carter, D. E., H. V. Aposhian and A. J. Gandolfi. 2003. The metabolism of inorganic arsenic oxides, gallium arsenide, and arsine: a toxicochemical review. Toxico. App. Pharmaco. 193:309-34. Lin, C., M. Wu, Y. Hsueh, S. S. Sun, and A. Cheng. 2005. Tissue distribution of arsenic species in rabbits after single and multiple parental administration of arsenic trioxide: tissue accumulation and the reversibility after washout are tissue-selective. Cancer Chemother. Pharm. 55:170-78.

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CHAPTER III

RESULTS AND DISCUSSION

Observations

Larvae developing on the median lethal dose (MLD) liver were the first to enter prepupal migration and pupation stages before the other groups. Half median lethal dose (0.5 MLD) and control treatment group larvae followed, while the chronic and twice the median lethal dose (2 MLD) treatment groups were last to migrate and pupate. Trends remained similar for both generations. The control treatment group emerged from pupae first. Emergence in the 0.5 MLD followed, and then in the chronic treatment group and lastly in the 2 MLD treatment group. Reproductive output or larviposition, occurred at similar times for each group, but was at a maximum in the control, chronic and 0.5 MLD groups. The MLD and 2 MLD treatment groups in both generations of experimental flies demonstrated delayed larviposition.

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Figure 3.1 Pupation differences observed between groups.

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Fly growth rate and development trends

Mixed effects ANOVA determined that there was a significant difference in overall mean larval length for generation one. Mixed effects ANOVA at each eight hour time point revealed significant differences between treatment groups at 32, 40, 48, 56, 64 and 72 hours (P<.05). Post-hoc analyses revealed significant difference at 32, 40, 48 and 56 hours. Mixed effects ANOVA determined that there was an overall significant difference duration mean larval length in generation two. Mixed effects ANOVA at each eight hour time point revealed significant differences between treatment groups at all hours except 64, 72 and 80 hours (P<.05). Post-hoc analyses revealed a significant difference at the same time as did ANOVA.

Figure 3.2 Generation one mean body length (mm) of Sarcophaga bullata feeding on experimental rabbit livers from larviposition to migration (n=20).

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Figure 3.3 Mean larval body lengths of Sarcophaga bullata at 8 hours. There were no significant differences between treatment groups.

Figure 3.4 Mean larval body lengths of Sarcophaga bullata at 16 hours. There were no significant differences between treatment groups.

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Figure 3.5 Mean larval body lengths of Sarcophaga bullata at 24 hours. There were no significant differences between treatment groups.

Figure 3.6 Mean larval body lengths of Sarcophaga bullata at 32 hours. Letters denote the results of post-hoc analyses.

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Figure 3.7 Mean larval body length at 40 hours. Letters denote the results of post-hoc analyses.

Figure 3.8 Mean larval body lengths of Sarcophaga bullata at 48 hours Letters denote the results of post-hoc analyses.

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Figure 3.9 Mean larval body lengths of Sarcophaga bullata at 56 hours. Letters denote the results of post-hoc analyses.

Figure 3.10 Mean larval body lengths of Sarcophaga bullata at 64 hours. There were no significant differences between treatment groups.

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Figure 3.11 Mean larval body lengths of Sarcophaga bullata at 72 hours. There were no significant differences between treatment groups.

Figure 3.12 Mean larval body lengths of Sarcophaga bullata at 80 hours. There were no significant differences between treatment groups.

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Figure 3.13 Mean body length (mm) of Sarcophaga bullata feeding on liver of control and liver of As treated rabbits for generation two. There are significant differences between groups at every stated hour except hours 64, 72 and 80 hours.

3.14 Mean larval body lengths of Sarcophaga bullata in detail at 8 hours. Letters denote the results of post-hoc analyses.

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3.15 Mean larval body lengths of Sarcophaga bullata in detail at 16 hours Letters denote the results of post-hoc analyses.

Figure 3.16 Mean larval body lengths of Sarcophaga bullata at 24 hours. Letters denote the results of post-hoc analyses.

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Figure 3.17 Mean larval body lengths of Sarcophaga bullata at 32 hours. Letters denote the results of post-hoc analyses.

Figure 3.18 Mean larval body lengths of Sarcophaga bullata at 40 hours. Letters denote the results of post-hoc analyses.

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Figure 3.19 Mean larval body lengths of Sarcophaga bullata at 48 hours. Letters denote the results of post-hoc analyses.

Figure 3.20 Mena larval body lengths of Sarcophaga bullata at 56 hours. Letters denote the results of post-hoc analyses.

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Figure 3.21 Mean larval body lengths of Sarcophaga bullata at 64 hours. There were no significant differences between treatment groups.

Figure 3.22 Mean larval body lengths of Sarcophaga bullata at 72 hours. There were no significant differences between treatment groups.

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Figure 3.23 Mean larval body lengths of Sarcophaga bullata at 80 hours. There were no significant differences between treatment groups.

Tukey’s HSD post-hoc analysis determined significant differences between groups from 32-56 hours in both generations one and two. This growth trend suggests that it takes a period of 24 hours before As affects the growth rate of Sarcophaga bullata, and also that the mean larval length of all S. bullata treatment groups stabilize after 56 hours. Prior to migration, larvae reached a maxima length in which the stop growing. Since the MLD treatment group reached their maximum length first, but did not migrate until 24 hours later, all other treatment groups were able to reach their maximum growth lengths before MLD migration. Additionally, Tukey’s HSD also determined a significant difference in the S. bullata treatment groups mean larval length in generation two at 8, 16 and 24 hours. The difference in length from 8 - 24 hours may be attributed to a smaller standard error from generation one to generation two. This difference in standard error can be explained either by increasing measurement accuracy, or because As caused more variability in mean larval growth length over time.

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Emergence and metamorphosis trends

Emergence trends were derived from means for each population of Sarcophaga bullata over time for generations one and two, respectively (figures 3.24 and 3.25). Numbers of flies emerged were recorded for each cage and averaged together based on treatment group. The relationship between metamorphic time and concentration of total As in experimental rabbit liver in a linear regression model is demonstrated in figure 3.26.

Figure 3.24 Emergence trends demonstrated for generation one of Sarcophaga bullata feeding on liver of control and As treated rabbits.

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Figure 3.25 Emergence trends demonstrated by for Sarcophaga bullata feeding on liver of control and As treated rabbits

Figure 3.26 Regression analysis of metamorphic time as a function of total As concentration found in experimental rabbit liver in parts per million in generation one (left) and generation two (right).

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Table 3.1 Table of statistics for regression analysis of metamorphic time as a function of total As concentration found in experimental rabbit liver for generations one and two. Generation 1 DF slope lower CI upper CI p‐value 8 30.878 18.82066 42.93455 3.5 × 10‐4 Generation 2 DF slope lower CI upper CI p‐value 8 17.978 10.39113 25.56502 5.98 × 10‐4

Analysis indicated a strong positive correlation between the amount of time pupae spent in metamorphosis, and arsenic (As) concentration in food source. This positive correlation suggests detoxification mechanisms present at the metamorphic life stage of S. bullata. There were no alkylated species of As found in S. bullata upon IC-ICP-MS analysis at Applied Speciation. However, both pentavelent and trivalent forms of As were present. Sarcophaga bullata does not possess the ability to methylate As in the excretion process, but may possess the ability to reduce arsenate to arsenite. Alkylating As is the first step of As metabolism in human beings (Hayakawa et al, 2005). However, since there are very low concentrations of total As in the imago of S. bullata, but total As is present in puparia, there is excretion of As after emergence. This is congruent with the reports of Aoki and Suzuki (1983) who discovered Sarcophaga peregrine excretes cadmium immediately after emergence. Further research suggests what mechanism of As metabolism S. bullata possesses, and when that metabolism is at its peak.

Mortality

Pearson’s Chi square analysis identified significant dependence of mortality on treatment groups at all life stages, with the exception of the pupal stage of generation one.

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Table 3.2 Pearson’s Chi square analysis of mortality per dose groups. Generation 1 mortality Generation 2 mortality larvae pupae Total larvae pupae total χ2 22.4053 7.2507 17.0351 22.3987 18.3487 30.2602 p‐value 0.00768 0.611 0.04817 0.007698 0.03134 0.000396

There was a negative correlation among both generations in the linear regression analyses. This negative correlation increases from a slope of -3.26 to -4.66 in generation one, and a slope of -3.7 to -7.6 in generation two when the 2 MLD is excluded from all treatment groups. The slope decreases (there is a greater negative correlation) when the 2 MLD group is excluded, suggesting that the negative correlation is only at these sublethal levels of As exposure. If the concentration of As is higher, this trend could change where death is positively correlated to concentration of As in rabbit liver. Somewhere between 1.9 ppm As in liver and 2 ppm As in liver, there is a threshold at which sarcophagids can no longer benefit from As.

Figure 3.27 Regression analysis of percentage mortality as a function of total As concentration in experimental rabbit liver in generation one (left) and generation two (right)

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Figure 3.28 Regression analysis of percentage mortality as a function of total As concentration in experimental rabbit liver in generation one (left) and generation two (right) excluding 2 MLD treatment groups.

Table 3.3 Table of statistics for regression analysis of mortality as a function of total As concentration found in experimental rabbit liver in generations one and two. Generation 1 DF slope lower CI upperCI p‐value total 8 ‐3.258 ‐6.2489 ‐0.26764 0.03624 minus 2xMLD 6 ‐4.658 ‐12.0244 ‐3.17063 0.0563 Generation 2 DF slope lower CI upperCI p‐value total 8 ‐3.699 ‐8.0186 0.61988 0.08369 minus 2xMLD 6 ‐7.598 ‐12.0244 ‐3.17063 0.00569

Results of Pearson’s Chi square analysis suggest that the number of deaths within each generation of experimental flies (in larval and pupal stages) is dependent upon treatment group. Pairing the results from Pearson’s Chi square analysis with the negative correlation between mortality and the total concentration of As in rabbit liver, and the closer correlation between mortality and amount of total As in rabbit liver when the 2 MLD group is excluded. This trend suggests S. bulla exposure to sublethal concentrations of As creates a hormetic trend in which mortality decreases as the concentration of total As increases until the 2 MLD treatment group’s concentrations of As in rabbit liver. There are similar to reports of the calliphorid,

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Phormia regina, when exposed to a non-essential metal salt, cadmium chloride (Nacarella and others, 2003). Future research continuing this experiment may include a 4 MLD treatment group to determine if the hormetic trend continues, and also where the total As concentrations are too great for the insect to overcome. This mortality trend can also be explained by As treatment of S. bullata midgut parasites, where the concentration of As does not adversely affect S. bullata, but may cause mortality to harmful midgut parasites.

Arsenic in tissues

There was As found in all experimental rabbit livers, although very low in the control rabbits. This could be background levels of As in rabbit feed, or contamination of samples. There were no detectable concentrations of As metabolites in control rabbit livers. Total As concentrations followed the following trend: The greater the amount of As with which the rabbit was dosed, then the greater the concentration of As was present in rabbit liver samples. There were no detectable amounts of As metabolites found in larvae exposed to 50 mg/kg BW arsenic trioxide that was mixed directly into pureed beef liver. (Table 3.4). Trends of total As concentrations in puparia and imago tissue were similar to that in treated rabbit livers where puparia and imago tissues exposed to more As had greater total As concentrations (Table 3.5).

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Table 3.4 Table of total As concentrations and As metabolite concentrations. As (III)- trivalent arsenic, As (V)- pentevalent arsenic, MMA- monomethylatedarsonic acid, DMA dimethylatedarsinic acid, ND- no data. Sample ID As(III) As(V) MMAs DMAs Additional As Total As Species Control A 0.088 0.041 ND (< 0.049) ND (< 0.032) ND 0.030 Control B ND (< 0.045) ND (< 0.052) ND (< 0.071) ND (< 0.047) ND 0.020 Chronic A 0.028 ND (< 0.032) ND (< 0.044) 0.169 ND 0.281 Chronic B 0.040 0.076 ND (< 0.045) 0.130 ND 0.156 0.5 MLD A 0.165 0.052 0.133 0.337 0.053 0.755 0.5 MLD B 0.049 0.026 0.052 0.173 ND 0.346 MLD 0.393 ND (< 0.045) 0.306 0.456 0.073 1.90 MLD 0.290 0.088 0.258 0.621 0.077 1.88 2 MLD 0.549 0.089 0.439 0.614 0.165 2.60 2 MLD 0.663 0.061 0.624 0.597 0.062 2.03 Larvae vs As alone 1.99 0.045 ND (<0.052) ND (< 0.034) ND 5.09

Table 3.5 Table of total As in experimental liver and insect tissues. SD- standard deviation Concentration of As in sample is in µg/g = (ppb concentration) × (dilution factor) × (volume of digestion in mL) × 1/1000 × 1/g, p = pupae, i = imago Sample ID Mean total SD As SD AsO End Dilution Sample Total As As in ppb dilution factor weight (g) concentration AsO (mL) (ppb) Blank ‐0.0074 0 0.01 50 0 0 0 10 ppb Spike 7.889 0.6 0.7 48 10 0.48 7.889 Control A p 0.403 0 0.01 24 20 0.12 1.612 Control A i 0.211 0 0.01 35 10 0.35 0.211 Control B p 0.232 0 0 22 20 0.11 0.928 Control B i ‐0.003 0 0 36 10 0.18 ‐0.006 Chronic A p 0.681 0 0.01 24 20 0.12 2.724 Chronic A i 0.188 0 0 48 10 0.48 0.188 49

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Table 3.5 continued Chronic B p 0.301 0 0 20 20 0.1 1.204 Chronic B i 0.086 0 0.01 40 20 0.2 0.344 0.5 MLD A p 1.217 0.01 0.01 20 20 0.1 4.868 0.5 MLD A i 1.043 0.01 0 42 10 0.42 1.043 0.5 MLD B p 1.265 0.01 0.02 22 20 0.11 5.06 0.5 MLD B i 0.22 0 0.01 34 20 0.17 0.88 MLD A p 4.187 0.02 0.05 20 20 0.1 16.748 MLD A i 0.623 0.01 0.01 38 10 0.38 0.623 MLD B p 5.68 0.04 0.05 26 20 0.13 22.72 MLD B i 0.73 0.01 0.02 50 10 0.5 0.73 2 MLD A p 6.898 0.02 0.09 22 20 0.11 27.592 2 MLD A i 0.8 0 0.01 40 20 0.2 3.2 2 MLD B p 3.259 0.02 0.04 20 20 0.1 13.036 2 MLD B i 0.361 0 0.01 40 10 0.4 0.361

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Figure 3.29 Regression analysis of total As concentration in puparia as a function of total As concentration in liver (top left), total As concentration in imago tissues as a function of total As concentration in liver (top right) and total As in concentration in imago as a function of total As concentration in puparia (bottom).

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Table 3.6 Table of statistics for regression analysis of total As concentration found in experimental rabbit liver and total As concentrations found in puparia and imago tissues. Liver versus puparia DF slope lower CI upper CI p‐value 8 0.0094 0.006854 0.01189 2.63× 10‐5 Liver versus imago tissues DF slope lower CI upper CI p‐value 8 5.9×10‐4 1.32×10‐5 0.001177 0.0461 Imago tissues versus puparia DF slope lower CI upper CI p‐value 8 0.6724 0.01347 0.121 0.0204

Linear regression analyses were performed to determine the relationship between the total As concentration in experimental rabbit liver, puparia, and imago tissues. These analyses revealed a positive correlation between total As concentration in experimental liver and total As concentrations in both puparia and imago tissues. ( Figure 3.30 top left and top right, respectively) Also, there was a positive correlation in the relationship between the total As concentration in puparia and imago tissues (Figure 3.30 bottom). The R2 value for As concentration in puparia tissues and As concentrations in rabbit liver (R2 = 0.90) reveal are greater positive correlation than the R2 value between As concentrations in imago tissues and As concentrations in rabbit liver (R2 = 0.41), and the R2 value for As concentrations in imago and As concentrations in puparia tissue (R2 = 0.51). This is likely a result of the low concentrations of As in the imago tissues. Decreasing As concentrations from puparia to imago stages may be explained by the excretion of As (as a non-essential metal) after emergence, similar to the findings of Aoki and Suzuki (1983).

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Reproductive Output

A linear regression model (Figure 3.30) demonstrates the relationship between total As concentrations in experimental rabbit livers and the number of offspring produced by each treatment group of flies. There is a negative correlation in generation one, but trends are not similar in generation two.

Figure 3.30 Regression analysis of number of offspring as a function of total As concentration in experimental rabbit liver in generation one (left) and generation two (right).

Table 3.7 Table of statistics for regression analysis of total As concentration in experimental rabbit liver, and number of offspring per experimental group. Generation 1 DF slope lower CI upper CI p‐value 8 ‐283.4 ‐557.897 ‐8.81411 0.04454 Generation 2 DF slope lower CI upper CI p‐value 8 32.62 ‐408.072 473.3021 0.86872

Although there is a negative correlation between reproductive output and total As concentration in experimental rabbit liver for generation one, there was no correlation observed for generation two. Therefore, there was not a correlation

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Literature Cited

Aoki Y., and K. T. Suzuki. 1984. Excretion of Cadmium and change in the relative ration of iso-cadmium-binding proteins during metamorphosis of fleshfly (Sarcophaga peregrine). Comp. Biochem. Physiol. 78:315-17. Hayakawa, T., Y. Kobayashi, S. Cui and S. Hirano. 2005. A new metabolic pathway of arsenite-glutathione complexes are substrates for human arsenic methyltransferase Cyt19. Arch. Tox. 79:183-191. Nascarella, M. A., J. G. Stoffaleno Jr., E. J. Stanek III, P. T. Kostecki, and E. J. Calabrese. 2003. Hormesis and stage specific toxicity induced by cadmium in an insect model, the queen blowfly, Phormia regina Meig. Environ. Pollut. 124:257- 262. Pickert, A. D., and N. A. Patterson. 1963. Arsenates: Effect on fecundity in some dipteral. Science. 140: 493-94.

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CHAPTER IV

CONCLUSIONS

There was a significant increase in mean larval growth length between MLD treatment groups and chronic treatment groups. This difference may cause up to a 16 hour variation in the accurate determination of the PMI of a person who was exposed to As antemortem. The increase in growth rate of the MLD treatment groups of Sarcophaga bullata may improve their ability to compete for food against other flies, which may be more sensitive to As. However, uncontrolled parameters may work antagonistically or synergistically with As. Since metamorphic time was positively correlated to the concentration of total As in experimental rabbit liver, and the concentration of total As was less in the imago than puparia, suggests there must be excretion of As after emergence. Sarcophaga bullata is able to almost entirely eliminate As after exposure in the larval stage. It is not known where the As is stored prior to excretion in S. bullata and is not known whether the larvae and adult possess the same As tolerance, although cockroach nymphs were found to be more tolerant than adult cockroaches (Dahm, 1959). Larval and pupal mortality were negatively correlated to sublethal concentrations of As. Future research is needed to determine what dosage of As in fly food causes negative effects on S bullata. Also, mechanisms to decrease mortality when sublethal As concentrations increase needs to be further investigated. As with growth, a reduction in mortality would make S. bullata more effective in competition for space and food with other species of flies. It is inconclusive whether As affects the reproductive output of S. bullata exposed in the larval stage. Generation one experimental flies were euthanized to utilize rearing facilities for generation two, and as a result, flies in generation two were allotted a greater amount of time to larviposit than generation one. Since the number of offspring was the contributing factor to reproductive output analysis, there was a greater number of offspring in the generation allowed a longer larviposition time.

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Future researchers may allow both generations of flies to larviposit for the same amount of time. Since As is significantly eliminated after emergence, reproductive output may not be affected by As (or other substances that can be excreted after emergence).

Literature Cited

Dahm, P. A. 1959. The mode of action of insecticides exclusive of organic phosphorus compounds. Ann. Rev. Entomol. 2:247-260.

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APPENDIX A

ARSENIC MSDS AND DIGESTION METHOD

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APPENDIX B

RAW DATA Table B.1 Rabbit liver weights Rabbit ID body weight (g) liver weight % body weight (g) Control A 2880 99.05 3.439236111 Control B 2680 65.59 2.44738806 Chronic A 2860 112.07 3.918531469 Chronic B 2930 111.66 3.810921502 0.5 MLD A 2520 72.13 2.862301587 0.5 MLD B 2960 89.75 3.032094595 MLD A 2660 69.03 2.595112782 MLD B 2820 70.34 2.494326241 2 MLD A 2720 63.27 2.326102941 2 MLD B 2560 71.46 2.79140625

Table B.2 Experimental rabbit doses. Rabbit ID Total Dose (mg) Control A 0 Control B 0 Chronic A 139.6 Chronic B 140 0.5 MLD A 24.9 0.5 MLD B 29.5 MLD A 52.9 MLD B 55.5 2 MLD A 110.7 2 MLD B 101.7

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Table B.3 Larval length data (Generation 1) Group Rabbit Hour Length Length Length Length Length Length Length Length Length Length 1 2 3 4 5 6 7 8 9 10 control A 8 4.37 4.19 2.17 4.41 3.53 4.04 3.62 2.59 4.18 ND B 8 4.58 4.59 3.78 3.76 3.75 4.19 3.29 3.28 3.25 ND chronic A 8 3.16 3.15 3.43 3.9 3.48 3.93 3.96 2.95 2.97 ND B 8 3.97 2.67 3.86 3.59 2.94 3.85 3.14 3.69 2.66 ND 0.5 MLD A 8 3 3.86 3.86 3.59 2.94 3.85 3.14 3.69 2.66 ND B 8 3.85 3.38 3.57 3.27 3.91 3.65 3.76 3.79 3.98 ND MLD A 8 3.46 3.6 3.27 3.5 3.59 2.74 3.9 3.58 3.28 ND B 8 4.46 4.39 3.37 3.67 3.36 3.12 3.58 3.31 3.22 ND 2 MLD A 8 3.31 3.52 3.97 3.51 3.7 2.71 3.11 2.52 3.47 3.41 B 8 3.98 3.35 3.73 3.95 3.14 3.98 3.15 3.51 3.73 ND control A 16 3.38 5.97 4.82 5.3 4 3.5 6.03 5.27 5.35 4.75 B 16 4.75 5.78 4.6 5.31 4.97 4.39 5.08 4.34 5.98 5.23 chronic A 16 5.39 5.39 5.23 4.62 3.98 4.68 5.05 4.32 5.1 4.76 B 16 5.49 5.54 5.32 4.01 5.25 5.61 4.84 4.89 2.62 5.32 0.5 MLD A 16 4.98 3.02 5.19 5.14 4.47 5.01 3.36 4.62 5.1 5.15 B 16 4.63 4.79 4.2 5.45 5.03 5.29 2.65 4.68 5.26 4.68 MLD A 16 4.69 4.58 4.3 4.83 4.38 4.73 5.69 4.46 5.46 4.8 B 16 4.45 4.57 4.99 3.99 4.44 4.44 4.87 4.33 5.52 4.29 2 MLD A 16 4.17 4.13 4.41 4.76 4.36 3.95 3.99 4.55 4.74 4.11 B 16 3.59 4.71 5.27 4.44 4.25 4.87 5.48 4.78 4.33 4.97 control A 24 6.68 6.71 5.87 5.64 5.41 5.17 5.84 6.92 6.1 3.98 B 24 3.13 6.46 7.05 7.39 7.1 7.66 2.84 4.35 7.15 6.67 75

Texas Tech University, Nina Dacko, May, 2011

Table B.3 continued chronic A 24 5.88 6.38 5.82 7.04 5.58 6.18 6.21 6.45 5.79 5.13 B 24 7.11 6.66 3.91 6.54 5.68 7.12 5.13 4.41 6.82 7.45 0.5 MLD A 24 6.13 7.68 6.09 5.07 7.3 4.24 7 6.89 5.86 5.97 B 24 6.91 5.12 4.87 6.2 6.73 6.69 5.13 6.36 5.78 5.08 MLD A 24 6.59 7.25 6.75 5.31 7.4 6.79 3.39 7.19 6.46 7.07 B 24 5.88 6.53 6.18 6.73 5.1 5.92 5.77 6.23 4.53 5.56 2 MLD A 24 6.82 5.75 5.12 6.29 6.22 6.65 6.49 4.63 5.31 5.42 B 24 6.73 6.63 5.09 6.45 7.46 6.46 6.56 7.43 5.47 7.5 control A 32 9.03 7.96 8.79 9.09 9.58 9.56 9.15 8.93 9.49 7.62 B 32 8.65 9.47 8.32 9.32 9.32 8.73 8.8 9.49 9.49 8.21 chronic A 32 8.13 7.91 8.7 4.51 7.15 8.15 9.21 8.94 7.68 7.16 B 32 8.18 9.48 8.65 9.43 8.77 7.54 4.69 7.95 7.42 9.42 0.5 MLD A 32 5.51 8.92 8.75 9.15 8.98 4.74 9.25 3.28 8.02 8.86 B 32 8.55 8.8 6.14 7.46 8.44 8.46 7.64 8.44 7.54 8.53 MLD A 32 8.79 8.36 8.28 9.28 7.53 9.41 4.28 9.31 9.33 8.34 B 32 6.66 8 9.22 6.14 7.26 7.46 8.57 8.14 9.6 8.41 2 MLD A 32 7.36 7.67 6.92 7.47 7.95 8.04 6.69 7.47 7.59 7.55 B 32 8.76 8.62 9.05 8.02 8.85 8.34 8.88 9.49 8.18 9.36 control A 40 10.29 9.88 9.34 11.44 9.13 10.45 11.2 11.37 10.97 11.28 B 40 9.61 11.4 10.28 11.7 11.25 10.96 10.64 10.88 10.67 10.82 chronic A 40 8.73 8.79 8.89 9.88 9.1 8.91 8.48 9.39 10.85 10.59 B 40 11.14 10.69 11.74 8.2 8.63 7.87 9.9 11.47 10.1 10.19 0.5 MLD A 40 9.48 10.76 11.12 9.35 9.69 10.47 11.39 11.12 9.66 10.1 B 40 10.13 8.79 10.48 10.03 10.27 9.29 8.01 10.45 10.47 10.81 MLD A 40 11.27 11.96 10.4 12.71 9.79 13.05 13 11.56 10.68 12.33

76

Texas Tech University, Nina Dacko, May, 2011

Table B.3 continued B 40 10.88 13.17 12.13 12.6 13.48 12.97 12.7 8.56 11.57 12.57 2 MLD A 40 9.44 10.88 9.83 7.61 10.21 10.53 10.76 10.54 10.79 9.99 B 40 13.32 12.64 12.66 10.83 11.88 11.16 9.08 10.06 10.9 13.5 control A 48 12.57 9.62 13.26 14.18 13.4 14.04 14.64 14.94 14.46 14.3 B 48 14.45 13.22 12.9 16 15.19 15.39 14.34 15.93 15.37 13.27 chronic A 48 13 14.7 14.14 15.42 13.39 13.01 11.98 13.98 12.66 10.27 B 48 15.02 12.75 12.73 12.85 13.33 13.89 13.88 13.58 9.46 8.37 0.5 MLD A 48 13.63 13.4 12.27 14.12 13.96 10.71 14.73 11.26 10.16 12.14 B 48 13.55 13.03 11.47 11.75 11.66 15.01 12.79 12.75 10.89 11.8 MLD A 48 16.22 14.28 14.01 16.66 13.83 16.11 14.85 16.56 16.22 16.57 B 48 15.49 17.02 16.47 16.17 16.53 16.73 16.91 16.3 14.04 15.39 2 MLD A 48 12.08 12.84 14.33 14.71 14.32 8.47 14.9 14.36 15.44 12.75 B 48 17.31 16.32 17.8 15.85 16.94 15.63 16.81 15.99 14.76 16.05 control A 56 18.34 18.47 17.32 17.53 18.53 18.75 18.29 18.2 17.88 18.5 B 56 18.63 19.36 19.53 19.67 18.75 18.45 19.03 16.28 15.28 18.37 chronic A 56 18.36 16.68 15.42 15.23 14.48 18.4 19.08 18.13 10.26 17.51 B 56 18.64 19.37 18.33 18.31 19.31 19.99 15.22 18.06 18.87 17.48 0.5 MLD A 56 19.05 18.18 16.85 17.47 19.9 20.4 18.41 17.51 18.05 16.82 B 56 17.38 17.93 17.1 17.35 18.26 16.83 17.53 17.04 15.7 18.02 MLD A 56 20.44 21.07 22.3 18.3 21.61 19.47 20.09 19.33 20.61 19.58 B 56 19.57 20.7 21.82 18.28 19.05 20.37 20.37 20.1 19.6 20.31 2 MLD A 56 15.77 18.13 17.37 18.66 18.83 17.57 17.39 18.06 18.85 17.55 B 56 18.8 18.78 20.3 17.7 20.32 18.25 15.78 13.48 21.1 18.97 control A 64 21.85 22.46 21.73 21.44 12.95 21.57 21.79 22.81 18.47 21.84 B 64 18.21 18.78 18.55 19.14 20.64 18.64 16.44 18.28 19.25 20.84

77

Texas Tech University, Nina Dacko, May, 2011

Table B.3 continued chronic A 64 20.38 20.3 19.7 20.47 21.22 17.56 19.15 10.76 21.57 20.3 B 64 18.84 20.7 22.48 21.89 22.21 20.29 21.44 21.3 19.95 21.2 0.5 MLD A 64 22.01 21.41 19.2 18.88 19.33 16.01 19.84 19.81 18.8 19.75 B 64 17.52 17.69 18.19 19.84 20.69 21.5 20.11 21.84 19.05 18.28 MLD A 64 20.4 21.16 21.67 19.73 20.88 21.64 22.38 22.39 20.39 23.31 B 64 20 19.66 21.62 20.66 19.99 20.36 21.07 21.37 22.41 21.4 2 MLD A 64 20.37 20.13 20.82 20.35 20.34 20.09 20.17 19.45 19.87 19.68 B 64 18.23 20 20.84 18 19.58 19.1 15.79 19.07 18 20.52 control A 72 22.31 22.02 21.73 22.22 21.93 22.51 22.53 22.39 22.47 22.48 B 72 17.71 17.39 17.62 19.62 18 18.52 19.81 18.74 19.24 19.74 chronic A 72 21.49 21.13 20.27 21.12 18.64 21.22 22 20.82 21.13 18.52 B 72 20.67 22.48 22.23 22.04 20.47 19.66 21.6 21.64 20.93 21.14 0.5 MLD A 72 20.23 19.65 20.85 21.32 20.98 20.32 20.31 21.89 20.97 21.69 B 72 17.22 19.94 21.85 19.12 20.83 20 21.69 18.33 19.6 21.34 MLD A 72 20.56 20.09 21.54 20.92 21.05 23.05 20.46 21.38 19.12 21.06 B 72 21.97 20.53 22.64 20.94 22.06 21.08 21.02 20.04 19.92 21.02 2 MLD A 72 20.01 21.77 21.75 21.27 21.27 21.25 21.8 21.65 21.67 20.83 B 72 19.63 20.42 21.16 20.92 21.07 20.04 21.4 21.8 18.89 20.51 control A 80 22.62 22.59 22.26 22.57 23.86 21.66 22.26 22.38 22.66 22.94 B 80 19.44 19.4 20.56 18.25 21.76 20.14 18.9 20.52 20.08 19.12 chronic A 80 22.58 21.12 20.92 21.7 21.81 20.36 22.1 21.36 21.32 20.32 B 80 23.59 22.85 21.8 17.04 23.34 22.14 23 13.86 23.29 21.5 0.5 MLD A 80 18.69 20.74 19.52 18.87 21.14 20.71 21.54 19.77 21.79 21.78 B 80 19.77 22.49 20.06 21.05 20.47 21.01 21.57 22.17 20.98 20.64 MLD A 80 22.32 20.05 23.34 21 20.16 23.13 19.96 22.13 20.11 21.65

78

Texas Tech University, Nina Dacko, May, 2011

Table B.3 continued B 80 20.86 19.78 20.46 21.52 21.93 22.7 20.21 20.94 20.56 21.57 2 MLD A 80 13.5 18.64 19.66 21.52 20.99 20.73 19.86 21.02 21.92 22.53 B 80 17.6 19.34 19.65 20.85 20.3 18.92 20.3 18.72 17.25 22.84

Table B.4 Larval length data (Generation 2) Group Rabbit Hour Length Length Length Length Length Length Length Length Length Length 1 2 3 4 5 6 7 8 9 10 control A 8 3.08 3.28 2.81 3.36 3.06 3.17 3.35 3.17 3.35 2.77 B 8 3.41 3.47 3.42 3.46 2.85 2.93 3.72 3.18 3.32 3.06 chronic A 8 2.86 3.41 3.05 3.48 3.27 2.7 2.47 3.4 3.49 3.32 B 8 3.12 3.03 3.15 3.13 3.46 2.97 3.03 3.08 3.15 3.33 0.5 MLD A 8 2.56 3.03 3.16 3.26 3.23 3.18 3.55 3.16 3.35 3.3 B 8 3.47 3.47 3.8 3.82 2.94 3.25 2.99 2.99 2.89 3.36 MLD A 8 3.92 2.99 3.65 3.95 3.74 3.79 3.51 3.88 2.93 3.59 B 8 3.39 3.2 3.64 3.25 3.19 3.62 3.77 3.74 3.59 3.59 2 MLD A 8 3.07 3.02 3.39 3.5 3.37 3.01 3.31 3.1 3.1 3.49 B 8 3.95 3.31 3.06 3.68 2.87 3.73 3.1 3.76 3.5 3.34 control A 16 4.31 4.67 4.14 4.31 4.67 4.72 4.37 4.15 4.47 4.39 B 16 4.31 4.41 4.25 3.97 3.91 3.91 4.41 4.95 4.66 3.9 chronic A 16 3.86 3.92 3.5 3.52 4.01 4.28 3.69 4.03 3.91 3.82 B 16 3.29 4.33 4.15 3.91 4.01 4.36 4.16 3.78 3.73 4.36 0.5 MLD A 16 4.18 4.36 4.44 4.23 4.18 4.23 4.48 4.1 4.7 4.82 B 16 4.2 3.94 4.19 3.9 4.49 4.05 3.98 4.55 4.43 4.67 MLD A 16 5.35 5.36 4.89 5.12 5.28 5.25 5.21 5.02 5.33 4.6 B 16 4.78 4.88 5.18 4.68 4.76 4.88 4.91 4.85 5.18 5.03 2 MLD A 16 3.39 4.38 3.7 3.5 3.91 3.11 4.15 3.28 4.2 3.64 79

Texas Tech University, Nina Dacko, May, 2011

Table B.4 continued B 16 3.64 3.66 3.75 4.11 4.26 4.22 3.51 3.61 3.65 3.86 control A 24 5.55 6.82 5.73 6.01 5.64 5.32 5.57 5.44 5.72 5.69 B 24 5.16 6.27 4.97 4.76 5.66 5.55 5.28 5.88 5.24 4.67 chronic A 24 4.72 4.72 3.29 4.79 4.86 4.8 5.25 4.54 4.37 4.54 B 24 4.59 6.33 5.17 5.82 5.48 5.58 5.3 5.3 5.88 5.65 0.5 MLD A 24 4.83 5.57 5.44 5.91 5.18 5.51 6.08 5.69 5.18 4.76 B 24 5.08 5.18 4.95 5.78 5.02 5.74 4.95 5.18 5.02 5.3 MLD A 24 6.2 5.6 6.36 7.17 6.81 6.23 5.19 6.99 6.83 5.06 B 24 5.89 6.38 5.4 5.65 6.37 6.28 6.16 6.07 5.46 5.49 2 MLD A 24 5.73 4.28 4.6 5.34 4.13 4.41 4.96 4.52 5.4 5.27 B 24 4.81 4.81 5.96 5.69 4.86 5.06 5.06 5.12 6.19 5.09 control A 32 6.94 7.68 6.58 5.92 7.51 7.69 5.39 7.78 8.17 6.24 B 32 6.93 7.28 7.1 6.97 6.52 6.5 6.73 8.3 6.97 5.72 chronic A 32 7.68 5.17 6.98 7.02 6.16 6.47 7.01 6.57 7.33 5.3 B 32 7.8 6.83 8.16 7.83 4.97 6.48 7.72 7.76 7.46 4.72 0.5 MLD A 32 7.96 7.59 7.12 8.59 5.96 7.82 6.98 7.35 7.27 7.74 B 32 7.38 8.31 7.1 6.18 7.1 6.94 8.39 8.73 6.39 7.45 MLD A 32 8.29 9.69 9.27 9.22 8.45 9.01 9.83 8.94 9.8 9.85 B 32 7.69 7.9 8.42 7.48 7.87 7.83 7.24 8.61 7.99 8.16 2 MLD A 32 6.2 5.83 6.09 7.54 7.59 7.5 6.18 6.19 7.74 4.92 B 32 7.04 6.17 5.89 6.29 7.67 6.85 6.22 7.91 6.27 6.15 control A 40 9.21 9.22 6.92 9.14 9.43 9.17 9.15 9.5 9.25 8.44 B 40 8.22 6.02 7.86 9.06 8.38 8.87 8.87 9.32 9.95 8.72 chronic A 40 7.8 6.83 8.31 7.38 8.24 8.27 7.96 8.3 7.4 7.72 B 40 9.87 8.79 8.49 9.01 9.43 8.86 9.88 9.04 8.92 9

80

Texas Tech University, Nina Dacko, May, 2011

Table B.4 continued 0.5 MLD A 40 8.5 8.24 8.65 8.41 9.35 8.78 8.98 8.85 8.69 9.25 B 40 8.79 8.56 8.86 8.05 8.57 8.77 6.56 9.28 8.42 9.25 MLD A 40 10.18 9.65 10.91 11.06 10.13 10.76 10.14 10 10.22 10.5 B 40 9.04 9.45 10.2 9.05 9.87 9.6 9.32 9.2 9.89 9.34 2 MLD A 40 7.7 8.24 7.49 6.4 8.17 9.04 9.81 8.27 6.58 8.36 B 40 7.43 8.04 9.1 8.34 8.66 9.14 8.22 7.38 8.2 8.37 control A 48 9.34 9.1 10.06 9.7 9.76 9.78 10.09 9.56 10.07 11.03 B 48 9.91 10.49 9.78 9.66 9.97 10.3 9.88 10.53 9.03 9.78 chronic A 48 7.79 7.97 8.65 8.55 10 8.31 9.71 9.8 7.19 7.55 B 48 10.01 8.96 9.87 12.06 10.01 9.77 10.7 8.07 10.86 11.79 0.5 MLD A 48 10.48 10.14 10.01 10378 10.76 10.48 9.89 9.23 10.15 10.34 B 48 9.32 10.38 9.67 10.31 10.26 7.52 9.08 9.75 9.07 9.79 MLD A 48 11.27 10.76 13.34 13.1 11.75 11.27 13.31 12.96 13.12 11.95 B 48 10.39 12.22 11.44 12.71 12.8 12.48 12.87 11.44 11.5 11.91 2 MLD A 48 10.1 9.9 8.04 8.05 9.95 8.69 9.78 10.79 10.91 9.71 B 48 9.74 10.01 10.67 10.44 10.62 10.72 10.89 8.78 8.82 11.03 control A 56 13.58 11.76 14.51 12.08 14.16 13.9 13.13 14.29 13.3 11.85 B 56 14.18 10.29 14.69 14.97 14.02 14.59 14.04 11.82 14.58 12.49 chronic A 56 14.25 11.29 12.42 11.32 11.45 12.27 10.96 10.15 12.18 13.51 B 56 12.05 11.35 13.64 13.37 14.33 14.07 13.83 13.65 11.32 11.82 0.5 MLD A 56 14.81 14.92 13.67 16.34 13.47 13.44 15.37 15.6 14.32 13.92 B 56 12.99 13.04 13.36 12.73 16.07 14.28 13.08 14.36 13.93 14.55 MLD A 56 16.03 17.37 17.85 16.48 17.77 17.03 17.7 17.63 17.25 16.54 B 56 15.72 14.49 16.42 13.46 16.45 14.45 15.44 15.41 16.13 16.83 2 MLD A 56 13.17 15.28 16.68 13.32 14.62 12.74 12.64 14.45 12.27 10.58

81

Texas Tech University, Nina Dacko, May, 2011

Table B.4 continued B 56 16.68 16.46 14.04 14.17 14.65 13.13 16.39 14.57 14.32 15.21 control A 64 14.82 15.74 14.44 18.18 17.53 15.74 14.72 16.2 17.83 17.99 B 64 16.21 16.06 16.52 18.15 15.9 18.64 17.52 15.06 17.35 16.83 chronic A 64 13.02 15.14 15.58 16.43 14.53 17.53 11.79 15.2 14.63 14.07 B 64 18.06 16.66 17.39 17.12 18.13 14.11 14.37 16.82 17.43 15.63 0.5 MLD A 64 15.92 15.45 18.35 15.21 16.81 16.37 15.68 15.29 16.54 15.91 B 64 13.31 16.87 18.55 16.08 16.09 16.13 15.75 15.72 16.91 11.63 MLD A 64 19.08 15.3 16.36 16.29 18.22 18.16 16.9 17.69 14.5 16.11 B 64 16.61 16.91 14.74 18.74 16.61 16.29 17.56 18.31 19.66 17.21 2 MLD A 64 16.94 14.52 14.53 17.79 14.88 17.71 18.51 15.1 17.81 14.55 B 64 16.42 14.43 18.12 17.23 19.52 16.07 18.67 17.71 17.53 16.53 control A 72 16.4 18.57 17.7 16.98 17.72 15.56 19.05 18.78 17.86 16.57 B 72 19.52 17.05 19.2 19.35 17.58 17.44 19.17 18.59 17.02 19.38 chronic A 72 17.78 16.47 19.05 19.19 17.38 19.75 18.38 18.81 17.61 21.05 B 72 16.93 20.13 16.54 17.83 18.71 15.23 18.58 18.9 18 18.58 0.5 MLD A 72 18.39 19.42 17.83 19.84 18.78 17.29 17.73 19.89 20.87 18.09 B 72 18.07 18.44 17.84 18.6 18.3 17.4 19.9 18.97 18.45 19.01 MLD A 72 19.07 16.82 19.5 19.27 18.83 18.1 20.19 19.95 17.7 18.99 B 72 17.53 17.16 14.7 19.76 19.63 19.92 18.76 19.56 19.71 18.6 2 MLD A 72 19.72 16.84 16.82 18.2 17.23 15.49 20.06 19.22 19.26 18.11 B 72 15.8 18.11 16.78 19.21 20.29 18.42 19.66 19.35 18.08 18.22 control A 80 19.03 19.02 20.04 19.44 20.24 20.7 21.36 19.59 19.82 19.82 B 80 17.45 19.88 18.76 18.48 19.51 19.3 19.02 20.22 18.61 14.83 chronic A 80 19.74 19.3 16.03 18.63 18.84 20.81 19.79 20.22 20.38 20.64 B 80 19.66 21.52 20.23 22.1 22.03 20.55 20.85 20.61 19.79 22.5

82

Texas Tech University, Nina Dacko, May, 2011

Table B.4 continued 0.5 MLD A 80 18.3 20.04 19.42 17.14 20.06 17.72 19.27 19.95 17.76 18.47 B 80 17.59 17.82 18.15 19.16 18.05 19.24 18.43 19.01 19.33 18.74 MLD A 80 20.16 16.72 22.29 18.68 20.52 19.42 21.93 14.08 19.62 21.15 B 80 19.13 19.15 19.6 19.05 18.05 19.3 19.74 19.72 18.07 20 2 MLD A 80 19.7 20.85 20.11 21.26 18.16 19.59 19.47 21.09 20.21 19.99 B 80 19.8 19.99 19.06 19.71 20.67 19.66 21.27 19.03 20.01 19.44

83

Texas Tech University, Nina Dacko, May, 2011

Table B.5 Amount of time spent in pupae as a function of As concentration in rabbit liver generation 1 total as Amount of Time in Pupae 0.03 354 0.02 306 0.281 330 0.156 354 0.755 338 0.346 338 1.9 394 1.88 394 2.6 410 2.03 386

Table B.6 Amount of time spent in pupae as a function of As concentration in rabbit liver generation 2 total as Amount of Time in Pupae 0.03 304 0.02 304 0.281 320 0.156 320 0.755 304 0.346 304 1.9 352 1.88 344 2.6 344 2.03 344

84

Texas Tech University, Nina Dacko, May, 2011

Table B.7 Mortality within each life stage generation 1 Rabbit larval mort pupal Total mort Control A 17 4 21 Control B 16 6 22 Chronic A 5 5 10 Chronic B 14 3 17 0.5 MLD A 12 7 19 0.5 MLD B 12 2 14 MLD A 7 4 11 MLD B 6 2 8 2 MLD A 5 7 12 2 MLD B 6 6 12

Table B.8 Mortality within each life stage generation 2 Rabbit larval pupal Total mort mort Control A 9 5 14 Control B 17 10 27 Chronic A 12 6 18 Chronic B 13 7 20 0.5 MLD A 12 3 15 0.5 MLD B 13 6 19 MLD A 4 2 6 MLD B 3 4 7 2 MLD A 8 11 19 2 MLD B 4 7 11

85

Texas Tech University, Nina Dacko, May, 2011

Table B.9 Reproductive Output as a function of As concentration in liver generation 1 Rabbit # Offspring Control A 1233 Control B 955 Chronic A 1386 Chronic B 751 0.5 MLD A 1260 0.5 MLD B 320 MLD A 773 MLD B 630 2 MLD A 103 2 MLD B 353

Table B.10 Reproductive Output as a function of As concentration in liver generation 2 Rabbit # Offspring Control A 914 Control B 1726 Chronic A 1112 Chronic B 726 0.5 MLD A 693 0.5 MLD B 391 MLD A 1720 MLD B 1504 2 MLD A 238 2 MLD B 1407

86