The use of selected genotoxicity assays in green-lipped mussels (Perna viridis): A validation study in Hong Kong coastal waters Stanley Y.M. Siu, Paul K.S. Lam, Michael Martin, Christopher W. Caldwell, Bruce J. Richardson * Department of Biology and Chemistry, Research Centre for Coastal Pollution and Conservation, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong article info Keywords: PAHs Organochlorine pesticides PCBs Genotoxicity assays 8-hydroxy-2 0 -deoxyguanosine (8-OHdG) DNA strand breakage Alkaline agarose gel electrophoresis Comet assay Micronucleus (MN) assay Dose–response relationships abstract In Hong Kong, discharges of domestic and industrial wastewaters and disposal of contaminated muds into coastal waters have resulted in high levels of persistent organic contaminants in the water column, biota and bottom sediments. Although the acute toxicity of effluents has been tested by government reg- ulators using mortality-based bioassays, the sublethal effects of many of the pollutants, including envi- ronmental carcinogens, have not been well studied. The objective of the present work was to examine the efficiency and efficacy of using: (1) DNA strand breaks (comet assay and alkaline agarose gel electro- phoresis assay); (2) oxidative DNA damage (levels of 8-hydroxy-2-deoxyguanosine in cellular DNA) and (3) micronuclei (MN) formation as early warning toxicological indicators of potential environmental car- cinogen exposure. Green-lipped mussels (Perna viridis) were transplanted to study sites and retrieved after 4, 8, 12, 16 and 30 days. The bioaccumulation of organic pollutants, including the polycyclic aro- matic hydrocarbons (PAHs), organochlorine pesticides (OC pesticides) and polychlorinated biphenyls (PCBs) was observed, and compared with biomarker assays. Some significant dose- and time-dependent inductions of the genotoxic biomarkers, including 8-OHdG, DNA strand breaks and micronuclei were observed. In particular, several very strong (r > 0.9) and significantly positive relationships were found at all sites during days 0–12. In addition, significant strong relationships between certain organic contam- inants (carcinogenic PAHs and 4 common OC pesticides) and micronuclei were detected at the polluted sites, Tsim Sha Tsui and Sai Wan Ho. The results suggest that the use of micronuclei as a biomarker is potentially effective, especially at the more polluted sites. Nonetheless, further research is required to validate their effectiveness under varying field conditions, especially to elucidate responses under lower contaminant loads, interactions between the environmental and physiological factors affecting organ- isms in differing coastal environments, and changes in biotransformation and DNA repair enzymes in test organisms. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Because Hong Kong is one of the most densely populated areas and one of the busiest ports in the world, its coastal environments have come under severe stress not only due to local influences, but also to the rapid development taking place in the Pearl River Delta. Marine pollution in the Hong Kong environment has resulted from industrial contamination by a variety of organic and inorganic con- taminants, discharges of largely untreated domestic and industrial wastewaters, and the disposal of contaminated mud through dredging and filling operations (Wu, 1988; Connell et al., 1998a,b). Relatively high concentrations of petroleum hydrocar- bons, PAHs and OC pesticides have been found in Hong Kong’s nearshore sediments, with the heaviest contamination in highly urbanized and developed areas, especially within Victoria Harbour and associated typhoon shelters (Zheng and Richardson, 1999; Richardson and Zheng, 1999; Richardson et al., 2003). PCBs are also widespread in Hong Kong sediments, with concentrations again centred on urbanized locations (Richardson and Zheng, 1999; Rich- ardson et al., 2000, 2001; Wong and Poon, 2003). These data indi- cate the need for continuing chemical monitoring along with observation of contaminant-related effects in local biota. Substances capable of modifying the genetic material of living organisms are of obvious environmental concern (especially in the dynamic Hong Kong environment), and several analytical tech- niques have been developed to detect the potentiality of genetic damage (Shugart, 1999, 2000). Alkaline agarose gel electrophore- sis, in which cleaved DNA fragments migrate according to their size, has been a common and effective technique for the determi- nation of single DNA strand breakage in nucleated animal cells (Theodorakis et al., 1994). Recent developments have resulted in the relatively simple, quick, low cost and sensitive use of the comet assay for the detection of strand breaks (Wilson et al., 1998; Akcha 0025-326X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2008.03.006 * Corresponding author. Tel.: +852 2788 7042; fax: +852 2788 7406. E-mail address: bhbrucer@cityu.edu.hk (B.J. Richardson). Marine Pollution Bulletin 57 (2008) 479–492 Contents lists available at ScienceDirect Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul