Persistent Organic Pollutants and Heavy Metals in the Green Sea Turtle, Chelonia Mydas

Abstract

The chemical contamination of sea turtles is an emerging area of conservation research. Chemicals, such as persistent organic pollutants (POPs) and heavy metals, have a wide range of harmful effects on animals and humans and are beginning to be reported in sea turtle populations around the world. However, prior to the present study, research on chemical contamination in sea turtles had generally been limited to studies on deceased animals. Furthermore, the analytical methods used in these studies had limited sensitivity and reported small numbers of compounds. The main objectives of this thesis were therefore to further develop methods for measuring POPs in sea turtles and to systematically investigate some of the important aspects of accumulation and transfer of POPs and heavy metals in the green sea turtle, Chelonia mydas. Gas chromatography with electron capture detection (GC-ECD) has generally been used to analyse POPs in sea turtle studies. However, GC-ECD relies on relative retention time for identification and can therefore not distinguish between co-eluting compounds. Furthermore, the limit of detection is relatively high (> 1 ng g-1) and these methods are therefore often unable to detect POPs in low trophic level organisms. More recent methods combining GC-ECD and gas chromatography with mass spectrometry (GC-MS) have reported a large number of POPs in sea turtles at trace concentrations. However, these methods required multiple injections of each sample into a complex arrangement of multiple gas chromatographs. This can only be replicated in well equipped and highly funded laboratories. It was therefore the first objective of this thesis to develop an equally accurate and sensitive method requiring a single injection of each sample onto a simple instrument setup. A method using gas chromatography with coupled mass spectrometry (GC-MS/MS) was developed on a Varian 3800 gas chromatograph fitted with a Saturn 2200 mass spectrometer and a 1079 programmable temperature vapourising (PTV) injector. Using calibrants and mass-labelled internal standard and recovery solutions obtained from the National Institute of Standards and Technology (NIST), South Carolina, USA, a GC-MS/MS method was established for 83 polyclorinated biphenyls (PCBs), 23 organochlorine pesticides (OCPs) and 19 polybrominated diphenyl ethers i i (PBDEs). Sample preparation was modified from previous studies and involved accelerated solvent extraction with dichloromethane, followed by gel permeation chromatography and Florisil clean-up procedures. Recoveries were generally > 60% and standard reference materials were reported to within 60 and 70% of the reference and certified values, respectively. The coefficients of variation of pooled samples were < 20%, although generally < 5%, and the limit of detection ranged from 5 to 35 pg g-1. This method therefore provided an accurate way of measuring a large number of POPs at trace concentrations in C. mydas. This method was then used to investigate a number of important accumulation and transfer aspects of chemical contamination in C. mydas. The earliest studies on chemical contamination in sea turtles sampled tissue from dead and stranded animals. However, it is of more interest to investigate the contamination of living sea turtle populations. Blood and carapace sampling have increasingly been used as non-lethal methods for analysing POPs and metals in sea turtles. However, there was very little information on how well blood and carapace samples represented the chemical contamination of internal tissues. To address this issue, blood, carapace, liver, kidney and muscle samples from 16 C. mydas that died at the Sea World Sea Turtle Rehabilitation Program were analysed for POPs and heavy metals. Heavy metal and POP levels in the blood and carapace were significantly correlated with internal tissue concentrations. Furthermore, these relationships were not affected by sex or age. While it must be considered that these C. mydas were in rehabilitation, blood and carapace samples are good predictors of the internal contamination of C. mydas. This information therefore provides scientists with reliable non-lethal methods for estimating chemical contamination in living sea turtle populations. Information on the accumulation of chemical contaminants in sea turtles was also limited prior to this study. The biology of sea turtles indicates that chemicals may accumulate through feeding and/or maternal transfer during vitellogenesis. The present study investigated the chemical contamination of C. mydas from different foraging areas. Satellite telemetry tracked the movement of three C. mydas nesting at Ma’Daerah, Peninsular Malaysia to three different foraging areas in Southeast Asia. Furthermore, the egg chemical contamination profiles of the C. mydas from these different foraging areas were significantly different. This suggested the use of ii i multivariate contaminant analysis to assess the variation of foraging area locations of a nesting C. mydas population. The use of contamination profiles to assess foraging ground variation in a nesting population was further supported by chemical analysis of eggs from 11 C. mydas nesting at Ma’Daerah, Peninsular Malaysia. The egg POP contamination profiles from the 11 turtles were separated into six groups. This indicated that these C. mydas nesting at Ma’Daerah may have migrated from six distinct foraging areas. However, investigation into the variation in chemical profiles of C. mydas from the same foraging areas must be investigated before this concept can be validated. There are a number of factors such as age, sex and specific foraging range that may lead to variations in C. mydas contamination from the same foraging area. The accumulation of POPs in C. mydas via maternal transfer to eggs and hatchlings was also investigated in the present study. Maternal blood, eggs and hatchling blood were collected from the 11 C. mydas nesting at Ma’Daerah. There were significant correlations in POP concentrations between maternal blood and eggs, indicating transfer of these chemicals to eggs during vitellogenesis. These results also indicated that egg sampling could be used as a relatively non-invasive method for determining POPs in adult female C. mydas. There were also significant correlations in POP concentrations between eggs and hatchling blood, indicating further transfer of these chemicals to hatchlings during development. Furthermore, as egg POP concentration increased the mass:length ratio of hatchlings decreased. This indicated a subtle influence of POPs on the development of C. mydas hatchlings that may compromise the duration of offshore dispersal and affect predator avoidance. The implications of chemical contamination on the conservation of C. mydas populations and on human health in communities that consume sea turtle eggs were investigated in Peninsular Malaysia. A sample of 55 C. mydas eggs was purchased from markets on the east coast of Peninsular Malaysia. According to the vendors, the eggs had been collected from sites ranging from adjacent beaches to thousands of kilometres away in Borneo Malaysia. The concentrations of POPs and metals in the eggs were variable among nesting areas and markets. A screening risk assessment indicated that the reported arsenic concentrations posed a relatively high risk of iv disruption to embryonic development. Furthermore, the large number of compounds detected was thought to increase the risks to embryonic development of C. mydas eggs. The concentrations of coplanar PCBs also posed a considerable risk to human health, with the consumption of a single C. mydas egg estimated to represent 3-300 times the acceptable daily intake (ADI) of these compounds. The large number of compounds detected was also expected to further contribute to the health risks of consuming C. mydas eggs. Incidentally, the human health risks associated with consuming C. mydas eggs may contribute to the conservation of this species in Southeast Asia. Public awareness campaigns highlighting the dangers associated with consuming C. mydas eggs could reduce the collection of eggs ..