The pattern of organochlorines in mussels Mytilus edulis L. from the south west Baltic Sea

The concentration of organochlorines (PCB, DDT and its metabolites, HCH isomers and HCB) were determined in mussel, Mytilus edulis from the south west Baltic Sea. The harbor Kiel Innenförde showed the highest contamination with PCB (487 g/kg dry weight), the Gelting bathing beach area the lowest contamination (38 g/kg). The highest contamination with DDT was measured in Lübeck bay, which may be explained by the long term application of DDT in the former German Democratic Republic.Penta- and hexa-chlorinated PCB were selectively accumulated in Mytilus edulis whereas highly lipophilic PCB with more than seven chlorine atoms were found in negligible concentrations. The accumulation of these compounds in mussels is determined by equilibrium partitioning with ambient water.     

Analysis of nodularin-R in eider (Somateria mollissima), roach (Rutilus rutilus L.), and flounder (Platichthys flesus L.) liver and muscle samples from the western Gulf of Finland, northern Baltic Sea

Nodularin (NODLN) is a cyanobacterial hepatotoxin that may cause toxic effects at very low exposure levels. The NODLN-producing cyanobacterium Nodularia spumigena forms massive blooms in the northern Baltic Sea, especially during the summer. We analyzed liver and muscle (edible meat) samples from common eider (Somateria mollissima), roach (Rutilus rutilus L.), and flounder (Platichthys flesus L.) for NODLN-R by liquid chromatography/mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA). Thirty eiders, 11 roach, and 15 flounders were caught from the western Gulf of Finland between September 2002 and October 2004. Eiders from April to June 2003 were found dead. The majority of samples were analyzed by LC-MS and ELISA from the same sample extracts (water:methanol:n-butanol, 75:20:5, v:v:v). Nodularin was detected in 27 eiders, nine roach, and eight flounders. Eider liver samples contained NODLN up to approximately 200 microg/kg dry weight and muscle samples at approximately 20 microg/kg dry weight, roach liver samples 20 to 900 microg NODLN/kg dry weight and muscle samples 2 to 200 microg NODLN/kg dry weight, and flounder liver samples approximately 5 to 1,100 microg NODLN/kg dry weight and muscle samples up to 100 microg NODLN/kg dry weight. The NODLN concentrations found in individual muscle samples of flounders, eiders, and roach (1-200 microg NODLN/kg dry wt) indicate that screening and risk assessment of NODLN in Baltic Sea edible fish and wildlife are required for the protection of consumer's health.     

Temporal and spatial variations of selected biomarker activities in flounder (Platichthys flesus) collected in the Baltic proper

The aim of the study was to examine temporal (monthly) and spatial changes in the activities of a battery of biomarkers and evaluate the influence of gender and environmental factors on those activities. Abiotic factors not correlated with chemical pollution—water temperature, salinity, and oxygen concentration—were evaluated.The activities of the following biomarkers—muscular ChEs (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)), hepatic ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST) and catalase (CAT), and gross indices—condition factor (CF), hepatosomatic index (HSI), and gonadosomatic index (GSI)—were measured in individual flounder Platichthys flesus. Female flounder were collected once a month from April 2003 to April 2004 at Sobieszewo (Gulf of Gdańsk). In December 2003 flounder of both sexes from nine stations in the Baltic Proper were caught and tested.Strong monthly (attributed mainly to spawning, and less—to pollution) and geographical (attributed to pollution) variations in biomarker activities, as well as gender differences, were demonstrated. Correlations between the enzymes with the abiotic properties of the environment were less important.     

Comparison of accumulation of micropollutants between igenous and transplanted zebra mussels (Dreissena polymorpha)

Zebra mussels (Dreissena polymorpha) were exposed at 12 canals and lakes situated in Flanders (Belgium), in cages for six weeks during the summer of 2002. Accumulation of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), hexachlorobenzene, and trace metals were measured in the transplanted mussels and levels compared to levels in indigenous mussels. Additionally, zebra mussels were exposed at a small lake in the vicinity of Antwerp (Belgium), and accumulation of contaminants was followed for an extended period from December 2001 to April 2002. Analysis of the pollutants in the indigenous mussels showed that the selected sites displayed a wide range of pollution from near to background to very high levels of metals and/or organic contaminants when compared to the literature. For organic contaminants and for most metals, comparison of levels between caged and resident mussels revealed no significant differences. Only for cadmium and nickel, significant differences were observed, with levels being either higher (cadmium) or lower (nickel) in caged mussels. For organic contaminants, significant correlations between levels in caged and resident mussels were found with r2 values up to 0.98. For some metals, no or poor correlations were found. At most sites, concentrations of those metals were of the same order of magnitude and comparable to levels in mussels from unpolluted sites. This might explain the absence of significant correlations. When mussels were exposed for an extended period, the concentration of some pollutants increased, whereas others decreased with time. Only in the case of certain metals did levels differ significantly because of the slow depuration of metals already present in the transplanted mussels. This is an additional indication that measured concentrations in transplanted mussels indeed reflected the local situation. With this study, we were able to prove the applicability of transplanted mussels as a biomonitoring tool for the accumulation of pollutants over a wide range of pollution.     

Genetic and physiological responses of flounder (Platichthys flesus) populations to chemical contamination in estuaries

We sampled and analyzed European flounder (Platichthys flesus) from two highly contaminated estuaries (Seine and Loire, France) and one moderately contaminated estuary (reference site: Ster, France). Significant and convergent modifications of the allelic frequencies for the loci phosphoglucomutase (PGM), glucose phosphate isomerase 2 (GPI-2), mannose phosphate isomerase (MPI), and aspartate aminotransferase (AAT-2) were evident for fish in the contaminated sites versus fish from the reference site. Back-calculation from otoliths showed that the average growth rate of fish between the first and the second winter was greater at the reference site (approximately 150 mm/year) than at the contaminated sites (approximately 100 mm/year). Flounder from the reference site also had a higher condition factor (somatic wt/(fish length)3) compared to fish from the two contaminated sites. However, the observed pattern of growth rate and condition factor might be biased by particular environmental conditions other than contaminants and must be confirmed by more extensive study. Flow cytometry analysis of fish blood revealed a significant difference in the frequency of abnormal profiles for fish from the Seine (20%) versus from the Ster (3%). We interpret this result as a marked genotoxic effect of contaminants on fish in the Seine system. Some genotypes, such as PGM 85/85, appeared to be linked to the measured components of fitness, particularly to DNA integrity. Thus, these genotypes might be considered to be more tolerant to pollutants. The frequency of the PGM 85 allele was clearly elevated in flounder from the more contaminated sites, compared to flounder from the reference site.     

Effects of season,stock, and laboratory protocols on survival of zebra mussels (Dreissena polymorpha) in bioassays

A series of experiments were conducted to determine the effects of maintenance method (fed or starved), stock location, season, mussel size, and rate of acclimation to temperature on the responses (mortality) of zebra mussels in bioassays. Mussels maintained on a diet of crushed Chlorella are more tolerant to Bayer 73^® and more sensitive to sodium hypochlorite than starved mussels. Variability in LC50s of zebra mussels is high during the first 60 days in the laboratory, after which the resistance of mussels to both hypochlorite and Bayer 73^® declines with reductions in body condition. Zebra mussels collected during the early summer and late fall are more tolerant to both hypochlorite and Bayer 73^®. There is significant variation in tolerances to biocides depending on the stock, such that stocks from locations with more degraded water quality have increased tolerances. Acclimating mussels from 4 to 20°C at rates of 2 and 10°C d^–1 does not significantly affect tolerance to biocides. In general, LC50s of mussels vary by only 2–3×, suggesting that mussels from any location, any season, and maintained under any maintenance protocol can be used in range-finding tests. Comparisons of results among studies requires knowledge of mussel stock, collection season, and laboratory maintenance protocols.     

Long-term exposure of European flounder (Platichthys flesus) to the flame-retardants tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD)

Tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD) are widely used flame retardants that have increasingly been found as contaminants in the aquatic environment. In the present study, European flounder (Platichthys flesus) were chronically exposed to TBBPA; (105 days) and HBCD (78 days), in a wide range including environmentally relevant concentrations. TBBPA was administered via the water, whereas HBCD was administered in food and sediment, or in sediment alone. Chemical analysis of muscle showed an average increase in internal concentrations of approximately two orders of magnitude for both compounds tested. Animals exposed to HBCD via sediment alone (8000 microg/g total organic carbon, TOC) showed a proportional increase of alpha-HBCD in muscle compared to animals exposed via food and sediment. In both studies, exposure to the test compounds did not affect general health and toxicity parameters (behavior, survival, growth rate, relative liver and gonad weight). Hepatic microsomal enzyme activities (TBBPA: EROD; HBCD: EROD, PROD, and BROD) were not induced by any of the tested chemicals. Aromatase activity in male gonads showed a mild increase with rising TBBPA levels. There were no morphological and immunohistochemical indications for increased production of the yolk precursor protein vitellogenin (VTG) in animals exposed to TBBPA and HBCD; immunochemical analysis of plasma VTG levels showed no dose response in animals exposed to TBBPA. In animals exposed to TBBPA, levels of the thyroid hormone thyroxin (T(4)) increased with internal concentrations of the test compound, possibly indicating competition of TBBPA for plasma protein binding. Triiodothyronin (T(3)) levels were not affected and histology showed no signs of altered thyroid gland activity. Other organs investigated (liver, gills, kidney, skin, and gonads) revealed no histological changes related to TBBPA or HBCD exposure. Overall, the present results indicate limited endocrine effects of these widely used flame retardants in a test species representative of European estuaries at environmentally relevant exposure levels and at internal levels up to 4300 ng TBBPA/g wet weight, and 446 microg HBCD/g lipid weight in flounder muscle.     

Relationship between sex steroid and vitellogenin concentrations in flounder (Platichthys flesus) sampled from an estuary contaminated with estrogenic endocrine-disrupting compounds

High concentrations of vitellogenin (VTG; egg yolk protein) have previously been found in male flounder (Platichthys flesus) from several UK estuaries; these levels have been ascribed to the presence of estrogenic endocrine-disrupting compounds (EDCs). Gonadal abnormalities, including intersex, have also been recorded in these estuaries. However, there is no firm evidence to date that these two findings are causally linked or that the presence of estrogenic EDCs has any adverse population effects. In the present study, we examined the relationship between concentrations of VTG and sex steroids (11-oxo-testosterone in males and 17beta-estradiol in females) in specimens of flounder captured from the estuary of the River Mersey. We first questioned whether the high concentrations of VTG in male and immature female flounder were indeed caused by a direct effect of exogenous EDCs and not indirectly via the endogenous secretion of 17beta-estradiol. The data favored the direct involvement of estrogenic EDCs. We then questioned whether the presence of estrogenic EDCs not only stimulated inappropriate VTG synthesis but whether it might also have had a negative effect on endogenous steroid secretion. It should be noted that the predicted consequences of a drop in steroid secretion include smaller gonads, smaller oocytes, fewer numbers of sperm, and depressed spawning behavior. This question was more difficult to answer because of the strong effect of the seasonal reproductive cycle and stage of maturation on steroid concentrations. However, matched by month of capture and stage of maturation, both 17beta-estradiol in females and 11-keto-testosterone in males were in most cases significantly lower in those years when VTG concentrations were higher.     

Fast and easy method for total DNA extraction and gene amplification from larvae,spat and adult mussels Mytilus trossulus from the Baltic Sea

Three nuclear DNA markers that diagnostically differentiate mussels within the Mytilus edulis complex (M. edulis, M. trossulus and M. galloprovincialis) are commonly used in taxonomic investigations: Glu5’, ITS and EFbis. As a rule, DNA extraction is performed before amplification. It is a time consuming process in the case of traditional methods based on chloroform and phenol extraction or relatively expensive using kits with ready spin columns. Moreover, DNA isolation from larvae is problematic, because of the small amount of tissue available. In this report we describe a simple, fast and inexpensive method of DNA extraction and gene amplification from larvae, spat and adults of the Baltic mussel Mytilus trossulus. The extraction method is adapted from that of Wang et al. (2006) and is based on digestion of tissue or whole animals in STE solution and direct gene amplification. On the basis of the results of routine analyses of mussels carried out in our laboratory we have concluded that the method we propose gives results that are consistent with standard methods, without requiring expensive reagents/equipment and is time saving.     

Combined effect of body size,season, and location on trace element levels in mussels (Mytilus edulis)

Specimens ofMytilus edulis (mussels) were collected at one-month intervals over a one-year period from two locations in Burrard Inlet, British Columbia, Canada and less frequently from other locations in the S.W. region of B.C., and analyzed for concentrations of Mn, Fe, Cu, Zn, Br, Pb, and Sr using X-ray energy spectroscopy. Size and condition of the mussels were recorded at the same time. The data were analyzed using multiple linear regression with size and season as predictor variables and trace metal contents as criterion variables. The values of the regression coefficients changed according to whether or not the mussels were collected from a polluted environment in disagreement with previously published studies. Principal component analysis of the correlation matrix of the variables yielded three principal components one of which can be used to ascertain whether or not mussels were collected from either a polluted or an unpolluted environment.     

Stimulation of oxyradical production of hepatic microsomes of flounder (Platichthys flesus) and perch (Perca fluviatilis) by model and pollutant xenobiotics

Stimulation of hepatic microsomal NAD(P)H-dependent hydroxyl radical (·OH) production by model compounds, viz. menadione (2-methyl-1,4-naphthoquinone) and nitrofurantoin (N-(5-nitro-2-furfurylidene)-1-aminohydantoin), and pollutant xenobiotics, viz. benzo[a]pyrene (BaP) diones (products of microsomal BaP metabolism), duroquinone (tetramethyl-1,4-benzoquinone—present in pulp mill effluent), and the pesticide lindane (-hexachlorocyclohexane), was examined in flounder Platichthys flesus. Duroquinone was also studied in perch Perca fluviatilis, a freshwater species used in studies of pulp mill effluents in the aquatic environment. Microsomal ·OH production was detected by the oxidation of the scavenging agent 2-keto-4-methiolbutyric acid (KMBA), using FeCl₃/EDTA as a promotor of the Haber-Weiss reaction (O ₂ ^– +H₂O₂=·OH+OH^–+O₂). All xenobiotics tested, except lindane, showed synergistic interactions with ferric/EDTA indicative of redox cycling of the xenobiotic. Inhibition of menadione- and nitrofurantoin-stimulated ·OH production by superoxide dismutase (50% inhibition) and catalase (80%) indicated respectively the involvement of O ₂ ^– and H₂O₂ in ·OH production. Maximal rates of KMBA oxidation (Vmax in nmol ethylene/min/mg protein) were similar for NADH and NADPH for menadione (4.58–4.61) and duroquinone (0.26–0.3 [flounder] and 0.93–0.99 [perch]), higher for NADPH than NADH for nitrofurantoin (1.21 and 0.77), and higher for NADH than NADPH for BaP diones, decreasing in the order 1,6-dione (1.12 and 0.14), 3,6-dione (0.75 and 0.25), and 6,12-dione (0.31 and 0.09). Rates for lindane, lacking a redox cycling structure, were low (0.01–0.05). Apparent K_m (app. K_m) values for xenobiotic were 1–2 orders of magnitude lower for BaP diones than the other compounds. App. K_m was lower for NADH than NADPH for 3,6-dione (1.23 and 1.66 M) and 6,12-dione (0.85 and 1.81 M), but the reverse of this was found for the 1,6-dione (1.41 and 0.78 M). App. K_m values were almost identical for menadione and duroquinone and lower for NADPH (32–44 M) than NADH (346–382 M). The reverse was seen for nitrofurantoin, viz., 76 M (NADH) and 269 M (NADPH). Hepatic 1000 g supernatants of P. flesus metabolized BaP to oxyradical-generating products, moreso for -naphthoflavone-induced than control fish, and production was reduced by UDP-glucuronic acid for the latter but not the former. The studies indicate a widespread potential for contaminant-stimulated oxyradical generation via redox cycling and other free radical interactions of xenobiotics and their metabolites.