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.

As, Cr, Hg, Pb, and Cd Concentrations and Bioaccumulation in the Dugong Dugong dugon and Manatee Trichechus manatus: A Review of Body Burdens and Distribution.

The death of dozens of manatees Trichechus manatus recently in Tabasco, Mexico, has captured international attention. Speculation about possible causes include water and food contamination by metals. Although federal authorities have ruled out water chemical pollution, the cause of these deaths is still awaiting conclusive laboratory results. Present work seeks to summarize information currently available on non-essential metals and those of great toxicological relevance in Sirenia (dugongs and manatees), highlighting its body distribution, presence in blood, and its relationship with their geographical distribution, gender and age, whenever possible. This paper focuses on the five elements: As, Cr, Hg, Pb and Cd, which are commonly considered as threats for marine mammals and reported in Sirenia. Some of these metals (Cr and Cd) were thought to be related to the recent deaths in Tabasco. All five elements are accumulated by Sirenia at different levels. Metal presence is associated to their diet but does not necessarily imply adverse effects for dugongs and manatees. Toxicological aspects and the human consumption risk in case of any illegal or traditional consumption in some cultures are discussed. Important toxicological research areas that need to be addressed are highlighted.

PCDD/F and PCB levels in different tissues from dugongs (Dugong dugon) inhabiting the Queensland coastline.

Previous studies on PCDD/Fs and PCBs in dugong (Dugong dugon) blubber reported unexpectedly elevated TEQ levels. This study analysed archived blubber, muscle, liver and faeces obtained from dugongs from two areas along the Queensland coast. All samples showed detectable levels of PCDDs and PCBs, while PCDFs were consistently near or below LOQ. PCDD levels in dugongs contributed to a large proportion (<95%) of sum TEQ levels in all tissues (blubber: 6.7-38 pg g-1 lw, muscle: 5.7-96 pg g-1 lw, liver: 3.3-42 pg g-1 lw, faeces: 203 pg g-1 lw). Liver/blubber tissue ratios show that PCDDs are preferentially accumulated in the liver with higher degree of chlorination. The same trend was not so obvious with PCBs, which occasionally showed higher hepatic sequestration of lower chlorinated PCBs such as PCBs 28 and 77. PCDD congeners were dominated by OCDD which is similar to the profiles from the dugongs' food source, namely sediment and seagrass.

Papillomatous Skin Netting of Cetaceans (Cetacea: Delphinapterus leucas, Balaena mysticetus, and Eschrichtius robustus) and Sirens (Sirenia: Trichechus manatus and Dugong dugon).

The structure of the papillomatous junction between epidermis and dermis (papillomatous netting, PN) in the skin of cetaceans (white whales, bowhead, and gray whales) and sirens (American manatee, dugong) was studied and compared using histophysiological and morphogeometric methods. The relative extent of PN development proved to be similar in members of both orders, but significant differences were found in PN configuration, the volume of "free area of grille", the degree of skin vertical compression, and skin density, which influence buoyancy. The differences are discussed from the viewpoint of species biology.

Microsatellite Analysis for Identification of Individuals Using Bone from the Extinct Steller's Sea Cow (Hydrodamalis gigas).

Microsatellite DNA can provide more detailed population genetic information than mitochondrial DNA which is normally used to research ancient bone. The methods detailed in this chapter can be utilized for any type of bone. However, for this example, four microsatellite loci were isolated from Steller's sea cow (Hydrodamalis gigas) using published primers for manatee and dugong microsatellites. The primers DduC05 (Broderick et al., Mol Ecol Notes 6:1275-1277, 2007), Tmakb60, TmaSC5 (Pause et al., Mol Ecol Notes 6: 1073-1076, 2007), and TmaE11 (Garcia-Rodriguez et al., Mol Ecol 12:2161-2163, 2000) all successfully amplified microsatellites from H. gigas. The DNA samples were from bone collected on Bering or St. Lawrence Islands. DNA was analyzed using primers with the fluorescent label FAM-6. Sequenced alleles were then used to indicate a difference in the number of repeats and thus a difference in individuals. This is the first time that H. gigas microsatellite loci have been isolated. These techniques for ancient bone microsatellite analysis allow an estimate of population size for a newly discovered St. Lawrence Island sea cow population.

Adaptive stress response pathways induced by environmental mixtures of bioaccumulative chemicals in dugongs.

To address the poorly understood mixture effects of chemicals in the marine mammal dugong, we coupled equilibrium-based passive sampling in blubber to a range of in vitro bioassays for screening mixtures of bioaccumulative chemicals. The modes of action included early effect indicators along important toxicity pathways, such as induction of xenobiotic metabolism, and some integrative indicators downstream of the molecular initiating event, such as adaptive stress responses. Activation of aryl hydrocarbon receptor (AhR) and Nrf2-mediated oxidative stress response were found to be the most prominent effects, while the p53-mediated DNA damage response and NF-κB-mediated response to inflammation were not significantly affected. Although polychlorinated dibenzo-p-dioxins (PCDDs) quantified in the samples accounted for the majority of AhR-mediated activity, PCDDs explained less than 5% of the total oxidative stress response, despite their known ability to activate this pathway. Altered oxidative stress response was observed with both individual chemicals and blubber extracts subject to metabolic activation by rat liver S9 fraction. Metabolic activation resulted in both enhanced and reduced toxicity, suggesting the relevance and utility of incorporating metabolic enzymes into in vitro bioassays. Our approach provides a first insight into the burden of toxicologically relevant bioaccumulative chemical mixtures in dugongs and can be applied to lipid tissue of other wildlife species.

Testosterone and tusks: maturation and seasonal reproductive patterns of live, free-ranging male dugongs (Dugong dugon) in a subtropical population.

Knowledge of male reproductive status and activity in free-ranging animals is vital to understanding reproductive patterns and population dynamics. Until now, almost all information regarding reproductive behavior of the dugong, a cryptic marine mammal, has relied on post-mortem examination. We examined the relationships between body length, tusk eruption (secondary sexual characteristic), seasonality, and group association on fecal testosterone metabolite concentrations in 322 free-ranging dugongs (159 males, 163 females) in subtropical Moreton Bay, Australia. Fecal testosterone concentrations demonstrated biologically meaningful differences in testicular activity between sexes and across reproductive/age classes, and were correlated with circulating concentrations in serum. Male dugongs have a pre-reproductive period that persists until a body length of 240 cm is achieved. Puberty apparently occurs between 240 and 260 cm body length when fecal testosterone levels increase fourfold (>500 ng/g) over juvenile levels, and is associated with tusk eruption. However, social maturity may be delayed until male dugongs are larger than 260 cm with well-developed tusks. In mature males, the lowest (<500 ng/g) fecal testosterone concentrations occur in the austral autumn months with maximal concentrations in September-October, coincident with the onset of a spring mating season. During spring, solitary mature males had fecal testosterone concentrations double those of mature males sampled within groups, potentially suggesting a mating strategy involving roving of reproductively active males. This study demonstrates that single-point physiological data from individuals across a population have value as indicators of reproductive processes. Our approach provides an efficacious non-lethal method for the census of reproductive status and seasonality in live male dugongs.

Diagnosing pregnancy in free-ranging dugongs using fecal progesterone metabolite concentrations and body morphometrics: a population application.

Assessing reproductive status and monitoring reproductive rates is important in the effective management of vulnerable marine mammal species such as the dugong (Dugong dugon). Knowledge of the reproductive physiology of this species is limited, and determining reproductive parameters (e.g., sexual maturation, pregnancy, and reproductive senescence) has been restricted by a lack of non-lethal methods for assessing reproductive status in free-ranging individuals. The aim of this study was to develop a method to identify pregnant individuals in a wild dugong population. Using an enzymeimmunoassay, we quantified concentrations of fecal progesterone metabolites (fP) in 322 dugongs, including confirmed pregnant females (n=10), presumed non-pregnant adult females (n=25), juvenile females (n=24), subadult females (n=41), adult females of unknown pregnancy state (n=63), and males of all sizes (n=159). External body morphometrics of each dugong were measured, and confirmation of pregnancy in adult female dugongs was determined by ultrasonography or observation of subsequent neonates. Concentrations of fP were different between sexes and reproductive size classes (P<0.001), and ∼30-fold higher in confirmed pregnant dugongs (2017-7760 ng/g) compared to presumed non-pregnant females (30-221 ng/g), juvenile females (29-195 ng/g), and males (24-261 ng/g) (P<0.001). Body measures of maximum and anal girths, and teat length were all greater in confirmed pregnant females than presumed non-pregnant females (all P<0.05). We evaluated a Discriminant Function Analysis (DFA) to provide a model for predicting pregnant and non-pregnant dugongs. Cross-validated results showed that the DFA correctly classified 100% of pregnant and non-pregnant females using fP concentrations, body length, fineness ratio (an index of body shape), and teat length (a female reproductive trait). Using the DFA model, we classified the pregnancy status of all female dugongs and identified a total of 30 females as pregnant and 133 females as non-pregnant from the sampled population over the sample period. Pregnant dugongs in the Moreton Bay population are characterized by fecal progesterone metabolite concentrations > 1000 ng/g, body length ≥ 260 cm, maximum girth ≥ 215 cm, anal girth ≥ 126 cm, and teat length ≥ 5 cm long. In summary, analysis of fP concentrations in combination with body morphometrics may be used to diagnose pregnancy in free-ranging dugongs, and provides a new tool to monitor breeding rates of wild sirenian populations.

Concentrations of organotin compounds in tissues and organs of dugongs from Thai coastal waters.

Concentrations of butyltin (BT) and phenyltin (PT) compounds were measured in organs and tissues of dugongs (Dugong dugon) from the coastal waters of Thailand. Concentrations of BTs and PTs were in the range of 14-14,468 and <1-30 microg kg(-1)(detection frequency: 79%), respectively. Although concentrations of BTs in dugongs were higher then reported concentrations in cetaceans and pinnipeds, PTs were lower in dugongs. In half of the dugongs in which measurements were made, the concentration of BTs in the liver was the highest among the all the tissues and organs tested. Dibutyltin (DBT) or monobutyltin (MBT) was found to be the dominant compounds among the BTs. The distribution in the body of PTs was not clear because of the lower levels of this compound. TPT was the dominant compound among PTs. The coastal area of Thailand is located off the Gulf of Thailand and the Andaman Sea. Concentrations of organotin (OT) compounds in dugongs collected from the Gulf of Thailand were compared to those from the Andaman Sea. No significant differences in BT or PT concentrations were observed between the two areas (p < 0.05). The concentrations of BTs and PTs in the livers of dugongs were decreased between 1998 and 2002, suggesting a decrease in OT concentrations in the surrounding environment.

Polychlorinated dibenzo-p-dioxins and dibenzofurans in Great Barrier Reef (Australia) dugongs (Dugong dugon).

Fat tissue samples from dugong (Dugong dugon) carcasses stranded at three sites along the Great Barrier Reef were analysed for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). Relatively high levels of PCDDs were determined in all three dugongs. In particular OCDD, the PCDD/F congener that is usually considered the least bioavailable of all 2,3,7,8 substituted congeners, was found at levels higher than reported for other marine mammals. Tissue accumulation of PCDDs by dugongs may be a consequence of sediment and/or seagrass ingestion during feeding, microbial biotransformation of PCDD precursors in the animal's hindgut or, alternatively, the possession of a selective degradation capability for PCDFs.