Implications from a field study regarding the relationship between polycyclic aromatic hydrocarbons and glutathione S-transferase activity in mussels

An aluminium smelter on the west coast of Scotland discharges an aqueous effluent containing polycyclic aromatic hydrocarbons (PAHs) at the head of Loch Leven. The loch also supports two mussel (Mytilus edulis) farms. Data are presented on burdens of PAHs in the soft tissues of mussels and the effect of these contaminants on glutathione S-transferase (GST) activity in mussel hepatopancreas. GST activity is shown to be correlated with total PAH burden and also with the concentrations of certain individual PAHs. These field data show that high molecular weight PAHs are closely correlated to GST activity, whereas low molecular weight PAHs are not. This suggests that 5- and 6-ring PAHs have a more pronounced role than 2- to 4-ring compounds in inducing GST activity in mussels from Loch Leven. It is proposed that it may be more appropriate to link GST activity with 5- and 6-ring compounds only, rather than with the total PAH burden.     

GSTA is a major glutathione S-transferase gene responsible for 4-hydroxynonenal conjugation in largemouth bass liver

We have previously shown that largemouth bass (Micropterus salmoides) has a remarkable ability to conjugate 4-hydroxy-2-nonenal (4HNE), a mutagenic and cytotoxic α,β-unsaturated aldehyde produced during the peroxidation of lipids. In addition, we have isolated a glutathione S-transferase cDNA (bass GSTA) that encodes a recombinant protein which is highly active in 4HNE conjugation and structurally similar to plaice (Pleuronectes platessa) GSTA. In the present study, HPLC-GST subunit analysis revealed the presence of at least two major GST isoforms in bass liver, with one peak constituting 80% of the total bass liver GST protein. Liquid chromatography mass spectrometry (LC–MS) and electrospray ionization analysis of the major bass GST subunit yielded a molecular weight of 26,396 kDa. Endo-proteinase Lys-C digestion and Edman degradation protein sequencing of this GST peak demonstrated that this protein was encoded by bass GSTA. Analysis of genomic DNA fragments isolated by nested PCR indicated the presence of a GST gene cluster in bass liver that contained GSTA, and was similar to a GST gene cluster characterized by Leaver et al., in plaice. Collectively, our data indicates the presence of a major GST in bass liver involved in the protection against oxidative stress. This GST is part of a gene cluster that may be conserved in certain freshwater and marine fish.     

Effects of three fungicides alone and in combination on glutathione S-transferase activity (GST) and cytochrome P-450 (CYP 1A1) in the liver and gill of brown trout (Salmo trutta)

In order to evaluate the gill glutathione S-transferase (GST) activity as a biomarker of effect of fungicide exposure in juvenile brown trout (Salmo trutta), the fungicides propiconazole {(R,S)-1-[2-(2,4-diclophenyl)-4-propyl-1,3-dioolan-2-ylmetyl]-1H-1,2,4-triazole} and fenpropimorph {(±)-cis-4-[3-(4-tert-butylphenyl)-2-metyl propyl]-2,6 dimetylmorfolinc} were administrated in the water separately and together in a static system (80 μg/l for each pesticide) for 5 days. The combined fungicides gave a significant decrease in gill GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB), whilst hepatic GST-activity was not significantly changed. Furthermore, continuous exposure to 540 ug/l thiabendazole{ 2-(thiazol-4′-yl)benzimidazole} in a flow-through system for 4 days significantly increased the gill glutathione S-transferase (GST) activity towards CDNB, whilst hepatic GST and cytochrome P450 (CYP 1A) activities were not increased by the treatment.     

Partitioning of polycyclic aromatic hydrocarbons between water and particles compared to bioaccumulation in mussels: a harbour case

Water and particles from three sites located in Halifax Harbour, near and further away from combined municipal sewage effluents (CMSE) were analysed for parental and alkylated polycyclic aromatic hydrocarbons (par and alkPAH). Bioavailability of PAH was compared for inter-tidal mussels collected at the same sites in April and November. The PAH fingerprint determined over 9 sampling times covering a period of 19 months differed more between phases (water and particles) and seasons, than between sites. In the spring, more alkPAH associated with diesel and gasoline were detected in the soluble phase, along with more bioaccumulation of alkPAH in inter-tidal mussels. A broader number of parPAH were detected in mussels collected in the fall. The mean sum of dissolved alkPAH concentrations was higher in water at the site closer to raw CMSE than at the other two sites and particles of that site. However, lowest bioconcentration factors (BCF) were determined in mussels of this more contaminated site. Similar biota-particle accumulation factors (BPAF) were determined for parPAH in mussels from the three sites, all lower than the BCF of alkPAH. The study indicates that sewage treatment plants will reduce the amount of parPAH especially larger than fluoranthene and pyrene from being deposited in the harbour; that alk naphthalenes and fluorenes present in water will continue to be discharged; that the disturbance of sediments can make particle-bound PAH available to mussels.     

Susceptibility to genetic damage and cell types in Mediterranean mussels

Micronucleus (MN) frequency is generally accepted as a marker of chromosomal damage and has been studied in a variety of cells and species. In previous work, we detected significant dose-related MN increases in the epithelial-like gill cells and agranular haemocytes of Mytilus galloprovincialis treated with benzo[a]pyrene, a well-known mutagenic pollutant. In addition, we have studied micronuclei and other nuclear abnormalities in mussels collected from the Venice lagoon (Italy). Frequency changes, possibly related to genotoxic/toxic stress, in both granular and micronucleated cells from gills and haemolymph, were detected. Environmental data suggest the effect of genotoxic pollutants and the importance of cell replication in the interpretation of micronucleus frequencies.     

Sources of polycyclic aromatic hydrocarbons in marine sediments from southern and northern areas of the Norwegian continental shelf

Variability in levels and sources of polycyclic aromatic hydrocarbons (PAH) in sediments from one large sea area off the coast of northern Norway (“North area”, NA) have been compared to similar data from another large area off the coast of southern Norway (“South area”, SA). Samples from NA were collected at the Norwegian continental shelf in south-western Barents Sea and north-eastern Norwegian Sea. Samples from SA were from the Norwegian Trench and the Skagerrak. Sediment cores have been dated, characterised by grain size distribution (GS) and organic carbon content (TOC), and the composition of PAH and geochemical biomarkers (alkanes and triterpanes) studied to provide an insight into the different sources of PAH. Generally, PAH levels are higher in sediments from SA compared to NA. A mixture of pyrogenic and petrogenic sources contribute to PAH levels in SA, while the contents of petrogenic PAH is negligible in surface sediments in NA. At some locations in NA, petrogenic PAH levels are elevated in the deepest sediment layers from pre-industrial times, indicating a natural input of petroleum through seepage. Occurrence of elevated levels of microbial hopanoids (hopenes) in the deepest sediment layers at some locations both in the north and the south indicate the presence of petroleum.     

DNA adducts in mussels and fish exposed to bulky genotoxic compounds

Biological and procedural factors can influence DNA adduct detection in aquatic organisms. Among them, functional structure and metabolic traits represent major biological determinants for adducts formed by lipophilic pro-mutagenic contaminants. In detecting DNA adducts through the 32P-postlabelling assay, efficiency in DNA purification, digestion, labelling, as well as adduct enrichment and quantification may explain differences between independent studies. Reference DNA adducts have been used to verify some 32P-postlabelling aspects. Data obtained for mussels and fish treated with benzo[a]pyrene (B[a]P) and environmentally exposed to genotoxins confirm the above assertions. Although the 32P-postlabelling assay cannot be proposed for routine biomonitoring it appears a reliable and very sensitive index of exposure to genotoxic pollutants in both fish and mollusks.     

Natural background and anthropogenic inputs of polycyclic aromatic hydrocarbons (PAH) in sediments of South-Western Barents Sea

Polycyclic aromatic hydrocarbons (PAH) were measured in sediment cores from 13 locations in South-Western Barents Sea as part of a detailed study of the Norwegian seabed under the MAREANO program. The generally low PAH levels found, an average around 200 ng g⁻¹ dry weight for sum PAH, indicate low inputs of petroleum hydrocarbons to the marine environment in the area. Differences in PAH composition and various PAH ratios indicate a natural, mostly petrogenic origin of PAH in sediments from the open sea locations, while the fjord locations show higher pyrogenic PAH contents with an increase towards upper sediment layers, indicating low inputs from human activities. Petrogenic PAH levels increase in deeper sediments at open sea locations, also when normalised to total organic carbon (TOC) contents, suggesting natural leakages of oil-related hydrocarbons in the area.     

Acute toxicity of heavy metals, tributyltin, ammonia and polycyclic aromatic hydrocarbons to benthic amphipodgrandidierella japonica

Benthic amphipod,Grandidierella japonica widely inhabits the Korean coastal waters and is developed as a standard test species for sediment toxicity tests. We exposedG . japonica to various pollutants including 4 kinds of inorganic metals (Ag, Cd, Cu and Hg), tributyltin [TBT], ammonia and 7 polycyclic aromatic hydrocarbon (PAH) compounds (acenaphthene, chrysene, fluoranthene, fluorene, naphthalene, phenanthrene and pyrene) to estimate the no observed effect concentration (NOEC) and the median lethal concentration (LC50) of each pollutant during the 96-hour acute exposure. Among all tested pollutants, TBT was most toxic toG. japonica, and Hg was most toxic among inorganic metals. The toxicity of pyrene toG. japonica was greatest among PAH compounds, followed by fluoranthene, phenanathrene, acenaphthene, fluorene and naphthalene. The toxicity of PAH compounds was closely related to their physico-chemical characteristics such as K_ow and water solubility.G. japonica responded adequately to pollutant concentrations and exposure durations, and the sensitivity ofG. japonica to various inorganic and organic pollutants was generally comparable to other amphipods used as standard test species in ecotoxicological studies, indicating this species can be applied in the assessment of environments polluted by various harmful substances.     

Plasma steroid levels in female flounder (Platichthys flesus) after chronic dietary exposure to single polycyclic aromatic hydrocarbons

The chronic effects of polycyclic aromatic hydrocarbons (PAHs) on ovary development, total hepatic lipids and plasma sex- and corticosteroid levels in female flounder (Platichthys flesus) were examined. Sexually mature feral female flounder were exposed via the diet to phenanthrene (0.5, 2.5 or 12.5 nmol/g food) or chrysene (0.4 nmol/g food) for 12 weeks, during the previtellogenic phase of the annual reproductive cycle. PAH exposure did not directly affect germ cell development since no structural and/or developmental differences were observed between control and exposed fish. On the contrary, all treatments resulted in altered plasma steroid levels. The most pronounced effect was the significant decrease in plasma 17β-estradiol to 19±11%, 27±7%, 63±20% and 61±12% in relation to control fish, respectively, in flounders exposed to 12.5, 2.5 or 0.5 nmol phenanthrene/g food and 0.4 nmol chrysene/g food. Impaired ovarian growth was not observed, most likely because experiments were ended before the period of vitellogenesis, even though a non-significant general decline in total hepatic lipids could be observed. Moreover, all exposed flounders, except fish fed with the highest amount of phenanthrene, showed a negative correlation between plasma 17β-estradiol and 17α-hydroxyprogesterone levels (r=−0.46). One possible explanation is that PAH action may be mediated by a specific inhibition of steroidogenic enzymes. These findings provide evidence that selected PAHs are antiestrogenic xenobiotics with the capability to impair female teleost reproductive function.     

CYP1A- and CYP3A-immunopositive protein levels in digestive gland of the mussel Mytilus galloprovincialis from the Mediterranean Sea

CYP1A-immunopositive protein can be elevated in response to planar PAHs and PCBs in Mytilus sp. digestive gland whilst CYP3A-immunopositive protein has been associated with testosterone 6β-hydroxylation in fish. Levels of CYP1A- and CYP3A-immunopositive protein were determined in Mytilus galloprovincialis digestive gland microsomes collected from 12 sites in the Mediterranean Sea during May and September 2001. CYP1A-immunopositive protein was significantly highest at contaminated sites whilst CYP3A-immunopositive protein was significantly lowest. A weak negative correlation (r²=0.21) was seen between CYP1A- and CYP3A-immunopositive protein. Little evidence of differences at the different sampling times was observed. These results confirm previous work indicating elevation of CYP1A-immunopositive protein in Mytilus sp. digestive gland at contaminated sites. Further study is required to characterise CYP3A-like expression in Mytilus and to elucidate the consequences of possible CYP3A-like down-regulation at contaminated sites.     

Effects of β-naphthoflavone on hepatic biotransformation and glutathione biosynthesis in largemouth bass (Micropterus salmoides)

We are investigating the effects of in vivo exposure of prototypical enzyme inducing agents on hepatic biotransformation enzyme expression in largemouth bass (Micropterus salmoides), a predatory game fish found throughout the United States and Canada. The current study targeted those genes involved in biotransformation and oxidative stress that may be regulated by Ah-receptor-dependent pathways. Exposure of bass to β-naphthoflavone (β-NF, 66 mg/kg, i.p.) elicited a 7–9-fold increase in hepatic microsomal cytochrome P4501A-dependent ethoxyresorufin O-deethylase (EROD) activities, but did not affect cytosolic GST catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB) or 5-androstene-3,17-dione (ADI). Glutathione S-transferase A (GST-A) mRNA expression exhibited a transient, but non-significant increase following exposure to β-NF, and generally tracked the minimal changes observed in GST–CDNB activities. Expression of the mRNA encoding glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in glutathione (GSH) biosynthesis, was increased 1.7-fold by β-NF. Changes in GCLC mRNA expression were paralleled by increases in intracellular GSH. In summary, largemouth bass hepatic CYP1A-dependent and GSH biosynthetic pathways, and to a lesser extent GST, are responsive to exposure to β-NF.     

不同流速和苗种规格对缢蛏平面流中间培育效果的影响

为了优化蛏苗集约化平面流中间培育技术,研究了不同进水流速和苗种规格对缢蛏中间培育效果的影响,并分析了集约化平面流中间培育系统的水质状况。结果显示,不同进水流速对缢蛏稚贝生长影响显著,稚贝生长速率随进水流速增加而增加,但成活率下降。通过流速与成活率和体质量日增生长量的线性回归分析,估算0.163L/s为适宜的进水流速。在适宜流速和相同放苗重量下,大规格苗种(8万粒/kg)生长速度显著高于小规格苗种(18万粒/kg),但因为小规格组放苗数量多,小规格组单位面积质量较其高出23.72%。除低流速组以外,平面流中间培育过程对叶绿素a和铵态氮有良好的去除效果,去除率分别达到36.99%和3.88%以上,这表明平面流集约化中间培育在利用海水池塘水体进行苗种中间培育的同时,也起到了池塘养殖水体的净化作用。综合认为,在养殖密度0.5kg/m²、流速0.163 L/s的培育条件下,可以保证水体自污染程度较低,缢蛏苗种生长较快,成活率在73.12%以上。     

Spatial and temporal variation of biomarkers in mussels (Mytilus galloprovincialis) from the Lagoon of Venice, Italy

A multiple biomarker approach was adopted in a seasonal study carried out in the Lagoon of Venice, with the double aim of evaluating the natural and anthropic stresses influencing the biological responses of mussel (Mytilus galloprovincialis) and of assessing the effects due to spatial rather than temporal variations.Biochemical (aldehyde dehydrogenase and catalase activities), cellular (neutral red retention time) and physiological (survival in air and condition index) biomarkers were determined in mussels collected in four differently impacted lagoon areas.Multivariate analysis showed that samples were distributed mainly according to temperature and four seasonal groups were identified. The combination of spatial and temporal information enabled us to distinguish physiological variations due to natural causes from those due to anthropic stress, and to identify the sampling period when several biomarkers are less influenced by both natural and endogenous factors.     

A laboratory and field comparison of sediment polycyclic aromatic hydrocarbon bioaccumulation by the cosmopolitan estuarine polychaete Streblospio benedicti (Webster)

Polycyclic aromatic hydrocarbon (PAH) bioaccumulation by the polychaete worm Streblospio benedicti (Webster) was measured under exposure to PAH-contaminated sediments in the field and for 28 d in the laboratory. Streblospio benedicti collected from field sediments contaminated at 2.94, 1.07, and l.52 μg g⁻¹ fluoranthene (FLU), benz[a]anthracene (BAA), and benzo[a]pyrene (BAP), bioaccumulated those PAHs at 1.53, 0.215, and 0.332 μg g⁻¹, while worms isolated from less contaminated field sediments (0.399, 0.228, 0.288 μg g⁻¹ FLU, BAA, and BAP) had FLU, BAA, and BAP body burdens of 0.543, 0.236, and 0.083 μg g⁻¹. Worms incubated for 28 d in PAH-spiked sediments (1.52, 0.991, 0.504 μg g⁻¹ FLU, BAA, and BAP) bioaccumulated those PAHs at 0.382, 0.966, and 0.602 μg g⁻¹, respectively. Data normalization to organism lipid and sediment organic carbon (biota-sediment accumulation factors [BSAFs] strongly suggest that Streblospio PAH bioaccumulation was directly related to percent sediment organic carbon, but BSAFs were substantially lower than predicted by equilibrium partitioning theory. BSAFs decreased with increasing PAH log K_ow, in worms collected from field sediments, but in spiked sediments BSAFs increased with increasing PAH hydrophobicity. This disparity may have been caused by insufficient spiked-sediment equilibration time (1.5h) in the case of the laboratory test sediments.     

Detecting a field gradient of PAH exposure in decapod crustacea using a novel urinary biomarker

Norwegian coastal waters are subject to PAH contamination from electrochemical industries such as aluminium smelters. Evidence of PAH exposure has been established in fish and bivalves. The present study tests the applicability of a novel crustacean PAH exposure biomarker to a PAH contamination gradient in the field (Karmsund Strait, SW Norway). Fluorescence analysis of urine samples collected from crabs at each site revealed 1-OH pyrene "equivalent" levels (indicative of pyrogenic PAH contamination) decreased with increasing distance from a point source of pyrogenic PAH (a large aluminium works). The assay was shown to be suitable for the detection of PAH exposure in wild crustacean populations, for discriminating between contaminated and clean sites and is also sufficiently sensitive to detect gradients of PAH contamination. The method provides a rapid, inexpensive and non-destructive measure of biologically available PAH in crustaceans.     

羊栖菜多糖的提取和抗氧化活性研究

羊栖菜(Sargassum fusiforme)是一种有"长寿菜"美誉的经济藻类,其中的多糖成分具有良好的抗氧化活性。本文依次用冷水和热水处理羊栖菜藻粉,得到两种多糖:冷水浸提多糖(SFCP)和热水煮提多糖(SFHP);随后对这两种多糖的理化性质进行比较,并通过测定二者对DPPH和羟自由基的清除能力评价其抗氧化活性。结果表明,两种多糖都是含有硫酸根的岩藻聚糖,但是SFCP中糖醛酸含量较高,分子质量分布集中,而SFHP中硫酸根含量较多,分子质量分布较宽,且二者硫酸根取代位置不同。两种多糖对DPPH和羟自由基均有一定的清除效果,且清除率与浓度存在剂量依赖关系。相比较而言,SFCP对自由基的清除效率较好。     

浒苔多糖的分级纯化及保湿活性研究

用DEAE-Sepharose FF离子交换凝胶层析柱对浒苔(Enteromorpha)多糖进行分级纯化,利用HPLC色谱分析了单糖组成,并以甘油和透明质酸为对照,研究其吸湿和保湿活性。结果表明,浒苔多糖主要为葡萄糖醛酸-鼠李糖-木糖-甘露糖聚合物;浒苔多糖主要由两种组分组成,其一组分木糖和鼠李糖含量摩尔比为1.11:1,另一组分木糖和鼠李糖含量比为1:3.28;浒苔多糖具有与透明质酸相似的吸湿和保湿能力。     

Birds, seals and the suspension culture of mussels in Bantry Bay, a non-seaduck area in Southwest Ireland

Concerns about the environmental impacts of mariculture have grown in recent years in response to the rapid expansion of the industry. The blue mussel (Mytilus edulis) is the main product of shellfish mariculture in the Northeast Atlantic and Baltic Sea, with approximately one third of the harvest cultured using suspended longlines within sheltered marine areas. The main aim of this study was to examine the interactions, and assess the impacts (if any) of mussel suspension culture on the seabird and seal community, employing a simultaneous study of culture and control sites. The study spanned a 20-month period (from November 2001 to August 2003) and encompassed six sites in Bantry Bay (Southwest Ireland).There was no significant difference in species richness between mussel and control sites. Similarly, species diversity did not significantly differ between the mussel and control sites although control sites were generally more diverse than mussel sites, the latter particularly dominated by large numbers of Laridae.Significantly higher numbers of Phalacrocoracidae, Laridae and Alcidae were recorded in mussel sites than in control sites. However, no significant difference was found between Gaviidae or common seal (Phoca vitulina) numbers in mussel and control sites. Seasonal patterns of abundance were similar in mussel and control sites, with peak numbers of most species groups occurring in spring.Mussel suspension culture does not appear to have an adverse effect on the abundance of seabirds or common seals in this area. The safe perching platforms provided by suspension culture floats, combined with a number of other factors, contribute to an increased abundance of a number of seabird species, particularly Laridae. The possible interactions between vertebrate predators and mussel suspension aquaculture are discussed and possible explanations for the increased seabird abundance observed in these areas are offered.