Heavy metal contaminants in eggs and hatchlings of olive ridley turtles (Lepidochelys olivacea) at a mass nesting rookery in India

Heavy metal pollution has emerged as a prominent threat in recent times with high metal levels widely reported in species across ecosystems. The threat of rapid biomagnification is particularly enhanced in species such as olive ridley turtles, which occupy a higher trophic position, increasing their exposure to heavy metals. In the current study, we examine the presence of heavy metals in adults, in-utero and oviposited eggs, hatchlings as well as the nesting beach at two important olive ridley rookeries in India – Devi and Rushikulya. We collected muscle and in-utero egg samples from stranded adult olive ridleys at the two rookeries, while oviposited eggs, hatchling and sand samples were obtained from hatchery nests at Rushikulya. We compared concentrations of 9 heavy metals (Cr, Mn, Ni, Co, Cu, As, Se, Cd, and Pb) across different sample types in an Inductively Coupled Plasma Mass Spectrometer (ICP-MS). We found mean metal concentrations of sand to be highest among all samples analysed, followed by muscle tissue. Arsenic was the most prominent metal in adult turtles suggesting bioaccumulation, while Selenium was found to be higher in egg components. Heavy metals (HM) were found in-utero eggs, providing evidence of maternal transfer. Most HMs were similar for in-utero and oviposited eggs, though a few metals were higher in in-utero eggs suggesting potential leaching out during development. Sand and hatchling samples show a high correlation for Mn suggesting potential environmental transfer. These findings emphasise the risk posed by heavy metals to adult and early life stages of olive ridleys and highlight the urgent need for mitigation of these threats.

Marine ecosystems, critical in supporting a significant part of global biodiversity, are particularly vulnerable to anthropogenic threats (Halpern et al., 2007). The wide-spread cumulative impacts of industrial overfishing, commercial harvest, shipping, sea-bed mining and coastal construction have fundamentally altered its trophic function (Pauly et al., 2005; Farina et al., 2020; Yao et al., 2025; Bulleri and Chapman, 2010). In recent decades, heavy metal pollution has emerged as a prominent threat. Metal compounds enter coastal and marine environments through activities like mining, smelting, industrial and agriculture discharge, and shipyard building, etc (Barakat, 2011; Mani and Bharagava, 2016; Yadav et al., 2017; Mishra et al., 2018), and are readily taken up by organisms like the microalgae due to their innate ability of biosorption (Lintner et al., 2025). Subsequently, these metals rapidly bio-magnify, accumulating along all levels of the trophic food chain (Gobert et al., 2017; Danovaro et al., 2023). Heavy metals (HMs) also interfere with the biological processes through additive, synergistic, or antagonistic effects (Sparling et al., 2010; Jakimska-Nagórska et al., 2011). For example, Ni and Cr demonstrate additive toxicity, while the combination of Zn-Ni-Cu shows synergistic effects (Khangarot and Ray, 1990). This suggests that high concentrations of certain metals can positively or negatively affect the metabolic pathways of other essential elements.

The presence of heavy metals has been widely reported in marine species including corals (Islam et al., 2023), microalgae and sponges (Méndez et al., 2021), molluscs (Anagha et al., 2022), cetaceans and fish (Zaharieva, 2024). Zn and Cu, for example, are vital micronutrients for molluscs as structural components of hemocyanin, a primary respiratory pigment. Hence, organisms maintain higher levels of these metals in their body (Jakimska-Nagórska et al., 2011). However, higher amounts of Cu can cause negative effects like lipid degeneration and formation of DNA adducts (Nesto et al., 2007; Xu et al., 2018). Conversely, metals like Hg and Pb serve no biological function and are potent metabolic inhibitors, causing loss of appetite, cerebral dysfunction and loss of coordination in humans as well as in fishes (Pandey and Madhuri, 2014; Balali-Mood et al., 2021). Mercury is often eliminated by the body with the help of selenium, as seen in the case of sea birds (Spiller, 2018; Vizuete et al., 2018).

Since sea turtles are known to forage on a range of species down the trophic chain, they are also affected by the biomagnification (Çelik et al., 2006; Cortés-Gómez et al., 2017; Perrault et al., 2019; Frossard et al., 2020; Dennis et al., 2020; Naghilou et al., 2025). Their dietary plasticity, longevity, trophic position and wide geographic distribution puts them at a greater risk for both bioaccumulation and biomagnification (Adel et al., 2017; Tanabe et al., 2022), but also makes them good bioindicators for understanding the extent of pollution in marine ecosystems (Martínez-López et al., 2021; Fraga et al., 2023). Although studies report HM accumulation in sea turtle scutes, muscle, liver, kidneys and blood (Caurant et al., 1999; Guirlet et al., 2008; Sinaei and Bolouki, 2017; Aymak et al., 2021; Abdallah, 2023), the breadth of physiological effects caused by high concentrations of these metals is not entirely known. Potential negative effects including immunosuppression (Day et al., 2007), endocrine disruption (Rie et al., 2005; Kitana and Callard, 2008), neurotoxicity (Pereira et al., 2013), genotoxicity and cytotoxicity (Speer et al., 2018), and reduced reproductive viability (Roe et al., 2004) have been reported in sea turtles and other reptiles.

Apart from trophic accumulation, metal pollutants also enter eggs through intra-ovarian maternal transfer and/or nest substrates (Al-Rawahy et al., 2007; Hopkins et al., 2013). During vitellogenesis, metals are passively transferred to eggs alongside other nutrients (lipids and proteins), leading to accumulation of heavy metals in egg yolk as well as, albeit to a lesser extent, albumin and egg shell (Russell et al., 1999). Further, metals can accumulate post oviposition through environmental transfer via the nest substrate (Al-Rawahy et al., 2007). Accumulation of heavy metals in eggs and hatchlings has been shown to have deleterious effects on hatching success (Bergeron et al., 2011; Hopkins et al., 2013), locomotory performance (Marco et al., 2004), thyroid functioning (Brasfield et al., 2004), survival and growth (Hamlin and Guillette, 2010). While numerous studies have reported the presence of heavy metals in adults and eggs (Sahoo et al., 1996; Ross et al., 2016; Cortés-Gómez et al., 2017), these values vary drastically between species, populations and even regional nesting sites. This observed difference is attributed to species-specific diet and trophic position, geographic distribution, remigration interval, age as well as region-specific pollution levels (Sakai et al., 1995; Gardner et al., 2006; Alava et al., 2011; Chan et al., 2025). Further, few studies so far have evaluated the presence and accumulation of heavy metals in early life stages, especially hatchlings.

Olive ridley turtles on the east coast of India represent a genetically and demographically significant population (Shanker et al., 2004, 2021). They feed in the coastal and offshore waters of Bay of Bengal, which has seen alarming increase in pollution load due to mining, untreated industrial waste, agriculture runoff and coastal construction (Khan et al., 2017; Khadanga et al., 2022; Mookan et al., 2023). In addition, major olive ridley nesting sites in Odisha such as Gahirmatha, Devi and Rushikulya, are located near river mouths (Bustard, 1976; Pandav et al., 1994), which potentially carry metals and other organic pollutants from upstream, further contaminating the nesting sites and nearshore waters. Hence, we aimed to elucidate heavy metal accumulation in adult olive ridley turtles, and determine the extent of metal transfer through maternal and environmental pathways.

In order to do this, we quantified and compared concentrations of heavy metals in the muscle of deceased adult turtles, in-utero and oviposited eggs, dead hatchlings from hatchery nests, and sand from two major nesting sites. Specifically, we a) assessed the HM load in deceased adult muscle tissue and their retained (in-utero) eggs (egg content, egg shells) from Rushikulya and Devi, to determine HM load in adults; in addition, their presence in in-utero eggs establishes maternal transfer b) compared the HM load in retained eggs (in-utero) and oviposited eggs (egg content & egg shell), to assess the changes in HM load during incubation, and c) examined HM concentrations among eggs, hatchlings, and sand, to assess potential environmental transfer.

Study area

The two study sites, Rushikulya and Devi, are located on the east coast of Indian peninsula, in the state of Odisha (Figure 1). Rushikulya is an active mass nesting beach, located at the mouth of Rushikulya river, that extends across 7 km and is bordered by nine fishing villages. Annually, roughly 100,000-500,000 turtles nest in an arribada that occurs once or rarely twice a year (Shanker et al., 2024). Devi nesting beach, located ~100 km north of Rushikulya, extends 5 km and is bounded by 2 rivers – Devi river and Keluni river, which constitute major distributaries of Mahanadi River. Devi was formerly a mass nesting site but has not witnessed an arribada since 1997. Both Rushikulya and Devi rivers are influenced by upstream industrial and mining activities, agricultural runoff and other urbanisation activities (Srichandan et al., 2016; Khadanga et al., 2022).

Sample collection

Adult turtles and in-utero eggs

Annually, olive ridley turtles in the region migrate to the east coast in November-December, where females remain in the nearshore waters until the end of nesting season in May. This period also coincides with peak fishing season, leading to increased interaction between motorised fishing vessels and olive ridleys (Rao et al., 2023). During this time, nesting beaches in Odisha witness a few thousand dead turtles washing ashore each season (Shanker and Manoharakrishnan, 2022). We opportunistically sampled fresh carcasses of female olive ridley turtles stranded along the Rushikulya and Devi nesting beaches between January and March 2024. Necropsy was performed on gravid dead turtles (Rushikulya n = 20; Devi n = 13) as per Aymak et al. (2021); oviducts of the adult turtles were cut using a scalpel blade, and about 7–8 eggs were collected at random from each turtle and stored in 300 ml containers. Muscle samples were also collected using scalpel blades and samples were stored in centrifuge tubes. Muscles tissues were only sampled from individuals from which eggs were collected. The egg and muscle samples were immediately transferred to a refrigerator and stored at below 0 °C and 4 °C, respectively.

Hatchery eggs and hatchlings

The Rushikulya mass nesting site has been monitored on a regular basis during the nesting season since 2008 (Shanker et al., 2024). As part of the long-term monitoring project, a few solitary nests are relocated every season to a hatchery located on the nesting beach. For the study, we sampled undeveloped eggs and dead hatchlings from hatchery nests that were excavated post emergence. We collected two eggs at random from the set of undeveloped eggs from 10 nests, and 2–7 dead hatchlings from the same nests based on availability during excavation. Sand samples were also collected from the same nest chambers. Dead hatchlings were cut in half and only the upper half was used for the analysis, without segregating them into different tissues. The lower half was utilised in a histology study for hatchling sex determination. Egg and hatchling samples from hatchery nests in Devi could not be collected due to logistical constraints.

Sand

In Rushikulya, sand samples were taken from hatchery nests post excavation at the same depth that the eggs were incubating. In Devi, sand sampling was done using a quadrat system where a 1m stick was used for marking 1m x 1m long quadrat in the sand. A shovel was used to remove the top 25cm of sand and samples were collected from 25 cm until a depth of 30 cm was reached. Sand samples from Rushikulya and Devi were sun dried and placed into air tight bags (Tanabe et al., 2022). All samples (muscle, egg, hatchling and sand) were then transferred at the end of the sampling period to a wet lab at Indian Institute of Science, Bangalore, using thermocol boxes and ice packs, where further heavy metal analysis was carried out.

Heavy metal analysis

Muscle samples were thawed outside until they were at room temperature. Following this, the samples were transferred from the centrifuge tubes onto an aluminium foil and dried in a hot air oven at 60 °C for 24 hours. Post drying, samples were grinded using sterile mortar and pestle and stored for further analysis. Similarly, egg samples (Rushikulya n = 10; Devi n = 10) were washed properly with distilled water to remove any additional materials on the surface. They were separated out into egg shells and egg contents (yolk + albumin), and placed on sterilised petri dishes in a hot air oven at 60 °C for a period of 2 days. Two eggs from one individual turtle were combined to form one sample representative. Egg shells and egg contents were then ground into a powder using sterile mortar and pestle. About 0.5g of each powdered biological sample was digested with 2ml of 69% HNO 3 (Merck, Darmstadt, Germany) and 2ml of 30% H 2 O 2 (Qualigens Fisher Scientific) as per Esposito et al. (2023). The samples were then kept in a hot air oven at 100 °C for 2 hours. From the original digestate, 0.4g was taken and diluted up to 5ml in falcon tubes using ultrapure water to achieve 1:100 dilution. For hatchery nests, a similar method was followed where two eggs separated out into shells and contents, then pooled and allowed to dry in the hot air oven at 60°C. Both eggs and hatchling samples were processed in the similar way as mentioned for the egg samples. For sand samples (Rushikulya n = 10; Devi n = 10), 0.5g of sand was placed in 2ml HNO 3 and allowed to digest at 100 °C. It was then diluted up to 5ml with ultrapure water (HPLC Grade, Fisher Chemical).

Samples were run once and their values were converted into ppm. Concentration of metals in the samples were quantified using Inductively Coupled Plasma Mass Spectrometer (ICP-MS, Thermo Scientific X-Series II) at the Centre for Earth Sciences (CEaS), IISc, Bangalore. For calibrating the machine, ICP grade multi-element standard solution was used (Merck, Darmstadt, Germany) to make calibration curves (0.1, 0.25, 0.5, 0.75, 1 ppm). A total of nine metals were analysed viz. Chromium (Cr), Manganese (Mn), Nickel (Ni), Cobalt (Co), Copper (Cu), Arsenic (As), Selenium (Se), Cadmium (Cd) and Lead (Pb).

Quality assurance and control

All apparatus and tools used were thoroughly washed in ultrapure water and 2% nitric acid before use. Standards were prepared using the same chemicals as those used for preparing biological samples. One of the standard solutions was run again to establish error percentage. The mean percentage error calculated was 1.84% across all runs and the value remained within the acceptable analytical range for metal analysis by ICP-MS (See Supplementary Table 1 for Limit of Detection (LoD) and Limit of Quantification (LoQ) values).

Statistical analysis

Descriptive statistics including mean ± SE of all metals across muscle, eggs, hatchlings and sand were calculated. The Shapiro-Wilk test was used for testing the normality of the data. To test the difference in HM profiles across different sample groups (muscle-eggs; in-utero and oviposited eggs; eggs-hatchlings-sand), we used a permutational multivariate analysis of variance (PERMANOVA) on standardised data, using site as a covariate where applicable. To further examine variation of metal composition across tissues, we performed a principal component analysis (PCA) for each of the sample groups mentioned above. The Kruskal-Wallis test was used to test the difference between sample means. We used the Spearman’s correlation to determine the relationship between individual metals in different samples. All statistical analyses were carried out in R Studio (2025) (version 4.5.2).

The mean weights for in-utero eggs and hatchery eggs were 23.14 ± 1.67 g and 24.93 ± 1.48 g, respectively. Metals recorded in the different samples analysed are given in Table 1 in µg/g dry weight (See Table 1 for descriptive statistics). Sand samples had the highest mean metal concentrations (5.16 ± 0.73 µg of metal/g), followed by muscle samples (4.38 ± 0.66 µg of metal/g), relative to other sample types. Mn concentration was exceptionally high in sand and dead hatchlings, As was high in muscles, while Se was high in the egg shell and egg contents of in-utero eggs (Supplementary Figure 2). From our correlation heat maps, Mn and Co show strong correlation between sand and dead hatchlings (0.88, 0.85). Individual metal correlations indicate that Cd and Pb, although non-essential in nature, are strongly correlated with Cu (0.78) Mn (0.91), Co (0.90) and Cr (0.87) which are essential (Supplementary Figures 3-5).

HM load in adult muscles and in-utero eggs

The HM profiles varied significantly between muscle, in-utero egg shells and egg contents from adult turtles in Rushikulya and Devi (Permanova pseudo - F = 2.37, p < 0.05), with muscle tissue showing higher HM load than egg shells and egg contents (Figure 2). Mean metal concentrations across the two study areas revealed As, Cd, Cr, Cu, Mn and Se to be slightly higher in Devi than Rushikulya although this difference was not significant (p = 0.35). Since there was no significant difference between the two study areas, the tissue samples from both the sites were combined for the PCA analysis (Figure 3A).

The PCA shows that muscle samples separate from egg shells and egg contents along Dim 2, which was associated with higher loading of As, and Cd. Egg shells and egg contents showed substantial overlap along Dim 1, which was associated with higher loadings of Mn, Co, Ni and Pb. Together, the two reduced dimensions, Dim 1 and Dim 2 account for 66% of variation between sample types.

HM load in in-utero eggs and oviposited eggs

The HM profiles of in-utero egg shells and egg contents varied significantly from oviposited egg shells and egg contents of hatchery nests from Rushikulya (Permanova pseudo - F = 3.03, p < 0.05). There was a significant difference between mean metal concentration across these sample types (p < 0.05). In general, egg shells had slightly higher HM concentrations than egg contents (Figure 4).

PCA revealed distinct metal profiles across sample types, with in-utero egg samples and oviposited eggs separating along both Dim 1 and Dim 2 (Figure 3B). Specifically, the egg shells of both the sample groups (in-utero and oviposited) varied substantially along Dim 2, with oviposited egg shells being associated with higher loadings of Mn, Co and Ni. On the other hand, egg contents of the two sample groups did not show strong separation. However, in-utero egg contents extended towards positive Dim 1 driven by higher loadings of Cr, Se, Cu, Cd, and As, while oviposited egg contents, similar to egg shells, show slight shift towards positive Dim 2. Overall, the oviposited eggs showed low variability and were separated from in-utero eggs, primarily driven by Mn, Co and Ni. Together, these two dimensions, Dim 1 and Dim 2 explained 71% of the total variation in the data.

HM concentrations in eggs, hatchlings, and sand from hatchery nests

Comparing between sample types within hatchery nests, the HM profiles of oviposited egg shells and contents, hatchlings and sand varied significantly (Permanova pseudo-F = 26.3, p < 0.05). Mean metal concentrations varied significantly between the groups (p < 0.05). Overall, sand had the highest HM concentrations, followed by hatchlings and eggs (Figure 5). The PCA revealed large variation in metal profiles, with sand forming a distinct cluster and showing substantial variation along Dim 1 driven by negative loadings of Co, Cr, Mn, Pb and As (Figure 3C). There was less variability in metal concentrations in sand, as indicated by the smaller ellipse. Hatchling samples showed high variability and separate from egg samples along negative Dim 2, which is associated with higher loadings of Cu, Se, As, Mn and Cd. Egg shells vary from egg contents along positive Dim 2, influenced by Ni. Overall, the first two dimensions, Dim 1 and Dim 2 explain 76% of the total variation.

Marine and aquatic environments are particularly susceptible to toxic heavy metals as they serve as end points to industrial and agricultural waste discharge or runoff (Panda et al., 2006). Odisha, located on the east coast of India, is one of the leading producers of essential metal ores like iron, chromite, manganese, bauxite, and coal (Lenka et al., 2025). It is also one of the largest cultivators of rice. Therefore, runoff from fertilisers and pesticides in rice fields as well as industries in the region has led to elevated levels of heavy metals in wetlands, rivers and other aquatic habitats (Reddy et al., 2013). For instance, Panda et al. (2025) reported high levels of Cu and Zn in wetland birds, prey, and soil showing evidence of biomagnification of these metals along the trophic chain. Further, Odisha’s extensive river systems including Mahanadi, Brahmani-Baitarani, Rushikulya, and Devi Rivers flow directly into the Bay of Bengal – potentially discharging heavy metals and threatening both freshwater and marine ecosystems (Rao et al., 2016; Rakib et al., 2022).

Olive ridley turtles on the east coast of India are of significant concern, as they represent the largest mass nesting population outside of Central America. They may be exposed to heavy metal pollution at both foraging grounds and breeding sites along the Odisha coast. Determining the pathways of heavy metal entry and accumulation, especially in early life stages, can help address this threat and inform management policies. To address this critical knowledge gap, we analysed the presence and accumulation of heavy metals in adults, in-utero and oviposited eggs, hatchlings and beach sand at two important olive ridley rookeries along Odisha coast.

Heavy metal accumulation in adults

Olive ridley turtles are omnivorous and opportunistic feeders that exploit a wide-range of prey, making them susceptible to significant heavy metal exposure and accumulation at their foraging sites (Hashmi et al., 2015). Upon migrating to nearshore breeding habitats, females may continue to forage at or near their nesting beaches (Behera et al., 2015), further increasing their exposure to heavy metals. We found high concentrations of HM in adult females at both the study sites, indicating significant exposure at the foraging sites. The overall concentration of heavy metals in Rushikulya and Devi showed minor differences, which was expected as turtles nesting at these two rookeries come from the same population and, hence, potentially use similar foraging sites (Pandav and Choudhury, 2006; Tripathy and Pandav, 2008; Kumar, 2016). Overlap of metal profiles of muscle and in-utero egg samples establish maternal transfer of certain heavy metals, although the sample types (muscle and eggs) show differential accumulation of metals.

Across both sites, we found a high concentration of Arsenic (8.66 ± 2.67 µg/g) in muscle tissues of adults, the highest reported so far for the species. Previous studies have documented As accumulation in various tissues of olive ridley turtles, specifically in liver, kidney and blood (Cortés-Gómez et al., 2014, 2018; Zavala-Norzagaray et al., 2014). However, there are no published studies reporting As concentrations in muscle tissues of olive ridley turtles, and therefore we are unable to draw a direct comparison for this particular element in the species. These studies do show higher accumulation of this metal in the liver compared to other tissues, and thus the muscle values reported by us may be an underestimation of the overall As load in the body. Moreover, high As in muscles indicate accumulation through potential chronic exposure and has been reported earlier in other species such as loggerheads (Aymak et al., 2021). Although As concentrations reported from rivers, deltas as well as coastal waters of Bay of Bengal are relatively low (Nath et al., 2021), high concentrations of As have been reported in various marine fish species from Bay of Bengal and Sri Lanka, areas that serve as major foraging sites for the population (Mukherjee and Bhupander, 2011; Moxness Reksten et al., 2021; Islam et al., 2022; Jayakody et al., 2024). We also found low but detectable levels of As in in-utero egg shells and egg content (1.34 ± 0.32, 1.37 ± 0.21 µg/g respectively), indicating maternal transfer of this non-essential metal.

The concentration of Cd found in muscle samples was low (0.67 ± 0.17 µg/g) compared to other studies on ridleys (Frías-Espericueta et al., 2006; but see Gardner et al., 2006). Concentration of Cd reported in our study is consistent with low Cd concentration reported from Bay of Bengal and surrounding areas (Mukherjee and Bhupander, 2011; Hasan et al., 2016; Srichandan et al., 2016). Previous studies indicate high accumulation of this particular metal in liver and kidney compared to muscle tissues (Gardner et al., 2006; Cortés-Gómez et al., 2014, 2018) and therefore, the current Cd values might not represent the total accumulation level. Similarly, the observed Cd levels in in-utero eggs (shell: 0.58 ± 0.14 µg/g; contents: 0.31 ± 0.11 µg/g) were relatively low, and are consistent with values previously reported for olive ridleys (Páez-Osuna et al., 2010) and other sea turtle species (Sakai et al., 1995, 2000; Godley et al., 1999; Day et al., 2005; Lam et al., 2006).

Although Se concentrations observed in muscle samples were substantially lower (3.9 ± 1.07 µg/g), we found very high concentrations of Se in in-utero eggs (shells: 21.15 ± 8.98 µg/g; contents: 12.60 ± 6.65 µg/g), well above the toxicity threshold for the metal. Previous studies have shown that Se tends to readily bioaccumulate in metabolically active tissues such as liver, kidneys and ovaries of many vertebrates through trophic transfer (Grillitsch and Schiesari, 2010). For instance, Hopkins et al. (2004) showed that African house snakes (Lamprophis fuliginosus) fed with seleno-D, L-methionine injected mice accumulated significant levels of Se in kidney, liver, and eggs. The elevated levels of Se observed in the current study may, therefore, indicate bioaccumulation of Se in adult females of the population. Further, the low Se levels detected in muscle samples might be an underestimation, as Se is known to accumulate more readily in liver and kidney, which were not analysed in the study. Moreover, limited data on Se concentrations in marine organisms from the region (Chatterjee et al., 2001), and lack of knowledge on the population’s primary foraging grounds, limits our ability to identify and confirm trophic accumulation. Given that Se accumulation has been linked to reproductive toxicity (Grillitsch and Schiesari, 2010), further studies are needed to assess Se exposure and its potential impacts on the population.

Lead is a non-essential metal known for its extreme toxicity even at low concentrations. Research on primates shows that lead levels in blood ranging from 3 µg/dl to 25 µg/dl – induced via dietary lead acetate – resulted in neural impairment and behavioural disorders (Rice, 1992; Needleman, 2004). In the current study, we found very low levels of Pb in the muscle as well as eggs. This can, be attributed to low levels of Pb reported in the offshore water of Rushikulya (Panigrahy et al., 2014; Srichandan et al., 2016). Similar low concentrations of Pb have been reported across most other sea turtle species (for review, see Cortés-Gómez et al., 2017), indicating that Pb accumulation in nearshore and offshore habitats is restricted. Essential metals, such as Mn, Cr, Ni and Co (see Table 1), were detected at low concentrations in both muscle and egg samples. These values fall within the threshold limits required for the normal physiological functioning Health Supplements Nutritional guide (2017).

Heavy metal in eggs (in-utero and oviposited)

The presence of HMs in in-utero eggs and the similarity in composition with oviposited eggs suggests that primary route of entry for these metals is maternal transfer. While maternal transfer of HMs to in-utero eggs has been documented in various other taxa, including birds (Burger, 1994), slider turtles (Burger and Gibbons, 1998) and alligators (Roe et al., 2004), there are no studies on reptiles comparing metal profiles of eggs pre- and post-oviposition. Although the two egg samples (in-utero & oviposited) formed slightly distinct clusters, significant overlap between egg shells and contents from both sources suggests a common biological origin and similar routes of transfer of certain metals (Frossard et al., 2020). PCA showed that HM concentration in egg shells was more variable than the egg contents, revealing a broader metal profile in case of egg shells and indicating that they might serve as a barrier to metal accumulation in egg contents. Further, they may also be more susceptible to environmental influx due to direct contact with the nest substrate.

The concentration of metals in eggs reflects the circulation level of metals in blood, thereby serving as a bioindicator of recent environmental exposure. Furthermore, it has also been suggested that metal levels in eggs should be monitored consecutively to establish any definitive trends (Burger and Gochfeld, 1993; Burger, 1994; Hashmi et al., 2015). Despite significant overlap between the two groups, we observed As concentration in in-utero eggs was approximately 2-fold higher (shells: 1.34 ± 0.32 µg/g; contents: 1.37 ± 0.21 µg/g) and Se concentration was almost 10-fold higher (shells: 21.15.2 ± 8.98 µg/g; contents: 12.60 ± 6.65 µg/g) compared to oviposited eggs (As, shells: 0.29 ± 0.02 µg/g, contents 0.50 ± 0.09 µg/g; Se, shells: 1.98 ± 0.39 µg/g, contents: 3.50 ± 0.63 µg/g). As and Se as elements have been known to be transferred maternally rather than environmentally in previous studies (Kubota et al., 2002; Roe et al., 2004). The presence of Se in eggs is expected as selenium is a fundamental part of selenocysteine and various other seleno-proteins which play an important role in egg development (Guirlet et al., 2008; Liu et al., 2023). On the other hand, we found low levels of Se in oviposited eggs in comparison to the former. Reptile eggs tend to develop pores during the late stages of embryonic development to allow gaseous exchange (Ackerman and Prange, 1972; Zhang et al., 2023). The permeability of the developing eggs could have, therefore, resulted in leaching out of some metals like Se. While hatchling samples from the study showed relatively high Se concentrations, this may represent a difference between the undeveloped eggs that were sampled for egg content and developed eggs from which hatchlings would have emerged.

We observed a high degree of selective specificity for certain metals, particularly nickel. Ni was relatively higher in oviposited eggs (almost 6-fold higher) compared to in-utero eggs, with higher concentrations in the egg shells (Table 1). Accumulation of Ni in egg shells has been reported previously (Páez-Osuna et al., 2010) with studies indicating that elevated nest temperatures can lead to increase in Ni absorption (Ho et al., 2014). Frossard et al. (2020) also found that Ni was the second highest metal in green turtle eggshells that were collected post hatching. Thus, the accumulation of Ni in oviposited egg shells may be attributed to the innate tendency of the metal to be adsorbed onto the eggshells from the surrounding substrate.

A decrease in concentrations of Cr (2-fold), Cu (2-fold), As (2-fold), Se (10-fold) and Cd (10-fold decrease in eggshells) were seen in the oviposited eggs compared to the in-utero eggs (Figure 6). This trend contrasts with the patterns observed for Ni, while Mn remained consistent across both sources. Other studies have observed higher metal concentrations in egg shells post-incubation (Frossard et al., 2020). Again, this might be due to the fact that our study utilised undeveloped eggs whereas Frossard et al. (2020) used eggs post development. In fact, studies have shown that the concentrations of metals increase in the egg shells of developed eggs in comparison to undeveloped eggs (Yalçin-Özdilek et al., 2011; Jian et al., 2025).

Heavy metals in hatchery nests

Previous studies have examined the effect of surrounding sediment contamination on developing eggs and found significant substrate transfer (Çelik et al., 2006; Tanabe et al., 2022; Aib et al., 2025). In Odisha, Sahoo et al. (1996) examined metals concentration in the freshly laid eggs of olive ridleys in Gahirmatha, and found them to be lower than hatchlings, indicating potential environmental transfer. While comparing eggs (shells and contents), hatchlings and sand from hatchery nests in Rushikulya, we found high metal load in sand, followed by hatchling, egg shell and egg content (see Table 1). We observed higher variability of metal profiles in hatchlings relative to eggs, similar to other studies (Frossard et al., 2020).

However, it is important to note that much of the high metal load observed in hatchlings is driven by a single metal, Mn (13.2 ± 3.73 µg/g). The observed Mn concentration in hatchlings was 5–10 times higher than the levels observed in eggs and muscle samples. This could be attributed to preferential accumulation of Mn in hatchling carapace and the difference in accumulation patterns across tissues (Frossard et al., 2020; Jian et al., 2025). We also found high Mn concentration in sand samples (31.74 ± 1.40 µg/g). The Mn levels detected in our study were comparable to those reported previously from the study site (Khan et al., 2021). Odisha is one of the leading producers of Mn, which may explain such high levels in the sand (Rout, 2023). We also observed a strong correlation (r = 0.88) between sand and dead hatchling tissue, suggesting potential environmental transfer of the metal. Frossard et al. (2020) also suggest that Mn may be environmentally transferred from the sediments. However, the bioavailable fraction of heavy metals varies significantly with substrate type (Yadav and Ramanathan, 2025) and lack of this data from the region precludes us from establishing environmental transfer of Mn. Since Mn can be toxic at extremely high levels, future studies should investigate extent and source of Mn accumulation in hatchlings.

Similarly, selenium was the second most abundant metal after Mn in dead hatchlings. Since we detected negligible levels of Se in sand, these elevated concentrations suggest that the metal is primarily sequestered through maternal transfer. Similarly, copper concentrations were significantly higher in hatchlings than egg shells, egg contents and sand. Liver is again the site for copper synthesis and storage of excess of Cu in form of Cu-metallothionein complex. It also helps in the production of ceruloplasmin – a major copper-carrying protein that helps in homeostatic regulation (Jian et al., 2025).

Although Cr concentration was low in egg shells, egg contents and hatchlings in the current study, Sahoo et al. (1996) reported high Cr levels (10 ± 1.7 mg/g) in egg shells and hatchlings (10.3 ± 3.0 mg/g) from Gahirmatha, another mass nesting site located ~150–250 km north of Devi and Rushikulya. Gahirmatha nesting site is near Brahmani-Baitarani river mouths which flows through Sukinda valley where chromite mining occurs, potentially explaining the high Cr values reported in the study. Hatchery sand had high concentration of Cr (6.92 ± 0.16 µg/g) again highlighting the effects of mining upstream of the rivers that flow into the ocean. Although Sahoo et al. (1996) reported environmental transfer of Cr, we found no evidence of this in our study. However, these results should be interpreted with caution, as only a part of the hatchling could be utilised for the study.

Arsenic concentrations remained negligible across eggs (shell and content), hatchlings and sand. This is in contrast to the significantly higher As levels observed in adult muscle tissues. Such a difference may reflect differential accumulation of the metal across tissue types. On the other hand, the lead concentrations observed in our sand samples were consistent with the concentrations of the metal observed in waters of Rushikulya (Panigrahy et al., 2014).

Physiological effects of heavy metals

While the immunological and physiological consequences of heavy metal exposure and accumulation in mammals and birds is well studied (Das et al., 2002; Koivula and Eeva, 2010; Khwankitrittikul et al., 2024), research concerning reptiles, particularly sea turtles, remains limited due to the difficulty in assessing long-term impacts of pollution in these long-lived species. Metals like Pb, As, Cd have important role in negating the effects of reactive oxygen species and metals like Se, Zn, Cu, Mn are associated with antioxidant enzymes.

A high level of Se was found to cause reproductive toxicity in fish and birds (Grillitsch and Schiesari, 2010). While the physiological consequences in turtles are not fully known, high concentrations of Se in in-utero eggs and hatchlings, as reported in this study, can potentially hinder embryonic development and survival. Existing research indicates that high levels of As and Se in green turtle eggs contribute to embryonic mortality and reduced hatching success (van de Merwe et al., 2009). The physiological consequences of heavy metal accumulation can be severe. Heavy metal buildup in the liver impairs detoxification processes and induces oxidative stress, particularly in species inhabiting highly polluted areas (Nagle et al., 2001; Marco et al., 2004; Roe et al., 2004). Within the reproduction system, heavy metals cause DNA damage and induce mutations, leading to reduced fecundity and increased embryonic mortality in fish and avian species (Gilani and Marano, 1980; Gilani and Alibhai, 1990; Jezierska et al., 2009). Chronic exposure can lead to systemic inflammation and compromised immunity in reptiles, threatening long-term survival and population viability of species exposed to heavy metal pollution.

This study, the first since the 1990s in the Bay of Bengal (Sahoo et al., 1996), addresses a major research gap by examining heavy metal presence in olive ridley turtles. Overall, we report high concentrations of Arsenic in the muscle tissue of adults and high levels of Selenium in in-utero eggs, both of which are the highest reported for the species. This indicates high chronic exposure in the case of As, and maternal transfer of Se. We also see high levels of Mn in hatchlings and sand, which indicates potential environmental transfer. The variability of metal profiles among sample types highlights tissue-specific accumulation patterns of heavy metals, especially among egg shells, egg contents and hatchlings. Overall, the level of non-essential metals was within permissible limits for the tissues we assessed. However, future studies should incorporate an assessment of maternal blood and hepatic tissues. Further, previous studies have alluded to the use of post-hatched egg shells as a non-invasive proxy for egg contents and metal load in the body (Burger and Gibbons, 1998; Naghilou et al., 2025). However, our results suggests that it is not a good proxy and that it is important to incorporate multiple tissue samples to accurately reflect metal load in the body. While olive ridley turtle nesting has increased dramatically in Odisha over the last decade, particularly in Rushikulya (Shanker et al., 2024), the impacts of pollution, and other factors such as climate change, can still affect the health of the population in the medium- and long-term. Thus, efforts need to be taken to monitor and mitigate the impacts of these threats.