Morten Sandvik

Contaminant accumulation and biomarker responses in flounder (Platichthys flesus L.) and Atlantic cod (Gadus morhua L.) exposed by caging to polluted sediments in Sørfjorden, Norway

Flounder (Platichthys flesus L.) and Atlantic cod (Gadus morhua L.) were subjected to caging at polluted sediments in a Norwegian fjord (Sorfjorden) for a period of 3 months. Three caging sites were located close to metal smelters, whereas a fourth site was located 30 km away as a reference. In sediment samples, polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and heavy metals were elevated at the innermost sites (1, 2 and 3) compared with the reference location (site 4). In fish, the biliary levels of fluorescent aromatic compounds (FACs) were elevated 5–20 fold in both species at the polluted sites. A two-fold difference in heavy metal levels was observed in cod (site 2 vs. 4), whereas no differences were seen in flounder. Pesticides bioaccumulated in a diffuse manner at all sites. In flounder at the innermost sites, plasma aspartate aminotransferase (AST) and hepatic cytochrome P450 1A (CYP1A) dependent 7-ethoxyresorufin O-deethylase (EROD) activity were elevated 4–5 and 5–10 fold, respectively, compared with the reference site. Both of these biomarkers were significantly correlated with FACs levels. For other biomarkers, the site effect was more marginal. The biomarkers seemed in general more responsive in flounder than in cod. The present study demonstrates biomarker measurements in caged fish as a promising approach for evaluating accumulation and effects of pollutants in marine teleosts.

A convenient and cost-effective method for monitoring marine algal toxins with passive samplers

Passive sampling disks were developed based on the method of MacKenzie, L, Beuzenberg, V., Holland, P., McNabb, P., Selwood, A. [2004. Solid phase adsorption toxin tracking (SPATT): a new monitoring tool that simulates the biotoxin contamination of filter feeding bivalves. Toxicon 44, 901-918] and protocols were formulated for recovering toxins from the adsorbent resin via elution from small columns. The disks were used in field studies to monitor in situ toxin dynamics during mixed algal blooms at Flødevigen in Norway. Examples are given from time-integrated sampling using the disks followed by extraction and high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis for azaspiracids, okadaic acid analogues, pectenotoxins, yessotoxins and spirolides. Profiles of accumulated toxins in the disks and toxin profiles in blue mussels (Mytilus edulis) were compared with the relative abundance of toxin-producing algal species. Results obtained showed that passive sampling disks correlate with the toxin profiles in shellfish. The passive sampling disks were cheap to produce and convenient to use and, when combined with HPLC-MS or enzyme-linked immunosorbent assay (ELISA) analysis, provide detailed time-averaged information on the profile of lipophilic toxin analogues in the water. Passive sampling is therefore a useful tool for monitoring the exposure of shellfish to the toxigenic algae of concern in northern Europe.

Biomarkers in flounder (Platichthys flesus): an evaluation of their use in pollution monitoring

Flounder (Platichthys flesus) is among the most common fish-species in Norwegian and European estuaries. It lives in or on sediments from which it also finds most of its food. The aim of the present work was to evaluate biomarkers in flounder for possible future use in monitoring programmes. There were clear biomarker responses in flounder following injection of model contaminants benzo[a]pyrene (B[a]P), PCB #156 and Cd, singly or in sequence. Cytochrome P4501A responded following injection of the organic contaminants and metallothionein (MT) following Cd injection. All groups receiving B[a]P, either singly or in combination with other contaminants, accumulated high levels of B[a]P-metabolites in bile. There was little change in glutathione-S-transferase activity (measured using CDNB as substrate) following the treatments. Starvation appeared to affect the response of hepatic MT to Cd, but none of the other biomarkers. PAH in sediments elicited strong biomarker responses in caged flounder, whereas sediment-associated metals appeared to be largely unavailable to flounder in this study.

Interaction of benzo[a]pyrene, 2,3,3',4,4',5-hexachlorobiphenyl (PCB-156) and cadmium on biomarker responses in flounder (Platichthys flesus L)

Interactive effects of a mixed pollutant exposure on biomarker responses were studied in European flounder (Platichthys flesus L.). The model chemicals, benzo[a]pyrene (BaP, 2.5 mg kg-1), 2,3,3′,4,4′5 hexachlorobiphenyl (PCB-156, 2.5 mg kg-1), and cadmium (cadmium, 1 mg kg-1), were administered to fish by subcutaneous injections. Biomarker responses were quantified both following administration of single chemicals and sequential combinations of the chemicals in pairs. Significant induction of CYP1A protein levels and corresponding ethoxyresorufin-O-deethylase (EROD) activities was observed in BaP and PCB treated flounder after 2 and 8 days, respectively. The strongest induction (44 fold) was caused by BaP. No further induction was observed after additional treatment with PCB 156. CYP1A induction caused by BaP was inhibited (40% compared with BaP treatment alone) in flounder pre treated with cadmium, whereas induction by PCB 156 appeared to be unaffected by pre treatment with cadmium. Flounder treated with...

Levels and patterns of persistent organic pollutants (POPs) in tilapia (Oreochromis sp.) from four different lakes in Tanzania: Geographical differences and implications for human health

In Tanzania fish is one of the most important protein sources for the rapidly increasing population. Wild fish is threatened by overfishing and pollution from agriculture, industries, mining, household effluents and vector control. To monitor possible implications for public health, the geographical differences of the occurrence and levels of persistent organic pollutants (POPs) in tilapia fish (Oreochromis sp.) from four different Tanzanian lakes were investigated in 2011. Concentrations of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), polybrominated diphenyls (PBDEs) and hexabromocyclododecane (HBCDD) were determined in pooled samples of tilapia muscle from Lake (L) Victoria, L. Tanganyika, L. Nyasa (also called L. Malawi) and L. Babati in Tanzania in 2011. Levels of Σ-DDTs (274 ng/g lipid weight (lw)) and sum of 7 indicator PCBs (Σ-7PCBs) (17 ng/g lw) were significantly higher in tilapia from L. Tanganyika compared to the other lakes. The highest levels of Σ-endosulfan (94 ng/g lw) were detected in tilapia from L. Victoria. Toxaphenes were detected in low levels in fish from L. Tanganyika and L. Babati. Results revealed a geographic difference in the use of DDT and endosulfan between L. Victoria and L. Tanganyika. Low ratios of DDE/DDT in tilapia from L. Tanganyika indicated an on-going use of DDT in the area. Median levels of ΣBDEs, including BDE-209, were highest in L. Victoria (19.4 ng/g lw) and BDE-209 was present in 68% of the samples from this lake. The presence of BDE-209 indicates increasing influence of imported products from heavy industrialized countries. The measured POP levels in the studied tilapia were all below MRLs of EU or were lower than recommended levels, and thus the fish is considered as safe for human consumption. They may, however, pose a risk to the fish species and threaten biodiversity.

Diversity of cyanobacteria and cyanotoxins in Hartbeespoort Dam, South Africa

The South African Hartbeespoort Dam is known for the occurrence of heavy Microcystis blooms. Although a few other cyanobacterial genera have been described, no detailed study on those cyanobacteria and their potential toxin production has been conducted. The diversity of cyanobacterial species and toxins is most probably underestimated. To ascertain the cyanobacterial composition and presence of cyanobacterial toxins in Hartbeespoort Dam, water samples werecollectedinApril2011.Inapolyphasicapproach,27isolatedcyanobacterialstrainswereclassifiedmorphologically and phylogenetically and tested for microcystins (MCs), cylindrospermopsin (CYN), saxitoxins (STXs) and anatoxin-a (ATX) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and screened for toxin-encoding gene fragments. The isolated strains were identified as Sphaerospermopsis reniformis, Sphaerospermopsis aphanizomenoides, Cylindrospermopsis curvispora, Raphidiopsis curvata, Raphidiopsis mediterrranea and Microcystis aeruginosa. Only one of the Microcystis strains (AB2011/53) produced microcystins (35 variants). Forty-one microcystin variants were detected in the environmental sample from Hartbeespoort Dam, suggesting the existence of other microcystin producing strains in Hartbeespoort Dam. All investigated strains tested negative for CYN, STXs and ATX and their encoding genes. ThemcyEgeneofthemicrocystingeneclusterwasfoundinthemicrocystin-producingMicrocystisstrainAB2011/53and in eight non-microcystin-producing Microcystis strains, indicating that mcyE is not a good surrogate for microcystin production in environmental samples. Additional keywords: Cylindrospermopsis, Hartbeespoort Dam, microcystin, Microcystis, Raphidiopsis, Sphaerospermopsis.

LC-MS Analysis with Thiol Derivatization to Differentiate [Dhb7]- from [Mdha7]-Microcystins: Analysis of Cyanobacterial Blooms, Planktothrix Cultures and European Crayfish from Lake Steinsfjorden, Norway

Kinetic studies showed that [Asp(3), Dhb(7)]MC-RR reacted with mercaptoethanol hundreds of times more slowly than MC-RR and a range of other [Mdha(7)]-containing microcystin congeners. The difference in reaction rate was sufficiently large that derivatization of microcystin-containing samples with mercaptoethanol, followed by LC-MS analysis, clearly discriminated between microcystins containing the isobaric [Dhb(7)]- and [Mdha(7)]-groups. Application of this approach, using LC-MS with both-ion trap and triple-quadrupole mass spectrometers, to water samples and Planktothrix cultures from Lake Steinsfjorden, Norway, demonstrated the presence of [Asp(3), Dhb(7)]MC-RR (5), [Asp(3)]MC-RY (14), and [Asp(3)]MC-LY (16), as well as analogues tentatively identified as [Asp(3)]MC-RR (4), [Asp(3), DMAdda(5), Dhb(7)]MC-LR (6), [Asp(3), Dhb(7)]MC-HtyR (8), [Asp(3)]MC-HtyR (9), [Asp(3), Dhb(7)]MC-LR (10), [Asp(3)]MC-LR (11), [Asp(3), Dhb(7)]MC-RY (15), and [Asp(3), Dhb(7)]MC-LY (17), together with low levels of several other analogues. This is the first use of this thiol-based LC-MS approach to identify Dhb-containing microcystins, and allowed identification of LC-MS peaks in a mixture of [Mdha(7)]- and [Dhb(7)]-congeners of [Asp(3)]MC-RR (4, 5), -RY (14, 15), and -LY (16, 17) in the samples from L. Steinsfjorden. This is also the first report of MC-RY-congeners outside of Africa, or in Planktothrix spp. Analysis of European crayfish (Astacus astacus) taken from L. Steinsfjorden revealed the presence of only trace levels of microcystins in the edible parts.

Estrogenic effects of selected hydroxy polychlorinated biphenyl congeners in primary culture of Atlantic Salmon (Salmo salar) hepatocytes.

Many persistent organic pollutants are known to have endocrine-disrupting effects in several aquatic and terrestrial species. In this regard, hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs) represent serious health and environmental concern because they are shown to act agonistic or antagonistic at hormone receptors (HRs) or to cause hormone-receptor-mediated responses. In the present study, salmon primary hepatocytes were used to study alterations in an estrogen signaling pathway resulting from exposure to four hydroxylated (4OH-CB 107, 4OH-CB146, 4OH-CB187, and 3OH-CB138) metabolites of PCB at different concentrations using quantitative real-time polymerase chain reaction. The effects of the PCB metabolites were compared to the mRNA expression in 17α-ethynylestradiol (EE2)-treated cells. Concentration-specific increase of vitellogenin (Vtg) mRNA transcription after exposure to OH-PCBs was observed. Decreased mRNA transcription was observed for zona radiata protein (Zr-protein) and cytochrome P450 side-chain cleavage (P450scc) enzyme. For estrogen receptor β (ERβ), the mRNA expression pattern was OH-PCB-metabolite congener-specific. A novel aspect of this study is that OH-PCBs produced effects on hepatic steroidogenic pathways by targeting the StAR protein and P450scc genes. Given that endocrine toxicology research mainly has focused on estrogenicity involving direct ER-mediated effects and that steroidogenic enzyme and proteins are highly tissue- and cell-type-specific and controlled by different promoters and second-messenger pathways, the present findings provide potential new targets for interaction with xenobiotics such as hydroxylated congeners of certain chemicals. The quantitative expression patterns of hepatic and extrahepatic steroidogenic genes and proteins after exposure to environmental contaminants are the subject of systematic investigations in our laboratory.

Differential Retention of Okadaic Acid (OA) Group Toxins and Pectenotoxins (PTX) in the Blue Mussel, Mytilus edulis (L.), and European Flat Oyster, Ostrea edulis (L.)

ABSTRACT This paper presents the results from an experiment, where uptake and elimination of diarrhetic shellfish toxins (DST) of the okadaic acid (OA) and pectenotoxin (PTX) groups were compared between blue mussels (Mytilus edulis) and European oysters (Ostrea edulis). Caged mussels and oysters were suspended in the water column and exposed to a dense bloom of Dinophysis acuta (500–2000 cells L-1) for 4 wk, which was followed by detoxification in the laboratory during 7 wk. Weekly sampling and analysis of OA-group toxins including fatty acid esters (‘DTX-3’) as well as PTX in individual shellfish and plankton samples were performed. The results showed that mussels rapidly accumulated OA-group toxins to levels about 10 times above the regulation limit (160 µg OA kg-1 mussel) whereas concentrations never reached this limit in oysters during the field exposure. Overall, levels were 10–50 times greater in mussels. The OA-group toxins were mainly in the form of esters (>90%) in oysters, whereas in mussels, the esters constituted only a minor proportion of total OA toxin levels. Reduction rates were estimated for each OA toxin to evaluate if faster elimination could explain the lower toxin retention in oysters. However, no consistent species-specific difference in reduction rates were observed, but esters of OA appeared to be reduced at a faster rate in oysters (t1/2 = 23 days) compared with mussels (t1/2 = 35 days). In both species, the free form of OA was eliminated at a faster rate (t1/2=15–17 days) compared with free DTX1 (t1/2 = 23–31 days) and DTX2 (t1/2 = 28–33 days). Slightly slower elimination rates were estimated for the ester forms (t1/2 = 23–42 days). Regarding PTX, PTX2 seco acid (PTX2 SA) was the major PTX detected in both species, but small amounts of PTX2, PTX12 and PTX12 SA were also found. As for the OA-group toxins, oysters generally contained lower total amounts of PTX compared with mussels, but the difference was much less apparent. Estimation of reduction rates of the different PTX compounds showed that these toxins were rapidly eliminated in both oysters and mussels (t1/2 = 6–13 days). We propose that differential rates of gut assimilation and/or biotransformation of the OA-group and PTX explain some of the observed differences in retention and toxin profiles between the bivalves, rather than differences in elimination rates. However, models related to differences in feeding rates, particle selection and behavioural response to toxic algae should be tested in future experiments to evaluate the importance of preingestive mechanisms to the differential toxin retention in these bivalves.From the industrial perspective, our results suggest that O. edulis may be regarded as a low-risk species for DST contamination, which should be taken into consideration by regulatory authorities in charge of sampling frequencies and monitoring programs for shellfish toxins.

Assessment of pollution in sewage ponds using biomarker responses in wild African sharptooth catfish (Clarias gariepinus) in Tanzania

The interactive effects of mixed pollutants in sewage wastewater on biomarker responses were investigated using wild male African sharptooth catfish (Clarias gariepinus) in Morogoro, Tanzania. A total of 58 fish were used, of which 21 were from Mindu dam (reference site) and 22, 9 and 10 from Mafisa, Mazimbu and Mzumbe sewage ponds, respectively. Liver somatic index (LSI) and gonadosomatic index (GSI) were significantly greater (two- to threefold) and (five- to sixfold), respectively, in fish from all sewage ponds. Haemoglobin concentration and gill filament 7-ethoxyresurufin O-deethylase (EROD) activities were significantly higher (1.2-fold and twofold, respectively) in fish from Mzumbe sewage ponds than in fish from Mindu dam, whereas liver EROD activity was significantly higher in fish from Mzumbe and Mafisa sewage ponds (5-fold). A HPLC method for determination of enzymatically formed p-nitrophenyl-glucuronide (PNPG) was developed and applied to measure UDP-glucuronosyl transferase (UGT) activities that was significantly higher in fish from all sewage ponds (2–2.5-fold) than in fish from Mindu dam. Kinetic characteristics and assay dependence of UGT were studied with microsomal preparations. Metallothionein (MT) content was significantly lower (three- to fourfold) in fish from sewage ponds than in fish from Mindu dam, and corresponded with cumulative levels of cadmium, lead and mercury. Condition factor, vitellogenin (Vtg), acetylcholinesterase (AChE) activities in plasma, eyes and brain, haematocrit, plasma protein and cytosolic glutathione S-transferase (GST) activities were comparable in fish from sewage ponds and Mindu dam. Although specific pollutants other than the metals were not identified by chemical analysis, application of a suite of biomarkers in C. gariepinus demonstrated that all sewage ponds were contaminated by pollutants of public health concern.