Identification of biomarkers in green mussel Perna viridis for mercury contamination at petroleum processing platforms in the Gulf of Thailand

Abstract

Contamination of mercury (Hg) from the production processes of natural gas production in the off-shore area is a major concern on the welfare of animals inhabited in the surrounding areas. In addition to the chemical methods commonly used for monitoring Hg contamination in the sea, sensitive techniques emphasized on determining the sublethal effects of Hg on living organisms is crucial for the monitoring program. In this study, bioassay for monitoring the change of Hg contamination in relation to molecular response of Green mussel, Perna viridis, in the surrounding areas of petroleum production platforms in the Gulf of Thailand was studied. Hg responsive genes including MT and 6 of its variants, HSP, and CYP4 genes were obtained and used for designing specific primers which were then used in semi-quantitative RT-PCR for determining mRNA levels of the target genes in tested mussels. The experiment was initially conducted by exposing mussels to Hg as HgCl2 at 0.1, 0.2, 0.5, and 1.0 µg/L under laboratory condition for 8 weeks. During the experiment, Hg concentrations in water and mussel tissues were analyzed. The result showed increasing level of Hg in both water and tissue coincide with the increase of applied doses. The Hg level in tissue was found to be thousand folds higher than that in water within 8 weeks of experiment. Expression levels of target genes were determined in the same samples. The result revealed that pvMT07, one of MT variants, was correlatively responded to the increasing level of Hg. Significant difference was detected at the concentration as low as 0.2 µg/L of Hg (P<0.05). The expression level of the other genes showed no significant difference within the range of applied concentrations. Field validation of the obtained bioassay was carried out by transplanting mussels to the surrounding areas of petroleum production platforms for 3 months. The result showed the correlation of pvMT07 expression with Hg level. These results confirmed that the expression of pvMT07 can be used as biomarker of Hg exposure at low level. Genotoxicity of Hg was investigated using single cell gel electrophoresis technique. Haemocytes and sperms of mussel were exposed in vitro with various doses of Hg as HgCl2 (0.001 to 10.0 μg/L) at 10, 30, and 60 min. The result of tail length and the tail moment of the assay showed that DNA damage was increased in corresponding to the increasing level of Hg. DNA damage was detectable after exposing to Hg level as low as 0.001 µg/L for 10 min. Sperm appeared to be more sensitive to Hg exposure than haemocyte. Hg monitoring techniques and knowledge obtained from this study will provide valuable information for the development of future Hg monitoring program in the marine ecosystem.