A. Dick Vethaak

Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.

Disturbance of host defence capability in the common shrimp, Crangon crangon, by exposure to harbour dredge spoils

Abstract Specimens of the common shrimp, Crangon crangon , were exposed to contaminated harbour dredgings in mesocosms for 12 days at 16 ± 2 ° C. The tanks contained either 100% dredged spoils from Rotterdam Harbour (the experimental tank), 5% dredged spoils mixed with 95% clean sand (the intermediate tank) or clean sand only (the reference tank). Animals exposed to the dredge spoils were found to display an elevation in recoverable haemolymph volume (RHV) and a reduction in total haemocyte count (THC). Related biochemical assays also indicated reduced blood cell phenoloxidase (PO) activity in animals sampled from the experimental tank. These results are indicative that immune capability and clotting are adversely affected in animals exposed to harbour dredge spoils. No differences were seen in antibacterial vigour (per unit protein) between the various groups of animals, although the decline in the THC means that antibacterial potential is probably reduced in vivo. Importantly, with the THC and RHV, a conventional dose-response function was not found in relation to the contaminant gradient used. For both parameters, animals in the intermediate tank showed a stronger response than those in the experimental tank. The finding that sediments containing as little as 5% dredge spoils have a marked effect on the host defence capability in C. crangon could have serious implications for the dumping of contaminated dredge spoils at sea.

Estrogenic effects in fish in The Netherlands: some preliminary results

Recently, a large-scale field study in The Netherlands has focused on the effects of estrogenic contaminants on feral fish populations. The freshwater bream (Abramis brama) and the estuarine flounder (Platichthys flesus) were sampled at a large number of locations in the spring and autumn of 1999. Concentrations of the yolk protein vitellogenin (VTG) in blood plasma of male flounders were small at most sites. At two sites, however, moderately elevated concentrations were found in autumn. Both sites were situated in the same industrial harbour zone also receiving effluent from sewage treatment works. At many sites VTG levels in male bream were significantly greater than at the control site. The greatest concentrations were observed in individuals collected from a small stream, close to the discharge of a relatively large municipal waste water treatment plant. This was also the only site where considerable intersex occurred; 37% of male bream exhibited ovotestes. Ovotestis was not observed in any of the male flounder captured. The results from The Netherlands are briefly discussed and compared with the well-known case studies in the UK.

Imposex induction in laboratory reared juvenile Buccinum undatum by tributyltin (TBT

Here we report a series of experiments on the development and occurrence of imposex in the common whelk, Buccinum undatum, under the influence of (chronic) exposure to butyltin compounds. The main objective of the experiments was to obtain more information about the effects of organotin compounds in the marine environment, which possibly relate to the reported decline of B. undatum in Dutch coastal waters. In these studies tributyltin (TBT) dose-dependently induced the development of male sexual organs in juvenile whelks. A TBT concentration >7 ng Sn/l induced imposex in juvenile whelks. Growth in TBT-exposed juvenile whelks was significantly reduced compared to the reference group at a nominal TBT dose ≥ 4 ng Sn/l in one of the exposure studies. After 5 years in the laboratory, egg-laying was only observed in reference aquaria. Thus, TBT might impair whelk reproduction through growth reduction. The results showed a sensitivity towards imposex development in different life-stages. Juveniles were the most sensitive, adolescent females also responded, but adult females did not respond to TBT exposure, although they dose-dependently increased their organotin (OT) body-burden when exposed. Environmental TBT during only the in ovo stage, did not result in an increased masculinisation compared to non-exposed developing whelks. Histological studies showed no sterilisation due to mechanical blockage of the (adult) female genital opening by sperm-duct tissue. Gonadal development in 2-year old juveniles was not observed. This implies that the differentiation of a penis and a vas deferens, which already occurred in the first few months after hatching, was not controlled by gonadal factors. No other sexual characteristics than those already visible with the eye were found. TBT inactivated CYP450 to its inactive form CYP420 in in vitro exposure studies with microsomal fractions of whelks. The studies have shown TBT to disrupt sexual development dose dependently in juvenile common whelks. TBT also dose dependently exerts an effect on enzymatic (CYP450) processes. Although no mechanical sterilisation was observed, reproduction might be impaired through growth reduction.

Differences in cypia response between flounder (Platichthys flesus) and plaice (Pleuronectes platessa) after long-term exposure to harbour dredged spoil in a mesocosm study

Abstract Flounder (Platichthys flesus) and plaice (Pleuronectes platessa) were exposed to three different levels of pollution involving contaminants from harbour dredged spoil in a large-scale mesocosm study lasting 30 months. Exposure of fish to PCBs and PAHs was estimated from liver concentrations of CB-153 and biliary levels of 1-OH pyrens in flounder. Both marker compounds were significantly elevated in fish from the polluted mesocosms in comparison to the reference mesocosm. Of the three parameters used to characterise CYPIA induction in flounder, only CYPIA-mRNA in female fish was significantly induced by pollution. However, significant induction of EROD activity and immunochemically measured CYPlA protein content was observed in plaice. To inivestigate in more detail the apparent lack of CYP1A induction, a parallel experiment was run in which flounder were injected with a single i.p. injection of benzo[a]pyrene (BaP) and sacrificed after 48 hours. Since this approach did result in significant induction of all the parameters used to characterise CYP1A induction (EROD, CYPIA, CYPIA mRNA), it appears that external factors may have prevented induction in the mesocosm experiments. Down-regulating effects of the possible presence of a compound with oestrogenic activity, or altered membrane-CYPIA or membrane-receptor interaction, could have contributed to this apparent lack of response, despite the intrinsic ability of flounder to increase CYPIA levels after exposure to a model inducer (BaP).

A weight-of-evidence approach to assessing the ecological impact of organotin pollution in Dutch marine and brackish waters; combining risk prognosis and field monitoring using common periwinkles (Littorina littorea)

In the present study an integrated ecological risk assessment based on multiple lines of evidence (LOEs) was evaluated in order to better assess the risk from TBT in Dutch harbours and open coastal waters. On the basis of spatial distributions of measured tributyltin (TBT) concentrations in sediments and suspended matter, predictions of the intersex index (ISI) in Littorina littorea and the ecological risk expressed as the Potentially Affected Fraction (PAF) of species were made. The results were compared to actual ISI measurements and presence of L. littorea in the field. The PAF calculated on the basis of TBT levels for open coastal waters ranged from 4.2% to 15.3%; for harbours it ranged from 3.5% to 26.9%. Significant intersex levels were observed only in waters where the risk was calculated above 10% PAF. This study suggests that the absence of L. littorea from some harbours with high ecological risk values can be explained by high TBT concentrations. A call is made for the use of integrated approaches like weight-of-evidence (WOE) to help practitioners improve ecological risk assessment.