Trace Metals in Eggs and Hatchlings of Pacific Leatherback Turtles (Dermochelys coriacea) Nesting at Playa Grande, Costa Rica
Abstract
Leatherback turtles in the Pacific Ocean have declined precipitously in recent decades. One issue that may be contributing to the declines of leatherbacks and impairing their recovery is low clutch viability resulting from high embryonic mortality. Here, we examine trace metal concentrations in eggs and hatchlings from leatherbacks nesting on the Pacific coast of Costa Rica to assess whether contaminant levels reflect variation in the time over which females accumulated contaminants and whether variation in egg contaminant concentrations among nests influences clutch viability and hatchling characteristics. Eggs and hatchlings of Pacific leatherbacks contained detectable concentrations of a suite of essential and nonessential metals including Cu, Cd, Fe, Mn, Ni, and Zn, but variables that likely reflect a female's degree of temporal exposure to contaminants (relative age of female, body size, and remigration interval) explained little of the variation in egg metal concentrations. Concentrations of Cu, Cd, Fe, and Ni in Pacific leatherbacks were higher or toward the upper range of metal levels examined in sea turtle eggs elsewhere, but we did not find evidence linking egg contaminant levels with clutch success or hatchling size or body condition. These results represent the most comprehensive measures of trace metal concentrations from leatherback turtles in the eastern Pacific Ocean.
Leatherback turtles (Dermochelys coriacea) in the Pacific Ocean have declined precipitously in recent decades, likely attributable to a combination of anthropogenic factors that threaten sea turtle populations worldwide, including (but not limited to) the direct harvest of eggs and females from nesting beaches, bycatch in fisheries, destruction and degradation of nesting beaches, and pollution (Chan and Liew 1996; Spotila et al. 2000; Sarti Martínez et al. 2007; Santidrián Tomillo et al. 2008). Leatherbacks are renowned for being the most fecund of all the sea turtles (Miller 1997; Wallace et al. 2007), although they have low hatching success relative to other species. Low hatching success results from high rates of early embryonic mortality (Bell et al. 2003), and once hatched, many individuals may never emerge from the nest (Santidrián Tomillo et al. 2009). The factors underlying low hatching and emergence success remain poorly understood, but regardless of ultimate causation, this low success certainly constrains population recruitment and the ability of the species to recover.
Of the many factors implicated in sea turtle declines, environmental contamination may potentially contribute to the leatherback's low hatching success. The embryonic life stage can be especially sensitive to environmental contaminant exposure in oviparous animals (Guillette et al. 2000; Roe et al. 2004; Hopkins et al. 2006). Contamination of sea turtles by trace metals is well documented (Sakai et al. 1995; Lam et al. 2006; Guirlet et al. 2008). Although many trace metals are essential for proper development, some essential and nonessential metals have been found in sea turtles at potentially harmful concentrations. For instance, eggs from the green turtle Chelonia mydas in the western Pacific Ocean had concentrations of As (0.35 µg/g), Se (8.31 µg/g), and Ni (0.45 µg/g) at levels potentially toxic to embryos (Lam et al. 2006; van de Merwe et al. 2009). Eggs from Atlantic leatherbacks contained 1.4 µg/g Se, also a potentially toxic concentration (Guirlet et al. 2008). Because of their pelagic nature and low trophic level of their gelatinous zooplankton prey (Davenport and Balazs 1991; James and Herman 2001), leatherbacks are not generally considered to be at high risk of biomagnification of potentially toxic contaminants (Guirlet et al. 2008). However, very little is actually known about environmental contaminant levels in leatherbacks or about the toxic effects of these chemicals in sea turtles in general.
Studies of environmental contaminants in leatherbacks have assessed pollutant concentrations in adults and eggs, but the majority has been focused on turtles in the Atlantic Ocean (Davenport et al. 1990; Caurant et al. 1999; Guirlet et al. 2008), with much less information for the critically endangered Pacific populations (but see Vasquez et al. 1997). To our knowledge, no studies have assessed the impact of contaminant exposure on reproductive parameters in leatherbacks. Here, we monitor nests and examine concentrations of trace metals in the eggs and hatchlings of leatherbacks nesting on beaches in the eastern Pacific Ocean with the following three objectives in mind: 1) to assess concentrations of essential and nonessential metals in whole eggs and hatchlings of Pacific leatherbacks for the first time; 2) to evaluate whether trace metal levels in eggs are influenced by the female's relative degree of temporal exposure; and 3) to investigate the relationships between trace metal levels in eggs and measures of clutch success and hatchling quality.
METHODS
Field and Hatchery Measures
During the 2002–2003 nesting season, eggs were collected from leatherback turtles nesting on Playa Grande (10°20′N, 85°51′W), a beach within the Parque National Marino Las Baulas (PNMB), located on the Pacific coast of Costa Rica. Research teams patrolled the beach every night from 1 October to 15 February to locate nesting females. Once oviposition began, curved carapace length (± 1 mm; CCL) was measured, and the turtle was scanned for the presence of a Passive Integrated Transponder (PIT) tag. All turtles nesting on beaches in the PNMB have been implanted with PIT tags since the 1993–1994 nesting season, giving them a unique and permanent identifier (Steyermark et al. 1996; Reina et al. 2002). If a PIT tag was detected, the turtle was classified as a “remigrant”. A remigration interval was calculated as the time in years since her most recent known nesting event in PNMB. Given the high observer coverage of the beach and the number of times an individual nests during a season, the probability of missing a turtle every time she nests is 0.107 (Santidrián Tomillo et al. 2007). Although individual females may move among localized nesting beaches between seasons, it is thought that females exhibit strong fidelity to the three beaches in PNMB (Santidrián Tomillo et al. 2007). Given the high detection probability, beach fidelity, and the maximum remigration interval of 7 years, Santidrián Tomillo et al. (2007) concluded that any unmarked turtles appearing after the 2000–2001 nesting season were new recruits to the population. Consequently, if no PIT tag was detected, the turtle was classified as a “new” nester, most likely representing the turtle's first lifetime nesting event.
Clutches from 14 remigrant turtles and 12 new nesting turtles were collected and relocated to a protected outdoor hatchery on Playa Grande. Clutches were only relocated to the hatchery if they were laid below the high tide line. For each female, two clutches were collected—one presumed to be the first nest of the season and the other from the female's fourth known nest of the season. These nests correspond to the early and middle periods of the nesting season at PNMB. Eggs were counted and a subsample of 20 from each clutch was weighed prior to burying the entire clutch in the hatchery. Mean egg mass was then multiplied by the number of eggs in the clutch to give an estimate of the total clutch mass. For a detailed description of the hatchery and protocols for burying and monitoring eggs, refer to Wallace et al. (2004).
Once hatchlings emerged, 20 from each nest were measured (± 1 mm; straight carapace length [SCL]) with digital calipers and weighed on an electronic balance. A body condition index was calculated as the deviation in observed mass from that predicted by the regression relationship between body mass and SCL of all hatchlings. All live hatchlings were immediately released on the nesting beach. Two days after hatching ceased, nests were excavated and the proportion of the clutch that hatched (hereafter hatching success) and proportion that emerged (hereafter emergence success) were determined. If no hatchlings emerged, nests were excavated 70 days after burial. One unhatched egg and one dead hatchling from each nest were retained for trace metal analysis. Dead hatchlings were only available for 7 new and 12 remigrant nests.
Trace Metal Analysis
Concentrations of 8 trace metals (Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) were analyzed using ICP-OES/AES (Inductively Coupled Plasma – Optical/Atomic Emission Spectrometer; Leeman Labs PlasmaSpec ICP 2.5) using the following procedures. Approximately 10 g of each homogenized whole egg or whole hatchling sample was digested in a mixture of trace metal grade sulfuric and nitric acid (10 ml each) before being heated. After digestion, samples were brought to a final volume of 100 ml using d-d water. Standard solutions for Cd, Cu, Fe, Mn, Pb, and Zn were made from 1000 ppm solutions from Leeman Labs, Inc. Solutions ranging from 0.005–100 ppm were prepared by serial dilution. Cr and Ni solutions were made by dissolving 0.76968 grams of Cr(NO3)3(H20)9 and 0.4049 grams of NiCl2(H20)6 in 100 ml of d-d water to provide standard solutions, followed by preparation of serial dilutions as described above. The 100-ppm standard solutions were used as peaking solutions to allow the machine to lock onto the appropriate wavelength of light. Once the appropriate wavelength had been acquired, the 10-ppm, 1-ppm, 0.1-ppm standard solutions and d-d water (blank) were analyzed with the ICP-OES/AES. Egg samples were run in groups of no more than 20 before running standard solutions again. All standards and samples were run in triplicate. The standards were used to determine the slope and y-intercept of the regression line used to determine actual metal levels in the egg samples using the following equation:
Metal levels below the range of our standard solution dilutions were classified as below the instrument's detection limits (BDL). Mean percent recoveries for metals in standards from Leeman Labs, Inc ranged from 93.5%–105.6%. Detection limits (ppm) were 0.4 for Cr; 0.1 for Cu; 0.01 for Cd and Pb; 0.05 for Mn; and 0.005 for Fe, Ni, and Zn.
Statistical Analyses
All statistical analyses were performed with SPSS 17.0 (Chicago, 2007). To satisfy the assumptions of normal distributions and equality of variances, all proportional variables were arcsine-transformed, and all trace element and turtle and egg size variables were log10-transformed. Statistical significance was accepted at the α ≤ 0.05 level except for multiple related comparisons, where the Dunn-Sidak method was applied to constrain the experiment-wide Type I error to 0.05.
To determine whether trace element concentrations in eggs varied between early and late nests, we used a repeated-measures multivariate analysis of variance (MANOVA) with element concentrations as dependent variables and nest sequence as the within-subjects factor. Next, we compared egg trace element concentrations between remigrant and new nesters using a multivariate analysis of covariance (MANCOVA), with element concentrations as dependent variables, female “group” as an independent variable, and log10-female CCL and total clutch mass as covariates. Also, we explored relationships between female CCL and element concentration of eggs and hatchlings using linear regressions. The Dunn-Sidak adjusted level of significance for these regression tests was α = 0.005. Using the first nests of remigrant turtles only, we then assessed relationships between egg trace element concentrations and remigration interval with regression. The Dunn-Sidak adjusted level of significance for these regression tests was α = 0.008.
To assess relationships between egg trace element concentrations and each of the dependent hatching variables (hatching success, emergence success, hatchling SCL, hatchling mass, and body condition index), we used multiple stepwise regression analyses. For analyses involving hatchling body size and condition, we used only data from the first nests because the fourth nests yielded considerably fewer hatchlings to measure (see Results). The Dunn-Sidak adjusted level of significance for each of these regression tests was α = 0.007.
RESULTS
We detected measurable amounts of all trace elements except Cr and Pb in eggs from leatherback turtles (Table 1). Fe and Zn concentrations in eggs increased with remigration interval (Fe: R2 = 0.44, F1,12 = 9.24, p = 0.01; Zn: R2 = 0.29, F1,12 = 4.94, p = 0.046; Fig. 1), but these relationships were not apparent after applying the Dunn-Sidak correction. Egg trace element concentrations did not vary between nests laid earlier and later in the season (Wilks' λ = 0.73, F6,20 = 1.23, p = 0.332); thus, we proceeded with analyses after combining nests into a single mean value for each female. Fe concentration in eggs decreased with female CCL (r2 = 0.173, p = 0.034; Fig. 2), although the correlation coefficient was low. Furthermore, this relationship (and that for all other elements) was not significant after applying Dunn-Sidak adjustments. After accounting for the potentially confounding covariates of female size and clutch mass, eggs from remigrant (presumably older) and new (presumably younger) nesting females did not vary in trace element concentrations (female CCL: Wilks' λ = 0.76, F6,17 = 0.90, p = 0.520; clutch mass: Wilks' λ = 0.68, F6,17 = 1.36, p = 0.285; group: Wilks' λ = 0.92, F6,17 = 0.25, p = 0.952).
Citation: Chelonian Conservation and Biology 10, 1; 10.2744/CCB-0837.1
Citation: Chelonian Conservation and Biology 10, 1; 10.2744/CCB-0837.1
For the first nests, hatching success was 0.38 ± 0.05 (range = 0–0.83), and emergence success was 0.30 ± 0.05 (range = 0–0.80). Hatchling SCL was 57.5 ± 0.4 mm (range = 54.6–61.2 mm) and mass was 40.8 ± 0.4 g (38.1–44.9 g). Trace metal concentrations in eggs did not explain a significant amount of the variation in clutch success or hatchling size and body condition for the first nests (R2 < 0.21, p > 0.173 in all cases). For the fourth nests, hatching success was 0.16 ± 0.04 (range = 0–0.84), and emergence success was 0.11 ± 0.04 (range = 0–0.78). Hatchling SCL was 53.9 ± 0.5 mm (range = 49.9–57.6 mm) and mass was 38.8 ± 0.7 g (35.0–46.7 g). Both emergence and hatching success decreased with increasing Cu concentration (F1,24 > 4.96, R2 > 0.23, p < 0.036), but these relationships were not significant after applying the Dunn-Sidak adjustment.
DISCUSSION
By using unhatched eggs and hatchlings that died prior to nest emergence, we were able to document the most comprehensive measures of trace metal concentrations from leatherback turtles in the eastern Pacific Ocean. The only other study that assessed trace metals or other potential environmental contaminants for leatherbacks in the region reports on egg shells remaining in nests after hatching (Vasquez et al. 1997). Measures from whole eggs and hatchlings reveal much more with regards to exposure of developing embryos and hatchlings to potentially toxic substances (Sakai et al. 1995). Our findings indicate that leatherback embryos accumulate a suite of essential metals including Cu, Fe, Mn, and Zn, as well as nonessential metals Cd and Ni. In part, such concentrations reflect the maternal transfer of metals to eggs but also may be influenced by exposure in the nest environment, a contamination route we did not measure. However, Nagle et al. (2001) concluded that trace metals are not incorporated into eggs from the nest substrate in appreciable amounts.
Trace Metals and Temporal Exposure
Several variables that reflect a female's relative degree of temporal exposure to contaminants failed to correlate significantly with metal accumulation as measured in their eggs. First, we expected that individuals with longer remigration intervals may accumulate higher concentrations of metals because of longer periods of exposure without opportunity for elimination. Female turtles transfer metals to eggs (Burger and Gibbons 1998; Nagle et al. 2001). Given the high reproductive output of leatherbacks, this route of elimination could be potentially significant for some metals, especially those that are essential micronutrients for embryonic development (Guirlet et al. 2008; but see Sakai et al. 1995). In Atlantic leatherbacks, concentrations of organic contaminants were higher in animals with longer remigration intervals (Guirlet et al. 2010). However, only concentrations of essential metals Fe and Zn increased with remigration interval in our study, and these relationships were not strong.
Second, we expected eggs from older and larger turtles to have higher metal concentrations than smaller and presumably younger individuals. However, egg metal concentrations did not increase with adult body size, and eggs from the presumably older remigrants had similar concentrations to the new nesters. Our inability to detect apparent age- and size-related variation in metal levels could reflect several factors. Though body size generally correlates with age, Price et al. (2004) concluded that major differences in body size likely result from variable juvenile and subadult growth rates. Similarly, our division of turtles into only two broad age groups overlooks wide variability in actual age of mature turtles. In support of our findings, Bishop et al. (1994) found that relative size was not a good predictor of contaminant levels in the freshwater turtle Chelydra serpentina, and hypothesized that individual variation in foraging and habitat preferences may be more important determinants of contaminant exposure (see also Bergeron et al. 2007). Leatherbacks nesting at PNMB traverse an enormous foraging area in the eastern Pacific Ocean between nesting events (Shillinger et al. 2008), including pelagic and near-coastal environments bordering many countries where spatial exposure to contaminants could vary considerably (Talavera-Saenz et al. 2006; Caut et al. 2008).
Trace Metals and Reproductive Effects
We also found little evidence that metal levels in leatherback eggs had any significant influence on clutch success, hatchling size, or hatchling body condition. Leatherbacks have a hatching success of approximately 50%, which is low relative to other sea turtles (Miller 1997). The mean hatching success for first (38%) and fourth (16%) nests in this study was considerably lower. Relocated leatherback nests have hatching success on average 6% to 10% lower than nests left in situ (Eckert and Eckert 1990; Garrett et al. 2010). Thus, our estimates of hatchling and emergence success should be viewed with caution when comparing across studies, but comparisons among relocated nests in this study are still warranted given their similar treatment. Despite the high level of individual variability in both metal concentrations and metrics of clutch and hatchling characteristics, metal concentrations of eggs were not strongly correlated with measures of clutch success, hatchling size, or hatchling body condition. It should be noted that our study lacked the proper controls to definitively link reproductive success with contaminant exposure. Such a study would compare reproductive success among individuals under controlled contaminant exposure levels (i.e., such as in the laboratory) or that differ in their spatial or temporal exposure to contaminants in the wild—both of which would be difficult for leatherbacks.
Comparisons to Other Studies
In light of the limitations of this study, it would be instructive to compare metal concentrations in leatherback eggs from PNMB with concentrations documented in other sea turtles and with reproductive effects thresholds for other oviparous vertebrates. Two essential metals, Cu and Fe, and two nonessential metals, Cd and Ni, were either higher than or toward the upper end of the range of concentrations in sea turtle eggs examined elsewhere (Sakai et al. 1995; Linder and Grillitsch 2000; Lam et al. 2006; van de Merwe et al. 2009), including leatherbacks (Vasquez et al. 1997; Guirlet et al. 2008). Leatherbacks are unique among sea turtles in several ways, potentially contributing to differences in metal concentrations. Leatherbacks have high hemoglobin concentrations in support of their endothermic metabolism, long dive duration, and high activity (Lutcavage et al. 1990). Such high hemoglobin (an iron-containing protein) could account in part for the relatively high Fe concentrations in leatherback eggs. Also, leatherbacks occupy a unique foraging niche, feeding primarily on gelatinous zooplankton (Davenport and Balazs 1991; James and Herman 2001). However, the relatively high Cd, Cu, Fe, and Ni levels in leatherback eggs are difficult to explain in light of the low trophic level of their diet, a situation where we would expect contaminant biomagnification to be low, although jellyfish can accumulate these metals at concentrations above that of the ambient environment (Templeman and Kingsford 2010). The higher concentrations of Cd and Cu in our study compared to leatherback populations in the Atlantic Ocean (Guirlet et al. 2008) indicate possible differences in metal exposure between ocean basins.
For both essential and nonessential metals, assessing the potential risk of elevated trace metal concentrations to leatherback eggs is hindered by the paucity of information on critical effects thresholds for trace metal contaminants in reptiles, and caution must be exercised in interpreting effects thresholds established for other species (Hopkins 2006). Concentrations of Cd in leatherback eggs (1.6 ppm) were 1.2–11-fold higher than no effects thresholds for chicken embryo mortality (Leach et al. 1979; Voleda et al. 1997). Likewise, Ni concentrations in leatherback eggs (1.9 ppm) were 11-fold higher than no effects thresholds for chicken embryos (Gilani and Marano 1980; Lam et al. 2006). For Cu and Fe, we found no evidence that abnormally high concentrations of these essential metals have reproductive effects in other oviparous reptiles or birds. Thus, based on the relationships between egg metal concentration and metrics of clutch success and hatchlings determined here, together with uncertainties of extrapolating effects across taxa and the paucity of controlled toxicological dose-response studies, we could not link low clutch viability in leatherbacks to the suite of potential environmental contaminants examined here. However, it should be noted that our analyses did not assess Se, an essential micronutrient that is maternally transferred to eggs in reptiles (Hopkins et al. 2004; Roe et al. 2004; Guirlet et al. 2008). Se can be embryotoxic in excessive concentrations (Heinz 1996; Lemly 1996), and Se can accumulate in sea turtles at levels known to be embryotoxic in other vertebrates (Lam et al. 2006; van de Merwe et al. 2009). It would be instructive for future studies to assess Se concentrations in Pacific leatherbacks given their high rates of embryonic mortality and declining populations. We conclude that factors such as maternal genotype, maternal health, or nest environment may have a more profound influence on clutch viability in leatherbacks than environmental contaminants at current exposure levels in the eastern Pacific Ocean (Bell et al. 2003; Wallace et al. 2004; Santidrián Tomillo et al. 2009).
Relationships between the trace metals Fe and Zn and remigration interval for leatherback turtles, Dermochelys coriacea, nesting at Playa Grande, Costa Rica. Note that relationships were not significant after Dunn-Sidak adjustments.
Relationship between Fe concentration and turtle body size (CCL) for leatherback turtles, Dermochelys coriacea, nesting at Playa Grande, Costa Rica. Note that relationship was not significant after Dunn-Sidak adjustment.
