An assessment of heavy metals in green sea turtle (Chelonia mydas) hatchlings from Saudi Arabia’s largest rookery, Ras Baridi

Introduction

The green sea turtle, Chelonia mydas, is classified as Endangered by the International Union on Conservation of Nature (IUCN, 2022). This conservation status is largely caused by incidental bycatch, direct poaching, entanglement, and boat strikes (Stanford et al., 2020). Additionally, anthropogenic activities generate environmental pollutants of concern due to their potential toxicity to marine fauna. For example, green sea turtles can be infected with a disease known as fibropapillomatosis (FP) which causes benign tumors that can grow large enough to inhibit sight, mobility, and foraging (Herbst, 1994). It is hypothesized that environmental contamination, including those from heavy metals, may be a cofactor (da Silva et al., 2014). In addition, sea turtles exposed to high concentrations of environmental pollutants, particularly heavy metals, have been shown to develop neurological and developmental disorders, cancer, and in more severe cases, death (Marsili & Fossi, 2003; Nordberg et al., 2007; Grillitsch & Schiesari, 2010). Since sea turtles are long-lived vertebrates with large home ranges, they are susceptible to the accumulation of heavy metals in their tissues (Sakai et al., 1995); because of this, they are often used as bio-indicators for environmental contaminants (Bruno et al., 2021). Sea turtle eggs, embryos, and hatchlings are vulnerable to heavy metal contamination as contaminants bioaccumulated in the female can be maternally transferred to the embryo and absorbed from the nest environment during incubation (Al-Rawahy et al., 2007; Hopkins, Willson & Hopkins, 2013). Sea turtle eggs incubate for 2 months, allowing sufficient time for contaminants to be absorbed into the developing embryo (Bustard & Greenham, 1968; Ackerman & Prange, 1972). Environmental contaminants can have significant impacts on embryotic development and can potentially contribute to embryo mortality (Hamlin & Guillette, 2010). Defects from these pollutants (i.e., thyroid dysfunction, reduced hatching success, eggshell thinning, and other abnormalities) are a potential source of mortality for turtle embryos, preventing survival to their juvenile life stage (Hamlin & Guillette, 2010).

In the past three decades, industrial activity and development have increased in Saudi Arabia’s coastal areas, introducing hazardous elements into the environment (Youssef & El-Sorogy, 2016; Al-Mur, Quicksall & Al-Ansari, 2017). In addition, rapid human population growth and urbanization have further increased pollution (Ozturk & Turkan, 1993; Badr et al., 2009). This is of particular concern in the region, as the population growth rate around the Red Sea is expected to double in the next 20–30 years (United Nations, 2017) with the development of large-scale “giga-projects” under the Kingdom’s Vision 2030 (PIF, 2017). Heavy metals may be introduced into the environment by aeolian transport (Olsen, Cutshall & Larsen, 1982) or directly from industrial pollution. This leads to their integration into sediments and the water column, where they are ultimately absorbed by living organisms (Olsen, Cutshall & Larsen, 1982; Claereboudt, 2004). Among these hazardous pollutants are elements with high molecular weight, such as chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), selenium (Se), cadmium (Cd), and lead (Pb). In high concentrations, these elements can have detrimental impacts on marine organisms (Khansari, Ghazi-Khansari & Abdollahi, 2005). Furthermore, there are additional environmental concerns as these pollutants do not biodegrade naturally (Briffa, Sinagra & Blundell, 2020). Despite reports suggesting sea turtles are considerably affected by heavy metal pollution (Lutcavage, Plotkin & Witherington, 1997; Sakai et al., 2000; Al-Rawahy et al., 2007), the majority of the work on heavy metal pollution to date has focused on salt marshes, estuaries, and mangrove systems.

Cement production is a significant source of heavy metal pollution (Achternbosch, Bräutigam & Gleis, 2003). Heavy metals such as Cd, Cr, Cu, Pb, and Zn are used in cement production and can be toxic to humans and animals, even at low concentrations (Al-Khashman & Shawabkeh, 2006; Kabata-Pendias & Mukherjee, 2007). The concentration of the heavy metals deposited varies depending on the wind velocity and particle size through the cement dust (Central Pollution Control Board (CPBC), 2007). The main transport pathway is via wind flowing over the cement kilns and releasing heavy metals into the air or settling as dust (Central Pollution Control Board (CPBC), 2007). In Oman, high concentrations of heavy metals have been recorded within a 0.5 and 2 km radius around a cement factory, affecting surrounding biota (Semhi et al., 2010). The severity of heavy metal pollution is dictated by the accumulation pathway, pollutant type, and vulnerability of the species exposed (Adekola, Inyinbor & Abdul Raheem, 2012).

Ras Baridi is the largest rookery for green turtles in the Red Sea, with an estimated 250 females nesting annually (Pilcher & Al-Merghani, 2000; Shimada et al., 2021). Ras Baridi is ~50 km north of the city of Yanbu and is located adjacent to the Yanbu Cement Factory. It has been proposed that cement dust from the kilns is blown onto the adjacent nesting beach, which compacts the sand, preventing hatchling emergence (Pilcher, 1999). Despite the potential negative impacts of cement dust at Ras Baridi first revealed 20 years ago (Pilcher, 1999), there have been no subsequent studies on the influence of the cement dust on incubating embryos on this nesting beach. Physical and environmental parameters of this nesting beach have been studied, including the sedimentology (Al-Mansi, Nawab & Sagga, 1991; Scott et al., 2022), temperature (Tanabe et al., 2020), sand compaction (Pilcher, 1999) and heavy metal concentrations in the sand (Tanabe et al., 2022) but heavy metal accumulation in sea turtle hatchlings is yet to be examined, with most studies worldwide conducted on stranded adults of this species (Bruno et al., 2021).

The bioaccumulation of heavy metals in green turtles has been identified for a number of populations (Godley, Thompsonà & Furnessà, 1999; Sakai et al., 2000; Lam et al., 2006), but to date, there have not been any studies conducted on Saudi Arabian turtle populations. As a result, information on the heavy metal biorisk in the Red Sea is limited. The present study was conducted at Ras Baridi to assess the heavy metal concentrations in the liver, muscle, and residual yolk in green turtle hatchlings to provide a baseline and the first report of these measurements in the Red Sea region. This study is a first step toward determining the impact of heavy metal contamination on endangered sea turtle populations in the Red Sea.

Methods

Freshly deceased green sea turtle hatchlings were collected in September 2021 from two beaches in Ras Baridi (24.258°, 37.571°). These turtles were presumed dead from the night prior to collection as they were still soft and had not been consumed by predators. The cause of death of these hatchlings is not known, but it is likely they emerged from their nest and were disoriented from the lights of the cement factory and were unable to make it to the sea by sunrise, eventually dying from heat exposure. Six hatchlings were collected from the beach adjacent to the cement factory (Fig. 1, blue), and 16 were collected from the beach ~1.5 km south (downwind) of the factory (Fig. 1, orange). In the summer in Ras Baridi, the prevailing wind direction is from North to South (Langodan et al., 2017). The GPS coordinates were recorded at the collection locations, and the hatchlings were immediately frozen for transport and stored at −20 °C until analysis. The hatchlings were weighed using a Mettler Toledo XS205 Dual Range Balance (accuracy ± 0.71 mg), and their straight carapace length and straight carapace width were measured with a Dual Reading Dial Caliper (Anytime Tools, accuracy ± 0.02 mm). This fieldwork was approved by King Abdullah University of Science and Technology who obtained coast guard permits for the site. Although no work with live animals was conducted in this study, the research team has approval from KAUST’s IACUC to work with turtles under protocol 19IACUC07.

Hatchlings were dissected, and a minimum of 0.1 g of liver, muscle, and residual yolk tissue were obtained from each individual. All tissue samples were weighed for wet mass and subsequently dried in a Binder incubator at 60 °C for 3 days. The samples were then re-weighed to measure the dry mass before homogenizing using a porcelain mortar and pestle. Each ground sample was digested with a mixture of 1.5 ml of 69% nitric acid (HNO₃) and 0.4 ml of 30% hydrogen peroxide (H₂O₂) using an Ultrawave digestive system (Table S1, also see Supplementary Methods for standard preparations). A minimum dry weight of 50 mg was attained by combining samples from the same location to ensure sufficient material was available for analyses (Table S2). The samples were left to cool before being diluted with Milli-Q water, and elemental analysis was quantified using an Agilent 8800 Inductively Coupled Plasma Mass Spectrometer (ICP-MS). The accuracy of the method was tested by analyzing a Certified Testing Material (ERM-CE278K) from European Reference Materials (Table S3). Quality controls were applied during the metal analysis including calibration blanks, matrix reaction blanks, continuous calibration verification, second vendor verification, duplicate samples, spiked blanks, and spiked samples once every 20 samples (Table S4). The concentration of Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Cd, and Pb was measured for each sample. These elements were chosen to mirror hatchling studies from other parts of the world (Stoneburner, Nicora & Blood, 1980; Sakai et al., 1995; Kaska & Furness, 2001).

All statistical analyses were conducted on R Studio Version 2021.09.0 (R Core Team, 2021), and statistical significance was assumed at p < 0.05. A Levene’s test and a Shapiro-Wilk were used to confirm equal variance and normality. Because some of the data did not meet the statistical prerequisites to conduct parametric tests (i.e., our data showed non-normal distribution), we conducted non-parametric tests. A Mann-Whitney U test was used to compare heavy metal concentrations between the sampled beaches anda Kruskal-Wallis test was used to compare heavy metal concentrations by tissue (liver, muscle, and yolk). Finally, a Spearman rank correlation coefficient was used to assess the relationship between hatchling size and mass with heavy metal concentrations. To contextualize the data, the average concentration of each heavy metal in each tissue (liver, muscle, and yolk) were descriptively compared to the average concentration of As, Cd, Cr, Cu, Fe, Ni, Pb, and Se (Tanabe et al., 2022) measured in the sand at the two sampled beaches. These sand samples were collected on May, 2021 (4 months prior to when the hatchlings were sampled).

Results

The Mann-Whitney U test revealed that As was the only metal to demonstrate a significant difference between beaches. The concentration of As in hatchlings’ tissues measured lower on the beach adjacent to the cement factory compared to the beach downwind from the factory (W = 472, p < 0.01) (Table S5). Because all other metals showed no significant differences between the two sites, the concentrations from both beaches were combined and the average heavy metal concentrations (dry weight, mg/kg) in the liver, muscle, and residual yolk were reported (Table 1).

Iron from the liver of hatchlings presented a maximum measurement of 630.8 mg/kg and an overall mean of 423.58 ± 111.89 mg/kg (Table 1). The concentration of Fe in the muscle was also high, averaging 280.47 ± 86.82 mg/kg. The mean concentration of Fe in the yolk was much lower at 43.72 ± 27.31 mg/kg. In the yolk, Zn exhibited the highest levels of all elements, with an average concentration of 56.12 ± 46.33 mg/kg (Table 1). Cd had the lowest average concentrations compared to the other elements, with the smallest concentration measured in the liver (0.01 ± 0.02 mg/kg) (Table 1).

We found significant differences in the concentrations of each heavy metal (Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Cd, and Pb) measured between hatchlings’ tissue (yolk, liver, and muscle) (p < 0.05, Table S6). We found that the yolk had the highest concentration of Cd. The liver had the highest concentrations of Cu, Fe, and Se, and the muscle had the highest concentrations of As, Co, Cr, Mn, Ni, Pb, and Zn (Fig. 2).

Lastly, we found no significant correlation between overall heavy metal concentration and hatchling mass (r(241) = 0.0914, p = 0.1563) or carapace length (r(241) = 0.0649, p = 0.3144). Though, when correlation was tested for each element separately, the only significant result found was Cd, which showed a negative correlation for both mass (r(43) = −0.6, p = 0.004) and carapace length (r(43) = −0.45, p = 0.036).

There was no consistent relationship found between heavy metal concentrations measured in the nesting environment to the concentrations measured in each tissue (liver, muscle, and residual yolk) (Fig. 3). Arsenic demonstrated higher concentrations in the sand at the site adjacent to the factory compared to the beach downwind, but this pattern was reversed in the tissues of the hatchlings (Fig. 3). Cadmium showed a similar result, but the concentration at the site downwind was higher than at the beach next to the factory, whereas the pattern was reversed in the tissues of the hatchlings (Fig. 3). Chromium and Ni had similar results to each other, which generally had lower measured sand concentrations than measured in the liver and muscle, but higher than in yolk (Fig. 3). Copper and Se concentrations in hatchlings’ tissues do not seem to have a relationship with the nest environment, as the mean concentration in the sand was lower than the mean concentration measured in the yolk, muscle, and liver (Fig. 3). Finally, Fe and Pb were much higher in the sand at these beaches compared to in the hatchlings’ tissues.

Discussion

Our findings provide the first measurements of heavy metals from green turtles in the Red Sea. We found no significant differences in heavy metal concentrations in hatchlings collected on the nesting beach adjacent to the cement factory compared to the beach 1.5 km downwind, with the exception of As. Furthermore, we found significant differences in heavy metal concentrations among hatchlings’ tissues (liver, muscle, and residual yolk). Seven of the heavy metals were measured highest in the muscle, three were highest in the liver, and only Cd was highest in the yolk. Also, we did not find many significant correlations between the heavy metal concentrations with the hatchlings’ mass or carapace length for most metals. There were no clear trends between the concentration of heavy metals measured in the hatchlings’ tissue and the sand at the beaches where they were found, but Fe and Pb potentially demonstrated a relationship between the sand and hatchlings’ tissue concentrations. Although our sample size is relatively small, these data provide an important initial assessment against which future measurements can be compared.

Arsenic was found to have significantly lower concentrations measured in the hatchlings’ tissues from the beach next to the factory compared to the beach 1.5 km downwind. When these concentrations were compared to the mean As concentration in the sand at these two beaches, there was no relationship, and the concentration in the sand was much higher at the factory compared to the site downwind (Tanabe et al., 2022). A study on As uptake by reptilian flexible-shelled eggs from contaminated nest substrates found that As concentration was significantly higher in eggshells compared to the embryos, suggesting that the eggshell may inhibit the passage of As into the eggs (Marco, López-Vicente & Pérez-Mellado, 2004).

Conclusion

These data reported in this study present baseline levels of heavy metals (Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Cd, and Pb) from green turtle hatchlings’ liver, muscle, and residual yolk tissue at Ras Baridi, Saudi Arabia’s largest turtle rookery. To our knowledge, this is the first study on heavy metal concentrations in green sea turtles from the Red Sea region. This baseline data is critical for biomonitoring efforts in Saudi Arabia before major coastal development occurs. Over the next decade, under Vision 2030 (PIF, 2017), multiple giga-projects aim to accommodate more than two million people on the Red Sea coast in the next decade (PIF, 2017), increasing the potential of heavy metal input into the surrounding environments. Biomonitoring of sea turtles for heavy metal concentrations (and their nesting and feeding grounds) should be conducted as a long-term assessment of the impacts of these developments; the present study will allow for post-development comparison. Sea turtles are flagship species that have broad appeal and significance in ecotourism efforts, including in the nascent tourism industry in Saudi Arabia. Improved understanding of the potential heavy metal concentrations throughout the Saudi Arabian Red Sea can inform national turtle conservation efforts and identify areas requiring mitigation.

Supplemental Information