연안 퇴적물 오염 평가 및 환경 독성 연구를 위한 다모류의 생체지표 활용

Abstract

The sediments in coastal environment serve as major repositories of pollutants as well as a source of contaminants for biota and overlying water. Due to these reason, it is possible to release various pollutants into the water column through the resuspension. The benthic organisms will be a good model for the monitoring of sediment pollution, as they inhabit sediment while directly affected by sediments. Polychaete, the most abundant taxon in benthic organisms, can easily be exposed to any polluted substances both in sediments and in water and accumulate several pollutants from sediment through their skin and intestine. Furthermore, their limited mobility also makes it possible for them to accumulate adjacent toxicant and indicate the localized environmental condition. They also play an important link in marine food web while they function as a food source for high trophic level organisms. Due to these advantages, many studies have used polychaete as bioindicator species; however, they are limited in distribution, mortality, and physiological changes such as reproduction rate, growth rate etc. The purpose of this study is to build a database for sediment assessment. Such is achieved by mining and evaluating biomarkers from benthic organisms as early warning signals to monitor sediment pollution in coastal environment. In this study, polychaete was selected as an indicator species. Several biomarkers from polychaete were evaluated to complement classical tiered tests; a sediment quality triad (SQT) was used as a guidelines for sediment pollution and community structures. The objectives of this study are as follows:

1) To evaluate sediment metal pollution of Lake Shihwa by integrating three lines of evidence community structure of macrobenthos, trace metal distribution, and the accumulated metal contents with mortality and metallothionein-like proteins (MTLPs) levels in polychaete and find useful species in polychaete associated with sediment and sensitive to heavy metals. 2) To obtain biomarkers more sensitive in polychaete P. nuntia, potentially useful biomarker genes were cloned and sequenced, while their modulations were measured at the messenger RNA with the comparison of enzyme activities in waterborn and sediment exposed polychaete. 3) To evaluate the susceptibility of potentially useful biomarkers in polychaete exposed to copper (Cu) and moderately polluted sediment along with several antioxidant enzymes.

As one of the approaches of sediment assessment, macrobenthic communities in Lake Shihwa were analyzed. Thus, it can be concluded that the macrobenthic community structure was poor in richness and diversity by showing that some species (polychaete Polydora ligni and bivalves Musculista senhousia) already known for opportunistic and/or indicator species of organic rich sediment were temporarily dominant. The surface sediments, especially the inner and some outer regions of Lake Shihwa seem to be affected by anthropogenic inputs according to the enrichment factor (EF) values. Particularly, Cu is the major element for sediment pollution in Lake Shihwa. Igeo values also have shown that Lake Shihwa is heavily polluted with metals. The sites adjacent to industrial complexes (St.1, St.2, St.3, and St.4) have high values of Igeo in Cu, Zinc (Zn), and Cadmium (Cd). It can be also explained that Lake Shihwa is proximal to the potential source of contaminants, particularly the industrial effluents. This is because the PLI values of Lake Shihwa are much higher when compared to its outside borders. The body burden of metals which can reflect metal bioavailability and MTLPs contents also measured in polychaete N. succinea collected from Lake Shihwa. The MTLPs contents in the polychaete, however, do not correspond with both sediment and polychaete metal contents, suggesting that the polychaete, N. succinea were stressed from metals along with other organic chemicals capable of inducing oxidative stress. From the comparison of toxicity data for P. nuntia and the inherent species, polychaete Cirratulus cirratus which has lived in situ in the sediment, demonstrates that there is a considerable difference in tolerance to metal exposure and metal binding protein between two species. The present study provides important information on the ecotoxicological assessment of polluted sediments using polychaetes. P. nuntia cultured in a clean laboratory environment appears to be a useful species in its sensitivity to heavy metals. To find potential biomarkers genes for environmental risk assessment in ecotoxicological and toxicogenomic studies, P. nuntia EST was set up by the pyrosequencing method. The candidate genes were annotated and listed as follows; 1) Antioxidant defense mechanisms: glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) 2) Molecular chaperone: heat shock proteins (Hsps) 3) Detoxification related gene: cytochrome P450 (CYP)

To validate candidate genes, the polychaetes collected from reference (aquaculture farm) and in-situ (coastal field) were compared. The transcript expression profiles of polychaete collected from coastal field were more highly expressed, as compared to those of the aquaculture farm. This finding suggests that massive transcript profiling may be useful for comparing different expression patterns of target gene families against different environmental factors simultaneously. In this study, GST and MT genes were characterized in the marine polychaete P. nuntia with emphasis on their novel isoforms and the potential to be a biomarker for evaluating oxidative stress. The result showed that GSTs genes might be a factor as antioxidant on Cd exposure. Among them, two GSTs (Pn-GST-sigma and Pn-GST-omega) showed significant upregulation, indicating that these two P. nuntia GSTs have high sensitivity for Cd exposure. The expression of seven GST isoforms in P. nuntia exposed to Cd can provide more specific and reliable information and understanding with total GST enzyme activity in this species. In case of MT gene, there were different expression patterns in P. nuntia according to the exposed elements. Particularly, it was found that Cd and lead (Pb) caused significant upregulation of the MT gene in P. nuntia. Regarding MTLPs and MT mRNA expressions, there were different expression patterns in P. nuntia. Finally, to identify and evaluate potentially useful biomarkers for oxidative stress as early warning indices in the polychaete, P. nuntia was exposed to Cu and was measured several biomarker enzymes (GST, GPx, MTLPs, and CAT) and genes (Pn-GSTs, Pn-CAT, and Pn-MT) with a cellular oxidative index, reactive oxygen species (ROS) level. Accumulated Cu concentrations in P. nuntia increased in a time-dependent manner. Intracellular ROS reached high levels 6 h after exposure in P. nuntia with an increase of GST activity and glutathione (GSH) content. Particularly, the GSH in polychaetes showed a positive correlation with Cu contents accumulated in P. nuntia. Also regarding long-term exposure, there was an obvious increase of Pn-GSTs, Pn-CAT, and Pn-MT genes with elevated concentrations of Cu and Cd in polychaete body, compared to initial levels. This suggests that P. nuntia in sediment was affected by metals as well as by other organic pollutants to induce oxidative stress genes and enzymes. These findings further suggest that oxidative stress is a potential modulator of defense system of P. nuntia. Several potential biomarker genes are available as early warning signals for environmental biomonitoring. In this study, biomarkers of polychaete P. nuntia as early warning signals were evaluated for use in sediment pollution assessment. These findings suggest the possibilities of polychaete as a bioindicator species and enhance understanding of biomarkers.