Philippe Tony Hoff

Toxicity evaluation of perfluorooctane sulfonate (PFOS) in the liver of common carp (Cyprinus carpio).

Perfluorooctane sulfonate (PFOS) has been manufactured for over 50 years in increasing quantities and has been used for several industrial and commercial aims. Due to persistence and bioaccumulation of this pollutant, it can be found worldwide in wildlife and humans. Biochemical effects of PFOS exposure are mainly studied in mammalian model species and information about effects on fish species remain largely scarce. This lack of toxicity data points out that there is an urgent need for the mechanistic molecular understanding of the mode of action of this pollutant. In the present study, common carp (Cyprinus carpio) was exposed through water for 14 days at concentrations of 0.1, 0.5 and 1 mg/l PFOS. Liver was selected as target tissue. Custom microarrays were constructed from cDNA libraries obtained with Suppression Subtractive Hybridization-Polymerase Chain Reaction (SSH-PCR) experiments. Microarray data revealed that the expression of several genes in the liver was influenced by PFOS exposure and real-time PCR was used to confirm these gene expression changes. The affected genes were mainly involved in energy metabolism, reproduction and stress response. Furthermore, the relative condition factor, the hepatosomatic index, and the available glycogen reserves of the exposed fish were significantly lower after 14 days of exposure than in the control fish. At all levels of biological organization, indications of a trade-off between the metabolic cost of toxicant exposure on one hand and processes vital to the survival of the organism on the other hand were seen. Our results support the prediction that increases in energy expenditure negatively affects processes vital to the survival of an organism, such as growth.

EXPOSURE PATTERNS OF PERFLUOROOCTANE SULFONATE IN AQUATIC INVERTEBRATES FROM THE WESTERN SCHELDT ESTUARY AND THE SOUTHERN NORTH SEA

Over the past decades little research has been conducted on the environmental behavior and effects of fluorinated organochemicals (FOCs). Recently it has been reported that perfluorooctane sulfonic acid (PFOS) is occurring worldwide. Little is known about the PFOS levels in organisms originating from the southern North Sea and the Western Scheldt estuary. In this study, we determined, for the first time, the PFOS-exposure levels in Crangon crangon, Carcinus maenas, and Asterias rubens from these ecosystems. Concentrations on a wet-weight basis in soft tissues of shrimp, crab, and starfish ranged from 19 to 520 ng/g, from 24 to 877 ng/g, and from 9 to 176 ng/g, respectively. These results show the existence of a PFOS pollution gradient in organisms along the Western Scheldt estuary, with the highest concentrations near Antwerp. The range of PFOS levels in shrimp and crab are slightly higher in coastal regions compared with sampling sites in open water. This study shows widespread distribution of PFOS in the Belgian and Dutch marine and estuarine environment at rather high concentrations.

Evaluation of the toxicological effects of perfluorooctane sulfonic acid in the common carp (Cyprinus carpio).

In the present study we evaluated the toxicological effects of a scarcely documented environmental pollutant, perfluorooctane sulfonic acid (PFOS), on selected biochemical endpoints in the common carp, Cyprinus carpio. Juvenile organisms were exposed to PFOS through a single intraperitoneal injection (liver concentrations ranging from 16 to 864 ng/g after 5 days of exposure) and after 1 and 5 days effects were assessed in liver and serum of the exposed organisms. The investigation of the hepatotoxicity of PFOS included the determination of the peroxisome proliferating potential (peroxisomal palmitoyl CoA oxidase and catalase activity) and the compounds influence on the average DNA basepair length (ABPL) by agarose gel electrophoresis. Total antioxidant activity (TAA), cholesterol and triglyceride levels were monitored in the serum. After 1 day of exposure the ABPL was significantly increased in the 270 and 864 ng/g treatment groups. After 5 days of exposure significant increases relative to the control were observed for the 16, 270 and 864 ng/g treatment groups. Enzyme leakage from the liver was investigated by measurement of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the serum. At 561, 670 and 864 ng/g PFOS a significant increase in serum ALT activity became apparent after 5 days of exposure with values ranging from 159 to 407% relative to the control. For serum AST activity a significant increase for the 864 ng/g treatment group was observed with a value of 112% relative to the control. Determination of the polymorphonuclear leukocyte migration into liver tissue as assessed through myeloperoxidase (MPO) activity in liver, was used as an indicator for inflammation. It appeared that inflammation was not involved in the observed membranous enzyme leakage for the 561, 670 and 864 ng/g PFOS treatment groups. The results of this study suggest that PFOS induces inflammation-independent enzyme leakage through liver cell membranes that might be related to cell necrosis. Furthermore, results show that PFOS does not significantly affects serum antioxidant levels nor does it clearly induce peroxisome proliferation in carp. This study also points out that PFOS might interfere with homeostasis of the DNA metabolism. The results of these biochemical analyses were used to perform an initial hazard assessment study indicating that PFOS levels observed in tissues of wildlife populations could induce a clear rise in serum transaminase levels indicative for disruption of hepatocyte membrane integrity.

Polybrominated diphenyl ethers (PBDEs) in freshwater mussels and fish from Flanders, Belgium

The levels and distribution of PBDEs in zebra mussels and several freshwater fish species (eel, carp and gibel carp) were investigated for different sites in Flanders, Belgium. In parallel, other organohalogenated contaminants, such as polychlorinated biphenyls (PCBs), p,p[prime or minute]-DDE and hexachlorobenzene (HCB) were also measured and their relationship with PBDEs was investigated. At most sites, individual PBDE congeners were present at detectable levels in mussel tissue, with the mean [summation operator]PBDE concentration ranging from 0.15 to 1.8 ng g(-1) wet weight (ww). The PCB concentrations in mussels ranged from 6.2 to 102 ng g(-1) ww. HCB and p,p[prime or minute]-DDE could be measured in mussels from most sites, mean values ranging from below the limit of quantification (LOQ) to 0.58 ng g(-1) ww and from 0.66 to 6.5 ng g(-1) ww, respectively. Except for one site (Blokkersdijk, Antwerp) where PBDEs were below the LOQ in carp muscle, all fish samples from other sites contained detectable PBDE levels, with the highest concentrations (14 +/- 14 ng g(-1) ww) being measured in eel liver from Watersportbaan (Ghent). The sampled sites covered a broad concentration range of organohalogenated pollutants with the highest values being consistently measured in eel liver. With few exceptions, all correlations between PBDEs and organochlorine pollutants for each species were low (r < 0.50) and most were statistically not significant (p > 0.05). This suggests that the exposure to contaminants arises from local sources possessing different signatures of PBDEs and organochlorine pollutants.

Cytosolic distribution of Cd, Cu and Zn, and metallothionein levels in relation to physiological changes in gibel carp (**Carassius auratus gibelio**) from metal-impacted habitats

In the present study the impact of environmental metal contamination in gibel carp (Carassius auratus gibelio) was investigated coupling disturbances in subcellular metal distribution to effect biomarkers. Gibel carp were caught at six different sampling sites in Flanders (Belgium), characterized by different degrees in environmental metal contamination. Tissue Cd, Cu and Zn concentrations and total metallothioneon (MT) levels were determined in gills, liver and kidney. Cytosolic metal distribution was measured in the main accumulating organs, liver and kidney, revealing tissue- and metal-dependent profiles. The MT pool dominated the cytosolic distribution profile. Although the importance of the MT pool increased with increasing environmental and cytosolic metal concentrations, also an undefined fraction of Cu and Cd fractions (probably free metal ions or metals bound to small organic complexes) increased at the most contaminated sampling sites. Disturbances in serum ion concentrations, serum alanine aminotransferase activity (ALT), hematocrite and condition factor were measured, as indicators of toxic effects. At the sampling site with the highest environmental Cd pollution a significant decrease in serum Ca(2+) concentrations and a significantly increased serum ALT activity were measured suggesting incomplete detoxification of Cd. Increased serum ALT concentrations suggested structural liver damage. The fact that the liver tissue, and probably also the kidney, could not cope with this high Cd burden in combination with the increased uptake of dissolved Cd through the gills most probably contributed to the perturbed serum Ca(2+) homeostasis.