Towards the development of standardised sea turtle primary cell cultures for toxicity testing.

Chemical contaminants are known to accumulate in marine megafauna globally, but little is known about how this impacts animal health. In vitro assays offer an ethical, reproducible and cost-effective alternative to live animal toxicity testing on large, long-lived or threatened species, such as sea turtles. However, using a cell culture from a single animal raise the question of whether the toxicity observed adequately represents the toxicity in that species. This study examined variation in the cytotoxic response of primary skin fibroblasts established from seven green (Chelonia mydas) and five loggerhead (Caretta caretta) sea turtles. Cell viability using resazurin dye was examined in response to exposure to five contaminants. The variation in cytotoxicity was generally low (within a factor of five) for both independent analyses of the same cell culture, and cell cultures from different individuals. This low within and between cell culture variation indicates that primary sea turtle cell cultures can provide a suitable approach to understanding toxicity in sea turtles. In addition, green and loggerhead turtle cells showed similar toxicity to the compounds tested, indicating that only subtle differences in chemical sensitivity may exist between sea turtle species. This study provides a framework for using species-specific cell cultures in future toxicological studies on sea turtles. Although in vivo studies are the gold standard for toxicological studies and species-specific risk assessments, the development of in vitro tools can provide important information when in vivo studies are not possible or practical. For large, endangered species such as sea turtles that are exposed to, and accumulate, a large number of contaminants, using validated cell cultures may facilitate the rapid assessment of chemical risk to these animals.

Species-specific bioassays reveal spatial variation in chemical contamination of green sea turtles.

The rapid increase of anthropogenic activity at shipping ports and surrounding coastal areas has been correlated with higher chemical contamination entering the surrounding marine environment. Chemical contaminants in marine environments can lead to significant health problems for green turtles (Chelonia mydas), especially when these contaminants accumulate in their foraging grounds. This study examined the exposure and toxicological effects of chemical contaminants on green turtle cells using a species-specific cell viability assay. Using the QuEChERs extraction, organic contaminants were extracted from 60 blood samples collected from green turtles in three foraging locations: Port Curtis, and two reefs (Heron Reef and Hoskyn-Fairfax Reefs) within the Capricorn Bunker Group of the outer Great Barrier Reef. Blood extracts were tested for cytotoxicity against primary green turtle fibroblast cells using an in vitro resazurin bioassay to assess cell viability. Extracts from Gladstone and Heron Reef indicated significant chemical contamination, at levels high enough to cause adverse health effects of green turtles. Very low toxicity values at the Hoskyn-Fairfax Reefs location indicate its potential to be established as a reference site for the southern Great Barrier Reef.

Dose metric evaluation of a cell-based bioassay for assessing the toxicity of metals to Dugong dugon: Effect of metal-media interactions on exposure concentrations.

Cell-based toxicity testing has emerged as a useful tool in (eco)toxicological research, allowing the ethical assessment of the effects of contaminants such as trace metals on marine megafauna. However, metal interactions with various dissolved ligands in the microplate environment may influence the effective exposure concentrations. Hence, the cells are not exposed to the nominal concentrations within the test system. This study aimed to establish and evaluate the effectiveness of cell-based bioassays for investigating the toxicity of selected metals in dugongs through the following objectives: (1) measure the cytotoxic potential of cadmium (Cd2+), and chromium (Cr6+) to dugong skin cell cultures, (2) investigate the interactions between media constituents and selected trace metals in cell-based bioassays, and (3) evaluate the risk to a free-ranging population of dugong based on effect values. Chromium was the most toxic of the metals tested (EC50 = 1.14 µM), followed by Cd (EC50 = 6.35 µM). Assessment of ultrafiltered (< 3 kDa) exposure media showed that 1% and 92.5% of Cr and Cd were associated with larger organic components of the media. Further, the binding of Cd to media constituents was calculated to underestimate Cd toxicity in cell-based assays by an order of magnitude. This understanding of metal partitioning in cell-based bioassays provides a more accurate method for assessing toxicity in cell-based bioassays. In addition, this study illustrated that dugong cells are more sensitive to Cr and Cd than other marine wildlife species. The chemical risk assessment found the dugong population in Moreton Bay to be at high risk from Cd exposure.

Understanding contaminant exposure risks in nesting Loggerhead sea turtle populations.

Queensland loggerhead turtle nest numbers at Mon Repos (MR) indicate population recovery that doesn't occur at Wreck Island (WI). Previous research illustrated that MR and WI turtles forage in different locations, potentially indicating risks differences. Blood, scute, and egg were collected from turtles nesting at MR and WI, with known foraging sites (from concurrent studies). Trace element and organic contaminants were assessed via acid digestion and in vitro cytotoxicity bioassays, respectively. WI turtles had significantly higher scute uranium and blood molybdenum compared to MR turtles, and arsenic was higher in WI turtles foraging north and MR turtles foraging south. Egg and blood titanium, manganese, cadmium, barium, lead, and molybdenum, and scute and egg selenium and mercury significantly correlated. Blood (75 %) extracts produced significant toxicity in vitro in turtle fibroblast cells. In conclusion, reducing chemical exposure at higher risk foraging sites would likely benefit sea turtles and their offspring.

Temporal changes in chemical contamination of green turtles (Chelonia mydas) foraging in a heavily industrialised seaport.

Port Curtis, a major shipping port, has undergone significant expansion in the last decade, with plans for further development into the future. These activities may result in an increase of contaminant concentrations, threatening local wildlife including sea turtles. This study used a species-specific in vitro bioassay to examine spatial and temporal differences in exposure to, and effects of, organic contaminants in green sea turtles foraging in Port Curtis. Blood was collected from 134 green sea turtles (Chelonia mydas) from five locations in the port over four years. Organic contaminants were extracted from blood, and the cytotoxicity of the extracts to primary green sea turtle cells was assessed. Results indicated spatially similar chemical contamination throughout Port Curtis, at levels significant to sea turtle health, and with signs that chemical contamination may be increasing over time. These results can provide valuable information on the health of green turtles as further development occurs.

Pelagic and coastal green turtles (Chelonia mydas) experience differences in chemical exposure and effect.

Green turtles foraging in coastal areas are exposed to land-based chemical pollutants that accumulate in the habitats to which they show high site fidelity. However, prior to coastal recruitment, they may be exposed to a different range of chemical threats. The recent development of species-specific in vitro bioassays for marine turtles allows for an effect-based assessment of toxicological endpoints. Blood was collected from green turtles of two life-stages, 'recent recruits' and 'coastal residents', in Hervey Bay and Moreton Bay. Organic contaminants were extracted from blood using the QuEChERS method, and cytotoxicity of the extracts measured in green turtle skin cells. Although not statistically significant, extracts from 'coastal residents' exhibited greater mean toxicity compared to 'recent recruits', possibly indicative of increased chemical accumulation from coastal habitat exposure. The bioassay results also indicated that turtles foraging in Hervey Bay are at greater risk of chemical exposure than those foraging in Moreton Bay.

Review of ecologically relevant in vitro bioassays to supplement current in vivo tests for whole effluent toxicity testing - Part 2: Non-apical endpoints.

Whole effluent toxicity (WET) testing uses whole animal exposures to assess the toxicity of complex mixtures, like wastewater. These assessments typically include four apical endpoints: mortality, growth, development, and reproduction. In the last decade, there has been a shift to alternative methods that align with the 3Rs to replace, reduce, and refine the use of animals in research. In vitro bioassays can provide a cost-effective, high-throughput, ethical alternative to in vivo assays. In addition, they can potentially include additional, more sensitive, environmentally relevant endpoints than traditional toxicity tests. However, the ecological relevance of these endpoints must be established before they are adopted into regulatory frameworks. This is Part 2 of a two-part review that aims to identify in vitro bioassays that are linked to ecologically relevant endpoints that could be included in WET testing. Part 2 of this review focuses on non-apical endpoints that should be incorporated into WET testing. In addition to the four apical endpoints addressed in Part 1, this review identified seven additional toxic outcomes: endocrine disruption, xenobiotic metabolism, carcinogenicity, oxidative stress, inflammation, immunotoxicity and neurotoxicity. For each, the response at the molecular or cellular level measured in vitro was linked to the response at the organism level through a toxicity pathway. Literature from 2015 to 2020 was used to identify suitable bioassays that could be incorporated into WET testing.

Review of ecologically relevant in vitro bioassays to supplement current in vivo tests for whole effluent toxicity testing - Part 1: Apical endpoints.

Whole effluent toxicity (WET) testing is commonly used to ensure that wastewater discharges do not pose an unacceptable risk to receiving environments. Traditional WET testing involves exposing animals to (waste)water samples to assess four major ecologically relevant apical endpoints: mortality, growth, development, and reproduction. Recently, with the widespread implementation of the 3Rs to replace, reduce and refine the use of animals in research and testing, there has been a global shift away from in vivo testing towards in vitro alternatives. However, prior to the inclusion of in vitro bioassays in regulatory frameworks, it is critical to establish their ecological relevance and technical suitability. This is part 1 of a two-part review that aims to identify in vitro bioassays that can be used in WET testing and relate them to ecologically relevant endpoints through toxicity pathways, providing the reader with a high-level overview of current capabilities. Part 1 of this review focuses on four apical endpoints currently included in WET testing: mortality, growth, development, and reproduction. For each endpoint, the link between responses at the molecular or cellular level, that can be measured in vitro, and the adverse outcome at the organism level were established through simplified toxicity pathways. Additionally, literature from 2015 to 2020 on the use of in vitro bioassays for water quality assessments was reviewed to identify a list of suitable bioassays for each endpoint. This review will enable the prioritization of relevant endpoints and bioassays for incorporation into WET testing.

Differences in marine megafauna in vitro sensitivity highlights the need for species-specific chemical risk assessments.

Sea turtles, dolphins and dugongs can be exposed to large mixtures of contaminants due to the proximity of foraging locations to anthropogenic inputs. Differences in accumulation and effect result in differences of chemical risk to these species. However, little is known about the effect of contaminants in marine wildlife. Cell-based, or in vitro, exposure experiments offer an ethical alternative to investigate the effect of contaminants in wildlife. Data from in vitro studies can then be placed in an environmental context, by using screening risk assessments, comparing effect data with accumulation data from the literature, to identify risk to populations of marine wildlife. Cytotoxicity of Cr6+, Cd2+, Hg2+, 4,4'-DDE, and PFNA were investigated in primary skin fibroblasts of green turtles, loggerhead turtles, hawksbill turtles, dugongs, Burrunan dolphins, and common bottlenose dolphins. The general order of toxicity for all species was Hg2+> Cr6+ > Cd2+> 4,4'-DDE > PFNA, and significant differences in cytotoxicity were found between species for Cr6+, Cd2+ and PFNA. For Cd2+, in particular, cells from turtle species were less sensitive than mammalian species, and dugong cells were by far the most sensitive. The results from the cytotoxicity assay were then used in combination with published data on tissue contaminant concentrations to calculate risk quotients for identifying populations of each species most at risk from these chemicals. Cr, Cd and Hg were identified as posing risk in all six species. Dugongs were particularly at risk from Cd accumulation and dolphin species were particularly at risk from Hg accumulation. These results demonstrate the importance of using species-specific effect and accumulation data for developing chemical risk assessments and can be used to inform managers of priority contaminants, species, or populations. Development of additional in vitro endpoints, and improving links between in vitro and in vivo effects, would further improve this approach to understanding chemical risk in marine megafauna.

Systematic review of reptile reproductive toxicology to inform future research directions on endangered or threatened species, such as sea turtles.

Threatened or endangered reptiles, such as sea turtles, are generally understudied within the field of wildlife toxicology, with even fewer studies on how contaminants affect threatened species reproduction. This paper aimed to better inform threatened species conservation by systematically and quantitatively reviewing available research on the reproductive toxicology of all reptiles, threatened and non-threatened. This review found 178 studies that matched our search criteria. These papers were categorized into location conducted, taxa studied, species studied, effects found, and chemicals investigated. The most studied taxa were turtles (n = 87 studies, 49%), alligators/crocodiles (n = 54, 30%), and lizards (n = 37, 21%). Maternal transfer, sex steroid alterations, sex reversal, altered sexual development, developmental abnormalities, and egg contamination were the most common effects found across all reptile taxa, providing guidance for avenues of research into threatened species. Maternal transfer of contaminants was found across all taxa, and taking into account the foraging behavior of sea turtles, could help elucidate differences in maternal transfer seen at nesting beaches. Sex steroid alterations were a common effect found with contaminant exposure, indicating the potential to use sex steroids as biomarkers along with traditional biomarkers such as vitellogenin. Sex reversal through chemical exposure was commonly found among species that exhibit temperature dependent sex determination, indicating the potential for both environmental pollution and climate change to disrupt population dynamics of many reptile species, including sea turtles. Few studies used in vitro, DNA, or molecular methodologies, indicating the need for more research using high-throughput, non-invasive, and cost-effective tools for threatened species research. The prevalence of developmental abnormalities and altered sexual development and function indicates the need to further study how anthropogenic pollutants affect reproductive output in threatened reptiles.

Combining analytical and in vitro techniques for comprehensive assessments of chemical exposure and effect in green sea turtles (Chelonia mydas).

Sea turtle populations foraging in coastal areas adjacent to human activity can be exposed to numerous chemical contaminants for long periods of time. For trace elements, well-developed, sensitive and inexpensive analytical techniques remain the most effective method for assessing exposure in sea turtles. However, there are many thousands more organic contaminants present in sea turtles, often at low levels as complex mixtures. Recently developed species-specific in vitro bioassays provide an effective means to identify the presence, and effect of, organic chemicals in sea turtles. This study used a combination of chemical analysis and effects-based bioassays to provide complementary information on chemical exposure and effects for three green turtle foraging populations (Chelonia mydas) in southern Queensland, Australia. Blood was collected from foraging sub-adult green turtles captured in Moreton Bay, Hervey Bay, and Port Curtis. Twenty-six trace elements were measured in whole blood using ICP-MS. Organic contaminants in turtle blood were extracted via QuEChERS and applied to primary green turtle skin fibroblast cell in vitro assays for two toxicity endpoints; cytotoxicity and oxidative stress. The trace element analysis and bioassay results indicated site-specific differences between foraging populations. In particular, turtles from Moreton Bay, a heavily populated coastal embayment, had pronounced cytotoxicity and oxidative stress from organic blood extracts, and elevated concentrations of Cs, Ag, and Zn relative to the other sites. Incorporating traditional chemical analysis with novel effects-based methods can provide a comprehensive assessment of chemical risk in sea turtle populations, contributing to the conservation and management of these threatened species.

Development and application of species-specific cell-based bioassays to assess toxicity in green sea turtles.

Despite the detection of a wide range of contaminants in the blood of green turtle populations foraging in three locations of northern Queensland - Upstart Bay, Cleveland Bay and the Howick Group of Reefs, little is known about the effects of these contaminants on turtle health. Newly developed cell-based bioassays using green turtle primary cell cultures provide an ethical, reproducible, and high-throughput method for assessing the risk of chemical exposure sea turtles. In this project, the toxicity of six priority metals (Mn, Co, Mo, As, Sb, Cu) and blood extracts from foraging turtles were tested in two bioassays adapted to green turtle primary skin and liver cells. Cytotoxicity of metals and blood extracts was measured in primary skin fibroblast cells using a resazurin assay. Glutathione-S-transferase (GST) activity was measured in primary skin fibroblasts and primary liver epithelial cells following exposure to metals and blood extracts. Arsenic, molybdenum, cobalt and copper were found to be cytotoxic to green turtle skin cells. Only manganese, cobalt and copper were found to alter GST activity, predominantly in skin cells, indicating a higher sensitivity of green turtle skin cells compared to liver cells. Effect concentrations of metals in both bioassays were above concentrations found in turtle blood. Turtle blood extracts from the three foraging grounds showed differences in cytotoxicity and GST activity. In both assays, blood extracts of turtles from Upstart Bay were the most toxic, followed by those from Cleveland Bay, then the Howick Reefs, suggesting turtles from Upstart Bay and Cleveland Bay may be at risk from current concentrations of organic contaminants. This study demonstrates that species-specific cell-based bioassays can be used effectively to assess chemical risk in sea turtles and their foraging grounds, and could be applied to assess chemical risk in other marine wildlife.

Cytotoxicity of organic and inorganic compounds to primary cell cultures established from internal tissues of Chelonia mydas.

Chemical contaminants have been found in the tissues of sea turtles from all over the world; however, very little is known about the effects. Recently, in vitro alternatives to live animal testing have been applied to sea turtles due to their ethical and practical benefits. While primary skin fibroblasts have been established for several species of sea turtle, cells from internal organs are lacking, though they may be more relevant due to the well documented accumulation of contaminants within internal tissues. This study established primary cell cultures from the small intestine, heart, liver, ovary and skin of green turtles (Chelonia mydas). Cells were exposed to ten contaminants typically found in sea turtles to examine potential variations in sensitivity among cells established from different organs. Differences between cells established from different animals were also examined, including a comparison of cells established from a turtle with fibropapillomatosis (FP) and healthy turtles. Loggerhead (Caretta caretta) primary skin cells were also included for species comparisons. Significant differences were found between the organ types, with liver and heart being the least sensitive, and skin being the most sensitive. Overall, variation between the organ types was low. Primary skin fibroblasts may be a suitable and representative cell type for in vitro turtle toxicology research, as it is relatively easy to obtain from healthy live animals. Skin cultures provide a more sensitive indication of effect, and could be used as an early warning of the potential effects of chemical contamination. Some species differences were found but no differences were found between cell cultures from an FP turtle and healthy turtles. When EC50 values were compared to accumulation values from the literature, inorganic contaminants, such as Zn, Cd, Cr, Hg, and Cu were identified as posing a potential risk to sea turtle populations around the world.

Primary green turtle (Chelonia mydas) skin fibroblasts as an in vitro model for assessing genotoxicity and oxidative stress.

Little is known about the effects of contaminants that accumulate in sea turtles. When in vivo exposure studies have ethical and logistical barriers, as is the case with sea turtles, in vitro tools can provide important information on the effects of contaminants. Several in vitro studies have assessed cytotoxicity of contaminants to sea turtles cells, however to gain a more refined mechanistic understanding of the effects of contaminants, sub-lethal effects also require investigation. Considering the complex mixture of contaminants that sea turtles are potentially exposed to, high throughput testing methods are necessary so that a large number of contaminants (and mixtures) can be rapidly tested. This study examined oxidative stress (reactive oxygen species production) and genotoxicity (micronucleus formation) in primary green turtle skin fibroblasts in response to 16 organic and inorganic contaminants found in coastal environments. Significant induction of oxidative stress was found with Cu, Co, Cr, and Hg. Significant effects on genotoxicity were found with Cu, Co, Cr, Hg, Pb and metolachlor. Effect concentrations from the bioassays were used in a simple risk assessment of turtles worldwide using accumulation values from the literature to identify populations at risk. Cu, Co, Cr and Hg were identified as posing the biggest threat to sea turtles. This study demonstrated the validity of using primary turtle cell cultures in the assessment of risk associated with a large number of contaminants using a high-throughput toxicity testing format.

Analysis of sugarcane herbicides in marine turtle nesting areas and assessment of risk using in vitro toxicity assays.

Agricultural processes are associated with many different herbicides that can contaminate surrounding environments. In Queensland, Australia, herbicides applied to agricultural crops may pose a threat to valuable coastal habitats including nesting beaches for threatened loggerhead turtles (Caretta caretta). This study 1) measured concentrations of herbicides in the beach sand of Mon Repos, an important marine turtle nesting beach in Australia that is adjacent to significant sugarcane crops, and 2) investigated the toxicity of these herbicides to marine turtles using a cell-based assay. Samples of sand from turtle nest depth and water from surrounding agricultural drains and wetlands were collected during the wet season when herbicide runoff was expected to be the greatest and turtles were nesting. Samples were extracted using solid phase extraction and extracts were analysed using chemical analysis targeting herbicides, as well as bioanalytical techniques (IPAM-assay and loggerhead turtle skin cell cytotoxicity assay). Twenty herbicides were detected in areas between sugarcane crops and the nesting beach, seven of which were also detected in the sand extracts. Herbicides present in the nearby wetland were also detected in the beach sand, indicating potential contamination of the nesting beach via the river outlet as well as ground water. Although herbicides were detected in nesting sand, bioassays using loggerhead turtle skin cells indicated a low risk of acute toxicity at measured environmental concentrations. Further research should investigate potentially more subtle effects, such as endocrine disruption and mixture effects, to better assess the threat that herbicides pose to this population of marine turtles.

The current state and future directions of marine turtle toxicology research.

Chemical contamination of marine turtles has been well documented in the literature, although information on the toxicological effects of these contaminants is poorly understood. This paper systematically and quantitatively presents the available marine turtle toxicological research (excluding oil chemicals and natural toxins) and the related fields of cell line establishment and biomarkers as indicators of exposure. Examination of the published literature identified a total of 49 papers on marine turtle toxicology, which were split into three categories: toxicity studies (n=33, 67%), cell line establishment (n=7, 14%), and publications using biomarkers (n=13, 27%). Toxicity studies were further broken down into four subcategories: those correlating contaminants with toxicological endpoints (n=16, 48%); in vitro exposure experiments (n=11, 33%); in vivo exposure experiments (n=5, 15%); and screening risk assessments using hazard quotients (n=3, 9%). In quantitatively assessing the literature, trends and gaps in this field of research were identified. This paper highlights the need for more marine turtle toxicology research on all species, particularly using high throughput and non-invasive in vitro assays developed for marine turtle cells, including investigations into further toxicological endpoints and mixture effects. This will provide more comprehensive species-specific assessment of the impacts of chemical contaminants on these threatened animals, and improve conservation and management strategies globally.