Persistent organic pollutant levels in eggs of leatherback turtles (Dermochelys coriacea) point to a decrease in hatching success

Chemosphere 146 (2016) 354e361

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Persistent organic pollutant levels in eggs of leatherback turtles (Dermochelys coriacea) point to a decrease in hatching success
s a, *, Bele
n Go
mara b, Daniel Gonza
lez-Paredes c, Jose
Ruiz-Martín d, Eva De Andre Adolfo Marco a
n de la Biodiversidad y Ecología Aplicada, Estacio
n Biolo
gica de Don ~ ana, CSIC, Americo Vespucio, 41092, Isla de la Cartuja, Departamento de Conservacio Sevilla, Spain b
lisis Instrumental y Química Ambiental (AIQA), Instituto de Química Orga
nica General, CSIC, Juan de la Cierva, 3, 28006, Madrid, Departamento de Ana Spain c Center for Marine Biodiversity & Conservation (CMBC), Scripps Institution of Oceanography, 9500, Gilman Drive, La Jolla, CA, USA d Departamento de Biología, Universidad de Sevilla, Av. Reina Mercedes, 41012, Sevilla, Spain a

h i g h l i g h t s
We quantify POP congeners in eggs of leatherback turtles.
We search for correlations between POP concentrations and reproductive parameters.
Sum of PBDEs was significantly and negatively correlated to hatching success.
Knowledge in this field could lead into a better management for this species.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 June 2015 Received in revised form 28 November 2015 Accepted 3 December 2015 Available online 29 December 2015

Sea turtles are susceptible to environmental pollution, since many harmful effects have been reported for different chemicals over the last two decades. In this context, persistent organic pollutants (POPs), such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are of particular concern due to their endocrine-disrupting nature. The aims of this study were to provide additional baseline data on PCB and PBDE concentrations in eggs of Dermochelys coriacea; and to investigate whether any of the congeners could compromise reproductive success in this species. A total of 18 nests from different females were studied during the nesting season of 2008 at Reserva Pacuare Beach, in the Caribbean side of Costa Rica. Reproductive parameters (viability, fertility and hatching rates) were calculated for all nests and hatchling morphometrics were successfully measured in 8 of them. Two to three fresh eggs per nest were taken for contaminant study. Different congeners of POPs were purified and identified using gas chromatography (GC) coupled to an ion trap detector (GC-ITD MS/MS), as described below. Mean ± SD concentrations were calculated for POP congeners within each nest and clustering was also evaluated. Correlations were performed searching for potential relationships with reproductive parameters. POP levels were similar to those reported in French-Guiana populations and slightly lower than those associated to Florida populations. Sum of PBDEs showed a negative correlation to the hatching success, suggesting potential harmful effects of these contaminants on the reproduction of leatherbacks. © 2015 Elsevier Ltd. All rights reserved.

Handling Editor: Myrto Petreas Keywords: Pollutants Reproduction Sea turtles Leatherback

1. Introduction The leatherback turtle (Dermochelys coriacea) is the largest of

* Corresponding author.
s). E-mail address: [email protected] (E. De Andre http://dx.doi.org/10.1016/j.chemosphere.2015.12.021 0045-6535/© 2015 Elsevier Ltd. All rights reserved.

the sea turtle species and the only marine reptile capable to dive up to 1000 m depth (Eckert et al., 1989). Leatherback females reach the age of maturity at about 9 years and nest on tropical and subtropical waters worldwide every two to three years (Zug and Parham, 1996). They are able to lay up to 11 nests about ten days apart within a single nesting season with clutches ranging from 65 to 86 eggs (Bell

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et al., 2003; Reina et al., 2002; Stewart and Johnson, 2006). Fertility rate in Dermochelys coriacea is usually higher than 90%. Nevertheless, hatch success has been reported to be lower than those of other sea turtle species with values < 60% (Arauz and Naranjo, 1994; Eckert and Eckert, 1990; Leslie et al., 1996; Reynolds, 2000). During the past three decades, concerns about this species have increased due to diminishing on nesting females of Pacific populations, which are cataloged as critically endangered under the IUCN Red List (Spotila et al., 2000; Wallace et al., 2013). North-west Atlantic nesting populations of D. coriacea appear to be increasing, probably due to an intensive program of protection and egg relocation carried out for more than 20 years at certain locations €ng (Dutton et al., 2005; Stewart et al., 2011b; Tiwari et al., 2013; Troe et al., 2004). Nevertheless, Caribbean nesting population sizes are considered to be very small if compared to those that nested in this coast in the past and threats to this species still persists (Tiwari et al., 2013). The causes of this relative low numbers may be due to biological limitations inherent in the reproduction of the leatherback turtle, the effects derived from anthropogenic activities or a combination of both. Currently, many of the hazards affecting leatherback turtles are linked to human activities such as fishery bycatch, egg poaching, plastic ingestion or contamination of foraging grounds (Alava et al., 2006; Kaplan, 2005; Kotas et al., 2004; Wallace et al., 2013). Furthermore, potential effects of persistent organic pollutants (POPs) may have serious consequences on leatherback population since they may impair some important features regarding to health, reproduction and offspring phenotypic quality. Among the studies about POP contamination, only a few researches have focused on sea turtles during last decades without optimistic results. For instance, there are evidences that certain levels of POPs may have negative effects on health parameters of sea turtles, that these contaminants are maternally transferred and that they may negatively interfere in the normal embryonic development and hatchling sizes (Guirlet et al., 2010; Keller et al., 2004; Van de Merwe et al., 2010; Stewart et al., 2011a, 2011b). We firmly think that an increasing knowledge on factors that could compromise reproductive success in D. coriacea can confer a broader view of population trends, and therefore, it can contribute to establish more-suitable strategies and policies that lead into a better management of this species. In this context, the aims of this study were two: 1) to provide additional baseline data about concentration of POPs, including both polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs), in eggs of the leatherback turtle; and 2) to investigate whether any of these compounds is correlated to viability, fertility, hatch success or hatchling morphometrics. 2. Methodology 2.1. Sampling Fieldwork was carried out along beaches at Pacuare Reserve, Costa Rica. Beaches are located on the Caribbean side, between geographic coordinates 10 120 28.1900 N, 83 150 55.4900 W and 10 100 00.5100 N, 83 140 01.4900 W. A total of 18 nests of different leatherback turtles were assessed during the period between June and August of 2008. Studied females laid their eggs from June 3 to June 8 (2008). Curved carapace length (CCL), curved carapace width (CCW), clutch size (number of potentially fertile eggs; termed viable eggs) and number of shelled albumin globes (SAGs) were determined at the time of oviposition (Bell et al., 2003). At the same time, one to three viable eggs were also collected from each nest (accounting for a total of 47 eggs). All of these eggs were stored and transported alive

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in an isotherm container (under the CITES legal framework) to the analytical laboratories in Spain within the 11 h following the last collection. During this process, eggs were at typical incubation temperatures between 28 and 32  C. Emergence began at early August. Marked nests were excavated, and contents of the nests were evaluated to count hatched and unhatched eggs. Hatching success rate was defined as proportion of hatched eggs to the total number of eggs (excluding SAGs) (Eckert and Eckert, 1990; Miller, 1985) and was measurable in only 11 of the 18 nests due to diverse factors such as intensive rains, floods, high tides, beach erosion, predation and/or poaching. Unhatched eggs were opened to determine embryonic stage as well as the number of infertile eggs. Fertile eggs were considered as hatched eggs plus eggs with dead developing embryos after nest emergence. Based on the literature (Bell et al., 2003; Chan et al., 1985; Chan and Liew, 1996; Leslie et al. 1996; Miller, 1999), fertility rate was calculated as fertile eggs divided by the clutch size, excluding SAGs. A new variable, termed viability rate was calculated as the number of viable eggs divided by the sum of both viable eggs and SAGs. Immediately following emergence, hatchling mass (±0.1 gr), straight carapace length (SCL) and straight carapace width (SCW) were successfully measured (±0.1 mm) in only eight of the clutches. Up to 10 hatchlings per nest were tried to be gauged, but it was not possible in all cases; so the number ranged from four to ten individuals, with one exception of one nest where just one individual was found and measured. 2.2. Pollutant analysis 2.2.1. Extraction and purification Fresh egg samples were opened in a clean Petri dish. Yolk subsamples of around 2.5% of the total sample were taken for lipid and carotenoid analyses (around 1 g for each determination from up to the 70e80 g of the whole egg yolk content). The rest of the yolk of every single egg (yolk plus egg white) was mixed thoroughly with a spatula and frozen at 20  C for its further lyophilization. The samples were kept lyophilized until the POP analyses. After the lyophilization, each lyophilized sample was again homogenized using an Omni-Mixer and kept in a clean glass container until its analysis. The extraction was carried out using 3 g of lyophilized samples (20 g of fresh sample approximately). Procedure described by Bordajandi et al. (2003), was followed. Briefly, extraction was carried out by matrix solid phase dispersion, freeze-dried egg sample was homogenized with 1:1 (w:w) silica gel: anhydrous sodium sulfate powder. The mixture was ground to become a fine powder, loaded into a column and spiked with a mixture containing 13C12-labeled congeners. Extraction was carried out with 400 mL of 1:1 (v:v) acetone: hexane mixture. Clean-up was carried out using a multilayer column filled with neutral silica, silica modified with sulfuric acid (44%) and silica modified with KOH (56%). The final fractionation was achieved by using Supelclean™ ENVITM-Carb solid phase extraction (SPE) cartridges (Supelco, Bellefonte, PA, USA). Two fractions were eluted, the first containing ortho PCBs and PBDEs and the second one containing non-ortho PCBs. For ortho PCB and PBDE fraction the final volume was 100 uL and for non-ortho PCB fraction the final volume was 10 uL. 2.2.2. Analysis of PCB congeners using GC-ITD (MS/MS) PCB congeners were determined by gas chromatography (GC) coupled to an ion trap detector (GC-ITD) in its tandem operation mode (MS/MS) using a Varian CP-3800 gas chromatograph coupled to a Saturn 2000 ITD (Palo Alto, CA, USA). The extracts were evaporated to dryness and diluted in a solution containing 13C12labeled PCBs 70, 111, 138 and 170 as recovery standards. A 4 mL

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aliquot was injected in the temperature vaporizing injector (PTV) mode (100  C, hold for 0.2 min, and then to 300  C at 200  C$min1; splitless time 2.0 min) using an apolar capillary column (VF-5MS 50 m  0.25 mm i.d., 0.25 mm film thickness, Varian). Helium was used as the carrier gas at a constant flow rate of 1 mL min1
mara et al., 2006a). Identification of PCBs was based on simul(Go taneous detection, at the appropriate retention time, of two ions of the product cluster for each congener, maintenance of the theoretical ratio among them within an appropriate range, and simultaneous detection of the two selected ions of the product cluster for the corresponding (or assigned) labeled congeners. Precursor ions were obtained by electronic impact (EI) at 70 eV and stored in the trap. Further fragmentation of the selected parent ions was achieved by collision-induced dissociation (CID) in the resonant mode, yielding to product ions corresponding to the loss of the two chlorine atoms. Instrument detection limits were in the range 0.07e0.4 pg mL1 for standards. Twenty PCB congeners were quantified including 7 ICES (International Council for the Exploration of the Sea) PCBs and all PCBs with a TEF (Toxic Equivalency Factor) value assigned (non-ortho and mono-ortho PCBs). PCBs were classified into three groups: Main PCBs (numbers 28, 52, 101, 138, 153, 170, 180 and 194), mono-ortho PCBs (numbers 105, 114, 118, 123, 156, 157, 167 and 189) and non-ortho PCBs (numbers 77, 81, 126 and 169).

blanks and BDE 209, due to the high levels of this BDE in house dust. Surrogate standards (prior to the extraction step) and injection standards (prior the instrumental determination for recovery calculations) were used, in order to comply with the adequate QC/ QA for the determinations of PCBs and PBDEs in real-life samples. Satisfactory precision were achieved when analyzing standard solutions, with relative standard deviations (RSDs) generally below 13% for the two families of compounds determined (PCBs and PBDEs) in both methods GC-ITD(MS/MS) and GC-ECNI-LRMS, except for BDE 209, whose RSD were lower than 19%. Further information related with ITD (MS/MS) and ECNI-LRMS conditions can
mara et al., 2006a, 2006b and 2007). Rebe found elsewhere (Go coveries found for labeled congeners added to samples before the extraction step (surrogate standards) were higher than 68% in all cases. The uncertainty of the measurements, calculated in two food samples (chicken and trout) from interlaboratory exercises, was lower than 15%. Moreover, the working group has participated in several international quality control studies for the analysis of POPs in different biological and food matrices, including palm oil, chicken, cod liver oil, reindeer, herring, trout, and adipose tissues from marine mammals (whale and polar bear) (Becher et al., 2004, 2005, 2007e2009, NIST/NOAA, 2003). The results were consistent at all times with the consensus means given by the inter-laboratory organizations.

2.2.3. Analysis of low-brominated (tri- to hexa-) BDEs using GC-ITD (MS/MS) GC-ITD in the MS/MS operating mode, using the isotope dilution
mara et al., 2006b) was use technique as described elsewhere (Go for analysis of tri- to hexa-BDEs (PBDE numbers 28, 47, 66, 85, 99, 100, 153 and 154). Analyses were performed on a GC CP-3800 (Varian Iberica, Spain) equipped with an ITD (Saturn 2000, Varian) and an 8200 CX autosampler (Varian). Samples were injected by a programmable PTV in hot splitless mode. A low bleed GC capillary column VF-5MS (FactorFour, 50 m  0.25 mm i.d., 0.25 mm film thickness) purchased from Varian (CA) was used for separation. Helium was used as carrier gas at a constant flow rate of 1.0 mL min1. Identification was done as previously described for PCBs, corresponding the product ion to the loss of two bromine atoms.

2.3. Statistical analysis

2.2.4. Analysis of high-brominated (hepta- to deca-) BDEs using GC-ECNI-LRMS The high brominated BDEs selected (PBDE numbers 183, 184, 191, 196, 197 and 209) were determined using a 6890N gas chromatograph coupled with a 5975 quadrupole mass spectrometer (Agilent, Palo Alto, CA) working in the electron capture negative ionization mode (ECNI). Standards and samples were injected in hot splitless mode (300  C, 1 mL; splitless time 2.0 min). A low bleed GC capillary column DB-5MS (15 m, 0.2 mm i.d., 0.2 mm film thickness) purchased from J&W Scientific (U.S.A.) was used for separation. All GC working conditions and the quantification
mara et al., 2007). method used are detailed previously (Go 2.2.5. Quality control and assurance (QC/QA) All POP analyses, such as blanks, recoveries, and parallel analyses, were complied with analytical standards as recommended by the EU Commission in the directive for measuring dioxins in food (Commission Regulation 1883/2006/EC). A method blank in each set of analysis (four analyses and one blank) was carried out and the results of each blank sample were subtracted from each corresponding four egg samples. To eliminate interferences in blanks, all the glassware, chemicals, solvents, and equipment used during extraction and cleanup procedures as well as the instrumentation used have been routinely checked. Special attention was paid to

For base-line data of the different compounds, all concentrations and proportions were averaged within each nest, and then using averages from each nest, averaged between all analyzed nests. Mean ± standard deviation (SD), median, minimum and maximum values were calculated for all compound groups. Grubbs' test was applied to searching for potential outliers. To evaluate those potential outliers, values were compared to the rest of eggs, to the eggs belonging to the same nest, to the patterns observed within the other nests and to other values reported in the literature. STATISTICA (v 8.0) was used to carry out statistical descriptions, normality tests, Grubbs' test and correlations. In order to evaluate differences among clutches and determine whether egg sampling was representative enough, analysis of similarity (ANOSIM) under BrayeCurtis index was performed, separately, for POPs with no data transformation using PRIMER (v 6.1.6). Each class, compound or group was entered as a separated variable and each egg was analyzed as an individual sample. To better illustrate the differences between and within clutches, a non-metric multi-dimensional scaling (NMDS) plot was also constructed using PRIMER (v 6.1.6) (Van de Merwe et al., 2010). Arcsine-transformation was applied to all proportional data such as reproductive rates (viability, fertility and hatching success) (Mosteller and Youtz, 1961; Bell et al., 2003). The mean (±SD) of each POP congener was also calculated for eggs within the same clutch for correlation analysis. ShapiroeWilk Normality test was performed for all variables in order to choose the best possible analysis. When variables were normally distributed and homoscedastic, parametric correlations (Pearson productemoment correlation) were used and for the rest of cases the Spearman rank order correlation was used. Bonferroni corrections were used when needed. 3. Results 3.1. Reproductive parameters Nesting female dimensions ranged from 140 to 164 cm for CCL and 105e117 cm for CCW (Table 1). Regarding to reproductive

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Table 1 Nesting details, reproductive and hatchling parameters of leatherback turtles. CCL and CCW are curved carapace length and width of nesting females, respectively. Clutch size is equal to number of yolked eggs; viability rate calculated as clutch size divided by clutch size plus SAGs; fertility rate calculated as the proportion of fertile eggs; hatchling rate as the proportion of hatched eggs. Hatchling morphometrics data are mean ± SD of four to ten hatchlings per nest. SCL and SCW are straight carapace length and straight carapace width, respectively. Nesting details

Reproductive parameters

Hatchling morphometrics

Tag

CCL (cm)

CCW (cm)

Clutch size

Viability rate

Fertility rate

Hatching rate

Weight (g)

SCL (mm)

SCW (mm)

mass:SCL (g mm1)

? 1151 D 10521 PN 1157 V 2242 VA 1040 VA 2942 VA 3174 VA 3650 VA 4893 VA 4898 VA 5857 VA 5897 VA 5928 VA 7754 VC 0418 VC 0419 VC 0476 VC 0489

156 153.5 148.5 140 164 150 150 153.5 163 148 153 146.5 148 153 158

109 108 106 108 113 110 105 107 108 107 112 106 113 114 113.5 112 117 113

() 73 99 76 78 85 59 69 96 77 79 74 82 80 56 80 73 100

() 0.63 0.66 0.66 0.67 0.79 0.69 0.63 0.66 0.65 0.90 0.61 0.67 0.73 0.52 0.84 0.60 0.75

() 0.96 () 0.88 0.78 () 0.76 0.96 0.78 () 0.78 0.95 0.95 () () 0.90 0.84 ()

() 0.44 () 0.71 0.05 () 0.70 0.67 0.55 () 0.71 0.47 0.24 () () 0.65 0.80 ()

() 49.5 ± () () () () 45.6 ± 46.1 ± 49.2 ± () 41.6 ± 50.5 ± 49.0 ± () () 43.5a () ()

() () () () () () 57.5 58.0 60.3 () 56.7 60.6 54.2 () () 56.4 () ()

() () () () () () 39.2 38.9 40.5 () 37.5 34.5 36.3 () () 38.0 () ()

() () () () () () 0.79 0.80 0.82 () 0.73 0.83 0.90 () () 0.77 () ()

162 159

1.5

1.3 2.4 1.5 2.4 1.7 2.8

± 1.8 ± 1.8 ± 3.6 ± 2.5 ± 0.6 ± 1.4

a

± 1.3 ± 3.0 ± 1.5 ± 1.3 ± 5.0 ± 2.0

a

± 0.03 ± 0.04 ± 0.06 ± 0.04 ± 0.01 ± 0.05

a

() Not measured. a Only one hatchling measured.

parameters, clutch size and viability rate were measured in 17 clutches, whereas fertility and hatching rate were measured in just 11 of them. VC0418 nesting female presented the lowest clutch size (56 eggs) and lowest viability rate (0.52), meaning that that female laid almost equal number of yolked eggs relative to SAGs. Unfortunately, neither the fertility rate nor hatching rate could be measured for that nest. The clutch size mean was approximately 80 yolked eggs per nest and the viability rate was quite low, around 70%. Although fertility rate was mainly high (86%), only an average of 54% of eggs hatched per clutch. Hatchling morphometrics are also shown in Table 1. Hatchling mass ranged from 41.6 to 50.5 g, and 54.2e60.6 mm for SCL and 34.5e40.5 mm for SCW. Mass:SCL ratio was also calculated, varying from 0.73 to 0.90 g mm1.

3.2. POP levels in eggs POP concentrations are shown in Table 2 and are expressed both by gram of wet mass and by gram of lipid weight (lipid normalized). These lipid normalized concentrations were almost 20 times the values expressed by wet weight in all different groups of congeners.

The 18 nests had a mean percentage of lipid content of 5.173 (SD ¼ 0.609, Min ¼ 3.61, Max ¼ 6.19, Median ¼ 5.271, N ¼ 18). The first remarkable feature of measured POP levels was the large variation presented in all cases. Standard Deviation (SD) by the majority of PCB groups was around or slightly higher than their mean value, whereas SD by PBDEs was two to three times the mean value. Therefore, the data were carefully checked for possible outliers that may be due to analytical errors. Thus, the Grubbs' Test was applied to identify these potential outliers (p < 0.05) in each of the POP congeners groups. All 47 eggs were taken into account for this test. When a potential outlier was identified at a general level, it was automatically compared with the concentration values of the rest of the eggs from the nest to which it belonged. If possible outlier values were inconsistent when compared the same nest eggs and regarding to the patterns observed in the other nests and other studies in eggs of D. coriacea (as well as in eggs of other sea turtle species), it was decided to remove it. Only one egg (VC 04193) was removed and definitively excluded for all the rest of statistical analysis, belonging to the group of tri- to hexa-BDEs. The concentration values of tri- to hexa-BDEs reached 20,859.3 pg g1

Table 2 POP concentrations in egg yolks of leatherback sea turtles from Costa Rica (values are expressed by gram of wet mass and by gram of lipid). For each nest an average value of every eggs was calculated. Data provided in the table are based on average values from each nest. N ¼ number of nests. Values obtained after removing VC 0419-3 egg, since it was detected as an outlier. Congener group

N

Pg g1 wet mass

ortho PCBs mono-ortho PCBs non-ortho PCBs P PCBs tri- to hexa-BDEs hepta- to deca-BDEs P PBDEs P POPs

18 18 18 18 18 18 18 18

4697.0 531.7 10.9 5239.5 542.3 43.0 585.3 5824.9

Mean ± SD ± ± ± ± ± ± ± ±

5043.3 344.5 6.9 5170.6 422.7 56.4 434.2 5342.2

ng g1 lipid Median

Min.

Max.

Mean ± SD

3927.0 448.6 10.4 4286.3 555.9 25.7 600.0 4584.4

1067.3 134.9 3.3 1220.2 57.1 11.2 74.7 1295.0

23,787.5 1152.2 31.8 24,612.9 7505.2 171.1 7676.3 25,274.3

91.5 10.6 0.2 102.3 10.7 0.9 11.6 113.9

± ± ± ± ± ± ± ±

89.9 7.8 0.1 93.3 7.9 1.3 8.3 96.5

Median

Min.

Max.

70.0 8.6 0.2 81.7 11.0 0.5 11.6 94.5

22.5 2.5 0.07 25.8 1.1 0.23 1.6 27.4

414.1 30.5 0.5 429.3 32.8 5.8 33.2 441.5

* Values obtained after removing VC 0419-3 egg, since it was detected as an outlier. Group of ortho PCBs includes congener numbers: 28, 52, 101, 138, 153, 170, 180 and 194; mono-ortho PCBs include congener Numbers: 105, 114, 118, 123, 156, 157, 167 and 189; non-ortho PCBs include congener numbers: 77, 81, 126 and 169; tri-to hexa-BDEs include congener numbers: 28, 47, 66, 85, 99, 100, 153 and 154; hepta-to deca-BDEs include congener numbers: 183, 184, 191, 196, 197 and 209.

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wet weight, being more than 10 times higher than the maximum proximal value in this study (1901.4 pg g1 wet weight) as well as than those reported in studies of D. coriacea (Stewart et al., 2011a, 2011b) and of Caretta caretta (Alava et al., 2011). After removing the outlier, both total sums of PBDEs were 585.3 pg g1 wet weight and 11.6 ng g1 lipid, where tri- to- hexacongener group accounted for more than 90% of the total PBDEs. Levels of PCBs were in both cases near one order of magnitude higher than PBDE concentrations (5239.5 vs. 585.3 pg g1 wet weight; and 102.3 vs. 11.6 ng g1 lipid) and accounted for almost the 90% of total POPs. Around 90% of total PCBs was represented by the ortho-PCBs group, averaging values of 4697.0 pg g1 wet weight and of 91.5 ng g1 lipid. The percentage of homologue groups for both PCB and PBDE congeners are shown in Fig. 1. POP profiles in the present study showed a high variability in concentrations comparing different eggs of the same clutch. The before ANOSIM-R value was 0.34 for POPs, with all p-values > 0.05. NMDS plot better illustrated the variation-grouping pattern within and between clutches (Fig. 2). However, despite of the high data variability, it does exist grouping in eggs of some clutches (e.g. clutches 3, 17 and 18) (Fig. 2). 3.3. Correlations with reproductive parameters Reproductive parameters and hatchling morphometrics were all normally distributed (SeW, p > 0.05), except for the fertility rate. Thus, all correlations related to normally distributed variables were

parametric (Pearson productemoment correlation) and all correlations related to the fertility rate were non-parametric (Spearman rank order). SeW normality test was also conducted for all variables taking into account the nest ranks covered by both variables; namely, hatchling parameters were only available for 7 to 8 nests, so SeW test was executed for all the other variables within those nests in order to choose the best correlation method. And for all possible combinations. Regarding to reproductive parameters, a biologically important significant correlation was found between PBDE concentrations and hatching success (Fig. 3). Interestingly, the sum PBDE levels were negatively related to the hatching rate both using wet (R2 ¼ 0.50) and lipid (R2 ¼ 0.41) contents of PBDE (Pearson correlation: r ¼ 0.70; p ¼ 0.016). No significant relationships were found between POP concentrations on hatchling size. 4. Discussion 4.1. Reproductive parameters According to this study, the leatherback turtle (D. coriacea) is characterized by spawning around 78.6 (±11.0) viable eggs per nest, which is consistent with other results reported to date (Bell et al., 2003; Eckert and Eckert, 1990; Reina et al., 2002; Stewart and Johnson, 2006). The global clutch size (adding viable eggs and SAGs) was about 115.3 ± 17.4, very similar to the results obtained by Eckert and Eckert (1990) (114.2 ± 22.9), who computed a total of

Fig. 1. Congener profiles for both PCBs (A) and PBDEs (B) in eggs of 18 leatherback turtle nests from Costa Rica, expressed as percentage of contribution of each congener to the total concentration.

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Fig. 2. 2D-NMDS plots of leatherback egg concentration profiles of POPs. Eggs of the same clutch are indicated by the same symbol, and each clutch has an assigned id. Nongrouping of eggs from the same clutch indicates that variation within clutches is larger than variation among clutches. Stress ¼ 0.1 suggests good fit; Stress ¼ 0, poor fit.

Fig. 3. Relationship showing a significant negative correlation between lipid-normalized PBDE concentrations and Arcsin-transformed hatching rate in Dermochelys coriacea. Linear regression equation: y ¼ 1.6388x þ 75.733.

217 in situ nests. Fertility and hatching success rates also showed values within the ranges reported in other studies, accounting here for 86.7% (±0.08%) and 54.5% (±22.9%), respectively (Arauz and Naranjo, 1994; Eckert and Eckert, 1990; Leslie et al., 1996; Rafferty et al., 2011; Reynolds, 2000). In this study, it was considered important to obtain the viability rate, in order to establish whether exist mechanisms or interactions at physiological level that may influence the process of vitellogenesis and, therefore, the reproductive success in leatherbacks. In addition to these parameters, this study contributes with new data regarding the size and mass of neonates, which will serve as a baseline to be compared with subsequent studies.

4.2. Variability within nests According to the results of ANOSIM, it was demonstrated that for the analysis of all different POPs, the sample size per nest was insufficient. While the internal variability of some nests was higher compared to the values found in other nests, there were nests that showed a remarkable degree of clustering, as shown in the NMDS plots. For analysis of POPs, Alava et al. (2006) suggested a range of 3e11 eggs per nest (depending on the degree of adjustment that is intended to get) to obtain more accurate data about the mean POP concentration of each nest, in eggs of loggerhead turtle (C. caretta). Nevertheless, Van de Merwe et al. (2010), through this same ANOSIM, concluded that 3 eggs per nest were more than enough to

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get a good measure of each nest, since their data did not show excessive variability in eggs of green turtle (Chelonia mydas). Their results agreed with those obtained by Keller (2013), where the mean-nest relative standard deviation was 10%. In this study, however, we cannot ensure that 3 eggs were sufficient to represent the average values of each nest, since the mean-nest relative standard deviation was above 30%. Possibly, there are many factors influencing this variability, such as the degree of exposure to such compounds which different populations are subjected, the distribution of POP concentrations in tissues of each individual sea turtles and among species (concentrations provided here were approximately 10 times higher than those reported by Van de Merwe et al. (2010)), or physiological differences of each species, as the mechanisms of assimilation, mobilization and introduction of POPs in the yolk may be different, depending on the species and amount of POPs exposed to. 4.3. Health risks of POPs in hatching success and species comparisons The potential effects that POPs may have on reproduction and health of wild animals have been reported by several studies throughout the literature. In recent years, some of these studies have focused on sea turtles, due to the harmful effects that these contaminants may have on reproductive success, compromising the future of some of these species. Results obtained by Keller et al. (2004) suggested that POPs affect health parameters and may have sublethal effects in the loggerhead sea turtle (C. caretta). Moreover, Guirlet et al. (2010) and Stewart et al. (2011a, 2011b) found that both PCBs and PBDEs are maternally transferred to eggs and hatchlings in leatherback turtles. Similar results were found by other authors with one more finding, showing a relationship between POP concentrations and reduction in size parameters of hatchlings of the green (Van de Merwe et al. (2010) and the loggerhead (Keller, 2013) sea turtles. Thus, there is evidence that certain levels of POPs can have negative effects on reproduction success of leatherbacks. In this study, fresh weight concentrations of PCBs, obtained from leatherback turtle eggs of Caribbean nesting populations, were 5068.9 ± 6615.5 pg g1 wet mass, which were very similar to those provided by Guirlet et al. (2010) in French Guiana (6980 ± 5020 pg g1 wet weight) and slightly lower than those reported by Stewart et al. (2011a) in Florida (8450 ± 7590 pg g1 wet weight). These comparisons suggest that foraging areas in northern latitudes of the American coasts might be highly polluted than those located at more central or southern latitudes, and it is consistent to the study carried out by Alava et al. (2011). This hypothesis would be consistent taking into account the level of industrial development and urbanization of each country associated to those latitudes. Thus, it is expected that contamination levels will be higher in waters surrounding countries with a longer history of waste-water discharges from industrial or urban activities. All these aspects should be considered when delimiting potential areas of conservation. PBDE concentrations reported here (533.8 ± 371.6 pg g1 wet weight) are also slightly lower than those reported by Stewart et al. (2011a) (845 ± 630 pg g1 wet weight). Compared to eggs of other species, the values cover a wide range. For example, Van de Merwe et al. (2010) showed values ten times smaller, and with relatively little variation, both PCBs (554 ± 54.6 pg g1 wet weight) and PBDEs (129 ± 8.15 pg g1 wet weight) in eggs of green turtles (C. mydas) of populations from Malaysia. This could be expected due to the higher trophic level occupied by D. coriacea and the vast geographical separation of the populations sampled. In this context, the same statement could be applied for C. caretta which it also shows a predatory trophic level.

For instance, Alava et al. (2011) reported concentrations of PCBs in eggs of C. caretta ranging from 32.4 to 1460.5 ng g1 lipid and PBDE concentrations of 1.08e13.5 ng g1 lipid. For PCBs, the concentrations in the present study showed values within that range (100.7 ± 122.4 ng g1 lipid), although the concentrations of PBDEs were slightly lower (10.8 ± 7.9 ng g1 lipid). A strong negative correlation between sum of PBDE concentrations and hatching success rate was found. This is the first study that provides evidence of this relationship in any sea turtle species. Moreover, maternal identity has been identified as one of the main factors determining hatching success in this species, by speculated intrinsic mechanisms that may affect embryonic mortality (Rafferty et al., 2011). Keeping this assumption in mind, it is feasible to think that each female that is subjected to POPs contamination (by food intake and/or contamination exposure) has also its own physiological mechanisms of detoxification that could lead to a different but inherent hatching success. Perhaps, those females that presented higher levels of embryonic mortality (reported by Rafferty et al., 2011) had higher levels of PBDEs as well. At certain levels, these endocrine disrupting chemicals may be interfering on the mechanisms that ensure the entire embryonic development, and therefore, the hatching success. It should also be noted that some environmental factors may influence hatching success of in situ nests, such as humidity, pH, sand type and moisture, predation or the presence of bacteria, insects and/or fungi (Marco et al., 2015; Patino-Martínez et al., 2014; Sarmiento-Ramírez et al., 2010). Nevertheless, hatching rate values obtained in this study from 11 nests perfectly agree with those obtained by Eckert and Eckert (1990), who analyzed a total of 217 in-situ nests. However, PBDE concentrations obtained in some eggs showed high variation with respect to the average values of the same nest. Therefore, it would be necessary to analyze a higher number of eggs per nest in order to obtain more accurate and representative means of each nest, and to draw conclusive results. Another factor to consider would be the fact that, in this study, an egg that had extremely high levels of PBDEs was annulled for all other statistical analysis because it was considered as an outlier. However, such considerations, although well argued, could skew the data. Despite all the uncertainties that surround it, in this study we found a significant and negative relationship between PBDE concentration levels and hatching success in eggs from wild populations of leatherback nesting turtles. We firmly believe that more studies are needed to confirm this, in order to obtain accurate information about the potential and adverse effects that these compounds may cause on populations of D. coriacea, as well as in other sea turtle species. Acknowledgments We thank all the volunteers from Pacuare Reserve, Didier
n and Hilda Denham; Zaida Ferna
ndez and Carlos Herna
ndez Chaco from Endangered Wildlife Trust. Financial support came from the BBVA foundation (BIOCON06/056), Consejo Superior de Investigaciones Científicas of Spain (CSIC) and the International Uni
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