Examining maternal and environmental transfer of mercury into American alligator eggs

Abstract

American alligators are exposed to mercury (Hg) throughout their natural range and may maternally transfer Hg into their eggs. Wildlife species are highly sensitive to Hg toxicity during embryonic development and neonatal life, and information on Hg transfer into eggs is critical when attempting to understand the effects of Hg exposure on developing oviparous organisms. To examine Hg transfer in alligators, the objectives of the present study were to 1) determine Hg concentrations in yolk (embryonic and neonatal food source) from wild alligator eggs collected from three locations - Yawkey Wildlife Center SC (YWC), Lake Apopka FL (LA), and Lake Woodruff FL (LW); 2) examine the relationship between THg concentrations in wild alligator nest material and egg yolk at Merritt Island National Wildlife Refuge, FL; 3) examine the Hg concentrations in wild maternal female alligators (blood) and the THg in corresponding egg yolks and embryos across three nesting seasons at a single location (YWC), and evaluate the relationship between nesting female THg concentrations (blood) and their estimated age and number of nesting years (YWC); and 4) assess the transfer of biologically-relevant Hg concentrations (based on Hg measured in maternal female blood) into embryos using an egg-dosing experiment. Mean total Hg (THg) concentrations observed at each site were 26.3 ng/g ± 11.0 ng/g (YWC), 8.8 ng/g ± 5.1 ng/g (LA), and 22.6 ng/g ± 6.3 ng/g (LW). No relationship was observed between THg in alligator nest material and corresponding yolk samples, nor between THg in maternal alligator blood and estimated age and number of nesting years of these animals. However, significant positive relationships were observed between THg in blood of nesting female alligators and THg in their corresponding egg yolk. We observed that 12.8% of the maternal blood THg is found in the corresponding egg yolk, and a highly significant correlation was observed between the two sample types (r = 0.66; p < 0.0001). The egg dosing experiment revealed that Hg did not transfer through the eggshell at developmental stage 19. Overall, this study provides new information regarding Hg transfer in American alligators which can improve biomonitoring efforts and may inform ecotoxicological investigations and population management programs in areas of high Hg contamination.

1. Introduction

Mercury (Hg) is a pervasive and persistent heavy metal that has adverse behavioral, neurochemical, and reproductive effects in wildlife (Scheuhammer et al., 2007). Although naturally occurring, Hg enters the environment through a variety of anthropogenic sources including fossil fuel combustion and metal production, among others (Hanisch, 1998; Pacyna and Pacyna, 2005; Ullrich et al., 2001). In aquatic ecosystems, bacteria convert inorganic Hg to the more lipophilic and toxic methylmercury, which readily bioaccumulates and biomagnifies in food webs, posing particular risks to animals occupying higher trophic levels (Chen et al., 2005; Chumchal et al., 2011; Lavoie et al., 2013; Ullrich et al., 2001).

Crocodilians (crocodiles, alligators, caiman, gharials) are long-lived, apex predators that live in lowland swamps, rivers, lakes, and other wetlands that are frequently subject to Hg contamination. As a result, Hg has been detected in numerous crocodilian species throughout their global range, and is believed to be the heavy metal of greatest concern to these animals (Almli et al., 2005; Buenfil-Rojas et al., 2015; Eggins et al., 2015; Nilsen et al., 2016; Rainwater et al., 2007).

The American alligator (Alligator mississippiensis) occurs in the southeastern United States, where Hg contamination in wetlands is common and considered to be one of the most serious environmental threats to wildlife in the region (Elsey and Woodward, 2010; Facemire et al., 1995). Information regarding the exposure and response of alligators to Hg is important because alligators are keystone species, providing multiple ecological services critical to the normal function of the aquatic systems they inhabit (Burtner and Frederick, 2017; Hall and Meier, 1993; Kushlan and Kushlan, 1980; Mazzotti et al., 2009; Nifong and Silliman, 2013). As such, Hg (or other pollutant) contamination in alligators may reflect or result in adverse effects on the habitats these animals help support. In addition, wild alligators are often harvested and consumed by humans through state-regulated hunting seasons (Elsey et al., 1999; Hord et al., 1990; Ruckel, 1993; Smith et al., 2017; Tipton et al., 2017). Data regarding Hg accumulation in alligators is therefore vital for identifying potential human health risks from the consumption of contaminated alligator meat.

Because wildlife species are highly sensitive to Hg toxicity during embryonic development and neonatal life, information on Hg transfer into eggs is critical when attempting to understand the effects of Hg exposure on developing oviparous organisms. To date, several studies have documented Hg concentrations in wild, free-ranging alligators while comparatively few have examined Hg in alligator eggs (Rainwater et al., 2002; Stoneburner; Kushlan, 1984; Xu et al., 2006). To our knowledge, no study has examined the transfer of Hg into alligator eggs and embryos. To address this data gap, the objectives of the present study were to:

1. To determine the Hg concentrations in yolk (embryonic and neonatal food source) from wild alligator eggs collected from three locations - Yawkey Wildlife Center SC (YWC), Lake Apopka FL (LA), and Lake Woodruff FL (LW),

2. examine the relationship between THg concentrations in wild alligator nest material and egg yolk at Merritt Island National Wildlife Refuge, FL (MINWR),

3. examine the THg concentrations in wild maternal female alligators (blood) and the THg in corresponding egg yolks and embryos across three nesting seasons at a single location (YWC); and evaluate the relationship between nesting female THg concentrations (blood) and their estimated age and number of nesting years (YWC), and

4. assess the transfer of biologically-relevant Hg concentrations (based on Hg measured in maternal female blood) into embryos using an egg-dosing experiment.

2. Methods

2.1. General sample collection

In June 2011, 2013, and 2014, alligator nests were located by helicopter, airboat, or on foot at LW, LA, and MINWR in Florida, and the YWC in South Carolina (Fig. 1 and Fig. S1). Alligators from these sites exhibit varying environmental contaminant profiles, but Hg has not been examined. Alligator eggs and nest material (e.g., comprised primarily of vegetation and soil) were collected from nests, placed in industrial bus pans, and transported to the laboratory, where nest material was replaced with damp sphagnum moss, and eggs were placed in commercial incubators at 32 °C (Thermo Scientific, Waltham, MA, USA, Forma Environmental Chambers, model #3920, Fig. S2) (Bangma et al., 2017a, 2017b; McCoy et al., 2015). Nest material samples were stored in WhirlPaks (Millipore Sigma, St. Louis, MO) and frozen at −20 °C until analysis.

Fig. 1.

Left: Locations of the four sites sampled for egg yolk and/or nesting material in this study. The inset highlights Florida and South Carolina in the United States of America as the area of study. Center: Locations (triangles) of alligator nests sampled at Yawkey Wildlife Center, SC (YWC) during 2011, 2013 and 2014. Right: Locations (triangles) of alligator nests sampled at Lake Woodruff, FL (LW) in 2015. GPS coordinates are provided in Tables S2 and S3.

In June of 2011 (n = 5), 2013 (n = 4), and 2014 (n = 7), whole blood samples were collected from adult YWC female alligators captured at nests (putative maternal females; Wilkinson et al., 2016) (Table S2). At YWC we had the unique ability to monitor alligator nests daily, and capture females for sampling when they visited their nest. We did not have this ability at the other sampling sites, so only YWC was used for the maternal paired samples. Blood was collected from the post-occipital venous sinus (Myburgh et al., 2014) with 1 inch 18.5-gauge needles (BD, Franklin Lakes, NJ, USA) and 60 mL Luer-lock syringes (BD), transferred to lithium-heparin Vacutainer tubes (BD), stored on ice in the field, and transported to the laboratory where they were stored at −80 °C until analysis. A series of body measurements including total length and snout-vent length (SVL) was measured for each female alligator sampled, and each animal was released at its capture location (Wilkinson et al., 2016).

All fieldwork and sample collections were performed under permits issued from the Florida Fish and Wildlife Conservation Commission, South Carolina Department of Natural Resources, and United States Fish and Wildlife Service.

2.1.1. Sample collection for determination of Hg in yolk

Yolk provides nutrition to developing alligator embryos but may also be a source of environmental contaminants if pollutants are transferred from the maternal female into yolk during vitellogenesis (Morafka et al., 2000; Noble, 1991; Rauschenberger et al., 2004a, 2004b, 2007). To examine potential Hg transfer from yolk to developing alligator embryos, we measured THg concentrations in yolk from wild alligator eggs collected in 2011 from LW, LA, and YWC. Eggs were collected as described above and incubated until reaching developmental stage 19–21 (25–30 days post-oviposition), at which time they were opened with clean dissection scissors and yolk was collected using a 10 mL Luer-lock syringe (BD)(Ferguson, 1987). Yolk was then transferred to a 5% nitric acid rinsed 50-mL centrifuge tube (Falcon), homogenized by gentle rocking, aliquoted into 2 mL cryovials (Corning, Corning, NY, USA), and stored at −20 °C until analysis (Table S3).

2.1.2. Sample collection for determination of Hg in nest material and corresponding eggs

Crocodilian eggs may be exposed to environmental contaminants in the nest environment during incubation, and laboratory studies have demonstrated the ability of some pollutants to transfer from nest material into reptile eggs (Cañas and Anderson, 2002; Marco et al., 2004; Wu et al., 2000). To examine potential Hg transfer from wild alligator nest material into corresponding eggs and embryos, we measured THg concentrations in nest material samples from alligator nests collected in 2014 at LW (n = 6), LA (n = 6), MINWR (n = 5), and YWC (n = 5) (Fig. 1). Samples of sphagnum moss (n = 3) used for incubation were also analyzed for comparison. Prior to analysis in the DMA-80, nest material (including sphagnum moss) samples were cryo-homogenized using a benchtop Cryomill (Retsch, Haan, Germany). Nesting material homogenates were then aliquoted into 2 mL cryovials (Corning, Corning, NY, USA) and analyzed. Egg yolk samples were collected from nests corresponding to nest material samples at YWC (n = 3) and MINWR (n = 4) and were processed as described in section 2.1.

2.1.3. Sample collection for determination of Hg in maternal female alligators

To examine the potential for maternal transfer of Hg into eggs and developing embryos, we measured THg concentrations in blood samples from putative maternal females captured at nests as well as in yolk and embryos collected from the same nests at YWC in 2011 (n = 18 eggs, 5 nests), 2013 (n = 11 eggs, 4 nests), and 2014 (n = 45 eggs, 7 nests) (Fig. 1, Table S2). Yolk was processed as described in Section 2.1. Embryos were collected from eggs using stainless steel dissection instruments and placed directly into cryovials without further processing due to the early developmental stage and small size of the embryos. All samples were placed on wet ice for no longer than 2 h before being stored at −80 °C until analysis.

2.1.4. Sample collection for the determination of transfer of Hg through eggshell

Alligator eggs used in this experiment were collected from LW during June of 2014 (Fig. 1, Table S3). Eggs were transported to the laboratory, and one egg from each nest (n = 2) was opened to determine the embryonic stage of that nest (Ferguson, 1987). Eggs were incubated at 32 °C as described above. Due to a lower-than-expected number of alligator nests located at LW in 2014, the experiment was designed to accommodate only two nests.

2.2. Instrumental method & quantification

For all experiments described below, the mass fraction of total Hg (THg) was determined with a direct Hg analyzer DMA-80 (Milestone Scientific, Shelton, CT). The method parameters for direct combustion atomic absorption spectroscopy measurements for the determination of THg were: 90 s ramp to 200 °C; 30 s hold; 90 s ramp to 650 °C, 180 s hold.

The instrumental and procedural blanks for the analysis of THg were measured concurrently with the samples. The procedural blanks were made with high-purity de-ionized water to mimic the processing of the samples. Aliquots of approximately 0.10 g were run alongside the experimental samples. The concentrations of the procedural blanks were below the limit of detection (LOD) and therefore were not subtracted from any experimental samples.

2.3. Quality control

National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 955c Toxic Metals in Caprine Blood Level 02 (4.95 ng/g ± 0.76 ng/g) and Level 03 (17.8 ng/g ± 1.6 ng/g), NIST Egg Reference Material QC04-ERM-1 (101 ng/g ± 3 ng/g), and of SRM 1573a Tomato Leaves (34 ng/g ± 4 ng/g) were run as control materials for the analyses, chosen for their similarity to the sample matrices. All replicated measurements of the control materials throughout the following experiments were within range of the certified or target values and had a % relative standard deviation of 5% or less. The measurement uncertainty is represented by the standard deviation of the measurements (Table S1).

All experiments performed herein conform to the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) at the Medical University of South Carolina.

2.4. Experimental design of egg-dosing study

To examine the amount of Hg that could transfer through the alligator eggshell, a laboratory dosing approach was tested for feasibility. Previous studies have reported successful results after topically applying chemical solutions to alligator eggshells (Cruze et al., 2015; Guillette et al., 1995; Gunderson et al., 2003; Hamlin et al., 2010). However, topical application as a dosing method has come under scrutiny in the field of environmental toxicology, as few studies provide quality control measurements to determine if the applied dose actually transferred through the eggshell (Muller et al., 2007). We examined the rate and amount of topically applied methylmercury-cysteine dissolved in Milli-Q water that is transferred through the alligator eggshell and into the developing embryo. Pure water was chosen as the environmentally relevant vehicle for dosing as it would mimic the atmospheric deposition of Hg in rainfall (Guentzel et al., 1998, 2001). Methylmercury-cysteine was chosen as it is the most bioavailable form of methylmercury and is less volatile than the smaller Hg²⁺ or HgCl₂ compounds (Bridges et al., 2012; Roos et al., 2010). The pre-conjugation to cysteine was conducted because the natural conjugation in the blood of the developing embryo would not occur as it would in a live-feeding alligator because the available cysteine in the embryo is utilized for differentiation of the endoderm (Bennett, 1973; Roos et al., 2010). Cysteine conjugation during transfer through the egg compartments is possible but unlikely since cysteine is rate-limiting for protein synthesis, is less than 0.2% of the total weight, and is found in concentrations of approximately 0.001 mol in chicken eggs (a close genetic relative to the alligator) (Breuille and Obled, 2000; Janke and Arnason, 1997; Osonka et al., 1947). Applying methylmercury-cysteine to the fully formed calcium carbonate (CaCO₃) eggshell should not elicit a reaction as these are both strongly bound compounds that do not dissociate easily (Breuille and Obled, 2000; Roos et al., 2010; Simkiss and Tyler, 1958).

Twenty-eight eggs were collected from each of the two nests from LW; one egg from each was used to determine developmental stage, leaving a total of 54 eggs for the experiment. The eggs were divided into three groups (reference, low, and high dose) of 18 eggs comprised of 9 from each of the two nests collected (Fig. S1, Table S5).

Eggs were topically dosed with methylmercury-cysteine (described in the Supplemental Information) at stage 12 of embryonic development to mimic maternal transfer based on the correlation equation we derived from the paired female – nest sample analysis detailed in section 2.3 and reported in section 3.3 (y = 0.138x – 2.5534). Stage 12 (13–14 days post-oviposition) was selected as it was the latest developmental stage of the two collected nests, and therefore both nests could be dosed at the same developmental stage. Doses were designed to reflect the extrapolated egg yolk THg concentrations estimated using the equation in section 3.3 from the average of all adult female blood Hg concentrations previously reported by Nilsen et al. (2016) in the Everglades of south Florida, and the lowest single blood Hg concentration in the same group of animals (1079 ng/g, and 213 ng/g THg, respectively; described in detail in the Supplement).

Two eggs (one from each nest) were collected from each dose group at 24 h, 48 h, 7 d and 14 d after the solutions were applied. These time points were selected to determine the rate of transfer for methylmercury-cysteine through the eggshell into the various egg compartments (Fig. S3). Because only two replicates from each treatment were measured, the collection methods and observed results are commented on in the Supplemental Information. The remaining eggs (n = 10 per dose) were incubated to stage 27, the final embryonic stage prior to hatching (60–63 days post-oviposition) At stage 27, seven eggs per dose group remained (the others were cracked or otherwise compromised and did not contain a viable stage 27 embryo). Eggs were opened, and a blood sample was collected from the stage 27 embryo (Myburgh et al., 2014). Blood samples were vortexed for 30 s and divided into erythrocyte and plasma fractions that were frozen separately at −20 °C until THg analysis. The erythrocyte sample was used for determination of THg transferred into the embryo by the end of development, and the plasma sample was reserved for future analyses.

2.5. Statistical analysis

2.5.1. Hg in yolk

THg egg yolk data had a normal distribution but were not homoscedastic, even under a log₁₀ transformation. Since the assumptions of parametric statistics were not met, the Kruskal-Wallis test was used to determine the statistical differences in yolk THg concentrations among collection sites, and the Dwass, Steel, Critchlow-Fligner (DSCF) multiple comparison test was used to determine statistical differences between sites using “proc npar1way data = filename” “dscf” “class site” and “variable Hg” functions (SAS 9.4, SAS Institute Inc, Cary, NC).

2.5.2. Hg in alligator nest material and corresponding eggs

Due to uneven and low sample sizes, the non-parametric Kruskal-Wallis test was used to evaluate the difference in THg in nesting material among sites. Four of the five MINWR nest material samples had one matched egg yolk sample. One MINWR nest did not have a matched yolk sample and was not included in the analysis. Of the five YWC nest material samples, three were matched to at least eight yolk samples; the other two YWC nest material samples had no matched yolk samples and were not included in the analysis. THg concentrations in multiple yolk samples from the same nest were averaged, paired with THg concentrations in nest material from the same nest, and compared using the non-parametric Spearman correlation. Statistical analysis was conducted using SAS 9.4 software by using “proc npar1way data = filename”“class site” and “variable Hg”; and “proc corr data = filename” “outs = Spearman” “by year” and “var NestMaterialHg YolkHg” functions.

2.5.3. Hg in maternal female alligators

THg concentrations in female alligator blood were normally distributed, while THg concentrations in the paired yolk were not normally distributed, or homoscedastic. Log₁₀ transformation normalized these data but did not reduce the variance between samples. Since the assumptions of parametric statistics could not be met, the non-parametric Spearman correlation was used to determine the statistical relationship between the THg concentrations in maternal female blood and corresponding egg yolk and embryo samples. Due to the long-term monitoring effort at YWC, we had the unique ability to examine the relationship between Hg concentrations in the maternal female blood and the estimated age and number of nesting years of each female. Estimated age was calculated following the model previously developed for the YWC alligator population (Lawson et al., 2019; Wilkinson et al., 2016). To determine the estimated number of years each female has nested, the estimated age of each female was reduced by 16 (the average age at sexual maturity), and then divided by 2 (two years), the mean nesting frequency for female alligators at YWC (Wilkinson and Rainwater, unpublished data). To compare female blood Hg concentrations to the estimated number of nesting seasons and estimated age, the Spearman correlation was used. Statistical analysis was conducted using SAS 9.4 software by using the “proc corr data = filename” “outs = Spearman” “by year” and “var FemaleHg YolkHg EmbryoHg EstAge or EstNests” functions.

2.5.4. Transfer of Hg through eggshell

Stage 27 erythrocyte samples were evaluated for statistical differences in Hg concentration between dose groups and nests using the non-parametric Kruskal-Wallis test. Differences in THg concentrations by dose group between nests were not examined due to the low sample sizes in those subgroups. Statistical analysis was conducted using SAS 9.4 software by using the “proc npar1way data = filename”“class nest or dose” and “variable Hg” functions.

3. Results & discussion

3.1. Hg in yolk

Mean egg yolk THg concentrations from LA, LW, and YWC were 8.8 ng/g ± 5.1 ng/g, 22.6 ng/g ± 6.3 ng/g, 26.3 ng/g ± 11.0 ng/g, respectively (Fig. 2, Tables S6 and S7). THg concentrations in yolk were significantly higher at LW and YWC than LA (p < 0.0001), but not significantly different between LW and YWC (p = 0.26) (Fig. 2). This result was somewhat surprising, as LA is located adjacent to several urbanized and industrial areas and has a long history of environmental contamination, while LW and YWC are located in more remote and protected locations (Milnes and Guillette, 2008). Lower Hg concentrations in alligator yolk at LA suggests few or no direct anthropogenic Hg inputs there and that Hg in all three areas likely occurs naturally and/or from atmospheric deposition.

Fig. 2.

THg concentrations in alligator egg yolk samples collected at Yawkey (n = 43), Apopka (n = 37), and Woodruff (n = 49) in 2011. The mean THg concentration at Lake Apopka (LA) was statistically lower than those at the other two sites by the Wilcoxon Each Pair comparison indicated by an asterisk (p < 0.0001). Diamonds indicate the mean; horizontal lines indicate the median. The error bars are calculated using the first and third quartiles ± 1.5 × the interquartile range. The first and third quartiles are displayed on each side of the median line on the boxplots.

These data indicate THg is detectable in egg yolk at sites where the average Hg concentration is less than 200 ng/g in adult alligator blood samples (Nilsen et al., 2017a, 2019). Based on in yolk our measurements and the conversion of previously published Hg concentrations in liver (440 ng/g) to estimated blood concentrations (~85.5 ng/g) using the equation provided by Nilsen, 2017, LA may be the lowest site from which THg in the egg yolk and tissues of adult alligators has been measured. Maternal transfer of contaminants, particularly Hg, is known to widely occur in mammals and has been shown in certain amphibians and reptiles (Grandjean et al., 1992; Hopkins et al., 2006; Verreault et al., 2006). The assumption that maternal Hg transfer occurs in alligators is supported by the known maternal transfer of other xenobiotics such as selenium and organic contaminants, and by the Hg concentrations measured in female alligator reproductive organs and unpaired egg samples (Rauschenberger et al., 2004a; Roe et al., 2004; Sparling et al., 2010).

3.2. Hg in alligator nest material and corresponding eggs

Nest material

THg concentrations in nest material from MINWR (mean THg = 25.4 ng/g) was significantly higher than those at LW (p < 0.008), YWC (p < 0.01), and the sphagnum moss (p < 0.04) but not LA (mean THg = 19.9 ng/g; p = 0.32) (Fig. 3, Table S8). This was not unexpected, as MINWR and LA are in close proximity to development and industrial areas (potential Hg sources), while LW and YWC are located in more remote areas.

Fig. 3.

The boxplots of the THg concentration found in the nesting material of American alligators. Overall statistical significance was assessed using the Kruskal-wallis test; statistical significance in the Pairwise Two-Sided Multiple Comparisons was examined using the Dwass, Steel, Critchlow-Fligner Method and is indicated with an asterisk. Diamonds indicate the mean; horizontal lines indicate the median. The error bars are calculated using the first and third quartiles ± 1.5 × the interquartile range. The first and third quartiles are displayed on each side of the median line on the boxplots.

Paired samples

No significant relationship was found between THg concentrations in alligator nest material and corresponding egg yolk samples from the same nests (r = 0.56; p = 0.19). The line of best fit used to further describe the data demonstrated an r value = 0.5819 (Fig. S4). These data suggest Hg transfer from the nest material to egg yolk is unlikely, and rather the THg detected in egg yolk is the result of maternal transfer.

3.3. Hg in maternal female alligators

Developing eggs

Over the three years that nesting female blood and paired egg samples were collected at YWC, nesting females exhibited a mean THg blood concentration of 172 ng/g ± 41 ng/g. This value is similar to those observed in other adult alligators at YWC (Fig. 4, Table S11) (Lawson et al., 2019; Nilsen et al., 2019). While there was inter- and intra-nest variability, the pattern of THg concentrations observed in female blood and yolk THg suggested a consistent amount of THg deposition from maternal females to developing eggs. The mean THg concentrations in YWC yolk samples over the three years was 21 ng/g ± 8 ng/g (Table S11). Overall, we observed that 12.8% of the THg concentration in nesting female blood was detectable in corresponding yolk samples (% = Yolk Mean ÷ Female Blood Mean), which supports the idea that THg concentrations in developing eggs are low at sites where THg concentrations in blood of nesting females are also low (Fig. 4).

Fig. 4.

THg concentrations in individual nesting female alligator blood samples (filled markers) and corresponding egg yolk average THg concentration (hollow markers) from Yawkey Wildlife Center, SC in 2011 (female n = 5), 2013 (female n = 4), and 2014 (female n = 7). The annual mean for each year and sample type is demarcated with a color-matched hollow or filled bar. The grand mean of all measurements of each sample type is indicated with dotted line through the sample group. All nests had n = 2 or more eggs except the nest corresponding to the 2013 female farthest right, the last female of the 2013 group which had n = 1 egg available for analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Annual variation in THg concentrations in female blood and yolk was assessed between the years sampled. In 2011, the mean THg concentration in female blood was 191 n/g ± 36 ng/g, and the mean THg concentration in yolk was 28 ng/g ± 10 ng/g, making the estimated maternal transfer of THg in 2011 15.2% (Table S11). In 2013, mean THg concentration in nesting female blood was 161 ng/g ± 48 ng/g, while the mean THg concentration in yolk was 18.0 ng/g ± 4.8 ng/g (11.5% of the THg concentration in nesting female mean) (Table S11). In 2014, nesting females exhibited a mean THg concentration in blood of 163 ng/g ± 43 ng/g, and their yolk had a mean THg concentration of 18 ng/g ± 5.4 ng/g (11.5% of the THg concentration in nesting female blood) (Table S11).

In 2014, THg concentrations in blood of nesting females were paired with both yolk and embryo samples from their corresponding nests. Embryos from these females displayed a higher mean THg concentration (26.5 ng/g ± 12.7 ng/g) than yolk samples (18 ng/g ± 5.4 ng/g) however, the variability in embryo THg concentrations (12.7 ng/g) was double that of the yolk (5.4 ng/g, Table S11). This variability suggests there may be individual differences in the way yolk is assimilated into embryos. A nest-specific study examining complete clutches (all eggs in a nest; similar to Wu et al. (2000)) is needed to better understand in THg concentration variation in alligator eggs produced by the same female.

Across the three years when adult female blood and yolk from her corresponding eggs were sampled, a significant positive correlation (r = 0.66, p < 0.0001) in THg concentrations was observed between these tissues (Fig. 5). The female – nest pairs of each year had different correlation coefficients, with the greatest coefficient corresponding to 2011 (r = 0.93, p < 0.0001; Fig. 5). The correlation coefficient variations are not surprising, since different female – nest pairs were sampled each year (Wilkinson et al., 2016). In 2014, embryos were added to the correlation and the resulting relationship was not as strong as when only yolk and blood were examined (r = 0.37, p = 0.26; and r = 0.43, p = 0.003; respectively; Fig. 5), but the cumulative 3-year relationship that included the embryos was still highly significant (r = 0.58, p < 0.0001, Fig. 5). The weak correlation observed when embryos were included in the analysis could be due to the timing of exposure, since the embryos used in this study were from the first stages of embryonic development (stages 3–9 [3–9 days post-oviposition]). These stages were chosen for sampling in 2014 as they would most closely reflect the Hg concentration deposited by the nesting female. The 2011, 2013 eggs were sampled between developmental stages 19–21 (as part of other studies), when much of the yolk had been assimilated into the developing embryo. A stronger correlation may have been observed if the 2014 embryos were collected at a later developmental stage when more of the yolk had been incorporated, but the strong relationships observed in the 2011 and 2014 samples suggest Hg concentrations in yolk are reflective of the Hg concentration in the blood of nesting females regardless of developmental stage.

Fig. 5.

The relationship between THg concentrations in putative mother blood samples and corresponding egg yolk and embryos. The * denotes statistical significance in the Spearman Correlation. All female-nest pairs used in the correlation analysis had n = 2 or more eggs available for analysis.

These data indicate there is a relationship between the THg concentrations in the blood of nesting females and THg concentrations in their corresponding egg yolk. This suggests vertical transfer of Hg from adult female to offspring (maternal transfer) is occurring, which has not previously been observed for large reptiles (Fig. 5) (Sparling et al., 2010). However, only 12.8% of the THg concentration in female alligator blood was observable in egg yolk in this study. This suggests that unlike the high concentrations of contaminants maternally transferred in other wildlife species, maternal transfer and “off-loading” of Hg in alligators is minimal (Lyons and Lowe, 2013; Verreault et al., 2006).

To our knowledge, this is the first study to examine Hg in paired female – egg (blood-yolk) samples in crocodilians or any other large reptile. The correlation equation derived from these data, (y = 0.138x – 2.5534) can provide insight into the ability of alligators, other crocodilians, and potentially other oviparous wildlife to maternally transfer and off-load Hg into their eggs. This information could be particularly useful at locations with high Hg contamination, and with wildlife populations experiencing reproductive impairment.

Estimated age

Data from long-term alligator research at provided us the ability to estimate the age as well as the number of years (i.e., opportunities to offload Hg) a female may have nested (Lawson et al., 2019; Wilkinson et al., 2016). No statistically significant relationship was observed (r = 0.21; p = 0.44) between age and THg concentrations in nesting females (Tables S2 and S4). This result was unexpected, as Hg is known to bioaccumulate in long-lived species (Chumchal et al., 2011). However, nesting alligators appear to off-load a small but consistent amount of Hg during the nesting period (see above), so Hg concentrations in these nesting females may be more closely related to the number of times they have nested in their lifetime (range) (Tables S2 and S4). To examine this relationship, THg concentrations in nesting females were compared to their corresponding estimated number of nesting years. Again, a statistically significant relationship was not observed (r = 0.36; p = 0.17). Collectively, these results suggest that Hg accumulation in nesting female alligators at YWC does not correspond to age or number of nesting years. In a similar study examining Hg concentrations in blood of subadult and adult alligators (both males and females) at YWC, Lawson et al. (2019) observed the highest Hg concentrations in “middle-aged” (30–40 years old) animals and the lowest concentrations in the youngest and oldest individuals, possibly due to age-related changes in metabolism or foraging behaviors. To further investigate the influence of age and nesting frequency on Hg accumulation in adult female alligators, similar studies with larger sample sizes are needed particularly those that monitor individual females annually.

3.4. Transfer of Hg through eggshell

The goal of this experiment was to determine if topically applied Hg in an environmentally relevant vehicle can transfer through alligator eggs, the rate of transfer, and the fate/assimilation of the applied Hg inside the egg. Using the equation developed in section 3.3 (y = 0.138x – 2.5534) reference, low, and high dose groups were created based on extrapolated values from females in the Florida Everglades that are subject to high concentrations of Hg through their diet (Julian, 2013; Julian et al., 2015; Nilsen et al., 2016).

Despite the lack of Hg transfer observed in the initial 14 days (Fig. S5), the remaining eggs (n = 7 per dose group) were developed to stage 27 when erythrocyte samples were collected. The erythrocyte samples enabled us to determine if the dosed solution transferred through the eggshell and was incorporated into the embryo by the end of development. We did not observe a statistically significant difference in THg concentration between the reference, low and high dose groups (p = 0.4886, Fig. 6 left, Fig. S5). All dose groups from each of the two nests exhibited very similar erythrocyte THg concentrations (mean THg = 14.3 ng/g ± 2.2 ng/g, and 26.8 ng/g ± 3.8 ng/g; Fig. 6, Table S12). We did, however, observe a significant difference in erythrocyte THg concentrations between the two nests (p = 0.0002, Fig. 6 right, Fig. S5). However, we attribute this to differences in maternal transfer of THg (by different mothers) prior to egg collection in the field, not the topically applied Hg solutions.

Fig. 6.

Left Boxplots summarizing the dose group THg concentration eggshell transfer data analyzed with the Kruskal-Wallis test. Right Boxplots summarizing the nest THg concentration comparison analyzed with the Wilcoxon rank sum test. Diamonds indicate the mean; horizontal lines indicate the median. The error bars are calculated using the first and third quartiles ±1.5 × the interquartile range. The first and third quartiles are displayed on each side of the median line on the boxplots.

The lack of THg transfer in the dosing experiment was unexpected, as previous studies have dosed alligator eggs using the same method for a variety of chemicals (Cruze et al., 2015; Guillette et al., 1995; Guillette et al., 1994). However, the previous studies did not measure the amount of dosed chemicals that transferred through the eggshell, which is a widely discussed limitation in reptilian ecotoxicology. While the results of previous studies suggest that the dosed chemicals did affect embryos, without direct measurement of the chemicals in each egg compartment, there is no certainty that the affects observed were the result of the dosed chemicals. We propose the lack of THg transfer observed in the present study was due to the calcification of the egg shortly after oviposition (~24 h). Future studies examining environmental transfer of Hg into crocodilian eggs should consider aspects of egg architecture and biochemistry that may influence chemical movement across the eggshell barrier.

4. Conclusion

This study examined the transfer of Hg into alligator eggs by investigating 1) Hg concentrations in wild alligator egg yolk, 2) the relationship between Hg concentrations in wild alligator nest material and egg yolk, 3) the Hg concentration in wild maternal female alligator blood and the relationship between corresponding egg yolk and embryos; and their estimated age and number of nesting years, and 4) transfer of biologically relevant Hg concentrations through the alligator eggshell.

Mercury was detected in egg yolk from all sites examined, but mean Hg concentrations were significantly lower at the more urban and historically contaminated Lake Apopka compared to two less-urbanized (more remote) sites, suggesting low anthropogenic Hg inputs in the former. No relationship was observed between THg in alligator nest material and corresponding yolk samples, nor between THg in maternal alligator blood and estimated age and number of nesting years of laying females. However, significant positive relationships were observed between THg in blood of nesting female alligators and THg in their corresponding egg yolk. These results suggest that nesting female alligators do deposit Hg into their eggs but at consistently low percentages (12.8% in low Hg environments) relative to maternal body burdens. The relationship between THg in the blood of nesting females and THg found in corresponding eggs can be used to predict THg concentration in developing eggs at other at-risk locations through minimally invasive blood sampling of wild adult females. Finally, though this study demonstrated that Hg can be maternally transferred into alligator eggs, the egg dosing experiment suggested that Hg does not transfer through the eggshell (and therefore environmental transfer is unlikely), at least at developmental stage 19. However, we recommend more thoroughly revisiting this technique using larger sample sizes (more nests and eggs), a wider range of environmentally relevant Hg doses, and assessments at multiple embryonic stages. Overall, this study provides new information regarding Hg transfer in American alligators which can improve biomonitoring efforts and may inform ecotoxicological investigations and wildlife population management programs in areas of high Hg contamination.