Polycyclic musks in green-lipped mussels (Perna viridis) from Hong Kong

Six polycyclic musk compounds [Cashmeran (DPMI), Celestolide (ADBI), Phantolide (AHMI), Traseolide (ATII), Tonalide (AHTN), and Galaxolide (HHCB)] were analysed in marine green-lipped mussels (Perna viridis) from Hong Kong. ADBI, HHCB and AHTN were detected in almost all samples, while AHMI, ATII and DPMI were not detected. Concentrations of ADBI, HHCB and AHTN in mussels ranged from below detection limit-0.0743 (mean: 0.0246), 0.247-6.08 (mean: 1.15) and 0.0591-0.738 (mean: 0.190)mg/kg lipid weight, respectively. Mussels from two sampling sites in central Victoria Harbour contained the highest total polycyclic musk levels, suggesting that these waters are heavily influenced by domestic sewage. Concentrations of HHCB and AHTN detected in the mussel samples were the second highest and the highest levels, respectively, compared to global concentrations. A preliminary risk assessment indicated that HHCB and AHTN in mussels pose little or no threat to the health of shellfish consumers. Nevertheless, more comprehensive studies are required to further assess the ecological and human health risks associated with polycyclic musks.     

Organochlorines and dioxin-like compounds in green-lipped mussels Perna viridis from Hong Kong mariculture zones

Concentrations of persistent organic pollutants including polychlorinated biphenyls (PCBs), organochlorine (OC) pesticides and dioxin-like compounds were measured in green-lipped mussels, Perna viridis, collected from seven mariculture zones in Hong Kong between September and October in 2002 in order to evaluate the status, spatial distribution and potential sources of pollution in these areas. Concentrations ranged from 300 to 4400 ng/g lipid weight for total OCs and 170–1000 ng/g lipid weight for total PCBs (based on 28 congeners). Relatively smaller DDT concentrations in mussels compared with previous studies suggest reduced discharges of DDTs from nearby regions into Hong Kong waters. Detection of a mixture of HCH isomers in the mussels indicated that Hong Kong waters were predominantly contaminated by technical HCHs rather than lindane. Mussel samples from all sampling locations elicited significant dioxin-like activity in the H4IIE-luc bioassay. The greatest magnitude of dioxin-like response (39 pg TEQ/g wet wt.) was detected in mussels from Ma Wan in the western waters of Hong Kong, which is strongly influenced by the Pearl River discharge. Human health risk assessment was undertaken to evaluate potential risks associated with the consumption of the green-lipped mussels. Risk quotient (RQ) for dioxin-like compounds was greater than unity suggesting that adverse health effects may be associated with high mussel consumption.     

The use of selected genotoxicity assays in green-lipped mussels (Perna viridis): a validation study in Hong Kong coastal waters

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 regulators using mortality-based bioassays, the sublethal effects of many of the pollutants, including environmental 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 electrophoresis 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 carcinogen 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 aromatic 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 contaminants (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 organisms in differing coastal environments, and changes in biotransformation and DNA repair enzymes in test organisms.     

Okadaic acid, a causative toxin of diarrhetic shellfish poisoning, in green-lipped mussels Perna viridis from Hong Kong fish culture zones: method development and monitoring

Green-lipped mussels (Perna viridis) were collected from seven fish culture zones (FCZs) in Hong Kong and analyzed for okadaic acid (OA). A conventional HPLC method was modified by incorporating a proteinase K digestion step. Results suggest that a higher recovery (2.5 times higher) of OA was obtained from spiked samples after the addition of 1.08 mg proteinase K in comparison with samples incubated without the proteolytic enzyme. For the hepatopancreas (HP) of individual field-collected mussels, the additional digestion step can enhance OA extraction by 3.1 times. Spatial and temporal variations in OA concentrations in the mussels from various FCZs were investigated. The highest concentration of OA in mussel HP samples was 1164.9 ng/g HP wet wt. With respect to OA concentrations in whole mussel tissues from seven sites and four seasonal samplings, the concentrations were between 70.0 and 131.0 ng/g wet wt., which did not exceed the generally recognized international regulatory criteria (>200 ng/g) for OA.     

Field validation of antioxidant enzyme biomarkers in mussels (Perna viridis) and clams (Ruditapes philippinarum) transplanted in Hong Kong coastal waters

Green-lipped mussels, Perna viridis, and Manila clams, Ruditapes philippinarum were sourced from “clean” sites in the Hong Kong region, depurated in a laboratory using uncontaminated filtered seawater for 8 days, and transplanted to a suspected gradient of chemically polluted sites in Hong Kong. After 14- and 28-days of field exposure, several antioxidant parameters including glutathione S transferase (GST), catalase (CAT), glutathione peroxidase (GPx), and glutathione (GSH) were quantified in gill and hepatopancreas tissues. Whole body tissue concentrations of polycyclic aromatic hydrocarbons (PAHs), petroleum hydrocarbons (PHCs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs) were determined in pooled site samples. Chemical analyses indicated that: (a) clams had higher levels of PAHs, PHCs, DDTs and PCBs, whereas mussels had higher hexachlorocyclohexane (HCHs) and there was no difference between species for dieldrin and remaining OCs; (b) Kat O should not be continued as a “clean” reference site for Hong Kong, because of the levels of contaminants measured and (c) PAH concentrations in the current survey were similar to those previously measured. Toxicological conclusions were: (a) antioxidant responses were different between species; (b) CAT and GST have highest utility in clams for field use in Hong Kong, whereas CAT in both gill and hepatopancreas tissue showed most potential in mussels; (c) significant induction of antioxidant responses over day 0 (excluding GPx in both tissues, and GST in mussel hepatic tissue); (d) groups of contaminants do not consistently induce antioxidant responses and (e) organochlorines and PCBs correlated significantly with CAT and GST in clam hepatopancreas and with CAT in mussel gill and hepatic tissue. Multivariate statistical techniques indicated little relationship between the site patterns for antioxidant responses and the contaminant gradients identified in body burden analysis.     

Organochlorines and dioxin-like compounds in green-lipped mussels Perna viridis from Hong Kong mariculture zones

Concentrations of persistent organic pollutants including polychlorinated biphenyls (PCBs), organochlorine (OC) pesticides and dioxin-like compounds were measured in green-lipped mussels, Perna viridis, collected from seven mariculture zones in Hong Kong between September and October in 2002 in order to evaluate the status, spatial distribution and potential sources of pollution in these areas. Concentrations ranged from 300 to 4400 ng/g lipid weight for total OCs and 170–1000 ng/g lipid weight for total PCBs (based on 28 congeners). Relatively smaller DDT concentrations in mussels compared with previous studies suggest reduced discharges of DDTs from nearby regions into Hong Kong waters. Detection of a mixture of HCH isomers in the mussels indicated that Hong Kong waters were predominantly contaminated by technical HCHs rather than lindane. Mussel samples from all sampling locations elicited significant dioxin-like activity in the H4IIE-luc bioassay. The greatest magnitude of dioxin-like response (39 pg TEQ/g wet wt.) was detected in mussels from Ma Wan in the western waters of Hong Kong, which is strongly influenced by the Pearl River discharge. Human health risk assessment was undertaken to evaluate potential risks associated with the consumption of the green-lipped mussels. Risk quotient (RQ) for dioxin-like compounds was greater than unity suggesting that adverse health effects may be associated with high mussel consumption.     

Responses of the green-lipped mussel Perna viridis (L.) to suspended solids

Laboratory experiments were conducted to investigate the lethal and sublethal effects of suspended solids on the survival and physiological, behavourial and morphological changes of the green-lipped mussel Perna viridis collected from Tolo Harbour, Hong Kong. Results showed that P. viridis survived in all test conditions of suspended solids from 0 to 1200 mg/l over a period of 96 h. Physiological responses of the green-lipped mussel under 14-d exposure of suspended solids from 0 to 600 mg/l, followed by 14-d recovery in natural seawater, revealed no significant changes (p>0.05) in oxygen consumption and dry gonosomatic index for treatments in different concentrations of suspended solids and exposure time. Changes in clearance rate were only found to be significant (p<0.001) with exposure time. Responses in behavourial and morphological changes of the green-lipped mussel were also studied under similar experimental treatments and exposure time. Byssus production was significantly (p<0.001) related to exposure time. Gill damage, however, was significantly greater in treatments (p<0.001). Present findings suggested that P. viridis could tolerate a high level of suspended solids in the laboratory. There were dose-dependent effects of suspended solids on morphology of gill filaments. Implications of survival and responses of the green-lipped mussel to suspended solids in the marine environment are discussed.     

Influences of ammonia-nitrogen and dissolved oxygen on lysosomal integrity in green-lipped mussel Perna viridis: laboratory evaluation and field validation in Victoria Harbour, Hong Kong

Lysosomal integrity in mussels has been applied as a biomarker to detect the pollution of trace organics and metals in the natural environments. However, few studies have examined the effects of water quality on the response of lysosomal integrity, in particular total ammonia-nitrogen (TAN) and dissolved oxygen (DO). This study demonstrated that high level of TAN (2.0 mg/l) and low DO (2.5 mg O₂/l) could significantly reduce the lysosomal integrity in green-lipped mussel Perna viridis, respectively by 33% and 38%, whereas the mussel lysosomal integrity decreased by 70% in the combined treatment of TAN and low DO under laboratory conditions after one week. The mussel lysosomal integrity of all treatment groups could return to the control level after a three week recovery period. In the field validation in Victoria Harbour, Hong Kong during an one-year study period, lysosomal integrity in P. viridis identified the cleanest site east to the harbour, where the lowest TAN and highest DO concentrations were found. While lysosomal integrity in mussels seemed not affected by seasonal changes, approximately 40% of the variation of this biomarker could be attributable to the changes in TAN and DO in seawater. In conclusion, the response of the mussel lysosomal integrity can be confounded by both TAN and DO prevailing in the natural environments and thus caution must be exercised in relating the observed changes in lysosomal integrity to any specific pollutant in coastal water quality monitoring studies.     

DNA adduct formation and DNA strand breaks in green-lipped mussels (Perna viridis) exposed to benzo[a]pyrene: dose- and time-dependent relationships

Green-lipped mussels, Perna viridis, were exposed to 0, 0.3, 3 and 30 micrograms l-1 (nominal concentrations) B[a]P under laboratory conditions over a period of 24 days. Mussels were collected on day 0, 1, 3, 6, 12, 18 and 24, and the levels of DNA adducts and DNA strand breaks in their hepatopancreas tissues monitored. Mussels exposed to 0.3 and 3 micrograms l-1 B[a]P showed marked increases in strand breaks after 1 day of exposure. DNA strand break levels in these mussels remained high and significantly different from the control values until day 3 for the 0.3 microgram l-1 treatment group, and day 6 for the 3 micrograms l-1 treatment group. This was followed by a gradual reduction in strand breaks. After 12 days, the levels of both groups had returned to the same level as that of the control. No increase in DNA strand breaks was observable in mussels exposed to 30 micrograms l-1 B[a]P in the first 12 days of exposure, but a significant increase was observed from day 12 to day 24. Increasing B[a]P concentrations resulted in elevated DNA adduct levels after 3-6 days of exposure, but this pattern of dose-related increase disappeared after 12 days. These results indicate that a better understanding of the complex interactions between exposure levels and durations is crucially important before DNA adduct levels and DNA strand breaks in P. viridis can be used as effective biomarkers for monitoring genotoxicants in marine waters.     

Induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis

Biomarkers are generally applied to detect pollution in environmental monitoring. Such biological responses should accurately reflect the stress over time in a quantitative manner. As such, the initial and maximum responses induced by stress, as well as adaptation and recovery of these biomarkers, need to be fully understood or else erroneous false-negative or false-positive may be arrived. However, most of the biomarker studies only provided information on initially induced responses under different concentrations of toxicants, while biological adaptation and recovery were poorly known. In this study, the time required for induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis upon exposure to benzo[a]pyrene was investigated over a period of 62 days. Maximum induction occurred on day 6 when lysosomal integrity was significantly reduced by 51%, and no further change or adaptation was detected thereafter. When mussels were depurated in clean seawater after 18 days of exposure to benzo[a]pyrene, a gradual recovery was observed, with lysosomal integrity returning to its background level and showing a complete recovery after 20 days of depuration. Lysosomal integrity was significantly correlated with the body burden concentrations of benzo[a]pyrene and condition index of the mussels. The relatively fast induction (6 days) and recovery (20 days) without apparent adaptation suggested that lysosomal integrity in P. viridis can serve as a good biomarker in biomonitoring, as its response is not likely to generate both false-negative and false-positive results.     

Size effects of suspended particles on gill damage in green-lipped mussel Perna viridis

The green-lipped mussels Perna viridis were exposed to <500 μm suspended solids (SS) with concentrations of 0 (control), 250, 500, 750 and 1000 mg/L for 14 days, followed by transferring to clean, filtered seawater for 28 days. Results of scanning microscopy showed significantly higher damages to the cilia on the frontal surface of the gill filaments than that on the abfrontal surface in both demibranchs. Percent ciliary depletion varied with SS concentrations and time. No sign of recovery of the gill filaments was observed after the mussels were transferred to clean seawater. In a second experiment, mussels were exposed to SS with size range from <63, >125–<250 and >250–<500 μm at 600 mg/L, together with a control (0 mg/L) for 14 days, followed by transferring to clean, filtered seawater for 28 days. Results of scanning microscopy showed significant ciliary damages in both the ascending and descending lamellae under the three particle size groups as compared with the control. Percent depletion of frontal cilia was most serious for the >250–<500 μm size group and least for the <63 μm size group. However, percent depletion of abfrontal cilia was most serious for the >125–<250 μm size group and least for the <63 μm size group. No recovery of ciliary damages was observed. The effects of particle size of suspended sediments on the morphological damages of gill filaments in the green-lipped mussels were discussed.     

Biomonitoring of heavy metals and trace organics using the intertidal mussel Perna viridis in Hong Kong coastal waters

This paper presents the results of a 6-year (1998–2003) survey of trace toxics in the intertidal mussel Perna viridis conducted by the Hong Kong Environmental Protection Department. Concentrations of heavy metals and trace organics were measured in the soft bodies of P. viridis collected from five sites in Hong Kong waters, i.e. Wu Kai Sha (Tolo Harbour), Ma Wan (Northwest), Tsim Sha Tsui (Victoria Harbour), Tai Tam (Hong Kong South) and Lamma Island (Southern Waters) in order to establish the spatial patterns of contaminants in mussels. Among the metals analysed, Cd showed a significant concentration gradient in Hong Kong waters. The levels of Cd in P. viridis were significantly higher at Ma Wan as compared to the other sites studied. Ma Wan also had relatively higher concentrations of Pb. Mn concentrations were particularly prominent at Wu Kai Sha. Significantly higher concentrations of Hg and Cu were recorded at Tai Tam and Tsim Sha Tsui. Tai Tam and Wu Kai Sha had higher levels of V; whereas higher Ni concentrations were recorded at Lamma Island and Tai Tam. No clear spatial patterns for Al, As, Cr, Fe and Zn were observed.

Higher concentrations of PAHs in P. viridis were observed around urban centres impacted by sewage discharges (e.g. Tsim Sha Tsui); whereas higher PCB levels were found not only in Tsim Sha Tsui but also in less urbanised areas such as Lamma Island and Tai Tam, suggesting that these may be due to non-sewage related inputs. The study also shows that Northwest and Southern waters are subject to a higher degree of DDT pollution compared with other sites. Of the 17 dioxin compounds analysed, positive data were mostly recorded for two compounds which are of low toxicity (i.e. OCDD and 1,2,3,4,6,7,8-HpCDD) whereas the most toxic congeners (i.e. 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD) were not detected in the 6 years of monitoring. In general, the levels of OCDD in P. viridis were found to be higher in Tai Tam and Lamma Island in Southern Waters of Hong Kong.

This study found that the levels of some highly toxic heavy metals (i.e. Cd, Hg and Pb) in the mussel P. viridis did not exceed the recommended limits for shellfish as food in Hong Kong (i.e. Cd: 2.0 ppm; Hg: 0.5 ppm; Pb: 6.0 ppm wet weight). The levels of As and Ni in P. viridis were also well below the action limits set by the US FDA (i.e. As: 86 ppm; Ni: 80 ppm wet weight). DDT and PCB contaminations in P. viridis were below the concentrations of concern. Compared with data obtained in the 1980s, the current levels of DDTs in P. viridis were 4–16 times lower; whereas Pb concentrations recorded in Tsim Sha Tsui have also been lowered significantly. This is mainly related to reduction in local and regional pollution sources in the past 20 years.     

Immune responses to combined effect of hypoxia and high temperature in the green-lipped mussel Perna viridis

Flow cytometry was used to examine immune responses in haemocytes of the green-lipped mussel Perna viridis under six combinations of oxygen level (1.5 mg O2 l(-1), 6.0 mg O2 l(-1)) and temperature (20 °C, 25 °C and 30 °C) at 24 h, 48 h, 96 h and 168 h. The mussels were then transferred to normoxic condition (6.0 mg O2 l(-1)) at 20 °C for further 24 h to study their recovery from the combined hypoxic and temperature stress. Esterase (Est), reactive oxygen species (ROS), lysosome content (Lyso) and phagocytosis (Pha) were reduced at high temperatures, whereas hypoxia resulted in higher haemocyte mortality (HM) and reduced phagocytosis. For HM and Pha, changes were observed after being exposed to the stresses for 96 h, whereas only a 24 h period was required for ROS and Lyso, and a 48 h one for Est. Recovery from the stresses was observed for HM and Pha but not other immune responses.     

Field observations on correlation of fatty acid profiles between suspended particulate matter and green-lipped mussels in subtropical waters of Hong Kong

By analysis of the fatty acid profiles in mussel tissues and suspended particulate matter (SPM) in water, the present study showed a significant relationship of the trophic linkage between mussels and the SPM. At seven locations from inner to outer areas along the eutrophic Tolo Harbour and Tolo Channel, Hong Kong, the composition (as percentage of total fatty acids) of both monoenoic and polyunsaturated fatty acids in the tissues of green-lipped mussels Perna viridis and SPM in water had significant correlation (p<0.01). In particular, the composition of docosahexaenoic acid (DHA, 22:6n3) (as percentage of total fatty acids) in mussel tissues was statistically correlated with that in SPM (p<0.01), implying that mussels incorporate DHA, the biomarker of dinoflagellates. Principal component analysis further demonstrated that the fatty acid profiles of SPM were different among locations in the harbour, mid and outer channel of the study area, so were the mussel tissue fatty acid profiles. Cluster analysis of phytoplankton data also revealed the dominance of diatoms in the inner harbour and channel areas, whereas dinoflagellates were abundant in the outer channel waters. The possible implications of using benthic suspension feeders such as green-lipped mussels P. viridis for controlling phytoplankton abundance in coastal waters are discussed.     

Bioenergetic responses in green lipped mussels (Perna viridis) as indicators of pollution stress in Xiamen coastal waters, China

Samples of green lipped mussels (Perna viridis) were collected from three sites (Huoshao, I’Maluan and Tong’an) around Xiamen coastal waters, where levels of various trace organic pollutants have been studied. Samples were also collected at a “cleaner” reference site near Dongshan Island for comparison. Clearance rate, absorption efficiency and oxygen consumption of the mussels were measured under controlled laboratory condition; organic pollutants in their tissues were also analyzed. Scope for growth (SFG) was employed as a general biomarker to evaluate the stress of pollutants. Specimens from Tong’an site had the lowest SFG values (1.14 J/h/g); while specimens from the I’Maluan site and Huoshao site had SFG values of 5.01 and 6.72 J/h/g, respectively. Specimens from Dongshan (reference site) had a relatively high SFG value of 10.96 J/h/g. There was a significant negative correlation between the SFG of the different populations of green lipped mussels and the concentration of DDT in their tissues.     

Effect of size, tissue parts and location on six biochemical markers in the green-lipped mussel, Perna viridis

The effect of size and tissue parts as well as site locations on a suite of six anti-oxidative biochemical markers, viz. the enzyme degrading neural transmitters acetylcholinesterase (AChE), the Phase II transforming enzyme glutathione-S-transferase (GST), the anti-oxidative enzymes superoxide dismutase (SOD) and catalase (CAT), the lipid metabolite malondialdehyde (MDA) and the ratio of CAT/MDA, in the bivalve mollusc, Perna viridis, was studied. Field mussels were collected from the piers of University of Science and Technology (UST), Pak Sha Wan or Hebe Haven (PS) and Sai Kung (SK) in Port Shelter, Hong Kong. Statistically, ANOVA showed that the activity of all six biochemical markers were generally tissue dependent, size independent and location dependent. Tukey multi comparison test indicated that the biochemical activities fell into two groups, with SK the highest activity group in most cases. The significance of these findings in relation to long-term coastal water biomonitoring is discussed.