Development of matrix-specific standards for LA-ICP-MS zinc analysis in sand flathead (Platycephalus bassensis).

The use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to analyse soft tissues is limited because of the lack of suitable certified reference materials resulting in difficulties with calibration. In this study, several laboratory-prepared thin agarose sections were tested as matrix-specific standards. Our results showed 1 mm thin agarose sections were suitable as calibration standards for LA-ICP-MS Zn analyses in fish muscle, especially when the signal intensity of 66Zn was normalised to 13C. The thin agarose standard sections were used for LA-ICP-MS Zn analyses in the muscle of melanised and non-melanised sand flathead collected from a polluted estuary and assigned with different melanisation scores. Zn levels in melanised regions of fish muscle were determined to be significantly higher than non-melanised regions across all melanisation scores, though Zn levels in the melanised regions of muscle were not significantly different between sand flathead with different melanisation scores. In non-melanised regions of muscle from fish with melanisation and fish unaffected by melanisation, Zn levels were around baseline levels. Overall, this study has successfully developed matrix-specific standards for LA-ICP-MS analysis of soft tissues, thus allowing broader application of this analytical technique in future environmental pollution studies.

The use of image analysis techniques for the study of muscle melanisation in sand flathead (Platycephalus bassensis).

Muscle melanisation in sand flathead is visible as black spots in the normally white flesh of fish. It is widespread in Tasmania, including at the Tamar Estuary, with increasing frequency of reporting by recreational fishers. The phenomenon is more prevalent in areas impacted by heavy metal pollution and has been linked to heavy metal accumulation. In this study, image processing software ImageJ was employed to study the phenomenon and to establish an objective rating system. A longitudinal profile plot was used to study the greying of the fillet. The degree of melanisation was rated based on the percentage surface area melanised on the surface and in transverse sections of fillets. A muscle melanisation scoring system for sand flathead was established based on visual interpretation using the macroscopic melanisation scoring criteria: melanisation scores 0 = <0.5%, 1 = 0.5-5%, 2 = 5-20%, and 3 = >20% (% = melanised surface area in proportion to the whole fillet). A refined image analysis technique was developed to quantify the percentage of melanised muscle surface area and the muscle melanisation scoring system was statistically validated. Sand flathead fillet with higher melanisation score was shown to be linked to increased intensity of greyness and greater numbers and size of black spots on the surface of fillets and within the flesh. The greying and black spots were primarily concentrated at the anterior region of fillet and around the dorsal vertebrae zone on transverse section of fillets. Overall, findings from this study established the use of image analysis techniques to validate visual inspection and to give a standardised and objective method to determine the degree of melanisation in sand flathead. As muscle melanisation appears to be linked to heavy metal pollution, the standardised scoring system would facilitate future research for environmental pollution and monitoring purposes.

Histopathological effects of short-term aqueous exposure to environmentally relevant concentration of lead (Pb) in shorthorn sculpin (Myoxocephalus scorpius) under laboratory conditions.

Shorthorn sculpin (Myoxocephalus scorpius) has been used as a sentinel species for environmental monitoring, including heavy metal contamination from mining activities. Former lead-zinc (Pb-Zn) mines in Greenland resulted in elevated concentrations of metals, especially Pb, in marine biota. However, the potential accumulation of Pb and effects of the presence of Pb residues in fish on health of sculpins observed in the field have not been validated in laboratory experiments. Therefore, our aim was to validate field observation of shorthorn sculpin via controlled laboratory exposure to environmentally relevant concentrations of dissolved Pb. We evaluated the effects of a short-term (28 days) exposure to Pb on Pb residues in sculpin blood, gills, liver, and muscle and the morphology of gills and liver. The highest level of Pb was found in the gills, followed by muscle and then liver. Pb levels in liver, gills, and blood of Pb-exposed sculpins were significantly higher than those in control fish, showing that blood is suitable for assessing Pb accumulation and exposure in sculpins. Histopathological investigations showed that the severity score of liver necrosis and gill telangiectasia of Pb-exposed sculpins was significantly greater than in control fish. The number of mucous cells in gills was positively correlated with Pb concentrations in organs. Overall, the results validated field observation for the effects of Pb on wild sculpin and contributed to the improved use of the shorthorn sculpin as sentinel species for monitoring contamination from Pb mines in the Arctic.

Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia.

Tasmanian recreational fishers have reported the presence of dark pigmentations in the usually white fillets of southern sand flathead (Platycephalus bassensis), a phenomenon known as muscle melanisation. Based on histology, it is suggested that eumelanin and pheomelanin are involved in the occurrence of the phenomenon. A gross melanisation scoring system was validated through a comparison with an image analysis technique, that quantified the percentage surface area of the fillets affected by muscle melanisation. The occurrence of muscle melanisation was most severe in fish inhabiting Deceitful Cove, Tamar Estuary. This indicated that muscle melanisation in P. bassensis may be caused by yet to be identified site specific factors. No significant relationships were evident between the percentage surface area of melanised muscle with condition index, age, sex, maturation stage, fish weight, fish length and size of melano-macrophage centres in the liver or spleen. Overall, this study has provided critical information that will frame the direction and focus of future P. bassensis muscle melanisation research.

Long-term patterns in estuarine fish growth across two climatically divergent regions.

Long-term ecological datasets are vital for investigating how species respond to changes in their environment, yet there is a critical lack of such datasets from aquatic systems. We developed otolith growth 'chronologies' to reconstruct the growth history of a temperate estuarine fish species, black bream (Acanthopagrus butcheri). Chronologies represented two regions in south-east Australia: South Australia, characterised by a relatively warm, dry climate, and Tasmania, characterised by a relatively cool, wet climate. Using a mixed modelling approach, we related inter-annual growth variation to air temperature, rainfall, freshwater inflow (South Australia only), and El Niño-Southern Oscillation events. Otolith chronologies provided a continuous record of growth over a 13- and 21-year period for fish from South Australia and Tasmania, respectively. Even though fish from Tasmania were sourced across multiple estuaries, they showed higher levels of growth synchronicity across years, and greater year-to-year growth variation, than fish from South Australia, which were sourced from a single, large estuary. Growth in Tasmanian fish declined markedly over the time period studied and was negatively correlated to temperature. In contrast, growth in South Australian fish was positively correlated to both temperature and rainfall. The stark contrast between the two regions suggests that Tasmanian black bream populations are more responsive to regional scale environmental variation and may be more vulnerable to global warming. This study highlights the importance of examining species response to climate change at the intra-specific level and further validates the emerging use of growth chronologies for generating long-term ecological data in aquatic systems.