Accumulation and Effects of HMX in the Green Anole (Anolis

Accumulation and Effects of HMX in the Green Anole (Anolis carolinensis)

by

Lindsey E. Jones, 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

MASTERS OF SCIENCE

Approved

Scott T. McMurry, PhD

Philip N. Smith, PhD

Todd A. Anderson, PhD

George P. Cobb, PhD

Matthew B. Lovern, PhD

John Borrelli Dean of the Graduate School

August 2007

Copyright 2007, Lindsey Erin Jones

Texas Tech University, Lindsey Jones, August 2007

ii

ACKNOWLEDGMENTS I would like to thank all those who have helped and supported me in this effort.

First and foremost, I would like to thank Dr. Scott McMurry for his support and

guidance through both the laboratory and writing processes. I would also like to thank

the other members of my committee, Drs. Phil Smith, George Cobb, Todd Anderson,

and Matt Lovern. Without their advice, insights, and technical knowledge this project

would not be possible. I would also like to extend my deepest gratitude to all of my

fellow labmates and technicians who have helped and supported me throughout this

project, especially Xiaoping Pan, Baohong Zhang, Pamela Bryer, and Sabrina Multer.

Last, but definitely not least, I would like to thank my husband and the rest of my

family for their neverending source of strength and support.


iii

TABLE OF CONTENTS ACKNOWLEDGMENTS ii

ABSTRACT iv

LIST OF TABLES vi

LIST OF FIGURES vii

I. INTRODUCTION 1 References 10

II. ACCUMULATION, REPRODUCTIVE, AND DEVELOPMENTAL EFFECTS OF HMX IN THE GREEN ANOLE 15

Introduction 15 Materials and Methods 20 Results 30 Discussion 34 References 55

APPENDIX 79


iv

ABSTRACT The use and subsequent environmental contamination of energetic compounds

is an ever increasing international concern. Perhaps one of the greatest lapses in

knowledge, and therefore threats to the natural environment, of these compounds is

their toxicity to reptiles, particularly with respect to reproduction. To that end, a three-

part study was conducted in an effort to define the role of octahydro-1,3,5,7-tetranitro-

1,3,5,7-tetrazocine (HMX, High Melting Explosive), one of the top four explosive

compounds of the twentieth century, in the reproductive toxicology of the green anole

(Anolis carolinensis), a common reptilian model species. First, the acute oral toxicity

(LD50) was measured in adult anoles using the up-and-down method described by the

Organization for Economic Co-Operation and Development and United States

Environmental Protection Agency. Then, part two of the study used artificially

contaminated nesting media to assess accumulation of HMX into eggs at different

combinations of incubation time and concentration. Also, initial growth parameters

were measured for all hatchlings. The third part of the study was designed to assess

HMX transfer from the diet of adults to eggs. In addition to assessing accumulation,

hatching rates of eggs, growth rate of hatchlings, and signs of toxicity and abnormal

reproductive function in adults were monitored.

HMX was not acutely toxic to adult anoles and the LD50 was estimated at

greater than 2,000 mg HMX/kg body weight. A dose-dependent accumulation of

HMX into the egg was observed, though no significant developmental differences

were observed among treatment groups. Exposed adults also exhibited dose-


v

dependent accumulation of HMX, as well as treatment-related food aversion. From

these studies it can be concluded that HMX readily accumulates in reptiles both

directly (via ingestion of contaminated prey and/or contact with contaminated soil)

and indirectly (via maternal transfer). Further study to determine the effect of HMX

over two breeding seasons may be warranted.


vi

LIST OF TABLES 1. Comparison of HMX concentrations in soil at the initiation

of the artificial nesting study and at study termination (87 days later). 64

2. Comparison of mean (± SE) growth parameters of hatchling green anoles following exposure to HMX via soil. 64

3. Comparison of the target weekly dose, actual mean (± SE)

weekly dose of HMX per anole, and mean weekly dose of HMX given to an average-weight female green anole (2.8 g) over a 12-week study. 65

4. Comparison of the mean dose and percent recovery of HMX in

crickets injected once a week for ten weeks (n=10). 65 5. Comparison of the total number of green anole eggs per dose

group and the number of eggs per female per week. 65 A.1. Raw data from the egg exposure study. 80 A.2. Raw data from the maternal exposure study. 81


vii

LIST OF FIGURES 1. Mean (± SE) concentration of HMX in fertilized green anole

eggs and passive sampling devices (PSDs; C18) incubated for up to 30 days in soil spiked with a nominal concentration of 200 mg HMX/kg soil. 66

2. Comparison of the accumulation of HMX into passive sampling devices (PSDs) and green anole eggs incubated for up to 30 days in soil spiked with a nominal concentration of 200 mg HMX/kg soil. 67

3. Comparison of the accumulation of HMX into green anole

eggs at four nominal concentrations of HMX in soil. 68 4. Comparison of hatching success of green anole eggs across

treatment groups exposed to HMX via contact with contaminated soil for up to 41 days. 69

5. Mean (± SE) number of crickets consumed per week by female

green anoles in the experimental control, vehicle control, low, medium, and high dose groups. 70

6. Mean (± SE) percentage of initial body weight of female green

anoles in the experimental control, vehicle control, low, medium, and high dose groups. 71

7. Mean (± SE) concentration of HMX in the bodies of adult

female green anoles as a result of maternal exposure to HMX in food over 12 weeks. 72

8. Mean (± SE) concentration of the explosive, HMX, in the

green anole egg as a result of maternal exposure to HMX in food. 73

9. Comparison of hatching success of green anole eggs across

maternal treatment groups exposed to various concentrations of HMX in prey. 74

10. Comparison of weight-gain trends of green anole hatchlings

exposed to the explosive, HMX, via maternal transfer. 75


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11. Comparison of snout-vent length trends of green anole hatchlings exposed to the explosive, HMX, via maternal transfer. 76

12. Comparison of hatch success of green anole eggs exposed to HMX

via two transport mechanisms. 77 13. Comparison of accumulation of HMX into green anole eggs via

two transport mechanisms. 78


1

CHAPTER I

INTRODUCTION Since the infancy of the modern toxicological profession, concerned scientists,

environmentalists, and humanitarians alike have been and continue to be plagued by

the question of how to keep the interests of society from overwhelming the capacity of

the already struggling natural environment. Nowhere is that more true than in the area

of national defense. In the world of 2001-2006, the importance of explosive materials

has escalated dramatically as nations poise themselves to combat terrorism both

domestically and abroad. While the need for the actual war on terrorism may still be

controversial, the need for effective weaponry both offensively and defensively cannot

be denied.

Explosives technology, therefore, has yet again been brought to the forefront of

society, and with it comes the subsequent contamination of ecosystems worldwide. In

the United States and Canada, significant contamination has been noted on many

military installations, including firing ranges (Jenkins et al., 1999), storage facilities,

ammunition plants, and load-and-pack (LAP) facilities (ATSDR, 1997; Card and

Autenrieth, 1998), amounting to an estimated 1,200 explosives-contaminated sites in

the United States alone (Schmelling et al., 1997). Although each type of explosive

device has its own unique combination of chemical compounds, there are essentially

three chemicals that have dominated the scene in the last century: 2,4,6-trinitrotoluene

(TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX, Research Department


2

Explosive), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX, High Melting

Explosive) (Hawari et al., 2000).

HMX, first discovered as a byproduct of the Bachmann process used to

synthesize RDX in World War II, is a non-aromatic organic polynitramine explosive

that has been used in propellants, detonators, primers, rocket fuels/boosters, nuclear

devices, and plastic explosives (Anonymous, 2004; Yinon, 1990). The flexible cyclic

structure allows four different conformations (α, β, γ, and δ), though the β form is

most often used (Card and Autenrieth, 1998; Yinon and Zitrin, 1993). A colorless to

white crystalline solid at room temperature, HMX owes its massive power (estimated

to be 75% more powerful than TNT) to its large size (molecular weight = 296.2) and

high density (1.903 g/cu cm [beta form]) (Anonymous, 2004; Goebel, 2002; Hawari,

2000; ONeil, 2001). As its name implies, HMX has a high melting point of 276-

286°C and will violently decompose at 279°C.

HMX contamination has been noted worldwide, with particular emphasis in

the United States and Canada. HMX residues are typically found in groundwater

and/or soil as a result of military activities, including leakage from buried or

unexploded ordnances, residues from low-order (i.e., incomplete) detonations, and, to

a lesser extent, high-order (i.e., complete) detonations (Clausen et al., 2004; Davis et

al., 2006; Jenkins et al., 2002). Distribution of HMX residues across these affected

areas is extremely heterogeneous. For example, soil concentrations of HMX at

Canadian Forces Base Valcartier, Quebec, Canada, can range from 209 mg/kg to

1,140 mg/kg between adjacent 3 m2 quadrants (Jenkins et al., 1999).


3

Though HMX is notably successful in its intended use, the ramifications of

these detonations to wildlife are potentially more encompassing than simply the initial

loss of habitat. Three general routes of exposure to xenobiotics exist in this case

(namely ingestion, inhalation, and dermal exposure); however oral exposure is by far

the most likely since HMXs vapor pressure and Henrys Law constant are low

enough to make HMX unlikely to enter the air (USACHPPM, 2001), and experimental

evidence indicate that HMX is only mildly irritating upon dermal exposure (Cuthbert

et al., 1985). However, laboratory studies involving oral exposure to HMX have

shown toxic effects in the central nervous, hepatic, and renal systems in vertebrate

systems.

Central nervous system disturbances were noted in acute studies, often

occurring after a single dose. Toxic effects noted included hyperkinesia, hypokinesia,

and ataxia in rats and mice and mild convulsions, hyperkinesia, and mydriasis in

rabbits (Cuthbert et al., 1985). A no observable adverse effects level (NOAEL) for

central nervous system disturbances could not be identified in these studies, but the

lowest observable adverse effects limit (LOAEL) was determined to be 100 mg/kg/day

in mice (Greenough and McDonald, 1985a).

Although central nervous system disturbances were seen in both genders of

mice and rats equally, disturbances in the hepatic system have shown gender-specific

variation, with males being more sensitive than females. Main effects noted in in vivo

hepatic studies indicate that centrilobular enlargement, hepatocyte hyperplasia,

cytoplasmic eosinophilia, and centrilobular degeneration were of main concern


4

(Everett and Maddock, 1985; Everett et al., 1985; Greenough and McDonald, 1985b).

The NOAEL for hepatotoxicity was 50 mg/kg/day in rats, 90 mg/kg/day in female

mice, and 75 mg/kg/day in male mice (Everett et al., 1985; Greenough and McDonald,

1985b). The LOAEL established by the US Environmental Protection Agency,

therefore, is 150 mg/kg/day in males (USEPA, 1988).

A gender-related effect was also observed for renal toxicity, with females more

sensitive than males. Female rats exposed to 270 mg/kg/day for 13 days exhibited

focal tubular atrophy, dilation, and increased kidney weights (Everett et al., 1985). In

the same study, higher doses of 1,500 to 4,000 mg/kg/day for 13 days caused

decreased Ph, increased urine output, and crystal formation in the urine of male and

female mice (Everett et al., 1985). Because of these gender differences observed, the

Environmental Protection Agency established a LOAEL of 270 mg/kg/day for renal

toxicity in females (USEPA, 1988).

These data also suggest various, however somewhat mild, systemic effects

from exposure to HMX. Respiratory studies found reddening of the lungs in exposed

animals, but were fairly inconclusive as to the full respiratory effect of HMX (Everett

and Maddock, 1985; Cuthbert et al., 1985). Mice exhibited no significant

cardiovascular disturbances as a result of exposure to HMX, suggesting that the heart

is not a target organ for the chemical (Everett and Maddock, 1985; Greenough and

McDonald, 1985a). Similarly, no musculoskeletal effects were evident in mice

exposed to 90 mg/kg/day HMX (Everett and Maddock, 1985). Data also suggest that

HMX has mild hematological and gastrointestinal effects at doses in excess of 4,000


5

mg/kg HMX in rats, but that lower doses produce little to no effects (Cuthbert et al.,

1985; Everett and Maddock, 1985; Everett et al., 1985). In addition, an in vitro study

demonstrated that HMX was not cytotoxic or mutagenic to human lymphoblastic or

hamster lung cells (Lachance et al., 1999).

Studies also seem to indicate a certain degree of interspecies variation in toxic

response to HMX. For example, although central nervous system disturbances were

observed in mice at greater than 100 mg/kg, no disturbances were observed in studies

involving bobwhite quail at feed concentrations up to 10,000 mg/kg (Johnson et al.,

2005). Interestingly, not only are marine polychaetes (Neanthes arenaceodentata) or

amphipods (Leptocheirus plumulosus) insensitive to sediment concentrations of HMX

up to 1,000 mg/kg, but low level exposures of HMX (approximately 50 mg/kg in

sediment) in freshwater midges (Chironomus tentans) appear to have hormetic effects

(Lotufo et al., 2001; Steevens et al., 2002).

Though studies of individual organ effects have been revealing thus far, the

study of entire organ systems, especially the reproductive system, can provide a

broader base upon which to determine the full effect of HMXs toxicity.

Unfortunately, however, explosives data are especially scant in this area. To date,

there have been limited reproductive studies with HMX in vertebrates. In two studies

located on the reproductive effects of HMX in bobwhite quail (Colinus virginianus),

food repellency induced by HMX was more deleterious to the reproduction of the

quail than actual overt toxicity of HMX (Brunjes et al., 2007; Johnson et al., 2005). In

the few studies conducted with invertebrates, HMX was found to have no effect on


6

survival of benthic invertebrates (Leptocheirus plumulosus and Neanthes

arenaceodentata, dosed to 600 mg/kg HMX) nor on midges (Chironomus tentans) and

amphipods (Hyalella azteca, dosed to 200 mg/kg HMX) exposed to contaminated

sediment (Lotufo et al., 2001; Steevens et al., 2002); or earthworms (dosed to 500

mg/kg HMX), enchytraeids and colemboles (dosed to 1,000 mg/kg) exposed to

contaminated soils (Lotufo et al., 2001; Schafer and Achazi, 1999). Moreover,

Dodard et al. (2005) found juvenile production by Enchytraeus albidus and E.

crypticus to be unaffected by HMX soil concentrations up to 918 mg/kg. In contrast,

Robidoux et al. (2002) found HMX to be more reproductively toxic to the earthworm

(Eisenia andrei) than TNT or RDX when natural forest soils are contaminated, as

opposed to the artificial soils used in other studies. E. andrei EC10 and EC20 values for

reproduction ranged from less than 15.6 ± 4.6 to greater than 711.0 ± 19.9 mg/kg

HMX, respectively (Robidoux et al., 2002).

The near complete lack of reproductive toxicity information in vertebrate

organisms warrants the continued need for reproductive testing (ATSDR, 1997;

USACHPPM, 2001). Compromised reproductive success is, in fact, the culmination

of one or several alterations within the reproductive system (such as lower sperm

counts, alteration of sex steroids, deterioration of reproductive organs, etc.).

Depending on the severity of the disturbance, alterations of this system could lead to

more pronounced population-level disturbances, as suggested in Chinook salmon

(Oncorhynchus tshawytscha; Spromberg and Meador, 2005). Moreover, loss of


7

certain species could modify predator-prey relationships in the area, further

compromising the health of the ecosystem.

Utilizing an appropriate model for a reproductive study, therefore, is crucial in

determining the true risk of a toxicant to wildlife. Selection of a sentinel species in

particular could help define risk to several species even in varying genera. Data

received from such a study could alert risk managers, site managers, and other

researchers to the necessity of future testing in other vertebrates. Reptiles, though

historically underutilized, have received increasing attention as a sensitive

reproductive model (Sparling et al., 2000; Lovern et al., 2004).

Study of reptilian responses to toxicants is of crucial importance from an

ecological standpoint. Reptiles occur around the world and occupy various trophic

levels in their environment. Many reptiles show strong site fidelity and occupy

relatively small home ranges. These life history aspects increase the potential

susceptibility of reptiles to a greater contaminant load (Rainwater, 2003). Indeed,

because of this susceptibility and other environmental and anthropogenic stressors,

reptiles are considered to be the taxonomic class at greatest risk of extinction (Gibbons

et al., 2000).

Not only are reptiles sensitive, but they are also important from a toxicological

perspective in that their diverse life history traits lend the reptile to being a supreme

candidate for animal studies. Because of their various trophic positions, research

garnered from reptile studies can be extrapolated to not only top-level consumers, but

also to lower-level consumers (Hopkins, 2000). Furthermore, their relatively high


8

reproductive capacity and short incubation is highly advantageous in studies of

reproductive and developmental toxicants. Results from these assessments also have

high extrapolation potential to other oviparous species.

My research focused on the common green anole (Anolis carolinensis) for

several reasons. First, the life history of the green anole is well-documented (Crews,

1977; Licht, 1973; Andrews and Rand, 1974; Brattstrom, 1974). Green anoles have

been used for over one hundred years as laboratory models, with most of the recent

research focusing on behavioral responses (Hoeglund et al., 2004; Deckel, 1996;

Baxter et al., 2001; Long et al., 1986) and as a reproductive model (Lovern et al.,

2004).

Anolis as a genus is prolific and found in various parts of the world in different

altitudes and climates (Crews, 1977). Their wide distribution is beneficial when trying

to extrapolate the effects of HMX to other lizards in other regions, possibly

worldwide. This species is also easy to culture, in that they are small (adults are ca. 13

20 cm in total length and ca. three four g in weight) and readily breed in captivity

(Lovern et al., 2004).

A unique aspect of this genus, which is especially helpful in this study, is the

single-egg clutch size. This smaller clutch size actually increases the reproductive

potential of the lizard, which is capable of an oviposition every seven to ten days

during the breeding season (Licht, 1973). This also carries important research

implications, in that any chemicals passing from mother to offspring will be

concentrated into one egg, instead of split among eggs of larger clutches, as seen in the


9

common tern (Sterna hirundo; Nisbet, 1982). Though interclutch variation is likely to

remain considerable, as seen in avian species (Ormerod and Tyler, 1990; Reynolds et

al., 2004), the lack of intraclutch variation will likely reduce the overall variability of

the data.

Also, a significant aspect for using the green anole as a model in this study is

its close association to the soil. Though characterized as an arboreal lizard, green

anoles nevertheless feed on, and deposit eggs in, the soil (Lovern et al., 2004). Thus,

exposure to contaminants in soil may be through diet and/or via transport across the

egg shell during incubation.

My research project involved three phases which measured acute HMX

toxicity in adults, accumulation of HMX into eggs and whole bodies, and effects on

reproduction and offspring survival and growth. Although HMX was not expected to

be acutely toxic to adults at concentrations up to 2,000 mg HMX/kg body weight, it

was hypothesized that it would accumulate readily in living tissue due to its

lipophilicity. Due to this accumulation, some sublethal effects were expected in the

reproducing females, including an altered egg production rate. Furthermore, because

developing embryos are often the more sensitive life stage of an organism, more

dramatic effects such as altered growth and/or hatching rates were expected in

exposed offspring.


10

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15

CHAPTER II

ACCUMULATION, REPRODUCTIVE, AND DEVELOPMENTAL EFFECTS OF HMX IN THE GREEN ANOLE

Introduction The use of and subsequent contamination of the environment with energetic

compounds is an increasing international concern (Talmage et al., 1999; USACHPPM,

2001; ATSDR, 1997). Although most energetic compounds have been in use for the

better part of a century, their fate in the environment and the resultant toxicities at the

organismal, population, and ecosystem levels have yet to be fully characterized.

Indeed, knowledge gaps for in vivo toxicity are still apparent for even the most popular

high energy explosives, including 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitro-1,3,5,7-

triazacyclohexane (RDX), and 1-3-5-7-tetranitro-1,3,5,7-tetrazocine (HMX) (ATSDR,

1995a, b, 1997; Hawari et al., 2000; USACHPPM, 2000, 2001, 2002).

Of the information collected to date, explosive compounds have been shown to

be potentially potent disruptors of several biological systems. TNT is primarily a

hematological disrupter (USACHPPM, 2000), but also causes toxic effects in the

central nervous system (CNS) and reproductive systems of mice, rats, and dogs

(Homma-Takeda et al., 2002; USACHPPM, 2000). Likewise, RDX is a disruptor of

the CNS and a possible hepatotoxicant (evidenced by increased liver weights) in mice

and rats (USACHPPM, 2002). In addition, both TNT and RDX have caused

reproductive disturbances in the earthworm (Eisenia andrei) at 881 mg/kg soil and up

to 189 mg/kg soil, respectively (Robidoux et al., 2000).


16

In comparison to TNT and RDX, HMX has especially scant data regarding its

toxicity in biota. Because HMX and RDX often occur in tandem (one is the impurity

in the synthesis process for the other), almost no epidemiological studies of

manufacturing plant workers have been done on HMX alone. In fact, it wasnt until

2004 that a human case of poisoning with HMX was documented (Testud et al., 2006).

Likewise, relatively few single-toxicity tests have been done on HMX, outside the

initial acute and subacute studies with mice, rats, and rabbits (ATSDR, 1997). Of the

known studies, HMX has been shown to disrupt the central nervous, renal, and hepatic

systems at levels exceeding 200 mg/kg in orally exposed mice and rats (Cuthbert et

al., 1985; Everett and Maddock, 1985; Everett et al., 1985; Greenough and McDonald,

1985a, b; USEPA, 1988). Despite the need, few studies of HMX effects on vertebrate

reproduction have been conducted (ATSDR, 1997; USACHPPM, 2001). Of the

studies located, it appears that indirect toxicity of HMX (i.e., reduced food

consumption due to repellency) could cause greater reproductive disturbances than

direct HMX toxicity (Brunjes et al., 2007; Johnson et al., 2005).

Although effects data remain lacking, more conclusive information is available

on exposure and accumulation of HMX into plants and biota under field and

laboratory conditions. Indigenous plants, for example, from Wainwright Firing

Range, Alberta, Canada, including prairie grasses (Agropyron smithii, Bromus

sitchensis, and Koeleria gracilis), have shown preferential concentration of HMX into

leaves (Groom et al., 2002). Additionally, agricultural plants, such as alfalfa

(Medicago sativa), bush bean (Phaseolus vulgaris), canola (Brassica rapa), wheat


17

(Triticum aestivum), and perennial ryegrass (Lolium perenne), readily accumulated

HMX from Wainwright soil into their leaves in the laboratory, with concentrations in

scenescent leaves sometimes exceeding that of the soil, as seen in canola (Groom et

al., 2002). Uptake of HMX from contaminated water into hybrid poplar trees

(Populus deltoides x nigra, DN-34) also showed a preferential translocation of

accumulated HMX into leaves (Yoon et al., 2002).

Similarly, accumulation of HMX into biota has been noted in a variety of

species. Uptake into estuarine amphipods (Leptocheirus plumulosus) and marine

polychaetes (Neanthes arenaceodentata) from spiked sediment was dose-dependent,

as molar-equivalent tissue concentrations of HMX increased with elevated HMX

exposure up to 100 mg/kg (Lotufo et al., 2001). HMX accumulation has also been

documented in the sheepshead minnow (Cyprinodon variegatus), whose kinetic

bioconcentration factor calculated from the study was estimated to be 0.5 mL/g in

contaminated water (Lotufo and Lydy, 2005). Earthworms (Lumbricus terrestris and

Eisenia andrei) exposed to contaminated soil at concentrations up to 396 mg HMX/kg

soil for 28 days also exhibited significant accumulation up to 77 mg HMX/kg body

weight (Robidoux et al., 2004).

This accumulation, though not astounding, has important implications because

of documented concentrations and persistence of HMX in the environment. HMX

concentrations at military installations have reached up to 2,160 mg/kg in soil at the

Canadian Forces Base Valcartier anti-tank firing range, Quebec, Canada (Jenkins et

al., 1999) and up to 3,055 mg/kg in soil at the Joliet Army Ammunition Plant, Illinois,


18

USA (Simini et al., 1995). In addition to these high levels, HMX is also fairly

persistent in the environment (t1/2= 1332,310 days in soil, 1.4 days in pure water, and

70 days in lagoon water) (ATSDR, 1997; Jenkins et al., 2003). In fact, some natural

soil and indigenous plant surveys of artillery grounds have shown HMX to be more

persistent than other explosive compounds released to the area (Dodard et al., 2005;

Groom et al., 2002; Lotufo et al., 2001; Robidoux et al., 2004).

Interestingly, though this risk exists, there is a noticeable gap in information on

both the exposure and effects of HMX to reptiles (USACHPPM, 2000, 2001, 2002;

ATSDR, 1995a, b, 1997). Reptiles have long been under-represented in

ecotoxicological investigations of pollutants, though they play vital roles in their

ecosystems as occupants of various trophic levels (Hopkins, 2000; Lovern et al., 2004;

Sparling et al., 2000; Talent et al., 2002). Reptiles occupying higher trophic positions,

in particular, have been documented to be at considerable risk of bioaccumulating

certain pesticides and trace elements (Bishop and Gendron, 1998; Hall and Henry,

1992; Hopkins, 2000; Hopkins et al., 2005). Potentiating this risk is reptilian

metabolism, which is completely dependent on ambient conditions, and thereby can

potentially increase or decrease toxicity to the organism (Talent, 2005). Many reptiles

also have close ties to the soil, utilizing it for purposes of oviposition and foraging,

thus facilitating an increased risk of exposure and effects (since most HMX

contamination is found at depths up to 15 cm from the surface; Jenkins et al., 1999;

Monteil-Rivera et al., 2003). Finally, as development of offspring occurs in two

stages in most reptiles (pre- and post-oviposition), offspring are susceptible to two


19

very different exposure pathways. For these reasons, the reproductive system of

reptiles may be particularly sensitive to toxic chemicals, and as more reptiles are being

listed as endangered worldwide the responses of reptiles to environmental stressors

have been receiving greater attention (Gibbons et al., 2000). Therefore, information

on the toxicity of explosives to reptiles is warranted and would provide valuable

insight into HMX toxicity and the use of reptiles in toxicological investigations.

The green anole (Anolis carolinensis) is a good model for laboratory toxicity

studies. This species has been used extensively in the laboratory setting, is easy and

cost-effective to maintain, and more importantly studies have indicated that anoline

responses more closely imitate the sensitivities of birds and mammals rather than

those of other poikilotherms, perhaps allowing the data a broader range of

extrapolation (Hall and Clark, Jr., 1982; Lovern et al., 2004). In relation to HMX

toxicity, anoles could be considered at a higher risk because of their strong site

fidelity, small home range, and distribution in the United States which overlaps with

many known HMX-contaminated facilities (ATSDR, 1997; Conant and Collins,

1998). The green anole in particular has a relatively high reproductive capacity

throughout its summer breeding season, which is marked by its frequent oviposition

(up to one egg every 10 to 14 days) of single-egg clutches (Andrews and Rand, 1974).

The acute and reproductive toxicity of HMX exposure to green anoles was

investigated in three phases in the present study. First, acute toxicity of HMX in the

anole was determined using the LD50 up-and-down method described by Organization

for Economic Co-Operation and Development Guideline 425 and United States


20

Environmental Protection Agency Reference Method OPPTS 870.1100 (OECD, 2001;

USEPA, 2002). This method was chosen because it requires few animals (about five

animals per sex) and provides a good estimation of the LD50. Using data obtained in

the first study, dose levels were developed for use in subsequent testing.

Because of the oviparity of the anole, contact between the potential toxicant

and the embryo could occur either when the egg is in the soil or oviduct. Therefore,

accumulation of HMX via transfer from soil to egg and via maternal transfer from

food to egg was studied to test the hypothesis that accumulation of HMX into eggs

would increase in a dose-dependent fashion, culminating in a reduction in hatching

success, growth, and survival.

Materials and Methods

Lizard Housing and Husbandry Sixty-six adult female and twenty-four adult male green anoles were purchased

from Kandys Quality Reptiles in LaPlace, Louisiana on 26 April, 2005 and housed at

The Institute of Environmental and Human Health, Texas Tech University. Each

anole was housed in its own individual 37.85-L glass aquarium, complete with

sphagnum peat moss substrate, PVC pipe for hiding, wooden perch for access to the

heat lamp, and plastic container with moist peat moss for oviposition. Aquaria were

placed on metal racks, with each animal having access to its own 40-watt heat lamp

and a shared UVB bulb (one per four tanks). The UVB bulb, operated by a timer, was

on for two hours every morning (08:00 10:00) to aide in vitamin D3 synthesis. All


21

of the aquaria were misted automatically by a Rainmaker I Expanded Fully Automatic

Misting System (Ecologic Technologies, Inc., Pasadena, MD, USA) three times daily.

Aquaria were cleaned at least once a week of fecal matter, dead skin, and cricket parts,

and sterilized every six weeks with over-the-counter vinegar. The room and individual

heat lamps were on a 14:10 light:dark cycle with the aid of timers, and the room

temperature was held at 23±2°C to mimic the summer breeding season. At a

minimum of once per month, the anoles were paired for 24 hours for breeding

purposes. Due to known sperm retention in the female anole, further pairing was not

considered necessary (Fox, 1963), and less pairing could potentially increase the

chance of an infertile egg (Licht, 1973).

Anoles were randomly assigned an identification number upon arrival and

were acclimated for at least two weeks before the start of the studies. Prior to any

dosing with HMX, all anoles were given one large cricket each morning. To ensure

proper nutrition for the anoles, crickets were given OrangeCube (Fluker Farms, Port

Allen, LA, USA) every two days and dusted with additional calcium powder once per

week.

Acute Toxicity

Standard HMX (CAS No. 2691-41-0; 99.0% pure; molecular weight = 296)

was obtained from Supelco (Bellefonte, PA, USA) and suspended in a polyethylene

glycol (PEG) solution at a nominal concentration of 250 mg/mL. Following the

OECD up-and-down method, four anoles (2 male, 2 female) were dosed via gavage

with HMX at 2,000 mg/kg body weight, which is the highest recommended dose for


22

this test (OECD, 2001; USEPA, 2002). One female control animal was dosed via

gavage with a similar volume of PEG. Animals were observed at regular intervals

throughout the first day and then three times daily for the following 13 days. Any

signs of toxicity were noted and animals were euthanized if they showed any signs of

morbidity, obvious pain, or distress. As no observable effects were seen, eight more

anoles (4 male, 4 female) were dosed in a similar fashion. Following the completion of

the acute study, all anoles were sacrificed via carbon dioxide asphyxiation and stored

at -20 °C.

Egg Exposure Studies

Soil Preparation. Soil (75% sand and 25% organic potting soil, by volume)

was mixed for 30 min. in a small cement mixer and then sifted through a 2 mm sieve

prior to spiking with an HMX/acetone solution (Supelco; Bellefonte, PA, USA). Five

kg of soil was spiked with 20 mg/kg HMX (low), 8.5 kg of soil was spiked with 200

mg/kg HMX (medium), 5 kg of soil was spiked with 2,000 mg/kg HMX (high), and 5

kg of soil was spiked with 200 mg/kg acetone (control). These concentrations were

based on the anole acute toxicity study, which found no evidence of acute toxicity at

concentrations up to 2,000 mg/kg. In addition, all of these concentrations are in the

ecologically relevant range of HMX found in soil at military installations (Jenkins et

al., 1999). Soil was sprayed with the appropriate solution, hand mixed, then sprayed

again. Soil from each dose group was then mixed for 40 min. in a rock tumbler and

placed in a darkened hood overnight to allow evaporation of the acetone. Soil was

mixed again for approximately five minutes the next morning and seven samples taken


23

randomly from each dose group for analytical verification of the concentration (see

analysis section below). The remaining soil was left for a second night to further

evaporate the acetone and was then stored in sealed containers at -20°C until needed.

Egg/PSD Study. The first egg exposure study was designed to assess the time-

dependent uptake of HMX from soil into eggs and the reliability of passive sampling

devices (PSDs) to predict concentrations of HMX in eggs. Eighteen eggs and 18

PSDs were housed individually in cups (eggs) or glass jars (PSDs) containing 100 g of

medium HMX soil (283 mg/kg soil, SE=19.35) and 10 mL of distilled water. Eggs

and PSDs were incubated in separate containers to remove the possible bias of

accumulation competition between the two matrices. Each egg was buried

approximately one centimeter below the surface of the soil and cups covered with

cellophane and secured with a rubber band. Each PSD was placed flat approximately

two centimeters below the surface of the soil and the jar sealed with a screw-top lid.

Egg cups and PSD jars were placed in the incubator (maintained between 29 and

32°C) using a randomized block design, which was stratified so that each dose group

had equal representation on the various shelves of the incubator. Three eggs and their

paired PSDs were taken from the incubator on days 1, 3, 6, 12, 18, and 30 and stored,

along with the soil from the container, at -20°C.

Eggs from the unexposed colony of anoles were randomly placed into each of

the groups of both studies so that no more than one egg from each female was placed

into any one group. The PSDs were assembled as described by Zhang et al. (2006).

Briefly, approximately 500 mg of C18 (octadecyl sorbent, Fisher Scientific, Houston,


24

TX, USA) was sealed in a polyethylene Whirl-Pak bag (Nasco, Fort Wilkinson, WI,

USA) and cut to approximately 5 cm2 in size.

Egg Only Study. The second egg exposure study was designed to assess the

dose-dependent uptake of HMX by eggs incubated in soil during the normal

incubation period. Fifty-five unexposed eggs from different females were randomly

placed into one of the four treatment groups (14 each in control, low, and medium

dose groups, and 13 in the high dose group). Nominal concentrations of HMX in each

dose group were 0 mg HMX (control), 20 mg HMX (low), 200 mg HMX (medium),

and 2,000 mg HMX (high). Eggs were placed in containers and treated exactly as

described for the first study. Egg containers were weighed weekly and distilled water

was added to replace water loss due to evaporation. Eggs were incubated until 41

days or hatching and all neonates were sacrificed within 24 hr of hatching. Snout-vent

length (±1 mm), whole body length (±1 mm), and body weight (±0.001 g) were

measured for all the hatchlings, which were then stored at -20°C prior to analysis for

HMX residues.

Maternal Exposure Study

This study was designed to examine HMX deposition into eggs following

maternal exposure and subsequent effects of HMX exposure on hatching success and

neonate survival and growth. Reproductively-mature female anoles were randomly

assigned to one of four treatment groups (15 each), consisting of a solvent control

(cricket injected with a volume of polyethylene glycol equivalent to high dose group),

low dose (20 mg HMX/kg body weight via cricket), medium dose (250 mg HMX/kg


25

body weight via cricket), and high dose (500 mg HMX/kg body weight via cricket).

In addition, three anoles were put into a negative control group and were fed a plain

cricket with no solvent injection to elucidate any effects related to the vehicle. Lizards

were housed individually as described above. Aquaria were placed on racks using a

randomized block design that was stratified so that each dose group had equal

representation on each of the shelves.

The HMX/PEG solution was mixed in a glass vial using a magnetic stir bar

with continual mixing throughout the dosing procedure to maintain the suspension.

The solution was drawn into a glass Hamilton syringe, a 28 gauge needle attached, and

then primed with solution. In an effort to validate this delivery method, each week for

10 weeks of the dosing study one cricket from each dose group was injected with the

solution and immediately stored at -20°C for residue analysis.

Anoles were dosed three times weekly for twelve weeks, beginning 11 July

2005. All animals were treated similarly throughout the study and separate utensils

(i.e., syringes and probing utensils for turning the soil looking for eggs) were used for

all dose groups so that unintentional exposure did not occur. Each female was

weighed on 10 July 2005 to get an initial pre-dose weight for calculation of the dose

for the first two weeks of the study. Thereafter, females were weighed every two

weeks and their respective dosage amounts recalculated. HMX was delivered to the

anoles by injecting the appropriate amount of HMX/PEG solution into a live cricket,

which was then offered to the anole as prey. Because the crickets often died within a

few hours of injection, each female was offered a maximum of three dosed crickets


26

throughout the day, until she either ate one of the crickets or it was determined that she

refused to eat. If a female rejected three spiked crickets, she was offered an undosed

cricket on the following day in order to minimize any problems with starvation.

Anoles were fasted on the seventh day of the week to increase their chances of eating

on the next dose day. Females were observed daily for any signs of toxicity (e.g.,

lethargy, loss of appetite, etc.).

All cages were checked daily for eggs. The first egg oviposited from each

female between ten days and four weeks after the first dosing was immediately frozen

at -20°C for later analysis of HMX residues. All other potentially viable eggs were

placed in a plastic cup with 9 g of vermiculite and 9 mL of nanopure water (milli-Q

water at 18.3 MΩ) and the top secured with cellophane and a rubber band (Lovern and

Passek, 2002). Eggs were incubated for 48 days and those that did not hatch were

frozen at -20°C for later residue analysis.

Hatchlings were housed in 21-L aquaria set up as described for adults,

including sphagnum peat moss substrate, PVC pipe for hiding, and wooden perch for

thermoregulation. The hatchling aquaria were given access to individual heat lamps

and shared access to UVB bulbs (one per five tanks). The light:dark and UVB cycles

were on the same timers as the adults. Hatchlings were fed daily using pinhead

crickets, which, as with adult prey, were fed a diet of OrangeCube and dusted with

vitamins once per week. Hatchlings were monitored daily for behavior and weekly for

developmental parameters, including body weight, snout-vent length, and whole


27

length for nine weeks. At the end of the developmental period, all hatchlings were

sacrificed and stored at -20°C.

Sample Analysis

All samples (soil, egg, cricket, and lizard) were individually homogenized with

liquid nitrogen using a mortar and pestle and dehydrated with sodium sulfate.

Weighed lizard samples from the egg exposure study were air dried for one hour and

cleaned of any soil particles before homogenization. Un-hatched egg samples from

the egg exposure study were rinsed with nanopure water (milli-Q water at 18.3 MΩ,

Barnstead NANOpure infinity system, Dubuque, IA, USA), dried, weighed, and

opened to stage the embryos development prior to homogenization. Un-hatched egg

samples from the maternal transfer study were weighed and rinsed prior to

homogenization. Because accumulation was likely to be limited and egg mass was

typically low (0.1281 ± 0.0065 g), all egg samples were homogenized in their entirety,

with eggshell and membranes included.

The extraction process followed the methods of Pan et al. (2006b). All

samples were extracted with a Dionex Accelerated Solvent Extractor (Model 200, Salt

Lake City, UT, USA) using 100% acetonitrile (analytical grade, obtained from

Supelco, Bellefonte, PA, USA) as the extraction solvent. The extraction procedure

was as follows: 5 minute preheat, 5 minute heat, 5 minute static extraction at a

constant temperature of 100°C and pressure of 1500 psi. Extracts (15-20 mL/sample)

were then purged from the cells into glass collection vials using nitrogen gas. Extracts

were diluted to 25 mL with acetonitrile in clean volumetric flasks. Five mL from each


28

extract was used as a stock solution and the remaining 20 mL was concentrated to

approximately two mL using a vortex evaporator. Concentrates were cleaned using

styrene-divinybenzene (SDB) cartridges (Supelco; Bellefonte, PA, USA). Each SDB

cartridge was conditioned twice with three mL of acetonitrile, after which the sample

extracts were loaded and eluates were collected by gravity into plastic 15-mL Falcon

centrifuge tubes (Franklin Lakes, NJ, USA). SDB cartridges were then rinsed with

one mL acetonitrile three times and the eluates collected. The final extract volume

was adjusted to five mL with acetonitrile. One mL of the final concentrated volume

was then diluted with one mL of nanopure water and filtered with through a 0.20 µm

PTFE syringe filter into amber autosampler vials.

All samples were analyzed using liquid chromatography tandem mass

spectrometry (LC-MS) utilizing the method of Pan et al. (2006a, b). Briefly, the liquid

chromatography portion used a Finnigan system, which included a vacuum membrane

degasser, a gradient pump, and an autosampler (San Jose, CA, USA).

Chromatographic separation was achieved using a Supelco RP C18 column (4.6 x 250

mm, 5-µm packing, Bellefonte, PA, USA). Mass spectrometry analyses were

conducted using a Thermo-Finnigan LCQ advantage ion trap mass spectrometer

operated in negative ion mode. Helium was used as the dampening gas for the ion trap

and nitrogen was the sheath and auxiliary gas for the ion source.

The C18 from the passive sampling devices was extracted and filtered in the

same way as the egg and soil samples. After using the vacuum manifold, the

concentrated volume was adjusted to five mL. One mL of this concentrate was mixed


29

with four mL of nanopure water and two mL of this mixture was filtered into the

autosampler vial using a 0.20 µm PTFE syringe filter. These samples were analyzed

using the HPLC.

Statistical Analysis

The acute toxicity endpoint (LD50) was estimated using OECD guidelines. An

analysis of variance (ANOVA) was used to compare mean weight, length (whole and

snout-vent length), and incubation time of hatchlings in the egg exposure study,

accumulation of HMX in eggs and dosed mothers from the maternal exposure study,

and egg weights in both egg and maternal exposure studies. Tukeys multiple

comparison of means was used as a post hoc means separation test in all analyses.

Regressions and correlations between spiked soil and eggs (data were log +1

transformed) and between eggs and PSDs were analyzed using Pearsons product-

moment correlation. Developmental endpoints from the maternal exposure study

(SVL and body weight of hatchlings) were analyzed using an analysis of covariance

(ANCOVA), using time (measured in weeks post-hatching) as the covariate. Maternal

weight change and egg production rates were also analyzed using ANCOVA with

initial weight and pre-dosing production rate as the covariates, respectively. Hatch

success in both the egg and maternal exposure studies was analyzed using chi-square.

Data are reported as mean ± standard error.


30

Results

Acute Toxicity HMX was not acutely toxic to the adult green anole. One male out of the 13

anoles gavaged in the study died, although lethality was not attributable to HMX

toxicity. Following treatment with HMX and/or PEG, the other participants in the

study showed no obvious signs of toxicity and quickly resumed normal behavior and

eating habits. Weight change over the 14-day observation period was minimal (loss of

0.098±0.06 g in females (likely related to egg retention and/or oviposition) and gain of

0.078±0.05 g in males). From these results, the approximate LD50 for the green anole

is greater than 2,000 mg HMX/kg body weight.


Initial and post-study concentrations of HMX in soil used in the artificial

nesting studies closely approximated the targeted nominal concentrations of 20, 200,

and 2,000 mg/kg (Table 1). Concentrations of HMX decreased in all soil treatment

groups during the exposure period, ranging from a 10% decrease in the 20 mg/kg

group to about 40% in the 200 mg/kg group. Nonetheless, the final concentrations

remained similar to the nominal target concentrations (Table 1).

Egg/PSD Study. Accumulation of HMX in eggs and PSDs followed a time-

dependent uptake by day 30, although concentrations at day 30 were variable (Figure

1). There was an unexplainable spike in mean HMX concentration on day 6 in the

PSDs (22.70±12.56) which was inconsistent with the remaining data and consequently

removed from analysis, along with their accompanying eggs. With the exception of


31

the PSD data on day 6, there appears to be correspondence between HMX

concentrations in PSDs and eggs, especially by day 30, though correlation analysis

shows that the relationship is not statistically significant (p=0.1280, r2=0.77; Figure 2).

Egg Only Study. Accumulation of HMX into eggs incubated in spiked soils

showed a clear dose-dependent response, reaching a maximum mean concentration of

86.6±17.8 mg/kg in the 2,000 mg/kg soil group (p<0.001, Figure 3). Because not all

eggs developed to full term, two analyses were performed; one with all eggs

regardless of embryo development and one with only embryos at stage 35 or beyond

(Dufare and Hubert, 1961). In both cases, about 92% of the variation in HMX

concentrations in eggs was explained by the concentration of HMX in soil.

Of the 55 eggs incubated in this study, 27 hatched (7 control, 6 low, 9 medium,

5 high). None of the developmental parameters measured (body weight, snout-vent

length, whole length, or incubation time) differed (p>0.05) among treatment groups

(Table 2), nor were there any differences in hatching success among groups

(x2=2.1, df=3, p>0.05; Figure 4). However, low sample size may have precluded any

strong inference from these data.


Adults. Mean prey consumption per week during the 12-week exposure period

appeared to follow a dose-dependent response (experimental control2.2 crickets,

PEG control2.1 crickets, low2.0 crickets, medium2.0 crickets, high1.8 crickets;

Figure 5). Though highly variable and not statistically different between groups

(p=0.1975), anoles in the HMX treatment groups appear to consume fewer dosed


32

crickets per week than the control anoles (Figure 5). This lowered consumption did

not considerably alter the intended dose levels, however, as a clear separation of doses

was maintained between the treatment groups (Table 3). Reduced prey consumption

also coincides with patterns observed in the percent weight loss throughout the study

(Figure 6). Anoles in the three HMX treatment groups (low, medium, and high)

consistently maintained a lower percentage of their original body weight than did

either of the control group anoles, though there was no statistical significance in

weight change between groups (p=0.68). Percent recoveries from the dosed crickets

frozen for method validation show that the expected concentrations administered to

the anoles were accurate (Table 4), so that effects could justifiably be attributed to

each dose group.

Consistent with the estimated doses of HMX for each dose group, whole body

concentrations of HMX in anoles was dose-dependent (Figure 7). One outlier was

removed from the medium group for this analysis since its concentration (25.6 mg/kg)

was substantially higher than all other data points. Anoles in the high dose group had

a whole-body concentration of HMX nearly 3-fold greater than those in the medium

dose group, which was significantly greater than the control groups, but not the low

dose group (p<0.001). There was no significant difference in mean concentrations

between the control groups and the low treatment group (20 mg/kg).

With regard to egg production rates, the findings are unclear as to what effect

HMX might have on anoles. There was no statistical significance in the egg

production rates of the anoles among treatment groups before or after exposure began


33

(p=0.28). However, as seen in Table 5, egg production rates appear much lower in the

high and medium groups than in the control groups even before dosing. The fact that

the animals were placed in the room using a randomized stratified block design rules

out the influence of any external variables in the animal room. Therefore the reason

for this discrepancy remains unclear.

Eggs and Hatchlings. HMX was detected in eggs from all HMX-treatment

groups, and generally followed the similar dose-dependent response observed in the

maternal whole-body residue analysis (Figure 7). In an effort to control for the

variability in HMX consumption within each group, two statistical analyses were

completed; one in which the eggs were analyzed collectively (Figure 8a) and another

in which the eggs were pooled for each mother to compensate for those females who

consumed more on average (Figure 8b). Interestingly, the same statistical relationship

was observed regardless of whether the eggs were censored or not. The high treatment

group had a significantly greater concentration of HMX than all other groups.

Concentrations of medium treatment group eggs (maternal dose of 250 mg/kg) were

significantly different than the control groups, but was not different than the low

treatment group eggs (maternal dose of 20 mg/kg) (p<0.001; Figure 8a). Egg weights

were not considered in this analysis, since a separate analysis of variance found

weights to not be statistically different (p=0.1212).

Accumulation of HMX into the egg, however, had unclear effects on embryos

and hatchlings. Overall hatching success was low (<50%) for all treatment groups

except the experimental control (Figure 9). Furthermore, there were no significant


34

differences among groups in hatching success (x2 = 9.0, df = 4, p>0.05) or

developmental parameters (body weight (p≤ 0.001; Figure 10) and snout-vent length

(p=0.019; Figure 11) showed a significant interaction with age, but did not show

significance among treatment groups). Both of these parameters appeared to be

greater for the PEG control and high dose groups compared to the other groups with

increasing age.

Discussion

Acute Toxicity

The low sensitivity of the adult green anole to the effects of acute exposure to

HMX is not entirely surprising. Mice and rats also were relatively insensitive to single

doses of HMX (LD50 =1,700-3,200 mg/kg HMX and 5,500-6,400 mg/kg HMX,

respectively) (Cuthbert et al., 1985). However, assessing toxicity under ideal

laboratory conditions may be misleading. Toxic effects of HMX could fluctuate

during in situ exposure, where the anole is subject to various environmental stressors,

such as in the winter months as seen with pyrethrin exposure (Talent, 2005). It is

during these months that the reptilian metabolism slows considerably, compromising

the anoles ability to metabolize and/or excrete HMX.

The possibility of inconsistent toxicity in field situations notwithstanding, the

insensitivity of the anole to acute HMX exposure (estimated LD50 >2,000 mg/kg)

should be considered an adequate estimate of overall response under field conditions.

This low sensitivity is congruent with acute oral toxicities of TNT and RDX, with


35

LD50s in rats and mice reaching 1,320 mg/kg/day and 1,012 mg/kg/day, respectively,

in males and 795 mg/kg/day and 660 mg/kg/day, respectively, in females (ATSDR

1995a, b; Dilley et al., 1982). As with many chemicals, however, low lethality does

not preclude sublethal effects, as seen with all three of these compounds in other

species (Everett and Maddock, 1985; Greenough and McDonald, 1985a, b; Homma-

Takeda et al., 2002; Nipper et al., 2001; Peters et al., 1991; Robidoux et al., 2002;

USACHPPM, 2000).


Accumulation of contaminants from media, especially soil, into eggs is a

particular concern for most reptiles, though this exposure pathway has, until recently,

received little attention. Of the few studies located, reptile eggs have been shown to

accumulate contaminants from the soil, which to some degree is driven by the

structure-activity relationship between the concentration and log Kow of the

contaminant (Cañas and Anderson, 2002). HMX has a low log Kow (0.26) (ATSDR,

1997) which would seem to facilitate its uptake into incubating eggs. Indeed, it is

suggested that HMX should have the highest potential for accumulation relative to the

explosives RDX (log Kow=0.90; ATSDR, 1995), TNT (log Kow=1.60; ATSDR, 1995),

and CL-20 (log Kow=1.92; Monteil-Rivera et al., 2004).

Perhaps more germane to the present study are the trends associated with the

soft, pliable shells of most squamates. Because of the structure of this eggshell, in

particular, water and air are allowed to pass in and out of the egg throughout

incubation (Sexton et al., 2005). Consequently, there appear to be three possible


36

conduits into the egg: as vapor, in water, or via attachment to eggshell proteins

(Talent, 2002), all of which probably allow passive movement of HMX into the egg.

Entering the egg as a vapor probably accounted for only a portion of the

accumulation in the study, since the vapor pressure of HMX is so low. Moreover,

Cañas and Anderson (2002) noted that vapor pressure and Henrys law constants were

some of the worst predictors of accumulation of organochlorines into snake eggs from

contaminated nesting material. Compounding this problem is that the partitioning of

HMX between soil and air is not well understood, so it is unknown whether HMX is

consistent with the patterns shown in organochlorines. However, since it is likely that

HMX did accumulate to some extent via vapor phase, it is intuitive that greater

accumulation into the egg was noted during the later stages of incubation, due to the

increased number of eggshell pores as seen in the American alligator (Alligator

mississippiensis; Kern and Ferguson, 1997).

Uptake of HMX in water is also plausible, since it would also seem to favor slow

accumulation into the egg because of the low solubility of HMX in water. Increasing

the likelihood of this particular mechanism to transport HMX are the findings of

Yoshizaki and Saito (2002), who found that the thickness of the eggshell membrane

actually decreased throughout incubation in quail eggs, apparently increasing the rate

of water uptake through the membrane. Additionally, Alava et. al (2006) found that

the percent lipid content of late-stage yolk in Loggerhead sea turtle (Caretta caretta)

eggs increased to approximately 1.5 times that of early-stage yolk. Along with this

increase in lipid was an approximately doubled concentration of certain hydrophobic


37

organic pollutants (PCBs, DDTs, chlordanes, and dieldrin) in the late-stage egg on a

wet-mass basis (Alava et al., 2006). It was expected that the similar hydrophobicity of

HMX would also drive late-stage accumulation.

The final, yet less likely, option is sorption of HMX to the organic matrix of the

eggshell. Eggshell sorption has been suggested to be the preferential route of

movement for chemicals with a higher affinity for soil particles (and especially

organic matter; Talent et al., 2002). Sorbed chemicals could then be drawn into the

egg late in development, when embryos begin to absorb minerals from the eggshell

(Cai et al., in press). A potentially confounding factor in this third option is that

Monteil-Rivera et al. (2003) showed evidence that organic matter doesnt regulate the

sorption of HMX in soil, making the other two exposure pathways more likely.

Regardless of the exact mechanism(s) by which HMX is able to enter the egg, which

at this point remains unknown, any one or combination of the above mechanisms

appears to be a plausible explanation of accumulation in the present study.

Because of these proposed transport mechanisms, some accumulation of HMX

was expected to occur; therefore, the egg exposure studies were designed to provide a

realistic representation of field conditions, encompassing both a realistic HMX

concentration range and length of exposure. The concentrations used were intended to

represent not only the range of potential exposures in the field (reaching in excess of

2,000 mg/kg) (Jenkins et al., 1999; Simini et al., 1995), but also the range of possible

effects. Eggs exposed to the lowest concentration level were expected to exhibit little

effect, while potentially acute effects were expected at the highest concentration, since


38

2,000 mg/kg body weight was the estimated oral LD50 for adult anoles. Sublethal

effects, on the other hand, were expected at the medium concentration level because

disturbances were noted in earthworm (Eisenia andrei) reproduction tests at

concentrations ≥ 280 mg/kg HMX in artificial soil (Robidoux et al., 2001). Possible

effects due to toxicant exposure at any of these concentrations post-oviposition

(approximately stage 30 of the 40-stage development) could induce changes in certain

basic morphological traits in the developing hatchling, such as formation of the eyes,

limbs, tail, claws, pigmentation, etc. (Dufaure and Hubert, 1961), which would be

severely detrimental to the development and survivability of the hatchling.

Egg/PSD Study. Although traditionally used to monitor air pollutants, PSDs

have been an effective monitor in terrestrial environments for toxicants, including

PCBs, certain pesticides, and most recently explosive metabolites MNX and TNX

(Zhang et al., 2006). Despite the fact that no work has previously been done with

PSDs and HMX, the correlation was predicted to be as strong as with MNX and TNX

(r2 ≥ 0.82 for all tested soils), which are RDX metabolites (Zhang et al., 2006).

Accumulation of HMX into the PSD was somewhat unpredictable and

characterized by minimal accumulation until after 18 days of exposure. This lag

phase is lengthier than that noted in MNX and TNX studies using the same PSD

design (four to eight days), but is probably due to the same diffusion processes noted

in Zhang et al. (2006). There are a number of factors that could contribute to this

increase in lag time and variability in the present study, including lowered rate of

diffusion from medium to sorbent due to lower vapor pressure and/or greater log Kow


39

of HMX relative to MNX and TNX. Variability could also be due to experimental

parameters, such as either low moisture content of the soil or shortened exposure

period, given that Zhang et al. (2006) placed their PSDs in soil with 75% moisture and

had exposure periods of up to 60 days. In the present study, however, leaving the

PSDs in the soil for such a long period of time would be unrealistic, because at the

incubation temperature (29-32 °C) the anoles were expected to hatch within

approximately 34 days (Lovern et al., 2004).

Accumulation of HMX into the egg also occurred predominately in the latter

half of incubation with maximum concentrations likely not reached until the end of

incubation. This same late-stage increase in concentration has been seen in

loggerhead sea turtles (C. caretta) and white leghorn chickens (Gallus domesticus)

with organic pollutants (Alava et al., 2006; Bargar et al., 2001a). In both of these

studies, lipid content of the yolk increased dramatically between the beginning and

end of embryonic development, which resulted in a two-fold greater concentration of

POPs (turtles) and a five-fold greater concentration of PCBs and endosulfan

(chickens) (Alava et al., 2006; Bargar et al., 2001a). Therefore, although both studies

assumed accumulation was solely a function of maternal transfer, it can be suggested

by this study that a similar increase in lipid content of the egg caused the rapid

increase in accumulation of HMX during the final days of incubation and embryonic

development. In other words, the increased lipid content in the egg changed the

gradient and allowed greater accumulation of HMX toward the end of development.


40

Comparison of the accumulation rates by eggs and PSDs appears to show quite

variable uptake efficiency, with rapid accumulation in the PSD occurring almost one

week later than in the egg. Exact reasons for this discrepancy remain unclear,

especially since accumulation into both media appears to be a passive process. It has

been suggested from an aquatic study and column and batch studies that HMX does

not reach equilibrium, so the disparity does not appear to be a function of variability in

equilibration times (Lotufo and Lydy, 2005; Myers et al., 1998). It was further

suggested by Ainsworth et al. (1993) that RDX reached a steady state in sterilized soil

within four hours, so if HMX were to near equilibrium it presumably would have done

so before the end of the 12 to 18 day lag phase noted in the present study. Other

possible explanations might include simple differences in permeability of the PSD and

egg membranes, or subtle differences in uptake regimes (e.g., greater water uptake by

eggs than by PSDs). Because neither the use of PSDs in HMX-contaminated soil nor

HMX uptake into reptilian eggs is well documented, further research is warranted to

elucidate the optimal soil parameters necessary for a better correlation, such as percent

moisture, pH, organic matter, or even different concentrations of HMX in the soil, as

well as understanding the dynamics of contaminant uptake in soft-shelled eggs.

Egg Only Study. As expected, a distinct pattern of HMX accumulation into

anole eggs incubated in increasing concentrations of HMX-spiked soil was observed

(p<0.001). In fact, there appeared to be no threshold of concentration below which

only minimal uptake could occur; exposure to each of the concentrations in this study

showed significant accumulation compared to controls and other treatment groups


41

(p<0.001). Furthermore, HMX concentrations in the egg were strongly correlated

with HMX concentrations in the soil (r2=0.92). In order to control for increased

accumulation of HMX due to increased metabolic activity of viable eggs as opposed to

no activity in non-viable eggs, a second analysis was done in which the data were

censored to only include eggs containing an embryo at or beyond developmental stage

35 (Dufaure and Hubert, 1961). Interestingly, censoring the data did little to alter the

observed correlation, which seems to indicate that viability and/or metabolic activity

was not a determinant of accumulation in the egg. Therefore, the data suggest that egg

accumulation, at least in the anole, is a passive process.

This finding also raises the concern that the method of homogenization in the

present study could present a confounding factor. Because the entire eggincluding

the eggshell, associated membranes, and residual yolk sacwas homogenized with

the developing embryo or hatchling, the concentration of HMX believed to be

contacting the embryo might prove to be artificially inflated, as noted by Bargar et al.

(2001a) in white leghorn chickens (G. domesticus). Although the eggshell was rinsed

thoroughly before homogenization, it is possible that HMX could have accumulated in

the eggshell itself, rather than passing through it. Additionally, the inclusion of

embryonic membranes, including the eggshell and chorioallantoic membrane (CAM),

could have further skewed the results. Furthermore, Bargar et al. (2001a) has shown

that accumulation of lipophilic compounds (especially PCBs and endosulfan) in the

yolk and albumin of white leghorn chicken eggs can actually exceed that of both the

CAM and embryo, but may not ever be utilized by the embryo during development.


42

Therefore, whole egg analysis could potentially overestimate embryonic exposure if

any of these compartments were analyzed with the rest of the egg, especially in a

species in which the partitioning of toxicants is not well established. Nonetheless,

now that the overall accumulation of HMX into incubating eggs has been documented,

additional research determining the transport process(es) involved in HMX uptake and

the partitioning of HMX once it passes the outer eggshell layer will be able to further

elucidate the risk of these embryos to HMX exposure.

Minimal effects in hatchlings pertaining to HMX exposure were noted in this

study. Neither hatchability nor any of the developmental parameters measured (body

weight, snout-vent length, whole length, or incubation period) were significantly

different from controls. Additionally, no obvious deformities were noted in any of the

hatchlings. What effects may appear past the 24-hour observed period remain unclear

and extrapolation of these data to long-term effects is not recommended.

Future research into the effects following exposure to HMX via contaminated

soil contact could also include contact with natural soils. While the present study

utilized artificial soil and found no discernable effects, Robidoux et al. (2000; 2002)

found that natural forest soils actually increased reproductive toxicity of TNT and

RDX in E. andrei. According to the studies, a similar concentration of TNT in natural

forest soil decreased the productivity of cocoons by 41%, while cocoon production

was only decreased by 4% in artificial soil, thus highlighting the inherent differences

in behavior of contaminants in varying types of soil. Therefore, although anoline

eggs would probably not take in the same amount of nutrients from the surrounding


43

media as the earthworm, it would be interesting to determine whether and to what

extent different soil parameters, such as pH, cation exchange capacity, and organic

matter, would change the accumulation potential and/or effects in the developing

embryo.

It appears, therefore, that although anoline eggs are prone to accumulation of

HMX from contaminated nesting media, hatchlings appear to be fairly resistant to the

effects of HMX for the measured endpoints. Whether the observed lack of effect was

due primarily to the resistance of the anole embryo to toxic effects from HMX

exposure, or to some compartmentalization and/or transportation issue with the egg

itself remains unclear. However, from the present study it can be assumed that HMX-

contaminated media would pose little threat to developing embryos at concentrations

likely to occur in the environment.


Based on its low solubility in water and log Kow (0.26) it has been assumed

that HMX would be unlikely to bioconcentrate, accumulate, or magnify in the food

chain, though none of these behaviors have been extensively studied (ATSDR, 1997).

The only study located that analyzed wildlife living on or near sites known to be

contaminated with explosives (Aberdeen Proving Ground, Maryland, USA and Joliet

Army Ammunition Plant, Illinois, USA) found no detectable levels of HMX, RDX, or

TNT (among others) in either harvested deer muscle and liver tissues or turtle muscle

tissue (USACE, 1996). However, because of its persistence in the environment, as


44

noted previously, concern has been raised that concentrations could eventually

increase to levels that are toxic to plants and other biota.

In an attempt to address this lack of exposure data in reptiles, specifically, the

present study on maternal transfer was designed around a modified food chain. This

approach is a modification from that suggested by Hopkins et al. (2005), in that prey is

spiked, instead of allowed to feed on contaminated food. This modified approach

allows less variability in the data than the method used by Hopkins et al. (2005), while

sacrificing less ecological realism than a traditional gavage study. The benefit of

offering spiked prey is that it incorporates a biomarker of susceptibility into the study,

which will help to increase the extrapolation potential of the dataset as well as help

define the animals true risk of toxicity in the field (without the interaction term

between the toxicant and receptor (lizard and/or egg) there is no toxicity, regardless of

the concentration).

The rejection of contaminated prey was likely to play a large role in the dosing

portion of this study. Previous studies have noted that the green anole does have some

ability to discriminate prey based on taste (Stanger-Hall et al., 2001). Furthermore,

wasting of HMX-treated laboratory animals has been noted in past studies with quail

(Colinus virginianus), Fischer 344 rats, and earthworms (Eisenia foetida) (Brunjes et

al., 2007; Everett et al., 1985; Greenough and MacDonald, 1985b; Johnson et al.,

2005; Phillips et al., 1993). In male and female rats exposed to 504,000 mg of

HMX/kg body weight/day for 13 weeks, a non-significant yet apparently dose-

dependent decrease in body weight was observed (Everett et al., 1985). Likewise,


45

Greenough and MacDonald (1985b) noted a decreasing trend in body weight gain in

rats exposed to 370 mg HMX/kg body weight/day for 14 days. In particular, with

striking similarity to the present study, dietary dosing studies with quail showed

significant dose-dependent decreases in food consumption and concomitant weight

loss in treated animals (Brunjes et al., 2007; Johnson et al., 2005). Because of the

severity of the food aversion and loss of body weight, the effects of HMX on egg

production in the quail were not discernable in the Johnson et al., (2005) study.

Although these effects were deemed by the authors to be due solely to a palatability or

avoidance issue with the HMX-contaminated food itself, of note is a dermal uptake

study on RDX-contaminated soil and salamanders (Plethodon cinereus), whose dose-

dependent weight loss was attributed to a decreased ability of the animal to forage

(Johnson et al., 2004).

Therefore, in an attempt to alleviate this problem, anoles were dosed three

times weekly and the highest dose (500 mg HMX/kg body weight) was set at 25% of

the estimated LD50. The high end of the dose range was set at 500 mg/kg because

pilot data suggested rejection of crickets spiked with HMX at concentrations of 1,000

mg/kg. Anoles were also monitored daily to note frequency of prey consumption and

body weight was measured biweekly.

In addition to accumulation of HMX in adults, the possibility of maternal

transfer of HMX was also of interest, as the maternal transfer of toxicants has been

extensively documented in numerous species and with a variety of compounds.

Interest in the past has predominantly focused on the transfer of toxicants in mammals


46

and birds, with especially scant data on maternal transfer in herpetofauna (Sparling et

al., 2000). Of the available literature for reptiles, the focus has been on the uptake and

transfer of selenium (Se) and other trace elements from coal ash deposition and

organochlorines primarily from pesticide use (Burger and Gibbons, 1998; Hopkins et

al., 2005; Nagle et al., 2001; Roe et al., 2004; Russell et al., 1999).

Two seemingly common trends in these transfer studies in reptiles are selective

transfer of contaminants and a high degree of variability. Nagle et al. (2001), for

example, found accumulation of Se, arsenic (As), cadmium (Cd), and chromium (Cr)

in the adult slider turtle (Trachemys scripta), but only Se was transferred to offspring.

Likewise, Burger and Gibbons (1998) found limited transfer of Cd and Cr in sliders in

and around the Savannah River site. Although not a reptile study, Barger et al.

(2001b) found even greater specificity of maternal transfer capabilities of three PCB

congeners (105, 156, and 189) in white leghorn chickens (G. domesticus). While this

transfer selectivity is interesting to note, no meaningful inferences could be drawn

from these studies as to the ability of HMX to enter the egg relative to other

compounds.

The second common trend in the reptile transfer studies is the high variability

of the reproductive output data, which often compromises the power of the data and

makes trends more difficult to decipher (Hopkins et al., 2004; Nagle et al., 2001;

Russell et al., 1999; Van Meter et al., 2006; present study). There are several possible

explanations for this trend toward high variability, which could include a failure of all

studies to consistently normalize their data for lipid content. Russell et al. (1999)


47

noted that lipid normalization of the ratio of egg concentration of PCBs to maternal

muscle tissue concentration of PCBs actually decreased the inter-species variation of

this ratio in fish by 20-fold. A second explanation can be seen in changes of egg size

and composition. Fernie et al. (2000) found uptake of PCBs by American kestrels

(Falco sparverius) caused a shift in egg composition which produced a higher yolk

and lower albumin content in heavier eggs. The increased concentration of PCBs in

these heavier eggs was believed to be a result of the increase in lipid content. Another

explanation, which applies more to field studies, could be the pooling of eggs without

censoring for developmental stage, which has been shown to increase variation, as

seen in Alava et al. (2006). Field studies also had the disadvantage of not knowing the

full diet and range of the mother and did not take into account nesting media toxicants

(though Cañas and Anderson (2002) and the present study have shown accumulation

through soil to be another viable exposure pathway), all of which could have

drastically increased variability. Finally, inter-species variation could also play a role

in the high variability of transfer data, owing to the fact that differing amounts of lipid

stores often go into reproduction in different species (Russell et al., 1999).

Adults. The present study examined the effects of repeatedly dosing the adult

green anole and, via maternal transfer, its offspring with HMX. Chronic exposure to

HMX by adult green anoles at doses up to 500 mg HMX/kg body weight three times

weekly for twelve weeks did not directly cause mortality. Neither did it cause any

obvious treatment-related toxicity in the dosed animals. The low sensitivities of the

anoles to the direct toxicity of HMX at the levels tested was expected and, more or


48

less, desired, as the more subtle sublethal effects of reproduction were the overall

target in this study. The findings also suggest that the introduction of HMX to the

anoles sole source of food causes a dose-dependent weight loss among treatments,

though this weight loss is not significantly different from the controls. In comparison,

significant weight loss and food aversion has been noted in HMX feeding studies with

quail at concentrations exceeding approximately 100 mg/kg (Brunjes et al., 2007;

Johnson et al., 2005). The trends noted in the present study are less dramatic than

those in quail, most likely because an alternate food source was available for those

anoles that refused HMX-spiked prey, an option that was not available in the quail

studies. However, whether weight loss effects in the anole would become significant

over time, and if the trends already seen are due to food repellency via simple

reduction in food consumption (due to taste or smell) or adverse affects associated

with decreased foraging abilities or ingestion remains unclear at this time.

Weight loss and repellency notwithstanding, findings suggest a dose-dependent

accumulation of HMX into the body of the anole, with statistical significance seen at

doses exceeding 250 mg/kg three times weekly (or up to 750 mg/kg/wk). While

administered concentrations were fairly high, it is difficult at this point to determine

what detrimental effects this had on the reproducing female, as no discernable toxic

effects were seen in the females. Interestingly, though anoles in the high treatment

group had opportunity to receive up to 18,000 mg/kg by the termination of the study, a

mean accumulation of approximately three mg/kg was all that remained in the anole.

Possible reasons for such a low accumulation might include poor absorption through


49

the gastrointestinal tract (approximately 85% and 70% elimination of dietary HMX in

the feces within four days in rats and mice, respectively; ATSDR, 1997), or a

relatively high rate of excretion of HMX through feces and/or egg deposition. Though

little can be said about similar accumulation in other reptiles, comparable doses (100

mg/kg/day) in food for 14 days produced excitability and hyperkinesia in mice

(Greenough and MacDonald, 1985a), neither of which were noted in the anole.

Egg productivity did not appear to be affected by exposure to HMX though

this might be misleading, since egg production was low altogether. The lowered

production rates of the treatment groups could have been impacted by reduced food

consumption of the treatment groups, as seen in quail (Brunjes et al, 2007; Johnson et

al., 2005). However, an interesting trend with regard to the present study seems to

emerge when looking at the two control groups. Though not statistically significant,

all treatment groups which involved the vehicle (PEG; i.e., all treatment groups except

the experimental control) experienced a decrease in egg production rate following

onset of the dosing period. Unfortunately, because of poor sample size in the

experimental control group (n=3), no meaningful conclusions can be drawn from this

trend.

The addition of the consumption rate variable (i.e., modified food chain), on

the other hand, allowed some assumptions to be made about the movement of HMX

under normal field conditions. Specifically, although the results of the present study

show that HMX can readily accumulate into biota, it appears that under field

conditions HMX would not likely significantly bioaccumulate, due to its aversive


50

reactions in the females. Indeed, although the anoles in the high dose group consumed

up to 10,000 mg/kg HMX in prey over the 12 week period, on average they retained

only three mg/kg HMX (therefore a bioaccumulation factor of <0.001). Likewise,

HMX appears especially unlikely to biomagnify in the food chain, as it seems that

anoles in the field would first try to find other sources of prey, rather than those

carrying a heavy load of HMX, which is consistent with feeding studies in quail

(Brunjes et al., 2007; Johnson et al., 2005). This preferential feeding behavior,

coupled with the intense spatial heterogeneity of HMX at contaminated sites, is likely

to further decrease the significance of accumulation in both the animal and its

offspring as seen with DDE concentration in the eggs of prothonotary warblers

(Protonotaria citrea) and European starlings (Sturnus vulgaris; Reynolds et al., 2004).

Eggs and Hatchlings. The present data also suggest that HMX is able to enter

the egg via maternal transfer in a dose-dependent manner. Transfer also appeared to

be fairly uniform among females, since pooling the eggs to remove individual

maternal bias had no effect. Surprisingly, variability as a result of developmental

stage, which has been a significant source of variability in other studies (Alava et al.,

2006), did not seem to be a problem.

Of further note is the relative amount of HMX found in the egg when

compared to the final concentration of the female. In the low and medium treatment

groups, it appears that an almost identical concentration was observed between the

mother and egg (Figures 7 and 8). This rapid excretion of HMX into eggs is

consistent with HMX deposition noted in Brunjes et al. (2007) in quail. However,


51

when nominal administered doses reached 1,500 mg/kg/wk (high group), maternal

concentrations were approximately double that found in the egg. Whether the trend

represents an accumulation threshold in the mother, or the heightened maternal

concentration was solely due to an inadequate amount of time for excretion is

uncertain at this time. Future research could be warranted to determine the half-life of

HMX in the anole to further elucidate this finding. In relation to other organic

chemicals, it appears that HMX is less likely to accumulate in developing embryos

due to maternal transfer than other hydrophobic compounds such as PCBs (0.4 to

1.2% in the chicken [G. domesticus] and 10% in the arctic tern [Sterna pardisaea] and

herring gull [Larus argentatus]), TCDD (1.1% in ring-necked pheasant [Phasianus

colchicas]), and DDT (22.4% in S. pardisaea) (Bargar et al., 2001b; Lemmetyinen et

al., 1982; Nosek et al., 1992).

Calculated residue concentrations in the eggs of this phase of the study might,

again, be an overestimate of exposure due to the homogenization technique. Residues,

though only analyzed in unhatched eggs, are probably analogous to what would have

been seen in the eggs of hatchlings, as seen with maternal transfer of PCBs in

American kestrels (Fernie et al., 2000). However, as noted in Bargar et al. (2001a)

and above, some HMX, though present in the egg, was likely never in direct contact

with the embryo. Because further developmental data was desired, hatchlings were

not immediately sacrificed to determine residues and by the end of the growing period

likely depleted their HMX stores. Therefore, little can be said about the true


52

contaminant load of the embryo, though the reported residues can be used as a fairly

accurate estimate of exposure.

Though accumulation in some sense is apparent, the effects that this

accumulation of HMX may have on the developing embryo appear to be minimal. In

the present study, neither hatching success nor developmental parameters (body

weight, snout-vent-length) appeared significantly different among treatment groups.

Unfortunately, growth rates were inexplicably depressed in all hatchling anoles.

According to growth rates observed in Michaud (1990), hatchlings should have

reached a snout-vent length of between 36 to 42 mm by the termination of the nine

week growth period; however the mean growth rate for any of the groups was just

over 26 mm. Because growth rates were low in all groups and were not dose-

dependent, it is unlikely that HMX was a causal factor. However, when looking at the

observed data, an interesting trend seems to begin to emerge by approximately week

eight, in which the low and medium group hatchlings weight and length growth rates

begin to decline. Should the study be carried out further into the development of the

hatchlings and, perhaps, even into the reproductive maturation of the individuals, more

pronounced effects might be seen.

Interestingly, a comparison of the accumulation and hatch success between the

two exposure methods shows a higher rate of accumulation from contaminated nesting

media with a corresponding increase in hatching success (Figures 12 and 13).

Though this appears somewhat counterintuitive, it might be explained by the presence

of the vehicle in the maternal exposure study (PEG). During the egg exposure study,


53

eggs were exposed to HMX in the absence of PEG and had an overall hatch success of

approximately 49.1%. However, during the maternal exposure study, when eggs were

exposed to HMX in the presence of PEG, hatching success decreased to an average

success of approximately 34.9% in all treatment groups. The trend becomes most

apparent when comparing the hatching success of the groups exposed to the highest

concentrations of PEG (the PEG control group (22.7%) and the high treatment group

(23.1%)) to the group with no exposure to PEG (the experimental control group

(69.2%); Figure 12). Indeed, it appears that as the dose of PEG (not HMX) increases,

hatching success decreases. It is unlikely, therefore, that the decreases in hatching

success noted in the maternal exposure portion of the study were due to HMX

exposure.

From the data obtained from the different phases of the present study, it is

apparent that HMX is able to accumulate into living anoline tissues via direct (i.e.,

accumulation from diet or nesting media) and/or indirect (i.e., maternal transfer)

pathways. Although the levels of exposure were fairly high, the extent of

accumulation, especially in the developing embryo, appeared to be relatively minimal.

Also, effects on the exposed hatchlings (including hatch success, snout-vent length,

and body weight) were minimal. Excluding the decrease in food consumption and

body weight, the adult anoles also appeared to show minimal effects to the levels of

exposure used in this study.

Although HMX accumulation was evident and dose-dependent,

bioaccumulation levels were not presented in this study because they were particularly


54

low (less than 0.05 in eggs exposed via contaminated soil; less than 0.001 in adults

exposed via contaminated prey). On a global scale, only compounds with a

bioconcentration or bioaccumulation factor of greater than 5,000 or a log Kow of

greater than 5 ate considered bioaccumulative (AEAT, 1995). It appears, therefore,

that the surrogate for bioaccumulation factors (log Kow) was fairly accurate in

predicting the low bioaccumulation potential of HMX. This finding is consistent with

a recent study of HMX bioconcentration in marine mussels (Mytilus galloprovincialis;

Rosen and Lotufo, in press). Rosen and Lotufo (in press), went on to suggest that

HMX is indeed least likely to bioaccumulate in comparison to RDX and TNT.

Even though the data presented from this study note limited toxicity in green

anoles, further research is needed to more fully understand how the more realistic

conditions of the natural environment would influence the toxicity of HMX. For

example, the actual transfer rate from soil and/or prey to impacted predators in the

spatially heterogeneous contaminated areas seen in actual field conditions would be

important to note, as this would most likely add a greater degree of variation to the

data than seen in the present study, as suggested in Reynolds et al. (2004). In addition,

as mentioned previously, the long-term effects of HMX in hatchlings and adults

remain unclear, and subtle trends that were not significant in the present study may

reach significance over the course of two breeding seasons or more. Finally, other

environmental stressors that were excluded from the present study, such as limited

prey availability or less than ideal temperatures, could also heighten toxic effects.


55

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64

Table 1. Comparison of HMX concentrations in soil at the initiation of the artificial nesting study and at study termination (87 days later).

Pre-Study Post-Study Nominal Concentration

(mg/kg) Mean

(mg/kg) SE Mean (mg/kg) SE

0 ND ----- ND ----- 20 19.76 0.622 17.72 0.194 200 283.4 19.4 174.4 3.34 2000 2244 848 1761 22.4 Table 2. Comparison of mean (± SE) growth parameters of hatchling green anoles following exposure to HMX via soil. WL = whole length, SVL = snout-vent length, Incubation = incubation period, Control = 0 mg/kg HMX, Low = 20 mg/kg HMX, Medium = 200 mg/kg HMX, High = 2,000 mg/kg HMX. Analysis of variance showed no statistical difference among dose groups for each of the variables (p>0.05).

Treatment

Parameter Control (n=7) Low (n=6)

Medium (n=9)

High (n=5)

Body Weight (mg) 331.9 ± 11.23 318.9 ± 29.47 321.6 ± 14.02 345.2 ± 15.16WL (mm) 66 ± 1.4 64 ± 0.70 60 ± 3.2 67 ± 2.0 SVL (mm) 24 ± 0.29 24 ± 0.22 23 ± 0.28 24 ± 0.68 Incubation (d) 30 ± 0.81 29 ± 0.67 29 ± 0.26 30 ± 0.63


65

Table 3. Comparison of the target weekly dose, actual mean (± SE) weekly dose of HMX per anole, and mean weekly dose of HMX given to an average-weight female green anole (2.8 g) over a 12-week study. Although the eating habits of the individual anoles were highly variable, the distribution of doses was separated among dose groups. Dose groups are not statistically different (p=0.0279).

Table 4. Comparison of the mean dose and percent recovery of HMX in crickets injected once a week for ten weeks (n= 10).

Dose Group Mean HMX Dose (mg) SE

Mean Percent Recovery of

HMX SE

PEG Control ND ----- ND -----Low 53.70 5.09 103.3 9.79Medium 742.7 172 114.3 26.5High 1191 161 91.59 12.4

Table 5. Comparison of the total number of green anole eggs per dose group and the number of eggs per female per week. There was no treatment-related effect on egg production (p=0.28).

Pre-Dosing (10 Weeks) Post-Dosing (12 Weeks) Maternal Dose Group Eggs/Female/

Week (± SE) Eggs/Dose

Group Eggs/Female/ Week (± SE)

Eggs/Dose Group

Exp. Control 0.30 ± 0.14 9 0.36 ± 0.10 13 PEG Control 0.38 ± 0.11 30 0.26 ± 0.03 40 Low 0.45 ± 0.08 36 0.21 ± 0.02 38 Medium 0.33 ± 0.06 30 0.23 ± 0.06 27 High 0.21 ± 0.05 15 0.20 ± 0.05 26

Dose Group Target Dose per

Female (mg/kg/week)

Mean Individual Weekly Dose (mg of HMX)

Mean Actual Weekly Dose (mg/kg/week)

Exp. Control 0 0.00 ± 0.00 0.00 PEG Control 0 0.00 ± 0.00 0.00 Low 60 0.11 ± 0.01 39.7 Medium 750 1.25 ± 0.11 451 High 1500 2.31 ± 0.17 834


66

Figure 1. Mean (± SE) concentration of HMX in fertilized green anole eggs and passive sampling devices (PSDs; C18) incubated for up to 30 days in soil spiked with a nominal concentration of 200 mg HMX/kg soil. Each time period had three replicates.


67

Figure 2. Comparison of the accumulation of HMX into passive sampling devices (PSDs) and green anole eggs incubated for up to 30 days in soil spiked with a nominal concentration of 200 mg HMX/kg soil. Each time period had three replicates. Data were not significant (p=0.1280).


68

Figure 3. Comparison of the accumulation of HMX into green anole eggs at four nominal concentrations of HMX in soil: control (0 HMX; n=7), low (20 mg/kg HMX; n=7), medium (200 mg/kg HMX; n=10), and high (2,000 mg/kg HMX; n=10). Data were censored to include only developed embryos approximately passed stage 35 of development.


69

Figure 4. Comparison of hatching success of green anole eggs across treatment groups exposed to HMX via contact with contaminated soil for up to 41 days. Control = 0 mg/kg HMX, Low = 20 mg/kg HMX, Medium = 200 mg/kg HMX, High = 2,000 mg/kg HMX. Values above bars represent sample size (n). Differences were not significant among dose groups (p>0.05).


70

Figu

re 5

. Mea

n (±

SE)

num

ber o

f cric

kets

con

sum

ed p

er w

eek

by fe

mal

e gr

een

anol

es in

the

expe

rimen

tal c

ontro

l (un

inje

cted

cric

ket),

veh

icle

con

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0 H

MX

; PE

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ject

ion)

, low

(20

mg/

kg H

MX

), m

ediu

m (2

50 m

g/kg

HM

X),

and

high

(5

00 m

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HM

X) d

ose

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ps.


71

Figure 6. Mean (± SE) percentage of initial body weight of female green anoles in the experimental control (uninjected cricket), vehicle control (0 HMX; PEG injection), low (20 mg/kg HMX), medium (250 mg/kg HMX), and high (500 mg/kg HMX) dose groups.


72

Figure 7. Mean (± SE) concentration of HMX in the bodies of adult female green anoles as a result of maternal exposure to HMX in food over 12 weeks. Values above bars represent sample size (n). Bars with different letters are significantly different (p≤0.05).


73

Figure 8. Mean (± SE) concentration of the explosive, HMX, in the green anole egg as a result of maternal exposure to HMX in food. Maternal dose groups represent: experimental control (uninjected cricket), control (0 HMX; PEG injection), low (20 mg/kg HMX), medium (250 mg/kg HMX), and high (500 mg/kg HMX) dose groups. Bars with different subscripts are different at p<0.001. (a) Eggs analyzed without regard for mother. (b) Eggs were pooled by individual prior to statistical analysis.


74

Figure 9. Comparison of hatching success of green anole eggs across maternal treatment groups exposed to various concentrations of HMX in prey. Maternal dose groups represent: experimental control (un-injected cricket), PEG control (0 HMX; PEG injection), low (20 mg/kg HMX), medium (250 mg/kg HMX ), and high (500 mg/kg HMX). Values above bars represent sample size (n). Differences were not significant among dose groups (p>0.05).


75

Age (weeks)0 2 4 6 8 10 12

Wei

ght (

g)

0.1

0.2

0.3

0.4

0.5

0.6

Experimental controlPEG controlLowMediumHigh

Figure 10. Comparison of weight-gain trends of green anole hatchlings exposed to the explosive, HMX, via maternal transfer. Maternal dose groups represent: experimental control (un-injected cricket), PEG control (0 HMX; PEG injection), low (20 mg/kg HMX), medium (250 mg/kg HMX ), and high (500 mg/kg HMX).


76

Age (weeks)0 2 4 6 8 10 12

Snou

t-ven

t len

gth

(mm

)

20

22

24

26

28

30Experimental controlPEG controlLowMediumHigh

Figure 11. Comparison of snout-vent length trends of green anole hatchlings exposed to the explosive, HMX, via maternal transfer. Maternal dose groups represent: experimental control (un-injected cricket), PEG control (0 HMX; PEG injection), low (20 mg/kg HMX), medium (250 mg/kg HMX ), and high (500 mg/kg HMX).


77

Figure 12. Comparison of hatch success of green anole eggs exposed to HMX via two transport mechanisms. Eggs in the egg exposure study were incubated in spiked soil at four nominal concentrations of HMX (0, 20, 200, and 2,000 mg/kg). Eggs in the maternal exposure study were indirectly exposed to HMX via maternal transfer. Mothers were exposed to four nominal concentrations of HMX in prey (0, 20, 250, and 500 mg/kg).


78

Figure 13. Comparison of accumulation of HMX into green anole eggs via two transport mechanisms. Eggs in the egg exposure study were incubated in spiked soil at four nominal concentrations of HMX (0, 20, 200, and 2,000 mg/kg). Eggs in the maternal exposure study were indirectly exposed to HMX via maternal transfer. Mothers were exposed to four nominal concentrations of HMX in prey (0, 20, 250, and 500 mg/kg).


79

APPENDIX

RAW DATA FROM THE EGG AND MATERNAL

EXPOSURE STUDIES

Texa

s Tec

h U

nive

rsity

, Lin

dsey

Jone

s, A

ugus

t 200

7

80

Ta

ble

A.1

. R

aw d

ata

from

the

egg

expo

sure

stud

y.

* D

enot

es d

ata

that

wer

e ce

nsor

ed o

ut o

f ana

lyse

s as d

escr

ibed

in th

e te

xt o

f Cha

pter

2.

Conc

entra

tions

are

giv

en in

mg/

kg H

MX

.

Trea

tmen

t gro

ups:

Veh

icle

con

trol =

ace

tone

, Low

= 2

0 m

g/kg

HM

X in

soil,

Med

ium

= 2

00 m

g/kg

H

MX

in so

il, H

igh

= 2,

000

mg/

kg H

MX

in so

il

Abb

revi

atio

ns: N

D =

not

det

ecte

d

Mea

n (S

E) n

Pa

ram

eter

(Fig

ure

No.

) Ex

posu

re

perio

d V

ehic

le

cont

rol

Low

M

ediu

m

Hig

h

Egg

accu

mul

atio

n (1

, 2)

1 d

---

---

3.7

(0.5

)3 --

-

3 d

---

---

4.9

(0.9

)3 --

-

6 d*

--

- --

- 4.

3 (0

.9)3 *

---

12

d

---

---

4.5

(0.5

)3 --

-

18 d

--

- --

- 7.

8 (1

.5)3

---

30

d

---

---

12.6

(4.3

)3 --

- PS

D a

ccum

ulat

ion

(1, 2

) 1

d --

- --

- 5.

7 (2

.9)3

---

3

d --

- --

- 3.

0 (0

.8)3

---

6

d*

---

---

22.7

(21.

8)3 *

---

12

d

---

---

4.9

(0.3

)3 --

-

18 d

--

- --

- 4.

6 (0

.8)3

---

30

d

---

---

20.4

(16.

0)3

---

Soil

conc

entra

tion

(3)

0 d

ND

14

19.8

(0.6

)1428

3.4

(19.

4)14

2244

.2 (8

48.2

)13

≤

41 d

N

D14

17

.7 (0

.2)14

174.

4 (3

.3)14

17

60.7

(22.

4)13

Eg

g ac

cum

ulat

ion

(3)

≤ 41

d

ND

14

7.7

(0.7

)14

17.2

(2.0

)14

86.6

(17.

8)13

Pe

rcen

t hat

ch su

cces

s (4)

≤

41 d

50

.0%

14

42.9

%14

64

.3%

14

38.5

%13

Texa

s Tec

h U

nive

rsity

, Lin

dsey

Jone

s, A

ugus

t 200

7

81

Tabl

e A

.2.

Raw

dat

a fro

m th

e m

ater

nal e

xpos

ure

stud

y.

# Bod

y w

eigh

t is g

iven

in g

. Tr

eatm

ent g

roup

s: Ex

perim

enta

l con

trol =

no

prey

inje

ctio

n, V

ehic

le c

ontro

l = p

olye

thyl

ene

glyc

ol in

ject

ion

eq

uiva

lent

in v

olum

e to

Hig

h tre

atm

ent g

roup

, Low

= 2

0 m

g H

MX

/kg

body

wei

ght,

Med

ium

= 2

50 m

g H

MX

/kg

body

wei

ght,

Hig

h =

500

mg

HM

X/k

g bo

dy w

eigh

t.

Mea

n (S

E) n

Para

met

er (F

igur

e N

o.)

Expo

sure

pe

riod

Expe

rimen

tal

cont

rol

Veh

icle

co

ntro

l Lo

w

Med

ium

H

igh

Cric

kets

con

sum

ed p

er a

dult

(5)

1 w

k 2.

3 (0

.5)6

2.6

(0.2

)15

2.4

(0.2

)15

2.1

(0.2

)15

2.2

(0.2

)15

2

wk

2.2

(0.5

)6 2.

3 (0

.2)15

2.

5 (0

.2)15

2.

3 (0

.2)15

1.

8 (0

.2)15

3 w

k 2.

2 (0

.5)6

2.2

(0.2

)15

1.8

(0.2

)15

1.9

(0.2

)15

2.2

(0.2

)15

4

wk

2.2

(0.5

)6 1.

9 (0

.3)15

2.

1 (0

.2)15

2.

0 (0

.2)15

1.

7 (0

.2)15

5 w

k 2.

6 (0

.2)5

2.1

(0.2

)15

1.9

(0.2

)15

2.1

(0.2

)15

1.5

(0.3

)15

6

wk

2.6

(0.4

)5 2.

4 (0

.2)15

1.

9 (0

.3)15

2.

0 (0

.2)15

1.

5 (0

.2)15

7 w

k 2.

0 (0

.7)4

1.9

(0.3

)15

1.9

(0.2

)15

2.0

(0.3

)15

2.1

(0.2

)15

8

wk

1.7

(0.3

)3 1.

9 (0

.3)15

1.

9 (0

.2)15

1.

6 (0

.3)15

1.

6 (0

.2)15

9 w

k 2.

3 (0

.3)3

1.9

(0.2

)15

2.1

(0.2

)15

1.7

(0.3

)15

1.6

(0.2

)14

10

wk

2.0

(1.0

)3 1.

8 (0

.3)15

1.

5 (0

.2)15

1.

5 (0

.3)14

1.

0 (0

.2)14

11 w

k 2.

3 (0

.7)3

2.1

(0.2

)15

2.2

(0.2

)14

2.5

(0.1

)13

1.9

(0.2

)14

12

wk

1.7

(0.7

)3 2.

3 (0

.2)14

2.

1 (0

.2)14

2.

3 (0

.2)13

2.

0 (0

.2)14

M

ater

nal b

ody

wei

ght (

6)#

0 w

k 2.

9 (0

.2)6

3.2

(0.2

)15

3.3

(0.2

)15

3.2

(0.2

)15

3.2

(0.2

)16

2

wk

2.7

(0.3

)6 2.

8 (0

.1)15

2.

9 (0

.2)15

2.

8 (0

.2)15

2.

8 (0

.1)16

4 w

k 3.

1 (0

.2)5

2.7

(0.1

)15

2.7

(0.2

)15

2.6

(0.2

)15

2.6

(0.1

)16

6

wk

3.2

(0.2

)5 3.

0 (0

.1)15

2.

7 (0

.2)15

2.

7 (0

.2)15

2.

7 (0

.1)14

8 w

k 2.

9 (0

.2)3

2.9

(0.1

)15

2.5

(0.1

)15

2.5

(0.2

)15

2.6

(0.1

)14

10

wk

3.2

(0.2

)3 3.

1 (0

.1)15

2.

6 (0

.2)15

2.

9 (0

.2)13

2.

9 (0

.1)13

12 w

k 2.

7 (0

.1)3

2.7

(0.1

)14

2.4

(0.1

)14

2.5

(0.1

)13

2.6

(0.1

)13

Texa

s Tec

h U

nive

rsity

, Lin

dsey

Jone

s, A

ugus

t 200

7

82

App

endi

x A

.2. (

cont

inue

d).

Raw

dat

a fro

m th

e m

ater

nal e

xpos

ure

stud

y.

C

once

ntra

tions

are

giv

en in

mg/

kg H

MX

. # B

ody

wei

ght i

s giv

en in

g.

Trea

tmen

t gro

ups:

Expe

rimen

tal c

ontro

l = n

o pr

ey in

ject

ion,

Veh

icle

con

trol =

pol

yeth

ylen

e gl

ycol

inje

ctio

n

equi

vale

nt in

vol

ume

to H

igh

treat

men

t gro

up, L

ow =

20

mg

HM

X/k

g bo

dy w

eigh

t, M

ediu

m =

250

mg

HM

X/k

g bo

dy w

eigh

t, H

igh

= 50

0 m

g H

MX

/kg

body

wei

ght.

Abb

revi

atio

ns: c

onc

= co

ncen

tratio

n; N

D =

not

det

ecte

d

Mea

n (S

E) n

Para

met

er (F

igur

e N

o.)

Expo

sure

pe

riod

Expe

rimen

tal

cont

rol

Veh

icle

co

ntro

l Lo

w

Med

ium

H

igh

Mat

erna

l bod

y co

nc (7

) 12

wk

ND

5 N

D15

0.

4 (0

.1)15

1.

1 (0

.5)16

3.

1 (0

.9)16

Eg

g co

nc (8

a)

---

ND

6 N

D35

0.

7 (0

.4)28

0.

9 (0

.6)20

1.

5 (1

.0)21

Eg

g co

nc p

oole

d by

m

othe

r (8b

)

---

ND

3 N

D13

0.

6 (0

.1)12

0.

9 (0

.1)9

1.7

(0.4

)9

Perc

ent h

atch

succ

ess (

9)

---

69.2

%13

22

.7%

22

41.7

%24

40

.0%

15

23.1

%13

H

atch

ling

body

wei

ght

(10)

# <

24 h

r 0.

26 (0

.01)

8 0.

24 (0

.01)

5 0.

26 (0

.01)

10

0.25

(0.0

1)5

0.27

(0.0

2)3

1

wk

0.27

(0.0

1)7

0.25

(0.0

3)4

0.26

(0.0

2)8

0.23

(0.0

3)4

0.29

(0.0

1)2

2

wk

0.27

(0.0

5)7

0.24

(0.0

3)4

0.28

(0.0

2)8

0.29

(0.0

2)3

0.27

(0.0

2)2

3

wk

0.29

(0.0

4)6

0.26

(0.0

4)4

0.31

(0.0

2)6

0.32

(0.0

1)3

0.28

(0.0

5)2

4

wk

0.30

(0.0

2)5

0.34

(0.0

4)3

0.32

(0.0

2)6

0.33

(0.0

2)3

0.39

1

5 w

k 0.

31 (0

.03)

5 0.

37 (0

.02)

3 0.

33 (0

.02)

6 0.

33 (0

.02)

3 0.

371

6

wk

0.34

(0.0

3)4

0.42

(0.0

1)3

0.34

(0.0

2)5

0.34

(0.0

2)3

0.40

1

7 w

k 0.

39 (0

.02)

3 0.

42 (0

.03)

3 0.

35 (0

.02)

5 0.

37 (0

.02)

3 0.

461

8

wk

0.41

(0.0

4)3

0.44

(0.0

3)3

0.33

(0.0

5)5

0.36

(0.0

2)3

0.50

1

9 w

k 0.

42 (0

.03)

3 0.

48 (0

.05)

3 0.

37 (0

.04)

3 0.

38 (0

.04)

3 0.

431

Texa

s Tec

h U

nive

rsity

, Lin

dsey

Jone

s, A

ugus

t 200

7

83

App

endi

x A

.2. (

cont

inue

d).

Raw

dat

a fro

m th

e m

ater

nal e

xpos

ure

stud

y.

§ Le

ngth

is g

iven

in m

m.

Trea

tmen

t gro

ups:

Expe

rimen

tal c

ontro

l = n

o pr

ey in

ject

ion,

Veh

icle

con

trol =

pol

yeth

ylen

e gl

ycol

inje

ctio

n

equi

vale

nt in

vol

ume

to H

igh

treat

men

t gro

up, L

ow =

20

mg

HM

X/k

g bo

dy w

eigh

t, M

ediu

m =

250

mg

HM

X/k

g bo

dy w

eigh

t, H

igh

= 50

0 m

g H

MX

/kg

body

wei

ght.

Mea

n (S

E) n

Para

met

er (F

igur

e N

o.)

Expo

sure

pe

riod

Expe

rimen

tal

cont

rol

Veh

icle

co

ntro

l Lo

w

Med

ium

H

igh

Hat

chlin

g sn

out-v

ent

leng

th (1

1)§

< 24

hr

23.4

(0.5

)8 21

.8 (0

.9)5

22.4

(0.7

)10

22.0

(0.7

)5 22

.7 (0

.3)3

1

wk

23.7

(0.4

)7 23

.8 (0

.9)4

23.8

(0.3

)8 22

.3 (0

.8)4

24.0

(0.0

)2

2 w

k 24

.0 (0

.4)7

24.0

(0.9

)4 24

.1 (0

.3)8

23.7

(0.7

)3 23

.5 (0

.5)2

3

wk

24.3

(0.8

)6 24

.0 (0

.9)4

24.3

(0.4

)6 24

.0 (0

.6)3

23.5

(0.5

)2

4 w

k 24

.6 (0

.5)5

25.0

(1.5

)3 24

.7 (0

.4)6

24.0

(0.6

)3 25

.01

5

wk

24.8

(0.6

)5 25

.7 (0

.9)3

25.0

(0.3

)6 24

.7 (0

.7)3

26.0

1

6 w

k 25

.5 (0

.3)4

26.3

(0.7

)3 25

.2 (0

.4)5

24.7

(0.7

)3 26

.01

7

wk

25.7

(0.3

)3 26

.7 (0

.9)3

25.4

(0.5

)5 25

.3 (0

.3)3

26.0

1

8 w

k 26

.0 (0

.6)3

27.0

(0.6

)3 25

.4 (0

.5)5

25.3

(0.3

)3 27

.01

9

wk

26.0

(0.6

)3 27

.3 (0

.7)3

25.7

(0.7

)3 26

.0 (0

.6)3

27.0

1


PERMISSION TO COPY

In presenting this thesis in partial fulfillment of the requirements for a masters

degree at Texas Tech University or Texas Tech University Health Sciences Center, I

agree that the Library and my major department shall make it freely available for

research purposes. Permission to copy this thesis for scholarly purposes may be

granted by the Director of the Library or my major professor. It is understood that any

copying or publication of this thesis for financial gain shall not be allowed without my

further written permission and that any user may be liable for copyright infringement.

Agree (Permission is granted.)

________________________________________________ ________________ Student Signature Date Disagree (Permission is not granted.) __Lindsey Jones__________________________________ __July 27, 2007____ Student Signature Date

Documents

Accumulation and Effects of HMX in the Green Anole (Anolis