In vitro and in vivo antiestrogenic effects of polycyclic musks in zebrafish.

The polycyclic musks 6-acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN) and 1,2,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyran (HHCB) are used as fragrance ingredients in perfumes, soaps, and household cleaning products. They are known to be ubiquitously present in the aquatic environment, and because of their lipophilic nature, they tend to bioaccumulate in aquatic biota. In surface waters, concentrations between 1 ng/L and 5 microg/L have been found, depending mainly on the proportion of sewage effluents in the water. In fish, under normal environmental conditions, concentrations in the microgram per kilogram fresh weight (fw) range are found. In a previous study we showed that AHTN and HHCB exert mainly antiestrogenic effects on the human estrogen receptor alpha (ERalpha) and ERbeta in an in vitro reporter gene assay. In the current study, we assessed the in vitro antiestrogenic effects of both musks on zebrafish ERs. Antagonism was observed on zfERbeta, and more pronounced on the newly cloned zfERgamma. Using a transgenic zebrafish assay, we studied antiestrogenicity of the musks in vivo. Dose-dependent antagonistic effects were observed at concentrations of 0.1 and 1 microM AHTN and HHCB. GC-MS analysis showed that the musks bioaccumulated in the fish, with internal concentrations (15-150 mg/kg fw) which were roughly 600 times higher than the nominal test doses. To our knowledge, this is the first time that environmental contaminants are shown to be antiestrogenic in an in vivo fish assay that focuses solely on ER-mediated effects. This makes the transgenic zebrafish assay a promising tool for the rapid detection of both estrogenic and antiestrogenic effects of chemicals in fish.

Evaluation of Simple Treat 3.0 for two hydrophobic and slowly biodegradable chemicals: polycyclic musks HHCB and AHTN.

In the current study, predictions by Simple Treat 3.0, a fate model for organic chemicals in sewage treatment plants (STPs), are compared with actual measurements in three STPs. Two polycyclic musks, Tonalide (AHTN) and Galaxolide (HHCB), were used for model evaluation. Results show that Simple Treat 3.0 is able to predict the removal efficiency within a factor 4. Predicted concentrations of both chemicals within the different physical compartments of STPs show a high correlation (r(2)=0.80) with experimental values. Although predicted free concentration levels were similar to previously reported experimental data, the trends along the compartments showed an inverse relationship. This bias of the model can be caused by an underestimation of BOD-removal (solids), or an overestimation of bacterial growth, evaporation, or a combination of these three factors. Results show that Simple Treat 3.0 is a valid tool for the risk assessment of slowly biodegradable chemicals, but still some adjustments of the model could be incorporated from a scientific point of view.

Removal of two polycyclic musks in sewage treatment plants: freely dissolved and total concentrations.

In the current study, the removal of slowly degradable hydrophobic chemicals in sewage treatment plants (STPs) has been evaluated with emphasis on the combination of free and total concentration data. Free and total concentrations of two polycyclic musks were determined in each compartment of four STPs. The free concentration of the polycyclic musks remains virtually constant throughout all the compartments of the STPs with values between 0.21 and 0.57 microg/L for AHTN and between 0.79 and 2.0 microg/L for HHCB. Total concentrations of these fragrances are highly dependent on the volatile solids in a given compartment resulting in much more variation with values between 0.42 and 92 microg/L for AHTN and between 1.25 and 258 microg/L for HHCB. It is concluded that free concentrations of these hydrophobic chemicals in the compartments of STPs are mostly biodegradation mediated, while total concentrations are mediated by the concentration of solids. The combination of measurements of free and total concentrations can improve estimations regarding removal efficiency and removal pathways.

Bioconcentration and acute toxicity of polycyclic musks in two benthic organisms (Chironomus riparius and Lumbriculus variegatus).

In the current study, the bioconcentration behavior and acute toxicity of two polycyclic musks, Tonalide 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN) and Galaxolide 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexa-methylcyclopenta[gamma]-2-benzopyran (HHCB), were studied in two benthic organisms. Polycyclic musks are frequently used fragrances, and they have been detected in different compartments of the environment. The aim of this study was to fill some empirical data gaps for AHTN and HHCB for benthic organisms. Results show that differences exist between both organisms. Chironomus riparius exhibited bioconcentration factors (BCFs) for AHTN and HHCB substantially lower than predicted for nontransformed organics. The BCFs for both chemicals increased after coexposure of the organism to the cytochrome P450 inhibitor piperonyl butoxide. Thus, the low BCF values were the result of rapid biotransformation of AHTN and HHCB in the midge larvae. Bioconcentration kinetics indicated that both chemicals induced their own cytochrome P450-mediated metabolism. Acute toxicity of AHTN to midge larvae was reduced compared to predicted baseline toxicity and was similar for HHCB. Bioconcentration of AHTN and HHCB in the worm (Lumbriculus variegatus) is in agreement with predictions based on the octanol-water partition coefficients of these chemicals. Acute toxicity was found to be similar to predicted values for baseline toxicity. Summarizing, for AHTN and HHCB, acute toxicity and bioconcentration behavior in L. variegatus was in accordance with predicted data for nontransformed organics. In C. riparius, bioconcentration as well as toxicity were reduced.

Sorption kinetics and microbial biodegradation activity of hydrophobic chemicals in sewage sludge: model and measurements based on free concentrations.

In the current study, a new method is introduced with which the rate-limiting factor of biodegradation processes of hydrophobic chemicals in organic and aqueous systems can be determined. The novelty of this approach lies in the combination of a free concentration-based kinetic model with measurements of both free and total concentrations in time. This model includes microbial biodegradation activity of the chemical in the aqueous phase and chemical sorption kinetics with respect to organic carbon and aqueous phases. The time dependency of free and total concentrations of 7-acetyl-1,1,3,4,4,6-hexamethyltetrahydronaphthalene and 7-acetyl-1,1,3,4,4,6-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta(g)-2-benzopyrane in activated sludge was experimentally determined in vitro. Evaporation losses from the test system were also determined. Least-squares regression to optimize the model parameters resulted in a model that is in accordance with the experimental data. Additionally, the model shows that a comparison between the decrease of free and total chemical concentrations in time, in combination with an independent measurement of the organic carbon/water partition coefficient provides information aboutthe rate-limiting step of the degradation process. This information can be used by sewage treatment plant managers to decide whether the microbial biodegradation activity itself or the desorption from organic carbon to the aqueous phase should be improved.