Why are hatching and emergence success low? Mercury and selenium concentrations in nesting leatherback sea turtles (Dermochelys coriacea) and their young in Florida

Marine Pollution Bulletin 62 (2011) 1671–1682

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Why are hatching and emergence success low? Mercury and selenium concentrations in nesting leatherback sea turtles (Dermochelys coriacea) and their young in Florida Justin Perrault a,⇑, Jeanette Wyneken a, Larry J. Thompson b, Chris Johnson c, Debra L. Miller d,1 a

Department of Biological Sciences, Florida Atlantic University, Building 01, Sanson Science, 777 Glades Road, Boca Raton, FL 33431, United States Nestlé Purina PetCare, 801 Chouteau Ave., St. Louis, MO 63102, United States Loggerhead Marinelife Center of Juno Beach, 14200 US Highway One, Juno Beach, FL 33408, United States d The University of Georgia, College of Veterinary Medicine, Veterinary Diagnostic and Investigational Laboratory, 43 Brighton Road, Tifton, GA 31793, United States b c

a r t i c l e Keywords: Dermochelys coriacea Hatching success Emergence success Hatchling Mercury (Hg) Selenium (Se) Marine turtles

i n f o

a b s t r a c t Leatherback sea turtles (Dermochelys coriacea) have low hatching and emergence success compared to other sea turtle species. Postmortem examinations of hatchlings showed degeneration of heart and skeletal muscle that was similar to that found in other neonates with selenium deficient mothers. Selenium deficiency can result from elevated concentrations of bodily mercury. Ingested mercury is detoxified by the liver through mercury–selenium compound formation. In animals persistently exposed to mercury, the liver’s ability to detoxify this element may decrease, especially if dietary selenium is insufficient. We measured mercury and selenium concentrations in nesting female leatherbacks and their hatchlings from Florida and compared the levels to hatching and emergence success. Both liver selenium and the liver selenium-to-mercury ratio positively correlated with leatherback hatching and emergence success. This study provides the first evidence for the roles of mercury and selenium in explaining low reproductive success in a globally imperiled species, the leatherback sea turtle. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Mercury (hereafter termed Hg) is found in marine organisms ranging from primary producers to top carnivores and it accumulates in the body from both water and food sources (Caurant et al., 1999; Guirlet et al., 2008). While Hg is present naturally in the environment (Campbell et al., 2005), the largest source (75%) of this element is traced to emissions from anthropogenic sources, mainly from the burning of fossil fuels (Pacyna and Pacyna, 2002). Mercury and Hg compounds (i.e., methylmercury, dimethylmercury, etc.) affect various functional processes including reproduction, growth, development, vision, and hearing. Mercury has no known normal physiological role in the body (EPA, 1985). Miller et al. (2009) conducted postmortem examinations of dead-in-nest leatherback sea turtle (Dermochelys coriacea) hatchlings from Juno Beach, Florida, as well as hatchlings that died ⇑ Corresponding author. Tel.: +1 561 297 0146; fax: +1 561 297 2749. E-mail addresses: [email protected] (J. Perrault), [email protected] (J. Wyneken), [email protected] (L.J. Thompson), [email protected] (C. Johnson), [email protected] (D.L. Miller). 1 Present address: Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries University of Tennessee, Knoxville, TN 37996, United States. 0025-326X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2011.06.009

in the laboratory shortly after nest emergence and found heart and skeletal muscle pathologies that were similar to those seen in bovid neonates whose mothers were selenium (Se) deficient (Orr and Blakely, 1997). They suggested that these changes might reflect Hg toxicity or Se deficiency because, in many animals, ingested Hg is detoxified in the liver (where both Hg and Se are stored) through formation of Hg–Se compounds. Thus, persistently elevated Hg may eventually deplete Se stores (Se deficiency) if dietary levels are not compensatory (Cardellicchio et al., 2002). Selenium deficiency may lead to Hg toxicity. Selenium is a trace element and is found naturally in marine food sources that are high in protein (Se is largely associated with proteins), but it has low bioavailability (Caurant et al., 1996; DRI, 2000). Selenium has known enzymatic, antioxidant, thyroidal, and immune functions (Rayman, 2000); however, at higher concentrations, Se can produce adverse effects including reduced reproductive fitness, tissue lesions, physiological anomalies, and can lead to death (Ohlendorf et al., 1988; Hoffman, 2002). Worldwide, leatherback sea turtles have experienced a substantial population decrease (67%) resulting from habitat degradation, poaching, and fisheries bycatch (Pritchard, 1982; Spotila et al., 1996; Sarti-Martinez, 2000). It is difficult for these organisms to rebound from a population decline, as they have delayed

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maturity (Avens et al., 2009) and exhibit low hatching success (25.8–56.0%; Whitmore and Dutton, 1985; Leslie et al., 1996; Bell et al., 2003; Hilterman and Goverse, 2003; Hernández et al., 2007); however, average hatching success at some rookeries is as high as 67–73.3% in leatherbacks (Livingstone, 2006; Stewart and Johnson, 2006), which suggests variability within the species across populations. Despite this higher hatching success at some rookeries, the high mortality observed during early life history stages (Zug and Parham, 1996; Davenport, 1997; Bell et al., 2003) limits the potential recovery of this imperiled species. Although low hatching and emergence success in the leatherback turtle has been explored from several nest-based perspectives, the causes remain unidentified. Redfearn (2000) found that leatherback nest depth did not contribute to mortality in Florida. Bell et al. (2003) documented high early embryonic mortality in an eastern Pacific population and hypothesized that maternal reproductive health, chemical contaminants, or bacterial infection may be the cause. Wallace et al. (2004) determined that neither hypoxia nor high nest temperatures (within the thermal tolerance range) were correlated with low hatching success at that same rookery. Surprisingly, no hypotheses regarding the roles of toxicants (e.g., Hg or persistent organic pollutants [POPs]) on hatching and emergence success have been tested, despite known and potential negative impacts on health and reproductive success of other vertebrates (loons: Burgess and Meyer, 2008; panthers: USEPA, 2000; marine mammals: Beland et al., 1993). Leatherback sea turtles make long distance migrations from foraging grounds to nesting beaches, and then subsequently return to foraging grounds (Eckert et al., 2006; Hays et al., 2006). At those foraging grounds, non-essential (e.g., Hg) and essential elements (e.g., Se) can accumulate in the body through food (e.g., pelagic medusa; Bjorndal, 1997) and water intake (Caurant et al., 1999; Guirlet et al., 2008). Females returning to nesting beaches to lay eggs can transfer these elements to their offspring via egg components (i.e., albumen, yolk), as has previously been shown in leatherbacks (Guirlet et al., 2008) and other reptiles (Roe et al., 2004; Rainwater et al., 2005; Unrine et al., 2006). Elevated concentrations of these elements could impact leatherback turtle hatching and emergence success. Leatherbacks lay 6–11 nests per season depositing an average of 70–80 eggs with a mean interclutch interval of 9– 10 days (Miller, 1997; Bell et al., 2003; Stewart and Johnson, 2006). In this study, our objectives were to (i) identify or exclude Se deficiency as a factor affecting leatherback turtle nest success, (ii) determine how Hg and Se in hatchling blood, liver, and yolk sac are correlated with each other and with maternal blood Hg and Se concentrations, and (iii) characterize Hg and Se concentrations in nesting leatherbacks (blood), hatchlings (blood from live turtles and liver and yolk sac from dead turtles), and egg shells and shelled albumen globs (SAGs).

2. Materials and methods 2.1. Study period and site description Western Atlantic female leatherbacks and their young were sampled on the nesting beach during the 2007 and 2008 nesting seasons. Samples were collected along Florida’s east coast (Juno Beach and Jupiter Beach, FL), primarily in the 20 km area of peak nesting between Jupiter Inlet (26°560 3600 N, 80°040 1500 W) and the Lake Worth Inlet (26°460 2400 N, 80°010 5300 W, Fig. 1). This rookery has been monitored nightly for leatherback nesting activity since 2001 (Stewart, 2007). Nesting season runs from March through early July in southeastern Florida. Over 400 individual nesting leatherbacks have been identified in Florida since tagging programs began (K. Stewart, personal communication).

2.2. Sample collection 2.2.1. Nesting females Sampling in Florida was conducted in conjunction with the Loggerhead Marinelife Center’s (LMC) leatherback tagging project. The beach was patrolled on foot or with all-terrain vehicles each night to encounter nesting leatherbacks during the nesting season. From 10:30 p.m. to 5:30 a.m., night-vision scopes were used to identify turtle crawls and the stage of nesting before approaching a nesting turtle to minimize the chances of disturbance. Nesting females were approached once they entered a physiological ‘‘trance’’, which begins after oviposition commences (Dutton and Dutton, 1994). Individuals were identified based on their flipper tags and/or internal PIT (passive integrated transponder) tags. However, if neither of these tags were present, PIT and/or flipper tags were applied by the LMC staff. Blood was collected into 7 mL BD K3EDTA VacutainerÒ tubes (Becton–Dickinson and Co. Franklin Lakes, NJ, USA) using an 18 gauge venous collection needle fitted in a VacutainerÒ tube holder. Before insertion of the needle, the entire area was swabbed with a sterile 70% isopropyl alcohol swab. Approximately 5 mL of blood were taken from the femoral rete system (Dutton, 1996). The location of this vascular network was identified by palpation of the area approximately 10 cm posterior to the knee. Blood collection ceased when a complete sample was obtained or egg-laying terminated (after approximately 7–11 min). The venipuncture site was then disinfected with a new alcohol swab and pressure was applied until the blood site clotted. The samples were chilled immediately. After blood collection, the turtle’s minimum curved carapace length was recorded (from nuchal notch, to posterior tip of the caudal peduncle, CCLmin, after Wyneken, 2001). Blood was frozen at 20 °C, and later shipped overnight, on ice, to the Veterinary Diagnostic and Investigational Laboratory at the University of Georgia (UGA-VDIL, P.O. Box 1389, 43 Brighton Road, Tifton, GA 31793, USA) for analysis.

2.2.2. Hatchlings We monitored the progress of all nests and recorded any incidents of predation, sea water inundation, or wash outs due to storm. On nights when hatchlings were expected to emerge (55– 60 days after egg deposition), a 61 cm  61 cm  10 cm cage was placed on top of the chamber in order to retain the hatchlings so that blood could be collected. The cages were covered with mesh so that predators were excluded if emergence occurred in the absence of the beach patrollers. The cages were monitored every 30–60 min. Upon emergence, up to 10 normal hatchlings were selected and weighed to the nearest 0.5 g with a PESOLAÒ 100 g scale. Their straight carapace lengths (SCL), straight carapace widths (SCW), and body depths (BD) were also recorded to the nearest 0.05 mm using Vernier calipers. Body condition (mass:SCL ratio) was also calculated. The dorsal and lateral neck of each hatchling was swabbed with a sterile 70% isopropyl alcohol pad and blood was collected from the external jugular vein (dorsal cervical sinus; Owens and Ruiz, 1980) using a 1 mL BD SafetyGlide™ allergy syringe. Approximately 0.1–0.2 mL of blood was collected from each of the hatchlings and was pooled by clutch into a K3EDTA tube for Se (and Hg if the quantity was sufficient) analysis. This amount (0.1–0.2 mL) is less than 5% blood volume for turtles weighing greater than 40 g and is considered safe for collection (Jacobson, 2007; Strik et al., 2007). Blood was chilled on ice immediately after collection. All samples were frozen at 20 °C until they were later shipped overnight, on ice, to the UGA-VDIL for analysis. Hatchlings were observed for 1 h and released after blood collection.

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Fig. 1. Juno Beach/Jupiter Beach, FL, nesting beach. This area is located in northern Palm Beach County, and lies on the east coast of Florida in the western Atlantic Ocean.

2.2.3. Nest inventory The nests from the study females were excavated and the contents inventoried 3 days following the major emergence date to determine hatching and emergence success, which were calculated after Miller (1999): Hatching success: # hatched eggs : # hatched eggs þ # unhatched eggs þ # pipped live þ # pipped dead

Emergence success: # hatched eggs  # ðlive hatchlings in nest þ # dead hatchlingsÞ : total eggsðhatched eggs þ unhatched eggs þ dead pipped þ live pippedÞ

A pipped egg is one in which the turtle cuts through the egg shell, but does not exit the egg. Inventory data include the number of SAGs (Bell et al., 2003), but they are not counted as part of hatching success as they never contain yolks or embryos. Up to five dead-in-nest turtles/nest that were in good condition were collected, frozen, and sent to UGA-VDIL for later sampling of the liver and yolk sac. Because the sacrifice of endangered species was prohibited and deemed unethical, we have to assume that all individuals in a clutch (dead-in-nest and those that emerged) have similar Hg and Se concentrations. Previous studies support this assumption (Heinz et al., 1987; Sakai et al., 1995; Bryan et al., 2003). Additionally, three hatched eggshells and three SAGs were collected for Hg analyses (and Se analyses when available).

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2.3. Sample analyses All sample analyses were standardized by using the same laboratory equipment and procedures. Mercury concentrations were determined using a flow injection analysis system (FIAS 400, Perkin-Elmer, Norwalk, CT) by atomic absorption spectrophotometry (AAS, AAnalyst 100, Perkin-Elmer). To quantify total Hg, approximately 1 mL of blood (or 1 g of liver tissue, yolk, albumen, or shell) from each animal was transferred to a TeflonÒ microwave vessel and mixed with 5 mL of 65% nitric acid (HNO3, Fisher Scientific) and 2 mL of 30% hydrogen peroxide (H2O2, Fisher Scientific). The samples then were digested using a laboratory microwave oven (MARS5, CEM Corporation, Matthews, SC, USA) and heated to 200 °C under high pressure over 10 min. The samples were cooled to room temperature (1 h). Approximately 15–20 mL of distilled water was added to the flask so that the contents could be easily mixed. Potassium permanganate (KMnO4, 1%) was added until the purple color from the KMnO4 persisted for 2–3 s. The sample mixture was diluted to volume with water (25 mL). The solutions were capped, and inverted 10 times to mix. The use of HNO3 in AAS can result in artificially high Hg concentrations; however, the back titration of KMnO4 corrects this error. The average of triplicate analyses is reported. Appropriate blanks (deionized water), standards (1, 4, and 5 ppb solutions made from Fisher Scientific’s Mercury Reference Standard Solution, 1000 ppm ± 1%, Certified), and controls (UTAK Laboratories, Inc. Metals Level 1 Whole Blood Toxicology Control for blood; NIST Bovine Liver Standard Reference MaterialÒ 1577c for liver, yolk sac, and albumen) were used for each run. Blood Se was quantified using graphite furnace AAS; 250 lL of blood was mixed with a prepared diluent (0.2% nitric acid [Fisher Scientific] and 0.1% Triton X-100 [Acros Organics]). Liver tissue, yolk, and albumen were digested using same procedures as for Hg analyses. Appropriate blanks (nitric acid/Triton X-100 diluent), standards (50 and 100 ppb solutions made from Fisher Scientific’s Selenium Reference Standard Solution, 1000 ppm ± 1%, Certified) and controls (UTAK Laboratories, Inc. Metals Level 1 Whole Blood Toxicology Control for blood; NIST Bovine Liver Standard Reference MaterialÒ 1577c for liver, yolk sac, albumen, and shell) were employed for each analytical run (Table 1). The average of triplicate analyses is reported.

2.4. Statistical analyses Data were tested for normality using the Shapiro–Wilk statistic. Least-squares linear regressions were run with hatching and emergence success as the dependent variable and nesting female Hg or Se as the independent variables. A multiple regression analysis was carried out to determine if Hg and Se were significantly related to hatching and emergence success. Simple regression analysis was carried out in order to determine if nesting female blood Hg and Se concentrations correlated with body size (CCLmin), clutch size, or hatchling blood, liver, and/or yolk sac Hg and Se concentrations. In order to determine if Hg or Se concentrations were correlated

with hatchling mass, SCL, SCW, BD, or mass:SCL ratio, simple regressions were performed. Where data were not normally distributed, rank regressions were performed. Nesting female blood Hg and Se were compared to hatchling blood and liver Hg and Se concentrations using a Mann–Whitney U test. Hatchling blood Hg and liver Hg were compared using a Mann–Whitney U test, in addition to hatchling blood Se and liver Se. Simple linear regressions were used to determine if a relationship existed between hatchling blood, liver, and/or yolk sac Hg and Se concentrations and hatching and/or emergence success. Paired t-tests were carried out to determine if blood Hg or Se concentrations differed significantly in nesting females from the first nesting encounter to the second nesting encounter. Similarly, paired t-tests were carried out to compare hatchling blood and liver Hg or Se from subsequent nests from the same nesting female. Lastly, SAG albumen Hg concentrations were compared to yolk sac Hg concentrations using a Mann–Whitney U test (Sokal and Rohlf, 1995). Data were analyzed using Systat 12 Software (Systat, Inc., Evanston, IL, USA).

3. Results 3.1. Nesting female Hg and Se concentrations and hatching/emergence success Blood from 60 nesting females was sampled during two nesting seasons, with 39 in 2007, and 21 in 2008, yielding a total of 52 Hg samples and 71 Se samples. Fewer Hg tests (than Se) were run because an insufficient volume of blood was collected from some individuals. Eleven females were sampled during more than one nesting event during the nesting season: one turtle was sampled on three occasions for Se, 11 turtles twice for Se, and eight turtles were sampled twice for Hg. These subsequent nesting events were not necessarily sequential. Size, mean CCLmin of the turtles (mean ± SD = 152 ± 8 cm, range = 125–174 cm) did not significantly correlate with either blood Hg or Se concentration (p > 0.05). Additionally, the clutch size of the nest (mean ± SD = 72 ± 14, range = 34–102 eggs, n = 60 clutches) did not significantly correlate with either blood Hg or Se concentration (p > 0.05). During excavations in 2007 and 2008, 10 nests could not be located (due to washout or inundation) and two were predated. Neither hatching nor emergence success met the assumptions of normality; therefore the median and range are reported (hatching success: median = 59.7%, range 0–93.4%; emergence success: median = 50.8%, range = 0–93.4%). Maternal blood Hg and Se (Table 2) were not significantly correlated (p > 0.05). Neither maternal blood Hg nor Se significantly correlated with hatching or emergence success (p > 0.05). When Hg and Se were combined into a multiple regression, no significant correlation was observed (p > 0.05). Maternal blood Hg concentrations were negatively correlated (by rank regression) with days into the nesting season (r2 = 0.10, p = 0.02, Fig. 2). Day 1 of the nesting season was set as 1 March. No significant trends in Se concentrations were observed. Blood Hg concentrations in each of the eight females sampled more than

Table 1 Quality control results (ppm dry weight) attained with reference materials.

a b

Reference material

Element

Reference value

Attained value

n

UTAK Metals Level 1 Control

Hg Se

0.0006a 0.123b

0.0004 ± 0.0002 0.101 ± 0.020

8 4

NIST Bovine Liver SRMÒ 1577c

Hg Se

0.00536 ± 0.00017 2.031 ± 0.045

0.00488 ± 0.00474 2.865 ± 0.153

9 7

Expected range: 0.00051–0.00069. Expected range: 0.105–0.141.

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Table 2 Synopsis of Hg and Se concentrations (mean ± SD or range) in leatherback turtle tissues and eggs from the literature and this study. Values are in ppm (wet weight) and were converted from dry weight if indicated.

a b c d e f g

Location

Year

Tissue

Stage/sex/ maturity

CCL range (cm)

n

[Hg]

[Se]

Reference

Wales, UK Wales, UK Wales, UK Wales, UK Wales, UK Scotland, UK Scotland, UK Scotland, UK Scotland, UK Gabon, Africa French Guiana French Guiana Georgia/Massachusetts, USA California, USA California, USA St. Croix, USVI Juno Beach/Jupiter Beach, FL

1988

Liver Muscle Blubber Liver Muscle Liver Muscle Liver Muscle Blood Blood Egg Blood Blood Blood Blood Blood

Mature male, D/Sa

159

Mature male, E/Da

170

Mature male, E/Da

151

a

141

1 1 1 1 1 1 1 1 1 6 78 76 16 3 9 11 52 (Hg), 71 (Se)

0.12 ± 0.01b 0.04 ± 0.01b 0.06 ± 0.01b,c 0.37 0.013 0.26d 0.1d 0.09d 0.04d 0.200 ± 0.200 0.011 ± 0.003 0.012 ± 0.003 0.01 ± 0.00 0.014 ± 0.035 0.007–0.048 BDLe-0.013 0.003–0.088

0.45 ± 0.01b 1.20 ± 0.16b <0.03b,c 6.5 4.3 N/A N/A N/A N/A N/A 9.98 ± 0.05 1.44 ± 0.38 7.55 ± 2.83 N/A N/A N/A 0.39–21.27

Davenport et al., 1990 Davenport et al., 1990 Davenport et al., 1990 Godley et al., 1998 Godley et al., 1998 Godley et al., 1998 Godley et al., 1998 Godley et al., 1998 Godley et al., 1998 Deem et al., 2006 Guirlet et al., 2008 Guirlet et al., 2008 Innis et al., 2010 Harris et al., 2011 Harris et al., 2011 Harris et al., 2011 This studyf,g

1996 1993 1995 2001–2002 2006 2006 2007–2008 2005–2007 2005–2007 2007–2008

Mature male, E/D Nesting female Nesting female

146–158 143–170

Males/females, Ea Foraging males Foraging females Nesting females Nesting females

136–161.5 144–160 143–170 N/A 125–174

D, drowned; S, stranded; E, entangled in fishing gear. Reported as mg metal/kg dry weight; converted to ppm wet weight using dry tissue percentage given in Godley et al. (1998); SD given as four replicates were performed. Assume 45% moisture as given in Godley et al. (1998). Converted to ppm wet weight using dry tissue percentage given in Godley et al. (1998). BDL, below detection limit. Hg detection limit for this study: 0.000009 ppm. Se detection limit for this study: 0.00005 ppm.

Fig. 2. Trends in nesting female blood Hg (ppm wet weight) across the nesting season. The regression weakly shows that Hg concentrations (ppm wet weight) significantly decreased as the nesting season progressed. Nesting season begins March 1; our first sample was collected on April 22.

once tended to decrease, in subsequent samples, although not significantly so (mean decrease = 0.016 ppm, paired t-test: t = 2.025, df = 7, p = 0.08, Fig. 3a). Blood Se concentrations of nesting females showed no pattern of increase or decrease (paired t-test: t = 0.914, df = 10, p = 0.38, Fig. 3b).

for normally distributed data; median and range for non-normal data). We found no significant correlations between clutch size, hatchling mass, SCL, SCW, BD, or mass:SCL ratio and blood, liver or yolk sac Hg or Se concentrations (p > 0.05). Nesting female blood Hg was significantly higher than hatchling blood Hg (U0.05 (2), 52, 15 = 753.5, p < 0.001) and hatchling liver Hg (U0.05 (2), 52, 22 = 347, p = 0.008). Nesting female blood Se was significantly higher than hatchling blood Se (U0.05 (2), 71, 44 = 473, p < 0.001) and hatchling liver Se (U0.05 (2), 71, 18 = 98, p < 0.001). Female blood Hg did not significantly differ from hatchling yolk sac Hg concentrations (U0.05 (2), 52, 5 = 196, p = 0.06). Using simple regressions, neither nesting female blood Hg nor blood Se concentrations significantly correlated with that of hatchling blood, liver, or yolk sac Hg or Se concentrations (p > 0.05). Additionally, neither hatchling blood Hg and Se concentrations nor hatchling liver Hg and Se concentrations were significantly correlated (p > 0.05). Using paired t-tests, we found that neither hatchling Hg nor Se (blood or liver) decreased in subsequent nests (p > 0.05) from the same nesting female. Within hatchling samples, liver Hg was significantly higher (24fold) than blood Hg (U0.05 (2), 15, 22 = 330, p < 0.001). The reverse relationship was observed with Se; hatchling blood Se was 1.5-fold higher than liver Se (U0.05 (2), 44, 18 = 583, p = 0.0039). Hatchling liver Se positively correlated with hatching success (r2 = 0.35, p = 0.02, Fig. 4a) and emergence success (r2 = 0.46, p = 0.004, Fig. 4b). When the ratio of hatchling liver Se to liver Hg was compared to hatching success, a stronger, significant positive correlation was found (r2 = 0.55, p = 0.002, Fig. 5a) than for liver Se concentrations alone. A similar trend in emergence success was also found (r2 = 0.72, p < 0.001, Fig. 5b).

3.2. Hatchlings 3.3. Eggshells and SAGs A total of 301 live hatchlings were sampled from 53 nests. Fifteen pooled blood samples were of sufficient volume for Hg analyses and 43 for Se analyses. Twenty-two dead-in-nest hatchlings were sampled for Hg concentrations in the liver, while 19 were sampled for liver Se. Seven hatchlings were sampled for Hg concentrations in the yolk sac. Table 3 summarizes morphometrics and Hg and Se concentrations in hatchlings (mean ± SD and range

Mercury and Se concentrations in eggshells and SAGs (Table 4) were not correlated with Hg or Se concentrations in maternal blood or in dead hatchlings (p > 0.05) SAG albumen Hg concentrations were significantly lower than Hg concentrations in the yolk sacs of the hatchlings by two orders of magnitude (U0.05 (2), 7, 14 = 98, p = 0.0003).

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4.1. Baseline Hg and Se concentrations

Fig. 3. Changes in individual nesting female blood Hg ((A) ppm wet weight, mean ± SE = vertical bar) and Se ((B) ppm wet weight, mean ± SE = vertical bar) concentrations for subsequent samples. Turtles (A–H) correspond to the same individuals in (A and B). The numbers in parentheses represent the intervals (in days) between sampling. Individual (A) was sampled a third time for Se, 20 days after the second sample was collected. The third sample was 11.48 ppm lower than the second sample and is not shown in the figure.

4. Discussion This study is the first study to document Hg and Se concentrations in nesting female leatherback sea turtles from the continental U.S. (Florida). Our results provide baseline concentrations of these elements in nesting female leatherbacks, in addition to hatchling leatherback turtles. This is the first study to (i) document Hg and Se levels in hatchling sea turtles (for blood, liver, and yolk sac), (ii) provide evidence for the transfer of these elements from mother to offspring in this region, (iii) document Hg and Se concentrations in SAGs of nesting leatherbacks, and (iv) identify factors that may contribute to the low hatching and emergence success, a feature common in this species (Whitmore and Dutton, 1985; Leslie et al., 1996; Bell et al., 2003; Hilterman and Goverse, 2003; Hernández et al., 2007).

Mercury concentrations of leatherbacks from Florida ranged from 0.003 to 0.088 ppm (median = 0.026 ppm). These levels were similar in magnitude to those reported in western Atlantic loggerheads (Caretta caretta, Day et al., 2005) and Kemp’s ridleys (Lepidochelys kempii) from the Gulf of Mexico (Kenyon et al., 2001). This similarity was unexpected since loggerheads and Kemp’s ridleys feed at higher trophic levels (e.g., crustaceans, horseshoe crabs, mollusks, Bjorndal, 1997) than leatherbacks and therefore should accumulate higher Hg concentrations. A possible explanation for these findings might be environmental because satellite tagged leatherbacks have been observed feeding in waters also used by loggerheads and Kemp’s ridleys (Eckert et al., 2006; Evans et al., 2008). Also of interest, nesting female leatherback blood Hg samples from Gabon, Africa (Deem et al., 2006) were an order of magnitude higher than blood samples collected from nesting leatherback turtles in Florida (this study), French Guiana (Guirlet et al., 2008), St. Croix (Harris et al., 2011), and from leatherbacks in waters off Georgia and Massachusetts (Innis et al., 2010), and coastal California (Harris et al., 2011; Table 2). Together, these results suggest that leatherbacks from separate populations that may forage in diverse areas (TEWG, 2007) and, depending on location, accumulate Hg at higher or lower concentrations from their food and water. This trend was found in diamondback terrapins (Malaclemys terrapin) from South Carolina and Georgia, where one site (located near a site’s chlor-alkali plant Hg discharge) showed blood Hg concentrations that were an order of magnitude higher than for the other sites (Blanvillain et al., 2007). Also, there were population level differences in heavy metal levels (Hg, cadmium, lead) in loggerhead sea turtle eggs from Florida, Georgia and North Carolina, suggesting that distinct, non-interbreeding groups (demes; Mayr, 1977) are present and that heavy metal contamination of these organisms can occur at different concentrations associated with different foraging grounds and populations (Stoneburger et al., 1980). Selenium concentrations in Florida’s nesting population ranged from 0.39 to 21.27 ppm (median = 7.64 ppm). These values were similar to those seen in nesting leatherbacks from French Guiana (Guirlet et al., 2008) and to leatherbacks entangled in fishing gear (Georgia and Massachusetts, USA; Innis et al., 2010; Table 2), signifying that organisms in these areas/populations could be foraging in similar areas. Leatherback turtle Se concentrations are high compared to other reptiles for reasons that remain unknown (Innis et al., 2010). Monitoring of toxicants and other elements in the food chain of leatherbacks has not been conducted. The Se concentration of one moon jelly (Aurelia aurita) caught off the coast of Florida was 10.07 ppm (Perrault, unpublished data), which is similar to blood and liver concentrations of the leatherbacks in this study. The high concentrations of Se in leatherbacks are most likely due to diet. Further comparative studies of prey items and nesting and at-sea caught individuals are necessary to determine if populations differ in Se levels and if levels vary among and between nesting season(s).

Table 3 Body mass, morphometrics, and Hg and Se concentrations (blood, liver, and yolk sac) of hatchling leatherback sea turtles. Mercury and Se concentrations given in ppm wet weight.

Range Mean ± SD or Median n a b c

Mass (g)

SCLa (mm)

SCWa (mm)

BDa (mm)

Mass:SCL

Blood [Hg]

Blood [Se]

Liver [Hg]

Liver [Se]

Yolk sac [Hg]

31.0–53.5 43.7 ± 3.9 301

49.0–65.6 59.5 301

26.9–44.3 39.8 301

21.6–29.5 25.6 ± 1.3 301

0.61–0.91 0.73 301

0.0004–0.001 0.0007 ± 0.0003 16

1.21–6.97 3.17 ± 1.36 45

0.009–0.031 0.017 ± 0.007 22

0.97–8.29b 1.91c 19

0.004–0.083 0.048 ± 0.029 7

SCL, straight carapace length; SCW, straight carapace width; BD, body depth. 8.29 is an outlier (more than 3 SDs away from the mean) and was removed for statistical analyses; range without this data point = 0.97–4.54. Median without outlier is 1.78.

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Fig. 4. Linear regression between hatchling liver Se (log transformed, ppm wet weight) and (A) hatching success and (B) emergence success (black triangles). Hatching and emergence success increased with increasing Se concentrations.

Fig. 5. Linear regression between the ratio of hatchling liver Se:Hg (log transformed, ppm wet weight) and (A) hatching success and (B) emergence success. Hatching and emergence success increased with increasing Se:Hg ratios.

For reptile hatchlings, Hg concentrations are reported for only one species (Burger, 1992) and Se concentrations for only two species (Burger, 1992; Nagle et al., 2001). Burger (1992) reported skin and whole body Hg and Se levels (ppm dry weight) in pine snake hatchlings (Pituophis melanoleucus). Assuming 75% moisture content, the Hg values converted to wet weight (for comparison) were body: 0.033 ± 0.007 ppm; skin: 0.070 ± 0.012 ppm. Selenium concentrations were 0.686 ± 0.110 ppm and 0.487 ± 0.055 ppm for body and skin, respectively (Burger, 1992). The Hg concentrations in the Burger (1992) pine snake study were higher and the Se concentrations were lower when compared to values from marine turtles. Nagle et al. (2001) reported whole body Se concentrations (ppm dry weight) in hatchling red-eared sliders (Trachemys scripta) from a coal ash basin (ASH, polluted) and a reference site (REF). Assuming 75% moisture content (for comparative purposes), the converted values to wet weight ranged on average from 0.41 (REF) to 1.84 ppm (ASH). These values overlapped with the concentrations found in our leatherback hatchlings (blood and liver). Additionally, Nagle et al. (2001) allowed their hatchlings to live for 9 days post-hatching, which would allow the turtles to absorb

most of their yolk sac, changing the bodily concentration of the element from when it first hatched, as Se is present in the yolk. The variations observed between the sliders/snakes and leatherbacks are most likely due to multiple differences including tissues sampled (blood, liver, and yolk v. skin and whole body), habitat differences, and prey preference/availability. Red-eared slider adults are omnivorous and opportunistic, feeding on potentially any prey item ranging from fallen leaves to amphibian tadpoles (Cagle, 1950). Snakes and leatherbacks both feed at or near the top of their respective food chains (Pitman and Dutton, 2004); however, the leatherback food chain is much shorter than that of snakes. Fewer levels of the food chain would account for lower biomagnification of Hg. It can be assumed that Hg levels in eggs reflect Hg levels in nesting reptiles (from their diets prior to nesting). Therefore, the snakes could be expected to pass on greater Hg loads to their offspring, which may explain the lower concentrations of Hg that was seen in leatherbacks. Selenium is found naturally in the marine environment, often in high concentrations. Gelatinous zooplankton, particularly jellyfish, form the primary diet of leatherbacks. Jellyfish can exhibit high

Table 4 Mercury and Se concentrations (ppm wet weight) of eggshells, SAG shells, and SAG albumen from nesting leatherback sea turtles.

Median Range n a

Eggshell [Hg]

SAG shell [Hg]

SAG albumen [Hg]

SAG albumen [Se]

0.002 0.0009–0.016 15

0.001 0.0004–0.002 13

0.0003 BDLa–0.002 15

0.02 BDL–0.09 15

BDL, below detection limit.

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levels of Se (Perrault, unpublished data). Cnidaria is the only phylum that characteristically contains a particular species of Se (SelJ selenoprotein homologs) in their tissues (Stillwell and Berry, 2005). This finding, along with the large volume of food that leatherbacks are hypothesized to consume daily (Lutcavage and Lutz, 1986; Wallace et al., 2006a), could account for the higher concentration of Se in leatherback hatchlings than pine snake hatchlings (through maternal transfer). The reason for high Se concentrations is unclear; however, it may be that high Se concentrations in leatherbacks are physiologically necessary to offset the negative consequences associated with high Hg consumption due to high prey volume intake. Overall, studies on trace element concentrations in hatchling sea turtles are lacking and greater attention is needed in this area in order to define baseline concentrations of these elements. 4.2. Maternal transfer and bodily elimination Up to 10 months prior to oviposition and when conditions are favorable, follicular growth occurs in the ovary of sea turtles (Wibbels et al., 1990; Rostal et al., 1997). In Kemp’s ridleys, vitellogenesis occurs approximately 4–6 months before the mating period (Rostal et al., 1998); however, in leatherbacks, it is unknown exactly how far in advance this occurs (Rostal et al., 1996, 2001). Vitellogenin (VTG) is produced in the liver and is transported to the oocytes via the plasma (Heck et al., 1997). In the body, Hg and Se are stored in the liver and these elements (along with others including copper, iron, manganese, and zinc) are likely transported by vitellogenin or other egg proteins (e.g., lipovitellin; Unrine et al., 2006) from maternal liver to the egg (Richards, 1997; Nagle et al., 2001; Roe et al., 2004; Hopkins, 2006). Based on this evidence, if vitellogenesis occurs while the females are foraging, higher concentrations of contaminants and/or necessary nutrients may be sequestered in the follicles if mothers offload Hg and Se that bioaccumulate during routine food and water intake (Caurant et al., 1999; Guirlet et al., 2008; Innis et al., 2010). These compounds would then be passed onto the developing embryos at higher concentrations than if the females were fasting. We found that Hg burdens in nesting females tended to progressively decrease with each subsequent nest sampled throughout the season (Figs. 2 and 3a). In contrast, no noticeable trend was reported for the nesting turtles in the French Guiana population, even for females laying over six clutches (Guirlet et al., 2008). However, in the same study, they observed a decrease in blood Hg concentrations between the first and the second clutches, which is consistent with our findings. Because the Florida turtle nest over a wide range of available beach sites, just two of our sequential samples represented a single clutch interval. Blood Se levels showed no significant seasonal trends in either Florida or French Guiana. Blood Se concentrations, reported here, increased in 7 of 11 of the nesting turtles sampled more than once, and the average showed an increasing trend, although not significantly so (Fig. 3b). Selenium can be stored in blubber of marine organisms (ringed seals, Phoca hispida: Kari and Kauranen, 1978; short-finned pilot whales, Globicephala macrorhynchus: Stoneburger, 1978; leatherback sea turtles: Davenport et al., 1990), although usually in lesser concentrations than those of other tissues. Feeding, drinking (Casey et al., 2010), and fat metabolism during the nesting season could mobilize Se, causing a small increase in the concentrations in the blood (Day et al., 2005). Deem et al. (2009) observed that blood Hg concentrations in nesting loggerheads were greater than foraging loggerheads, indicating that nesting females mobilize Hg during the nesting season and probably deposit it into the albumen of their eggs. Therefore, hatchlings acquire Hg from both yolk absorption and ingestion of albumen. It is likely that, as embryos develop in ovo, Hg and Se

concentrations may change as a result of water and gas exchange between the egg and the nest environment (Roe et al., 2004; Guirlet et al., 2008). Therefore, the environment, as well as maternal input, may influence Hg and Se loads in developing sea turtles. We did not find significant correlations between maternal blood Hg and Se concentrations and hatchling Hg and Se concentrations. This was unexpected for Se, as Guirlet et al. (2008) found a significant correlation between maternal Se and Se concentration of the egg contents. Theoretically, this lack of correlation may reflect dilution due to uptake of water vapor (and possibly liquid water) from the environment during development (Ackerman et al., 1985; Kam and Ackerman, 1990; Wallace et al., 2006b). However, Nagle et al. (2001) found that there was no difference between bodily burdens of trace elements in red-eared slider (Trachemys scripta) hatchlings reared in substrate with elevated concentrations of trace elements and hatchlings reared in substrate from a reference site. This indicates that parchment-shelled turtle eggs (common to both T. scripta and D. coriacea) and egg membranes may act as a protective barrier to certain trace elements. If so, maternal input is the most important source of essential and non-essential elements to developing turtle embryos. This was verified by Nagle et al. (2001), when they found that hatchlings from mothers that resided in Se-laden waters had Se concentrations over four times that of those with mothers from the reference site. The lack of correlation between nesting females and hatchlings differs from trends found for other contaminants. For example, persistent organic pollutants (POPs including RDDTs, pp0 -DDE and PCB 153 + 105, PCB 180, PCB 138) in blood and eggs were positively correlated nesting leatherbacks from French Guiana (Guirlet et al., 2010). Blood sampled from nesting green turtles from Terengganu, Malaysia showed concentrations of POPs (RPCBs, RPBDEs, cHCH, and trans-chlordane, mirex) that significantly correlated with egg contents and blood of hatchling turtles (van de Merwe et al., 2010). The lack of correlation observed in our study may be explained by the toxicokinetics of Hg and Se in tissues when compared to POPs. POP concentrations in the blood at the time of nesting are thought to represent the contamination accrued at foraging grounds prior to nesting (4–10 months prior, Rostal et al., 1997; Guirlet et al., 2010), whereas Hg and Se in the blood may indicate more recent uptake (days–weeks prior). The lack of correlation between nesting females and hatchlings is consistent with this hypothesis as the majority of Hg and Se found in hatchlings comes from the yolk. Mercury concentrations in the yolk sacs of the hatchlings did not significantly differ from blood Hg concentrations in the nesting females, a finding comparable to that in Guirlet et al. (2008). Maternal blood Hg concentrations were extremely similar (0.001 ppm difference in means) to the concentrations of egg contents (yolk and albumen). Because all yolked follicles are formed before turtles arrive at the nesting beach (Rostal et al., 1996), leatherbacks may pass on similar concentrations of trace elements to their offspring in each subsequent clutch (we found no significant differences in Hg and Se concentrations in hatchlings from subsequent clutches). This would ensure that all hatchlings were equally provisioned with the necessary nutrients in their yolk sacs, and that no single clutch is burdened with a high contaminant load (Sakai et al., 1995), unless high concentrations of these compounds are deposited into the albumen during the nesting season, which is unlikely. We found significantly lower concentrations (two orders of magnitude) of Hg in the albumen of the SAGs (we did not test for Se in yolk sac or albumen) when compared to the yolk sacs of the hatchlings, indicating the yolk is the main source of this toxicant for the hatchlings. Sakai et al. (1995) found little intraclutch and interclutch variation in Hg concentrations of yolks of loggerhead sea turtle and green sea turtle eggs, respectively. Interclutch Se concentrations were shown to vary minimally in the eggs of

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captive bred mallards (Anas platyrhynchos; Heinz et al., 1987) and in eggs from a reference population of common grackles (Quiscalus quiscala; Bryan et al., 2003). We found significantly higher concentrations of Hg in the livers of hatchlings than in the blood. In other vertebrates, Hg is stored in the liver where it can be or has been detoxified; assuming hatchling leatherbacks process Hg similarly, this may account for the lower concentration in the blood. Interestingly, we found significantly higher concentrations of Se in the blood than the liver. Magat and Sell (1979) observed that Se binds to the yolk in hens, but, in mallards fed Se as selenomethionine (an amino acid containing Se), a greater proportion of this dietary Se was transferred to the eggs and was bound at greater levels in the albumen. Selenomethionine is a natural form of Se most often encountered by animals (Spallholz and Hoffman, 2002). It is not regulated homeostatically. Since embryos of oviparous species consume albumen during development (Romanoff, 1967; Palmer and Guillette, 1991), this could contribute to their total Hg and Se concentrations. In hatchlings, absorption of the yolk and ingestion of egg albumen/amniotic fluid may cause blood Se levels to be elevated over those in the liver when this form is present due to the lack of bodily regulation (Burk and Levander, 1999). Overall, blood Se concentrations in hatchlings were 4500 times higher than blood Hg concentrations; liver Se concentrations were 130 times higher than liver Hg concentrations (nesting leatherback blood Se:Hg: 907:1, Guirlet et al., 2008; green turtle liver Se:Hg: 12:1, Anan et al., 2001; hawksbill turtle liver Se:Hg: 56:1; diamondback terrapin liver Se:Hg: 1.4:1, Burger, 2002; Black-footed albatross liver Se:Hg: 0.78:1, Ikemoto et al., 2004a; common eider ducks liver Se:Hg: 7.9:1, Wayland et al., 2000; common eider ducks blood Se:Hg: 17.8:1, Wayland et al., 2000; ringed and bearded seal liver Se:Hg, <1:1, Smith and Armstrong, 1978, for review; Northern fur seal liver Se:Hg, 1:1, Ikemoto et al., 2004a; Dall’s porpose liver Se:Hg: 2.5:1, Ikemoto et al., 2004a; pilot whale blood Se:Hg: 3.9:1, Nielsen et al., 2000; sperm whale blood Se:Hg: 0.6:1, Nielsen et al., 2000). Sea turtles have higher Se:Hg ratios than other brackish and marine organisms. Therefore, nesting females pass on essential elements to their young at greater concentrations than non-essential and potentially harmful elements. We found measurable concentrations of Hg in eggshells and Hg and Se in the SAGs of leatherback turtles. Lam et al. (2006) and Burger (1994) reported Hg and Se in green turtle (Chelonia mydas) eggshells from Hong Kong and in avian eggshells from Long Island, New York, respectively. Egg-laying may provide a means of toxicant elimination in reptiles (Burger, 1994), although the amount excreted may be insignificant (Sakai et al., 1995; Guirlet et al., 2008). SAGs may also reduce toxicant loads slightly in nesting female leatherbacks without causing harm to the offspring. The albumen and eggshells are produced progressively during the nesting season. Together, these findings provide a plausible explanation for the decrease in Hg burden as the nesting season progressed. 4.3. Hatching and emergence success We found that the concentration of Se (a necessary nutrient) in the liver of hatchlings as well as the higher ratio of hatchling Se to Hg were both positively correlated with leatherback turtle hatching and emergence success at the Florida rookery. While we did not find a significant correlation between hatching and emergence success and maternal blood Hg and/or Se concentrations, this may not be surprising. Blood concentrations are more indicative of the current status in the body and of recent dietary intake. Evidence for feeding by gravid leatherbacks (Casey et al., 2010) suggests prey and seawater intake is low during the nesting season. Mercury concentrations are almost always higher in liver than blood, but this depends on the age, diet, and species being tested (Puls,

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1994; Blanvillain et al., 2007). In general, concentrations of these elements in the liver are better indicators of long term status in the body, but this sampling technique is not practical for live animals, particularly free-ranging wildlife. Day et al. (2005) found that Hg concentrations in the scutes of loggerheads were accurate predictors of the Hg concentrations in the liver based on stranded individuals. They also reported that blood and scute concentrations were highly correlated in loggerheads from the eastern Atlantic; these results provide guidance for sampling other cheloniids, but not leatherbacks. Because leatherbacks lack thick keratinous scutes (Deraniyagala, 1939), such a comparison cannot be made in leatherbacks. Miller et al. (2009) documented cardiac and skeletal muscle anomalies in dead-in-nest and captive reared leatherback hatchlings and post-hatchlings. They noted that the cardiac changes were similar to those seen in bovine neonates that were deficient in Se (a condition that can result from elevated Hg concentrations; Enjalbert et al., 1999). They hypothesized that maternal or hatchling Hg burden/Se status may have contributed to the muscular anomalies and to low hatching and emergence success of the nests. Our findings support the Miller et al. (2009) hypothesis. Both Se status and Hg load in leatherback sea turtle hatchlings correlated with hatching and emergence success (Figs. 4 and 5). Selenium deficiency is linked to muscular degeneration in a variety of animals (Dierenfeld, 1989; Enjalbert et al., 1999; Miller et al., 2009), which may explain decreased hatching/emergence success, as hatchlings must rely on adequate skeletal muscle performance to escape from eggs and the nest. Selenium is important in detoxifying Hg and other toxicants including cadmium and lead in the body (Naganuma et al., 1983; Sasakura and Suzuki, 1998; Ikemoto et al., 2004a). Mercury concentrations have been documented in a number of fish, birds, and mammals and its toxic effects include impairment of the nervous system, immune system, growth, and development (Zelikoff et al., 1994; Wiener et al., 2003; Day et al., 2007). In marine mammals and seabirds, methylmercury, the most toxic form, is converted to inorganic Hg (e.g., mercuric chloride, mercuric acetate, and mercuric sulfide) by Se (Iwata et al., 1982) and to a lesser extent, other mechanisms (reactive oxygen species: Yasutake and Hirayama, 2001; gut bacteria: Rowland, 1988). The inorganic forms most likely bind to metallothioneins and subsequently to proteins (high molecular weight substances, HMWS) in the liver (Ikemoto et al., 2004b). Decomposition of these Hg–Se bound HMWS produces an insoluble form (Ikemoto et al., 2004a) that can be excreted (Ralston and Raymond, 2010). While leatherback hatchlings have Hg and Se concentrations that suggest detoxification is likely, embryos and hatchlings may not be fully protected. Embryonic leatherbacks may be more sensitive to heavy metal toxicity, as Se may fail to protect the developing embryo during early developmental stages (e.g., before functional liver formation) as occurs in developing carp (Cyprinus carpio) and chicks (Huckabee and Griffith, 1974; Birge et al., 1976; Cuvin-Aralar and Furness, 1991). If true in leatherback hatchlings from Florida, such developmental limitations may explain the decrease in hatching and emergence success when Se was present in lower ratios when compared to Hg. In seabirds, methylmercury is the dominant form of Hg in the blood, whereas in the liver and kidney, it is generally present as the less toxic, inorganic form, suggesting that it has been detoxified by Se or metallothioneins (Clarkson, 1994; Thompson et al., 1990). Selenium, which is found at high concentrations in leatherbacks, could act as a protective mechanism against Hg in this species. Additionally, Se concentrations in hatchlings may be at the ideal range for the leatherbacks, allowing for an optimal standard metabolic rate for growth and development (Mitchelmore et al., 2006). Selenium in elevated concentrations can cause toxicity; however, few data exist on Se and Hg toxicity in reptiles. The Se concentrations

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observed in this study (for both liver and blood; adults and hatchlings) fell below or within the range of toxicity thresholds for Se predicted for other egg-laying vertebrates (3–16 ppm, fish: Skorupa, 1998; birds: Skorupa and Ohlendorf, 1991; Heinz, 1996; Lemly, 1996; Fairbrother et al., 1999) and were much lower than Se concentrations that caused anatomical and pathological anomalies in aquatic birds (eggs: 2.2–110 ppm; livers: 19–130 ppm; Ohlendorf et al., 1986). Mercury concentrations of the hatchlings (blood, liver, and yolk sac) were much lower than those predicted to harm waterbirds (5 ppm in liver, Zillioux et al., 1993), wild common terns (Sterna hirundo, 0.5–1.5 ppm; Fimreite, 1974), and developing captive bred mallard embryos (1 ppm; Heinz and Hoffman, 2003). Overall, Se’s protective effect against Hg and other toxicants and its importance to growth and development offers no surprise that it correlates with the reproductive success of marine organisms, including the leatherback sea turtle. 5. Conclusions This study is the first to correlate both maternal and hatchling contaminant loads with reproductive success in sea turtles and is also the first to document Hg and Se concentrations (blood, liver, and yolk sac) in any hatchling sea turtle species. It is also the first to provide evidence that SAGs of leatherbacks may help decrease the bodily burden of toxicants through elimination. Most importantly, we found that Se and the ratio of Se to Hg positively correlated with leatherback sea turtle hatching and emergence success. Therefore, the physiological protection of Se against Hg may allow for more live turtles to hatch and subsequently emerge from the nests. This increase in hatchling production could lead to an increase in the leatherback sea turtle population, which is extremely important for this globally imperiled species. Previous studies of toxicant levels in sea turtles have documented the effects of contaminants on clinical health parameters in subadult and adult sea turtles (POPs: Keller et al., 2004; Hg: Day et al., 2007; Hg and Se: Innis et al., 2008) and their effects on hatchling body condition (POPs: van de Merwe et al., 2010), but none have focused on nesting females and their consequences to reproduction. Further investigations of trace element levels and their toxicokinetics are needed for hatchling sea turtles to establish baselines, mechanisms of detoxification, and causal relationships. Acknowledgements The authors thank the Loggerhead Marinelife Center staff and volunteers, including S. Bergeron, S. Fournies, K. Garrido, and M. Merrill. K. Stewart provided valuable discussions regarding this project. J.E. Knowles provided assistance with maps. The authors also thank C.E. Proffitt for statistical advice and D. Scheurle for use of equipment. Lastly, the authors thank L. Bryan, T. Cook, E. Courtney, E. Dougherty, E. Eads, J. Lasala, M. Martin, A. Merrill, C. Ross, R. Timmons, and J. Yost for sample analyses and field assistance. The authors thank the reviewer for his or her thorough review and thoughtful comments on this manuscript. This study was supported, in part, by a Florida Sea Turtle License Plate Grant to D.L.M., The University of Georgia Veterinary Diagnostic and Investigational Laboratory, the FAU Nelligan Fund, and personal funds. All procedures were in adherence to Florida Fish and Wildlife Conservation Commission Marine Turtle Permit #073 conditions and FAU IACUC approval A07-03. References Ackerman, R.A., Seagrave, R.C., Dmi’el, R., Ar, A., 1985. Water and heat exchange between parchment-shelled reptile eggs and their surroundings. Copeia 1985, 703–711.

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