title: Development, validation and application of an in vitro toxicological model for sea turtles
Creators: name: Finlayson, Kimberly
Publisher: name: Griffith University
Year: 2018
Subjects: subject: In vitro toxicological
subject: Sea turtles
subject: Marine megafauna
subject: Cell cultures
subject: Blood extracts

description: Chemical contaminants accumulate in marine megafauna globally, including sea turtles. Logistical and ethical constraints around exposure experiments using large, long-lived, and often threatened species, has limited our understanding of how these pollutants may affect wildlife. The knowledge gaps are outlined in a systematic quantitative literature review (Chapter 2), which found that in vitro bioassays offer an ethical, reproducible, and cost-effective alternative for investigating the effects of contaminants. The development of an in vitro toxicological model can provide important information for conservation and management. This thesis aimed to develop, validate and demonstrate the applicability of an in vitro model for sea turtles. Cell cultures were established from skin and internal organs of green sea turtles including heart, small intestine, ovary and liver. Important questions concerning individual variation (Chapter 3) and variation between tissue types (Chapter 4) were addressed to select an ideal cell culture for further testing and validation. Variation in cytotoxic response was generally low between cell cultures established from different individuals. This suggests that one cell line can be used representatively. However the results highlight the importance of preliminary analysis in order to select a cell culture with an average response and this chapter provides a framework for doing so. Variation in cytotoxic response between tissue types was also generally low, though a clear pattern in organ sensitivity was apparent. This pattern identified skin as the most sensitive tissue type. This is particularly useful for future research, as skin can more readily be obtained from live, healthy turtles. Based on the results from Chapter 3 and 4, an ideal cell culture was selected for continued use. The usefulness of the selected cell culture was validated in two additional bioassays measuring oxidative stress and genotoxicity to test the effects of 16 model compounds (Chapter 5). Oxidative stress was measured through the formation of reactive oxygen species and genotoxicity was measured through the formation of a micronucleus. The results from cytotoxicity (Chapter 4), oxidative stress and genotoxicity assays (Chapter 5) were used in a screening risk assessment for wild turtle populations based on contaminant accumulation data from the literature. These screening risk assessments identified a number of locations where turtles may be at risk from current contaminant concentrations. Finally, the applicability of the in vitro model to broader ecological questions was demonstrated (Chapter 6). Blood extracts of turtles from three different foraging grounds were used in the bioassays to examine differences in exposure. This data, along with chemical analysis of trace elements, was used to assess risk from chemical exposure to these populations. Blood extracts from Moreton Bay turtles caused significantly stronger responses in both the cytotoxicity assay and oxidative stress assay, both of which were important factors contributing to differences between foraging grounds when combined with trace element data. The measured effect concentrations for blood extracts from Moreton Bay turtles were approximately half the concentration found in blood, indicating a higher risk associated with chemicals in blood from turtles in Moreton Bay compared to the other sites monitored. These results illustrate that using in vitro bioassay data can provide unique information into exposure and effects in sea turtles, and this data can be used to identify and prioritise populations at risk. This thesis has demonstrated that species-specific in vitro methods are suitable and useful in identifying chemical risk to sea turtles. In vitro methods can be used to understand the molecular initiating events of chemicals in sea turtles, data from these studies can be used in screening risk assessments, and finally, in vitro models can be used with biological samples to assess current concentrations and mixtures of contaminants. Altogether, species-specific in vitro models offer a promising avenue for sea turtles and other marine megafauna as well.