Relationship between heavy metal levels and muscle melanisation in sand flathead (Platycephalus bassensis) from Tasmania, Australia

Muscle melanisation in sand flathead (Platycephalus bassensis) is defined as the development of abnormal black spots in the normally white flesh of the fish. It is widespread in the state of Tasmania, Australia, and poses major concerns for the Tasmanian recreational fishing community, as the sand flathead represents the most commonly caught finfish species in the state with a very high recreational value. Sand flathead affected by muscle melanisation are commonly found in areas that contain sediments polluted by heavy metals, especially Zn. Thus, the main aim of this study was to study the relationship between heavy metal levels and muscle melanisation in Tasmanian sand flathead. Image analysis techniques were utilised to develop and validate a standardised muscle melanisation scoring system for sand flathead: melanisation scores 0 = <0.5%, 1 = 0.5-5%, 2 = 5-20%, and 3 = >20% (% = melanised surface area in proportion to the whole fillet). The scoring system standardised the assessment of muscle melanisation in sand flathead and allowed findings in the project to conform to a standardised system and facilitate comparisons. Higher melanisation scores corresponded to sand flathead fillets with significantly greater intensity of greyness and an increase in the number and size of black spots on the surface of fillets and within the flesh. The greying and black spots were primarily concentrated near the vertebrae where blood actively circulates, namely at the anterior region of the fillet and around the dorsal vertebrae zone on transverse section of fillets. Zn levels were measured in sand flathead muscle collected from various sites throughout Tasmania and in crabs, a major component of the fish‚ÄövÑv¥s diet, using flame atomic absorption spectroscopy (FAAS). An improved technique for extracting only the melanised regions of fish muscle and a reagent mixture and technique suitable for digesting whole crab using microwave assisted acid digestion, both limited in literature, were developed. Significantly elevated Zn levels were detected in the melanised regions of muscle, where it was found at 2.1‚ÄövÑv¨3.5 times higher levels than in non-melanised regions of muscle from fish with melanisation, or in muscle of fish unaffected by melanisation. In non-melanised regions of muscle from fish with melanisation and in muscle of fish unaffected by melanisation, Zn levels were around the normal baseline level of 5 mg/kg wet weight. Zn levels in crabs studied as a potential Zn source for the fish, were found to be 1.5 times higher in two of the crab species studied from a polluted area compared to the same species from a non-polluted area. To address the limitations associated with conventional methods of metal analysis for melanised muscle, a direct elemental analysis technique using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was employed. As certified reference materials for LA-ICP-MS analysis of soft biological samples were not readily available, laboratory-prepared spiked agarose films were developed as matrix-similar standards for the calibration and analysis of Zn in sand flathead muscle by LA-ICP-MS. This enabled Zn levels in melanised muscle to be determined with better accuracy and higher spatial precision. It also allowed sand flathead with minor levels of melanisation, which constitute the majority of affected fish, to be analysed for the first time and established that Zn levels in the muscle were related to the presence but not the severity of melanisation. Inductively coupled plasma mass spectrometry (ICP-MS) and LA-ICP-MS were used to study the short-term and long-term exposure to heavy metals in sand flathead and its association with muscle melanisation by analysing the whole blood and otoliths, respectively. Exposure to heavy metals were found to be unrelated to heavy metal levels in the whole blood, suggesting that metals, including Zn, are well regulated in sand flathead blood. The exposure patterns of heavy metals over the life of sand flathead did not differ significantly between the otoliths from sand flathead affected and unaffected by muscle melanisation, though higher Zn levels were present around the edge of the otoliths, suggesting excessive accumulation of Zn from the diet. Overall, this study discussed the links between muscle melanisation in sand flathead, Zn pollution in the environment and the levels of Zn in fish diet, muscle, whole blood and otoliths. It described how the accumulation of Zn in the melanised regions of muscle is potentially related to a detoxification system in which elevated uptake of Zn from the fish‚ÄövÑv¥s diet is absorbed into the blood. It is then selectively transported to the melanised regions of muscle that act as a sink to sequester the excess Zn and protect other fish tissues and organs. It is proposed to be a mechanism facilitated by melanin and enables the fish to cope with living in a polluted environment. As marine pollution is a common problem and muscle melanisation is reported in more wild fish species, findings from this study will benefit future muscle melanisation research in other fish species. The novel analytical methods and techniques described in this study are also transferable to other studies and will support future research effort. In Tasmania, findings from this study will also help guide and inform sand flathead management practices.