Sensitivity of turtles to anticoagulant rodenticides: Risk assessment for green sea turtles (Chelonia mydas) in the Ogasawara Islands and comparison of warfarin sensitivity among turtle species.

Although anticoagulant rodenticides (ARs) are effectively used for the control of invasive rodents, nontarget species are also frequently exposed to ARs and secondary poisonings occur widely. However, little data is available on the effects of ARs, especially on marine organisms. To evaluate the effects of ARs on marine wildlife, we chose green sea turtles (Chelonia mydas), which are one of the most common marine organisms around the Ogasawara islands, as our primary study species. The sensitivity of these turtles to ARs was assessed using both in vivo and in vitro approaches. We administered 4 mg/kg of warfarin sodium either orally or intravenously to juvenile green sea turtles. The turtles exhibited slow pharmacokinetics, and prolongation of prothrombin time (PT) was observed only with intravenous warfarin administration. We also conducted an in vitro investigation using liver microsomes from green sea turtles, and two other turtle species (softshell turtle and red-eared slider) and rats. The cytochrome P450 metabolic activity in the liver of green sea turtles was lower than in rats. Additionally, vitamin K epoxide reductase (VKOR), which is the target enzyme of ARs, was inhibited by warfarin in the turtles at lower concentration levels than in rats. These data indicate that turtles may be more sensitive to ARs than rats. We expect that these findings will be helpful for sea turtle conservation following accidental AR-broadcast incidents.

Integrated chemical exposure assessment of coastal green turtle foraging grounds on the Great Barrier Reef.

The Great Barrier Reef receives run-off from 424,000 km2 catchment area across coastal Queensland, incorporating diffuse agricultural run-off, and run-off point sources of land-based chemical pollutants from urban and industrial development. Marine biota, such as green turtles (Chelonia mydas), are exposed to these diverse chemical mixtures in their natural environments, and the long term effects on turtle and ecosystem health remain unknown. This study was part of a larger multi-disciplinary project characterising anthropogenic chemical exposures from the marine environment and turtle health. The aim of this study was to screen for a wide range of anthropogenic chemical pollutants present in the external and internal environment of green turtles, using a combination of traditional targeted chemical analyses, non-target suspect screening, and effect-based bioassay methods, while employing a case-control study design. A combination of passive (water) and grab (water, sediment) samples were investigated. Three known green turtle foraging sites were selected for sampling: two coastal 'case' sites influenced primarily by urban/industrial and agricultural activities, respectively; and a remote, offshore 'control' site. Water and sediment samples from each of the three sampling locations showed differences in chemical pollutant profiles that reflected the dominant land uses in the adjacent catchment. Targeted mass spectrometric analysis for a range of pesticides, industrial chemicals, pharmaceuticals and personal care products found the greatest detection frequency and highest concentrations in coastal samples, compared to the control. Non-target screening analysis of water showed clear differentiation in chemical profile of the urban/industrial site. In-vitro assays of sediment samples from the control site had lowest induction, compared to coastal locations, as expected. Here we present evidence that turtles foraging in coastal areas are exposed to a range of anthropogenic pollutants derived from the adjacent coastal catchment areas.

Assessment of Mercury Concentration in Turtles (Podocnemis unifilis) in the Xingu River Basin, Brazil.

Many studies on mercury contamination in aquatic biota deal with the effect of consuming metal-contaminated organisms on human health. In this study, we examined the factors that cause mercury contamination in Podocnemis unifilis in the Xingu River Basin of Mato Grosso and Pará States, Brazil. We quantified by atomic absorption spectroscopy with cold vapor the total mercury (THg) content in the liver and muscle samples of 50 Podocnemis unifilis specimens collected from the basin. The liver and muscle samples contained 134.20 ± 119.30 ng g−1 THg and 24.86 ± 26.36 ng g−1 THg, respectively. Each chelonian or meal has, on average, 5.34× more Hg than the highest level established as acceptable. From the results it can be inferred that, given the weekly consumption of chelonians, the riverine and indigenous communities in the Xingu River Basin are at risk of chronic consumption of Hg in amounts beyond the acceptable limit. The potential high risk to the health of this population is evident; however, the risk classification needs to be further studied.

First ecotoxicological assessment of Caretta caretta (Linnaeus, 1758) in the Mediterranean Sea using an integrated nondestructive protocol.

The loggerhead sea turtle (Caretta caretta) is a long-lived carnivorous reptile included in the IUCN Red List. The IUCN assessment for this species underlines the lack of information regarding pollution and pathogens and indicates as a priority effort to investigate and reduce the impacts of these threats. In this contest, the aim of our study was to conduct the first ecotoxicological assessment of this species in the Mediterranean Sea using a nondestructive integrated methodology. We set up and applied a monitoring protocol which includes endpoints, such as CYP1A, LPO, ENA assay, B esterases, never investigated before in this species. Seventy-five loggerhead turtles were sampled in a nondestructive way in Italian Sea Turtle Rescue Centers or free-ranging along the Spanish coasts. Blood, skin and carapace samples were used to test biomarker responses (CYP1A, VTG, LPO, Comet and ENA assay, BChE, GGT) and contaminant (OCs, PAHs, Pb, Cd, Hg) levels. Elaboration of experimental results was carried out taking also into consideration different age classes of the specimens. Among the main results obtained we should underline the statistically significant correlation between carcinogenic PAHs in blood and DNA fragmentation as well as between Cd in carapace and GGT in plasma. We also evidenced as youngest animals showed significantly higher DNA fragmentations, BChE inhibition and increase of GGT. Older specimens showed the highest levels of erythrocyte nuclear abnormalities which may indicate a long term toxicological stress. This study contributed to expand the knowledge about the ecotoxicology of C. caretta in the Mediterranean. The nondestructive protocol could also be applied to other marine ecosystems and other sea turtle species.

Standard metabolic rates of early life stages of the diamondback terrapin (Malaclemys terrapin), an estuarine turtle, suggest correlates between life history changes and the metabolic economy of hatchlings.

I estimated standard metabolic rates (SMR) using measurements of oxygen consumption rates of embryos and unfed, resting hatchlings of the diamondback terrapin (Malaclemys terrapin) three times during embryonic development and twice during the early post-hatching period. The highest observed SMRs occurred during mid to late embryonic development and the early post-hatching period when hatchlings were still reliant on yolk reserves provided by the mother. Hatchlings that were reliant on yolk displayed per capita SMR 135 % higher than when measured 25 calendar days later after they became reliant on exogenous resources. The magnitude of the difference in hatchling SMR between yolk-reliant and exogenously feeding stages was much greater than that attributed to costs of digestion (specific dynamic action) observed in another emydid turtle, suggesting that processing of the yolk was not solely responsible for the observed difference. The pre-feeding period of yolk reliance of hatchlings corresponds with the period of dispersal from the nesting site, suggesting that elevated SMR during this period could facilitate dispersal activities. Thus, I hypothesize that the reduction in SMR after the development of feeding behaviors may reflect an energy optimization strategy in which a high metabolic expenditure in support of development and growth of the embryo and dispersal of the hatchling is followed by a substantial reduction in metabolic expenditure coincident with the individual becoming reliant on exogenous resources following yolk depletion.

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.

Thyroid Hormones Reduce Incubation Period without Developmental or Metabolic Costs in Murray River Short-Necked Turtles (Emydura macquarii).

Metabolic processes are affected by both temperature and thyroid hormones in ectothermic vertebrates. Temperature is the major determinant of incubation length in oviparous vertebrates, but turtles can also alter developmental rate independent of temperature. Temperature gradients within natural nests cause different developmental rates of turtle embryos within nests. Despite temperature-induced reductions in developmental rate, cooler-incubated neonates often hatch synchronously with warmer siblings via metabolic compensation. The physiological mechanisms underlying metabolic compensation are unknown, but thyroid hormones may play a critical role. We applied excess triiodothyronine (T3) to developing eggs of Murray River short-necked turtle (Emydura macquarii)-a species that exhibits metabolic compensation and synchronous hatching-to determine whether T3 influences developmental rate and whether changes to incubation period incur metabolic costs. We measured heart rate, oxygen consumption and incubation period of eggs, and morphology and performance of hatchlings. Embryos that were exposed to T3 pipped up to 3.5 d earlier than untreated controls, despite no change in total metabolic expenditure, and there were no treatment differences in hatchling morphology. Hatchlings treated with T3 demonstrated similar righting ability to hatchlings from the control groups. Exposure to T3 shortens incubation length by accelerating embryonic development but without statistically increasing embryonic metabolism. Thus, T3 is a mechanism that cooler-incubated reptiles could use to accelerate their development to allow synchronous hatching with their warmer clutch mates but at little or no metabolic cost. Thus, metabolic compensation for synchronous hatching may not be metabolically expensive if T3 is the underlying mechanism.

Assessment trace elements concentrations in tissues in Caspian Pond Turtle (Mauremys caspica) from Golestan province, Iran.

Concentrations of cadmium, lead, zinc, and copper were measured in different organs and tissues of 15 Caspian Pond Turtle (Mauremys caspica) collected from Gharehsu River, Golestan province, Iran in June and July 2012. Mean concentrations (dry weight) of zinc and copper were 66.9 and 6.7µgg(-1) in liver, 147 and 3.4µgg(-1) in heart, 93.2 and 4.9µgg(-1) in shell, and finally 150.7 and 4.5µgg(-1) in muscle, respectively. Mean concentrations of cadmium and lead were 5.8 and 32.4µgg(-1) in liver, 2.9 and 20.9µgg(-1) in heart, 3.5 and 21.5µgg(-1) in shell, and finally 2.5 and 27.5µgg(-1) in muscle, respectively. On average, lead, cadmium, copper and zinc concentrations in the analyzed tissues were much higher than those reported in other freshwater turtle species. In particular, the mean concentrations of lead in liver and muscle of Caspian Pond Turtle was extremely high. To our knowledge, this is the first report into metal accumulation in tissues and organs of Caspian Pond Turtle from of the Gharehsu River in Golestan province, Iran.

Characterization of fructose-1,6-bisphosphate aldolase during anoxia in the tolerant turtle, Trachemys scripta elegans: an assessment of enzyme activity, expression and structure.

One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05). A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively). Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival.

Measuring energy expenditure in sub-adult and hatchling sea turtles via accelerometry.

Measuring the metabolic of sea turtles is fundamental to understanding their ecology yet the presently available methods are limited. Accelerometry is a relatively new technique for estimating metabolic rate that has shown promise with a number of species but its utility with air-breathing divers is not yet established. The present study undertakes laboratory experiments to investigate whether rate of oxygen uptake (VO2) at the surface in active sub-adult green turtles Chelonia mydas and hatchling loggerhead turtles Caretta caretta correlates with overall dynamic body acceleration (ODBA), a derivative of acceleration used as a proxy for metabolic rate. Six green turtles (25-44 kg) and two loggerhead turtles (20 g) were instrumented with tri-axial acceleration logging devices and placed singly into a respirometry chamber. The green turtles were able to submerge freely within a 1.5 m deep tank and the loggerhead turtles were tethered in water 16 cm deep so that they swam at the surface. A significant prediction equation for mean VO2 over an hour in a green turtle from measures of ODBA and mean flipper length (R(2) = 0.56) returned a mean estimate error across turtles of 8.0%. The range of temperatures used in the green turtle experiments (22-30 °C) had only a small effect on Vo2. A VO2-ODBA equation for the loggerhead hatchling data was also significant (R(2) = 0.67). Together these data indicate the potential of the accelerometry technique for estimating energy expenditure in sea turtles, which may have important applications in sea turtle diving ecology, and also in conservation such as assessing turtle survival times when trapped underwater in fishing nets.

Assessment of exposure to lead in humans and turtles living in an industrial site in Coatzacoalcos Veracruz, Mexico.

The intake of lead from the environment may occur thru various receptors. In order to measure lead levels absorbed, samples were taken from Children who live in three localities surrounding an industrial complex in Coatzacoalcos, Veracruz. Samples were also taken from turtles. Samples were analyzed and results were compared against the general population. In children tested, over 75% of all values were determined to be above CDC's safety levels of (10 µg/dL). The geometric mean lead concentration was 11.4 µg/dL, which is clearly higher around the industrial complex than in the general population. In turtles, lead blood levels in the exposed population were 2-fold above (24.2 µg/dL) those of turtles in the reference population (10.1 µg/dL). Lead levels observed represent a risk for both human and fauna health.

Low metabolic cost of locomotion in ornate box turtles, Terrapene ornata.

Evolution has produced a wide range of body plans, but for a given body mass, the energetic cost of transport (COT) of terrestrial animals falls in a relatively narrow range. Previous research indicates that the COT depends on the proficiency of minimizing mechanical work performed, efficiency of performing that work, and cost of generating force to support weight. Turtles are unique in that their protective shell and shoulder-girdle articulation may eliminate the need for the ;muscular sling'. In addition, turtles have slower, more efficient muscles than other vertebrates. However, slow locomotion may raise the COT by confounding mechanical-energy conservation via the inverted-pendulum mechanism. Our goal was to determine the metabolic COT and efficiency of a terrestrial turtle species during locomotion. We studied 18 ornate box turtles, Terrapene ornata. Walking speed was extremely slow (0.07+/-0.005 m s(-1)). The average minimum COT was 8.0+/-0.70 J kg(-1) m(-1) attained at approximately 0.1 m s(-1). Ornate box turtles consume only half the energy predicted by the allometric relationship for all terrestrial animals (15.9+/-0.35 J kg(-1) m(-1)), and, thus, appear to be very economical walkers. When walking up a 24 deg. incline turtles moved significantly slower (0.04+/-0.004 m s(-1)), but performed the extra work required to walk uphill with very high efficiencies (>49%). It appears that the co-evolution of a protective shell, the associated shoulder morphology, and very slow, efficient muscles produce both economical level walking and efficient uphill walking.

Oxidative cost to ventilation in a turtle, Pseudemys floridana.

Estimates of oxidative cost of ventilation for Pseudemys floridana were obtained by comparison of mean resting VO2 with that obtained while artificially ventilating the lungs with a constant air flow in excess of the normal VE, a circumstance during which the animals remained apneic due to reduction in alveolar PCO2. At a body temperature of 22 degrees C, the oxidative cost attributable to ventilation was 0.0047 ml O2/ml gas ventilated, a value about 10 times that of man. This cost of ventilation is then related to values of VO2 and VE at various body temperatures. The oxidative cost of ventilation, expressed as a percentage of resting VO2, is given by the equation: cost = 0.47 VE/VO2. Relative cost declines with body temperature since VE/VO2 (resting) declines according to the equation: VE(BTPS)/VO2 = -0.017 BT -0.025 log Wt + 2.01 where units are in ml, kg and min. At body temperatures of 10 and 37 C relative costs were 30 and 10% VO2, respectively. While the inverse relation between VE/VO2 and body temperature has important implications for regulation of arterial pH through influencing arterial PCO2, it has the additional meaning of reducing the relative oxidative cost of ventilation as VO2 increases.