D.G.J. Larsson

Ethinyloestradiol — an undesired fish contraceptive?

Environmental oestrogens are natural or synthetic substances present in the environment, which imitate the effects of endogenous oestrogen. Oestrogenic substances were identified by gas chromatogra ...

Prioritising pharmaceuticals for environmental risk assessment: Towards adequate and feasible first-tier selection.

The presence of pharmaceuticals in the aquatic environment, and the concerns for negative effects on aquatic organisms, has gained increasing attention over the last years. As ecotoxicity data are lacking for most active pharmaceutical ingredients (APIs), it is important to identify strategies to prioritise APIs for ecotoxicity testing and environmental monitoring. We have used nine previously proposed prioritisation schemes, both risk- and hazard-based, to rank 582 APIs. The similarities and differences in overall ranking results and input data were compared. Moreover, we analysed how well the methods ranked seven relatively well-studied APIs. It is concluded that the hazard-based methods were more successful in correctly ranking the well-studied APIs, but the fish plasma model, which includes human pharmacological data, also showed a high success rate. The results of the analyses show that the input data availability vary significantly; some data, such as logP, are available for most API while information about environmental concentrations and bioconcentration are still scarce. The results also suggest that the exposure estimates in risk-based methods need to be improved and that the inclusion of effect measures at first-tier prioritisation might underestimate risks. It is proposed that in order to develop an adequate prioritisation scheme, improved data on exposure such as degradation and sewage treatment removal and bioconcentration ability should be further considered. The use of ATC codes may also be useful for the development of a prioritisation scheme that includes the mode of action of pharmaceuticals and, to some extent, mixture effects.

Comparison of six different sewage treatment processes-Reduction of estrogenic substances and effects on gene expression in exposed male fish

Treated sewage effluents often contain a mixture of estrogenic compounds in low concentrations. The total combined activity of these, however, may be sufficiently high to affect the reproduction of aquatic vertebrates. The introduction of advanced treatment technologies has been suggested as a way to remove micro-contaminants, including estrogenic substances. In this study, one municipal influent was treated with six different processes in parallel on a semi-large scale in order to assess their potential to reduce substances that could contribute to estrogenic effects in male fish. The effluent from a conventional, activated sludge treatment line was compared to a similarly treated effluent with a final sand-filtering step. The addition of ozonation (15 g O(3)/m(3)), a moving bed biofilm reactor (MBBR) or both in combination was also evaluated. There was also a separate treatment line that was based on a membrane bioreactor. A small battery of hepatic estrogen-responsive genes was measured in the exposed fish using quantitative PCR. Concentrations of steroid estrogens and estrogenic phenols in the effluents were measured by GC-ECNI-MS. The ozonated effluents were the only tested effluents for which all measured biological effects in exposed fish were removed. Chemical data suggested that the MBBR technology was equally effective in removing the analyzed estrogens; however, elevated expression of estrogen-responsive genes suggested that some estrogenic substances were still present in the effluent. The membrane bioreactor removed most of the measured estrogens and it reduced the induction of the estrogen-responsive genes. However, fish exposed to this effluent had significantly enlarged livers. Given that the same influent was treated in parallel with a broad set of technologies and that the chemical analyses were combined with an in vivo assessment of estrogenic responses, this study provides valuable input into the assessment of advanced treatment processes for removing estrogenic substances.

Development of hepatic CYP1A and blood vitellogenin in eel (Anguilla anguilla) for use as biomarkers in the Thames Estuary, UK.

The potential of eel (Anguilla anguilla) as a monitoring species for the Thames Estuary, UK, was examined. Hepatic cytochrome P4501A [7-ethoxyresorufin O-deethylase (EROD) activity] and blood vitellogenin (Western analysis) were investigated as biomarkers of exposure to, respectively, organic contaminants and to contaminants showing estrogenic activity. Hepatic microsomal EROD activities in A. anguilla from seven sites in the Thames Estuary in May 1998 varied three-fold (111 +/- 24 to 355 +/- 42 pmol min-1 mg protein-1) (mean +/- S.E.M.) and showed correlation with salinity; however, the latter relationship was not maintained at other times of the year. The range of EROD activities was two- to eight-fold higher than the 37 +/- 8 pmol min-1 mg-1 for A. anguilla from the relatively clean Tamar Estuary. beta-Naphthoflavone treatment (5 mg kg-1 wet wt.; 2 days) of Thames A. anguilla produced a two-fold increase in hepatic microsomal EROD activity. Comparing the Thames EROD data with those for A. anguilla from well-characterised contaminated sites in the Netherlands (Van der Oost, R., Goksøyr, A., Celander, M., Heida, H., & Vermeulen, N. P. E. 1996. Aquatic Toxicology, 36, 189-222), the Thames is suggested to be moderately impacted by polycyclic aromatic hydrocarbons and related contaminants. 17-beta-Estradiol treatment produced the appearance of a plasma protein of 211 Kd app. mol. wt. (recognised by antibodies to vitellogenin of Morone saxatilis), but putative vitellogenin could not be detected in A. anguilla from selected sites in the Thames Estuary.

Contraceptive pill residues in sewage effluent are estrogenic to fish

Abstract The estrogenicity of sewage effluent water to fish has been described in several countries. Natural oestrogens have been confirmed as major causing agents in several British rivers. In a recent study (Larsson et al., Aquatic Toxicology, 1999, 91–97) we identified estrogenic substances by GC/MS in effluent water from a Swedish sewage treatment works receiving mainly domestic wastewater. Substances found include the synthetic estrogen used in contraceptives 17α-ethinylestradiol (4.5 ng/l), the natural estrogens estrone (5.8 ng/l) and 17β-estradiol (1.1 ng/l) and the weaker non-steroidal estrogens 4-nonylphenol (840 ng/l) and bisphenol A (490 ng/l). Ethinylestradiol exceeded levels shown to be estrogenic to fish by 45 times. The estrogenicity of the effluent water was investigated by introducing juvenile rainbow trout (Oncorhynchus mykiss) in cages downstream of the sewage treatment works. All estrogens indicated above were present in the bile of the fish, and the estrogen-inducible protein, vitellogenin, was found in large amounts in the plasma as determined by ELISA and Western blotting. At present we are studying effects further downstream on caged as well as wildcaught fish. Our studies suggest that a widely used synthetic estrogen affects the endocrine systems of fish exposed to sewage effluent water.

Induction of hepatic carbonyl reductase/20β-hydroxysteroid dehydrogenase mRNA in rainbow trout downstream from sewage treatment works—Possible roles of aryl hydrocarbon receptor agonists and oxidative stress

Carbonyl reductase/20beta-hydroxysteroid dehydrogenase (CR/20beta-HSD) serves both as a key enzyme in the gonadal synthesis of maturing-inducing hormone in salmonids, and as an enzyme protecting against certain reactive oxygen species. We have previously shown that mRNA of the hepatic CR/20beta-HSD B isoform is increased in rainbow trout caged downstream from a Swedish sewage treatment plant. Here, we report an increase of both the A as well as B form in fish kept downstream from a second sewage treatment plant. The two mRNAs were also induced in fish hepatoma cells in vitro after exposure to effluent extract. This indicates that the effects observed in vivo could be a direct effect on the liver, i.e. the mRNA induction does not require a signal from any other organ. When fish were exposed in vivo to several effluents treated with more advanced methods (ozone, moving bed biofilm reactor or membrane bioreactor) the expression of hepatic mRNA CR/20beta-HSD A and B was significantly reduced. Their abundance did not parallel the reduction of estrogen-responsive transcripts, in agreement with our previous observations that ethinylestradiol is not a potent inducer. Treatment with norethisterone, methyltestosterone or hydrocortisone in vivo did not induce the hepatic CR/20beta-HSD A and B mRNA expression. In contrast, both isoforms were markedly induced by the aryl hydrocarbon receptor agonist beta-naphthoflavone as well as by the pro-oxidant herbicide paraquat. We hypothesize that the induction of CR/20beta-HSD A and B by sewage effluents could be due to anthropogenic contaminants stimulating the aryl hydrocarbon receptor and/or causing oxidative stress.

Antibiotics and common antibacterial biocides stimulate horizontal transfer of resistance at low concentrations

There is a rising concern that antibiotics, and possibly other antimicrobial agents, can promote horizontal transfer of antibiotic resistance genes. For most types of antimicrobials their ability to induce conjugation below minimal inhibitory concentrations (MICs) is still unknown. Our aim was therefore to explore the potential of commonly used antibiotics and antibacterial biocides to induce horizontal transfer of antibiotic resistance. Effects of a wide range of sub-MIC concentrations of the antibiotics cefotaxime, ciprofloxacin, gentamicin, erythromycin, sulfamethoxazole, trimethoprim and the antibacterial biocides chlorhexidine digluconate, hexadecyltrimethylammoniumchloride and triclosan were investigated using a previously optimized culture-based assay with a complex bacterial community as a donor of mobile resistance elements and a traceable Escherichia coli strain as a recipient. Chlorhexidine (24.4μg/L), triclosan (0.1mg/L), gentamicin (0.1mg/L) and sulfamethoxazole (1mg/L) significantly increased the frequencies of transfer of antibiotic resistance whereas similar effects were not observed for any other tested antimicrobial compounds. This corresponds to 200 times below the MIC of the recipient for chlorhexidine, 1/20 of the MIC for triclosan, 1/16 of the MIC for sulfamethoxazole and right below the MIC for gentamicin. To our best knowledge, this is the first study showing that triclosan and chlorhexidine could stimulate the horizontal transfer of antibiotic resistance. Together with recent research showing that tetracycline is a potent inducer of conjugation, our results indicate that several antimicrobials including both common antibiotics and antibacterial biocides at low concentrations could contribute to antibiotic resistance development by facilitating the spread of antibiotic resistance between bacteria.

Skewed embryonic sex ratios in a viviparous fish: a result of endocrine disruption?

Abstract Sex ratios in catches of wild adult fish are often affected by sex related differences in growth, behaviour and survival. Therefore, such sex ratios may not reflect the relative number of males and females that are recruited to the population. However, this can be circumvented by studying unborn fish embryos. We have found skewed sex ratios among embryos from the viviparous eelpout ( Zoarces viviparus ) which could indicate exposure to endocrine disrupting chemicals. The sex ratio was close to 50/50 at two presumably clean sites from Kattegat/Skagerrak and two from the Baltic Sea. On the Swedish Baltic coast near a large pulp mill the relative number of female embryos was significantly lower (42%; P =0.006) but approached 50% further south in the discharge gradient. Treatment of females during early pregnancy with methyltestosterone inhibited oocyte development in all embryos, and instead testis-like tissue was formed. Thus, masculinization found in the field could be caused by exposure to androgen mimics or substances interfering with steroid-synthesis, activity or excretion. This may arise from exposure to the pulp mill effluents, since endocrine disruption in fish has been reported earlier near North American mills. Irrespectively of the cause, reduced numbers of female offspring may have negative impacts on the recruitment capacity of a population.

Seasonal variations in the activity of selected hepatic biotransformation and antioxidant enzymes in eelpout (Zoarces viviparus)

Abstract The eelpout Zoarces viviparus is used in Sweden for environmental monitoring of pollutant effects in fish. The activities of several hepatic biotransformation and antioxidant enzymes were monitored during the period of 12 months from August 1996 to July 1997 using newly caught feral eelpout from Kattegatt, off the west coast of Sweden. In females, the enzyme activities showed significant seasonal variations; CYP1A-activity, measured as ethoxyresorufin- O -deethylase (EROD), was lowest during the spawning season in August, possibly due to high estrogen levels. UDP-glucuronosyl transferase activity was lowest in November, while the glutathione- S -transferase activity markedly decreased during the winter. On the other hand, catalase activity peaked in April. The number of male fish was limited and the seasonal variations less clear. However, in both sexes, glutathione reductase and peroxidase activities were highest in the autumn and in May, respectively. The results are important for the ability to distinguish between pollutant effects and natural variations in the activity of selected enzymes in eelpout.

Carbonyl reductase mRNA abundance and enzymatic activity as potential biomarkers of oxidative stress in marine fish

Carbonyl reductase (CBR) is an enzyme involved in protection from oxidative stress. In rainbow trout (Oncorhynchus mykiss), the hepatic mRNA abundance of the two isoforms (A and B) is increased after exposure to treated sewage effluents, as well as after exposure with β-naphthoflavone (β-NF) and the pro-oxidant paraquat. In this study, we show that the same chemicals similarly increase the single known hepatic CBR mRNA level and CBR catalytic activity in the coastal living eelpout (Zoarces viviparus). Hepatic CBR mRNA abundance and catalytic activity were also compared between eelpout collected at contaminated and reference sites on the Swedish west coast, but no differences were observed. In conclusion, CBR is a potential biomarker candidate for monitoring the exposure and effects of AhR agonists and/or pro-oxidants in the marine environment, but more research is needed to investigate temporal regulation as well as dose dependency for different chemicals. The mRNA and enzymatic assays presented in this study provide two additional tools for researchers interested in expanding their biomarker battery.