Material and Methods

Green turtle eggs (n  =  110) were collected from 24 different clutches laid between 10 and 23 July during the 2004 nesting season at Şeyhhızır Beach, which is a 4.5-km section of the 14-km-long Samandağ Beach. It is reported that a female sea turtle builds its next clutch 13 days after building its previous clutch (Broderick et al. 2002). The clutches examined in this study were deposited within a 13-day period; therefore, all the clutches were assumed to belong to different females (Table 1).

Table 1. Sample size and clutching dates of eggs used in analyses.

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Due to sensitivity toward sacrificing individuals that are endangered species, only eggshells from eggs where the embryos had died naturally during development or remained after successful hatching were examined. Eggshells were individually put into sterile plastic bags after being carefully removed from their nest chambers and were transported to the laboratory. The eggshell contents were analyzed in 6 groups: infertile; first, second, third, and fourth developmental stages; and successful hatchlings. When there was no visible white spot, sign of an embryo or blastodisk, or blood formation, then the egg was assumed to be infertile. The developmental stages were modified from Miller (1985) and Kaska and Downie (1999). The classified developmental stages of green turtles are divided into 1) neurulation (0–6 days), when the embryo is only a white spot or a sign of an embryo or blastodisk or blood formation; 2) organogenesis (7–17 days), when no carapace exists and the eye is observable; 3) early growth (18–32 days), when the carapace exists with no pigmentation; and 4) late growth (33 days–hatching), when pigmented carapace exists. The eggshells of successful hatchlings also were used for analysis.

Preparation of the samples for chemical analyses followed the accepted acid digestion procedure (Hossner 1996). A sample of approximately 1 g from each eggshell was digested with laboratory-grade nitric acid in a screw Teflon vessel. The bombs were heated on a hot plate for 2 hours at 90°C during which the bombs were opened 3 times to release accumulated carbon dioxide gas. Before completion of heating, 2 mL of hydrogen peroxide was added into each cup. Elemental analyses were performed via inductively coupled plasma–atomic emission spectroscopy at the Central Laboratory of Mustafa Kemal University. Seven corresponding standards were tested before the measurement of element concentrations, and a regression coefficient of > 0.995 was achieved for each element before analytical readings. The eggshells were analyzed for their Ca, Cu, Mg, K, Na, Sr, and Zn contents. The lowest detection limits for Ca, Cu, Mg, K, Na, Sr, and Zn were 10, 1, 10, 10, 10, 0.5, and 1 mg/kg, respectively.

Statistical calculations were performed by using the SPSS 10.0 program. First, data were checked for normality, and a test for homogeneity of variance was completed by using Levene statistics. Ca and Cu were compared by using analysis of variance for comparison of these parameters during the developmental stages. Because the data for K, Mg, Sr, and Zn demonstrated heterogeneous distribution between groups, a nonparametric Kruskal-Wallis test was performed to assess changes in these chemical parameters during incubation. All statistical analyses were conducted with an experiment-wise error rate of 0.05.

Results

The mean eggshell Ca concentrations at different developmental stages were statistically different (Table 2). The eggshell Ca in late developmental stage eggs and successfully hatched eggs was found to be significantly different from calcium concentrations at all other stages (F  =  33.9, df  =  5, p < 0.001). A gradual decrease was observed during the developmental stages. The greatest decrease in eggshell Ca concentration occurred between the third and fourth developmental stages (−35%, p  =  0.007) and between the fourth developmental phase and successful hatching (−54%, p < 0.001). The decrease in Ca concentration in eggshells was 72% from the infertile to the hatchling (p < 0.001).

Table 2. Mean (± standard error) elemental composition of eggshells.

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The Cu concentration in hatchling eggshells was higher than that for other eggshells from infertile to third-stage eggs (p < 0.001). The largest increase (+47%) in Cu concentration was observed between the third and fourth stages of development (p  =  0.017) (Table 2).

The K decreased significantly from the second developmental stage to successful hatchling (Table 2). Compared with Ca, the K concentration decreased very little in the eggshell of the second and third stages. The decreasing K concentration between the second and third stages, third and fourth stages, and between the fourth stage and hatching were not significant (p > 0.05). Successfully hatched eggshell K varies from all the other stages (p < 0.05), which indicates that the main decrease in total K of the eggshell occurs after the fourth developmental stage. Overall, a decrease of only 21% was observed in K concentration between infertile and hatchling eggshells (p  =  0.003).

There were increases and decreases in the mean Mg concentrations of eggshells between stages. However, the mean Mg concentrations of eggshells in various developmental stages did not vary statistically (p  =  0.429). Even total increase of (+19%) observed in eggshells from infertile to hatchling was not statistically different (p  =  0.162).

The Na concentrations in eggshells at various development stages were significantly different (p < 0.001) (Table 2). Sodium concentration was constant during the first 2 development stages; there was not a significant decrease between the third and fourth developmental stages (−23%; p  =  0.388). However, a total decrease of 35% was recorded between infertile and hatchling eggshells (p  =  0.002).

Eggshell Sr concentrations at various development stages differed significantly (p < 0.001). Sr concentration varied significantly between the fourth developmental phase and successful growth (−56%, p  =  0.003) (Table 2). Overall, a 70% decrease was observed from the infertile to hatchling eggshells in Sr concentration (p < 0.001). The Zn concentrations in eggshells at various developmental stages showed statistically significant variations (p < 0.001) (Table 2). Specifically, Zn concentrations in eggshells during the second and third developmental stages were found to be statistically different (−24%; p  =  0.03) in this study. The total decrease in eggshell Zn concentration from infertile to hatchling eggshell was 52% (p  =  0.007).

Discussion

Eggshell samples were collected from the nests, including undeveloped eggs at the natural conditions. Some clutches included eggs that were only one developmental stage, others were 2 or 3 developmental stages. Therefore, during the study, there was no chance to control the sampling. There may be many reason that embryonic development is interrupted in a clutch. This study focused only on the element concentration of eggshell in which the embryo died. Moreover, the cause to stop the development of eggs may be effective in elemental concentration of eggshell.

Avian embryos use eggshell Ca for ossification during development (Romanoff 1967; Packard and Packard 1984). Based on our results, Ca appears to have been used in the third and fourth development stages, perhaps in the development of the carapace. As Sahoo et al. (1998) reported, Ca absorption from olive ridley turtle (Lepidochelys olivacea) eggshells primarily occurs after 40 days of incubation. This incubation time corresponded to late growth, the fourth developmental stage in our study, and the greatest decrease in Ca concentration occurred before this time. Our results for total Ca decrease (approximately 72%) in the eggshells of green turtles during embryonic development was greater than the decrease noted by Bilinski et al. (2001), Sahoo et al. (1998), and Bustard et al. (1969), who report figures of 43%, 60%, and 62%, respectively. However, Simkiss (1961) reported a 75% decrease in Ca concentration in the eggshells of leatherback turtles (Dermochelys coriacea) during embryonic development. Ca concentration of leatherback eggshells decreased almost 50% during incubation, similar to findings for Chelydra serpentina and Chrysemys picta (Packard and Packard 1984, 1986, 1989). Moreover, approximately 60% Ca loss was reported in olive ridley turtle eggshells during embryonic development (Sahoo et al. 1998).

Phillott et al. (2006) indicated that fungal invasion by Fusarium solani also caused depletion of the Ca concentration in green turtle eggshells during embryonic development. In the present study, all the specimens were collected from natural habitats, and fungal invasions were not observed. Therefore, the reason for Ca depletion in eggshells is not based on fungal invasion in this study. Nys et al. (2004) noted that the avian eggshell is a source of Ca for the developing embryo, and Bustard et al. (1969) mentioned that the eggshell is the major source of Ca for developing embryos among loggerhead sea turtles. The depletion could have resulted from absorption by the embryo during development. Alternatively, rain falling through the nests may have drained Ca and other elements from the eggshells.

The Ca concentration at this site was recorded as 278 ± 122 ppm in sand samples during the 2003 nesting season (Özdilek 2006). The mean eggshell Ca value exceeded the sand Ca concentration, which could mean that eggshells were taking up Ca from the sand or losing Ca to the sand. The eggshell is a medium for excluding potentially harmful elements, one of which may be Cu. Cu appears to be a nonessential element for incubation. Cu is removed from wastewater by binding to eggshells (Vijayaraghavan et al. 2005). In our study, there was no decrease in eggshell Cu concentration during development but rather, a slight increase in Cu concentration. Therefore, the reason for the increase could be absorption of this element in the eggshell during the late developmental stages. However, further research is required to determine why Cu increases in the eggshell during embryonic development.

Mg, another major inorganic element in eggshells, is effective on calcite morphology (Cusack et al. 2003). Sahoo et al. (1998) determined the relationship between yolk–albumen Mg concentrations and the concentration required by developing olive ridley hatchlings and eggshell Mg concentration. The Mg concentration in the eggshell was more or less stable during the embryonic growth stages in our study. Therefore, Mg concentration in the egg yolk might be sufficient for the developing embryo of green turtles. In fact, we found that the Mg concentration in green turtles eggshells was about 6 times that of olive ridley eggshells (Sahoo et al. 1998).

Sahoo et al. (1998) reported that the freshly deposited eggs of olive ridley have 490 mg/kg K in their eggshells, which is roughly a quarter of green turtle eggs (Sahoo et al. 1998). The beach sand K concentration in the current study was 180 mg/kg and was homogeneously distributed throughout the study area (Özdilek 2006). This amount is approximately a tenth of the concentration found in eggshells. Eggshells that yielded successful hatchlings had a decrease of about 54% Na by concentration. As Thompson and Speake (2003) and Thompson (1989) noted, Na is taken from outside the egg during growth, but this is probably not necessary for embryonic growth in reptiles. During the last 2 phases of incubation, Na concentration decreased by 7% and 31%, respectively. However, this decrease in Na is not statistically comparable with the decreases observed in Ca and Sr content of eggshells during the last 2 embryonic development phases. Sr is an essential element that plays a role in bone and skeleton development (Grynpas and Marie 1990; Dahl et al. 2001). Reduction of the mean Sr concentration of eggshells in the third developmental stage suggests that it could be used after this stage. Meyers-Shone and Walton (1994) indicated that Sr is not an essential element for life; it may have been transferred to the eggs in place of Ca. At the same time, Sr is used for normal shell formation and statolits in gastropods (Bidwell et al. 1986). The decrease in eggshell Sr was correlated with that of Ca in our study (Spearman rho, r  =  0.90, n  =  110, p < 0.001). Therefore, the reason for depleting Sr in eggshells during embryonic development could be the same as that for Ca depletion.

Özdilek and Yalçın Özdilek (2007) indicated a decrease in the Zn concentration of hatchling eggshells at Samandağ Beach. Similarly, after increasing during the first 2 stages, Zn concentration decreased in the current study. Zn chloride can be corrosive and may cause eroded mucous membranes (Kent 1998). Özdilek and Yalçın-Özdilek (2007) also indicated that Zn is a potentially corrosive element for the eggshell of green turtles. The conceptus preferentially accumulates Zn and Cu; an action that may be important for conceptus development, growth, and survival (Hostetler et al. 2003). It is interesting that another heavy metal, chromium, is important for the selection of nesting sites by the green turtle on the Samandağ coast (Yalçın Özdilek et al., unpubl. data, 2006). Therefore, in addition to Zn and Cu, the impact of other heavy metals, such as chromium, on the development of green turtles also needs to be investigated.

The eggshell is an important source in terms of essential elements and also trace elements for the embryo. Moreover, changes in the concentration of these elements during the developmental stages indicate embryo intake–outtake of elements during the embryological process. Some researchers have suggested the usage of waste eggshells as a means of removing heavy metals (e.g., Park et al. 2007), which means that the eggshells could be collecting some elements from the ambient. Further experimental studies are suggested to support the hypothesis that the eggshells collect some elements for transporting inside–outside the eggs.