Laboratory and field scale bioremediation of hexachlorocyclohexane (HCH) contaminated soils by means of bioaugmentation and biostimulation.

Hexachlorocyclohexane (HCH) contaminated soils were treated for a period of up to 64 days in situ (HCH dumpsite, Lucknow) and ex situ (University of Delhi) in line with three bioremediation approaches. The first approach, biostimulation, involved addition of ammonium phosphate and molasses, while the second approach, bioaugmentation, involved addition of a microbial consortium consisting of a group of HCH-degrading sphingomonads that were isolated from HCH contaminated sites. The third approach involved a combination of biostimulation and bioaugmentation. The efficiency of the consortium was investigated in laboratory scale experiments, in a pot scale study, and in a full-scale field trial. It turned out that the approach of combining biostimulation and bioaugmentation was most effective in achieving reduction in the levels of α- and ß-HCH and that the application of a bacterial consortium as compared to the action of a single HCH-degrading bacterial strain was more successful. Although further degradation of ß- and δ-tetrachlorocyclohexane-1,4-diol, the terminal metabolites of ß- and δ-HCH, respectively, did not occur by the strains comprising the consortium, these metabolites turned out to be less toxic than the parental HCH isomers.

Gammarus spp. in aquatic ecotoxicology and water quality assessment: toward integrated multilevel tests.

The amphipod genus Gammarus is widespread and is structurally and functionally important in epigean freshwaters of the Northern Hemisphere. Its presence is crucial, because macroinvertebrate feeding is a major rate-limiting step in the processing of stream detrius. In addition, Gammarus interacts with multiple trophic levels bu functioning as prey, predator, herbivore, detritivore, and shredder. Such a broad span of ecosystem participation underlines the importance of Gammarus to pollutants and other disturbances may render it a valuable indicator for ecosystem health. This review summarizes the vast number of studies conducted with Gammarus spp. for evaluating aquatic ecotoxicology endpoints and examines the suitability of this native invertabrate species for the assessment of stream ecosystem health in the Northern Hemisphere. Numerous papers have been published on how pollutants affect gammarind behavior (i.e., mating, predator avoidance), reproduction, development, feeding activity, population structure, as well as the consequences of pollution on host-parasite, predator-prey, or native-invasive species interactions. Some biochemical and molecular biomarkers have already been established, such as the measurement of vitellogenin-like proteins, metallothioneins, alkali-labile phosphates (in proteins), and lipogenic enzyme activities for assessing endocrine distribution and detoxification mechanisms.

Insecticides cause transcriptional alterations of endocrine related genes in the brain of honey bee foragers.

Bees are exposed to endocrine active insecticides. Here we assessed expressional alteration of marker genes indicative of endocrine effects in the brain of honey bees. We exposed foragers to chlorpyrifos, cypermethrin and thiacloprid and assessed the expression of genes after exposure for 24 h, 48 h and 72 h. Chlorpyrifos caused the strongest expressional changes at 24 h characterized by induction of vitellogenin, major royal jelly protein (mrjp) 2 and 3, insulin-like peptide (ilp1), alpha-glucosidase (hbg3) and sima, and down-regulation of buffy. Cypermethrin caused minor induction of mrjp1, mrjp2, mmp1 and ilp1. The sima transcript showed down-regulation at 48 h and up-regulation at 72 h. Exposure to thiacloprid caused down-regulation of vitellogenin, mrjp1 and sima at 24 h, and hbg3 at 72 h, as well as induction of ilp1 at 48 h. The buffy transcript was down-regulated at 24 h and up-regulated at 48 h. Despite compound-specific expression patterns, each insecticide altered the expression of some of the suggested endocrine system related genes. Our study suggests that expressional changes of genes prominently expressed in nurse or forager bees, including down-regulation of buffy and mrjps and up-regulation of hbg3 and ilp1 may serve as indicators for endocrine activity of insecticides in foragers.

Estrogenic activity of ternary UV filter mixtures in fish (Pimephales promelas) - an analysis with nonlinear isobolograms.

Numerous estrogenic compounds are present in aquatic environments, but currently it is not well understood how compounds that differ in maxima and slope of their individual dose-response curves contribute to the overall mixture effect. In order to better understand such interactions we investigated 3 commonly used UV filters, for their estrogenic mixture activity and analysed their joint effects by using the concentration addition (CA) concept. Thereby, we extended the method of isoboles for analysis of 3 compounds that differ in maxima and slopes of their dose-response curves. 3-Benzylidene camphor (3BC), benzophenone-1 (BP1) and benzophenone-2 (BP2) are estrogenic in fish and act as pure- or partial estrogen receptor alpha agonists. First we exposed juvenile fathead minnows for 14 days to six concentrations of each UV filter alone to determine vitellogenin (VTG) induction curves, calculate equi-effective mixture concentrations and predict mixture effects. For 3BC, BP1 and BP2 significant VTG-induction occurred at 420, 2668, and 4715 microg/L, respectively. BP2 displayed a full dose-response curve, whereas 3BC and BP1 showed submaximal activity of 70 and 78%, respectively. Second, we exposed fish to 6 equi-effective mixtures (EC-NOEC, EC1, EC5, EC10, EC20, EC30) of these UV filters. Significant VTG-induction occurred at EC5 and higher. Submaximal activity of 67% as compared to the control EE2 (100 ng/L) was reached. The curves for the observed and predicted mixture activity agreed for mixture levels (EC10 to EC30), however, at EC-NOEC, EC1 and EC5, lower activity was observed than predicted by CA. Detailed isobolographic analysis indicate additivity at EC10 to EC30, and antagonism at low levels (EC-NOEC to EC5). Our data show for the first time, that for compounds with differences in maxima and slope, considerably more mixture combinations are additive than previously thought. This should be taken into account for hazard and risk assessment of UV filters and xenoestrogens.

Endocrine disruption and chronic effects of plant protection products in bees: Can we better protect our pollinators?

Exposure to plant protection products (PPPs) is one of the causes for the population decline of pollinators. In addition to direct exposure, pollinators are exposed to PPPs by pollen, nectar and honey that often contain residues of multiple PPPs. While in legislation PPPs are regarded mainly for their acute toxicity in bees, other effects such as neurotoxicity, immunotoxicity, behavioural changes, stress responses and chronic effects that may harm different physiologically and ecologically relevant traits are much less or not regarded. Despite the fact that endocrine disruption by PPPs is among key effects weakening survival and thriving of populations, pollinators have been poorly investigated in this regard. Here we summarize known endocrine disruptive effects of PPPs in bees and compare them to other chronic effects. Endocrine disruption in honey bees comprise negative effects on reproductive success of queens and drones and behavioural transition of nurse bees to foragers. Among identified PPPs are insecticides, including neonicotinoids, fipronil, chlorantraniliprole and azadirachtin. So far, there exists no OECD guideline to investigate possible endocrine effects of PPPs. Admittedly, investigation of effects on reproduction success of queens and drones is rarely possible under laboratory conditions. But the behavioural transition of nurse bees to foragers could be a possible endpoint to analyse endocrine effects of PPPs under laboratory conditions. We identified some genes, including vitellogenin, which regulate this transition and which may be used as biomarkers for endocrine disruptive PPPs. We plea for a better implementation of the adverse outcome pathway concept into bee's research and propose a procedure for extending and complementing current assessments, including OECD guidelines, with additional physiological and molecular endpoints. Consequently, assessing potential endocrine disruption in pollinators should receive much more relevance.

Cytotoxicity and molecular effects of biocidal disinfectants (quaternary ammonia, glutaraldehyde, poly(hexamethylene biguanide) hydrochloride PHMB) and their mixtures in vitro and in zebrafish eleuthero-embryos.

Frequently used biocidal disinfectants, including quaternary ammonium compounds (QAC), glutaraldehyde and poly(hexamethylene biguanide) hydrochloride (PHMB), occur in the aquatic environment but their potential effects in fish are poorly known, in particular when occurring as mixtures. To investigate their joint activity, we assessed the cytotoxicity of three QACs (BAC, barquat and benzalkonium chloride), glutaraldehyde andPHMB by the MTT assay individually, followed by assessing binary and ternary mixtures in zebrafish liver cells (ZFL) and human liver cells (Huh7). We also analysed molecular effects by quantitative PCR in vitro and in zebrafish eleuthero-embryos employing a targeted gene expression approach. QACs displayed strong cytotoxicity in both cell lines with EC50 values in the low µg/ml range, while glutaraldehyde and PHMB were less cytotoxic. Most of the binary and both ternary mixtures showed synergistic activity at all equi-effective concentrations. A mixture containing all five compounds mixed at their no observed effect concentrations showed strong cytotoxicity, suggesting a synergistic interaction. Additionally, we determined transcriptional alterations of target genes related to endoplasmatic reticulum (ER) stress, general stress, inflammatory action and apoptosis. Induction of ER stress genes occurred at non-cytotoxic concentrations of barquat, glutaraldehyde and BAC in ZFL cells. Barquat and BAC induced tumor necrosis factor alpha (tnf-α). Similar transcriptional alterations were found in vivo upon exposure of zebrafish eleuthero-embryos for 120h. Glutaraldehyde led to induction of ER stress genes and tnf-α, while BAC additionally induced genes indicative of apoptosis, which was also the case with benzalkonium chloride at the highest concentration. We demonstrated strong cytotoxicity of QACs, and synergistic activity of binary, ternary and quintuple mixtures. Barquat and BAC let to induction of ER stress and inflammation in vitro, and BAC and glutaraldehyde at non-toxic concentrations in vivo, while benzalkonium chloride induced expression of tnf-α only.

Effect-based tools for monitoring estrogenic mixtures: Evaluation of five in vitro bioassays.

In vitro estrogen receptor transactivation assays (ERTAs) are increasingly used to measure the overall estrogenic activity of environmental water samples, which may serve as an indicator of exposure of fish or other aquatic organisms to (xeno)estrogens. Another potential area of application of ERTAs is to assist the monitoring of the potent steroids 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2) under the Water Framework Directive (WFD) watch-list mechanism. Chemical analysis of E2 and EE2 is currently hampered by limits of quantification being mostly above the proposed annual average Environmental Quality Standards (AA-EQS) of 0.4 and 0.035 ng/L, respectively. Sensitive ERTAs could circumvent current detection challenges by measuring total estrogenic activity expressed as E2-equivalent (EEQ) concentrations. However, the use of different ERTAs results in different EEQ concentrations for the same sample. Reasons for these differences are known, but it remains unclear how to use and interpret bioassay results in a harmonised way. The aim of this study was to compare the intra- and inter-day variability of EEQ measurements using five different ERTAs (YES, ERα-CALUX, MELN, T47D-KBluc and GeneBLAzer-ERα) with regard to their applicability as effect-based tools in environmental monitoring. Environmentally relevant artificial mixtures of (xeno)estrogens were prepared to represent samples with higher (i.e. multiple times the AA-EQS for E2) or lower pollution levels (i.e. around the AA-EQS for E2). Mixtures were tested either directly or following solid phase extraction (SPE). The SPE step was included, as environmental samples typically require enrichment before analysis. Samples were analysed repeatedly to test intra-day and inter-day variability. Estrogenicity was quantified using the 10% effect level (PC10) of the positive control (E2) and expressed as EEQ concentrations. The average coefficient of variation (CV) of EEQ concentrations for the five ERTAs and all samples was 32%. CV was lower for intra-day experiments (30%) compared to inter-day experiments (37%). Sample extraction using SPE did not lead to additional variability; the intra-day CV for SPE extracted samples was 28%. Of the five ERTAs, ERα-CALUX had the best precision and repeatability (overall CV of 13%).

The ultraviolet filter 3-benzylidene camphor adversely affects reproduction in fathead minnow (Pimephales promelas).

The ultraviolet (UV) filter 3-benzylidene camphor (3BC) is used in personal care products and in a number of materials for UV protection. 3BC has been shown in vitro and in vivo in fish to be estrogenic, but possible effects on fertility and reproduction are unknown. In this study we evaluate whether 3BC affects reproduction of fish Pimephales promelas. After a preexposure period of 21 days, reproductively mature fathead minnows were exposed to increasing concentrations of 3BC for 21 days in a static-renewal procedure. Actual 3BC concentrations decreased to 23% of initial levels and median concentrations were 0.5, 3, 33, 74, and 285 microg/l. 3BC affected reproduction in a dose-dependent manner with weak effects on fecundity at 3 microg/l, a significant decrease at 74 microg/l, and a cessation of reproduction at 285 microg/l. 3BC was accumulated in fish with an average bioconcentration factor of 313 +/- 151. Dose-dependent demasculinization in secondary sex characteristics of male fish and dose-dependent induction of plasma vitellogenin occurred, which was significant at 74 microg/l and higher. 3BC had a profound and dose-dependent effect on the histology of gonads of male and female fish at 3 microg/l and higher. At 74 and 285 microg/l, oocyte and spermatocyte development was inhibited in male and female gonads. Testes of exposed males had much fewer spermatogenic cysts, and ovaries of exposed females had much fewer mature but more atretic, follicles. This study shows significant effects of the UV filter 3BC on fertility, gonadal development, and reproduction of fish after short-term exposure that may have negative consequences on the population level.

Comparison of in vitro and in vivo estrogenic activity of UV filters in fish.

In this work, we evaluate whether in vitro systems are good predictors for in vivo estrogenic activity in fish. We focus on UV filters being used in sunscreens and in UV stabilization of materials. First, we determined the estrogenic activity of 23 UV filters and one UV filter metabolite employing a recombinant yeast carrying the estrogen receptor of rainbow trout (rtERalpha) and made comparisons with yeast carrying the human hERalpha for receptor specificity. Benzophenone-1 (BP1), benzophenone-2 (BP2), 4,4-dihydroxybenzophenone, 4-hydroxybenzophenone, 2,4,4-trihydroxy-benzophenone, and phenylsalicylate showed full dose-response curves with maximal responses of 81-115%, whereas 3-benzylidene camphor (3BC), octylsalicylate, benzylsalicylate, benzophenone-3, and benzophenone-4 displayed lower maximal responses of 15-74%. Whereas the activity of 17beta-estradiol was lower in the rtERalpha than the hERalpha assay, the activities of UV filters were similar or relatively higher in rtERalpha, indicating different relative binding activities of both ER. Subsequently, we analyzed whether the in vitro estrogenicity of eight UV filters is also displayed in vivo in fathead minnows by the induction potential of vitellogenin after 14 days of aqueous exposure. Of the three active compounds in vivo, 3BC induced vitellogenin at lower concentrations (435 microg/l) than BP1 (4919 microg/l) and BP2 (8783 microg/l). The study shows, for the first time, estrogenic activities of UV filters in fish both in vitro and in vivo. Thus we propose that receptor-based assays should be used for in vitro screening prior to in vivo testing, leading to environmental risk assessments based on combined, complementary, and appropriate species-related assays for hormonal activity.

Multiple hormonal activities of UV filters and comparison of in vivo and in vitro estrogenic activity of ethyl-4-aminobenzoate in fish.

UV filters have been detected in surface water, wastewater and fish, and some of them are estrogenic in fish. At present, little is known about their additional hormonal activities in different hormonal receptor systems despite their increasing use and environmental persistence. Besides estrogenic activity, UV filters may have additional activities, both agonistic and antagonistic in aquatic organisms. In our study, we investigate a series of UV filters for multiple hormonal activities in vitro in human receptor systems and evaluate the predictive value of these findings for the activity in fish in vitro and in vivo. First we systematically analysed the estrogenic, antiestrogenic, androgenic, and antiandrogenic activity of 18 UV filters and one metabolite in vitro at non-cytotoxic concentrations with recombinant yeast systems carrying either a human estrogen (hERalpha) or androgen receptor (hAR). All 19 compounds elicited hormonal activities, surprisingly most of them multiple activities. We found 10 UV-filters having agonistic effects towards the hERalpha. Surprisingly, we identified for the first time six UV filters with androgenic activities and many of them having pronounced antiestrogenic and antiandrogenic activities. As much as 17 compounds inhibited 4,5-dihydrotestosterone activity in the hAR assay, while 14 compounds inhibited estradiol activity in the hERalpha assay, indicating antiandrogenic and antiestrogenic activity, respectively. In particular, the antiandrogenic activities of phenyl- and benzyl salicylate, benzophenone-1 and -2, and of 4-hydroxybenzophenone were higher than that of flutamide, a known hAR antagonist. In a second series of experiments, we investigated the predictive power of the hERalpha assay for aquatic organisms by further investigating the estrogenic UV filter ethyl 4-aminobenzoate (Et-PABA) in vitro and in vivo in fish. Et-PABA showed estrogenic activity in a recombinant yeast system carrying the rainbow trout estrogen receptor (rtERalpha) with higher activity than in the hERalpha assay. In addition, Et-PABA induced vitellogenin after 14 days of exposure in juvenile fathead minnows at 4394mug/L. Our study shows estrogenic activity of this UV filter in fish both in vitro and in vivo. In conjunction with in vitro human receptor-based systems our results give a more detailed picture about distinct hormonal activities of UV filters occurring in aquatic systems. We conclude that receptor-based assays are important for in vitro assessment of UV-filters prior to or concurrently with in vivo assays, which ultimately provide data for the environmental risk assessment of these important personal care products.

In vitro bioassays to screen for endocrine active pharmaceuticals in surface and waste waters.

In the context of the European Water Framework Directive (WFD) it is fully recognized that pharmaceuticals can represent a relevant issue for the achievement of the good chemical and ecological status of European surface water bodies. The recent European Directive on the review of priority substances in surface water bodies has included three pharmaceuticals of widespread use (diclofenac, 17α-ethinylestradiol (EE2), 17ß-estradiol (E2)) in the European monitoring list, the so-called watch list. Endocrine active pharmaceuticals such as EE2 and E2 (also occurring as natural hormone) can cause adverse effects on aquatic ecosystems at very low levels. However, monitoring of these pharmaceuticals within the watch list mechanism of the WFD and national monitoring programs can be difficult because of detection problems of most routine analytical methods. With proposed annual average Environmental Quality Standards (AA-EQS) of 0.035 ng/L and 0.4 ng/L, respectively, the estrogenic pharmaceutical EE2 and the natural hormone E2 are among those substances. Sensitive in vitro bioassays could reduce the current detection problems by measuring the estrogenic activity of environmental samples. In a short review article the application of this approach to screen and assess the risks of endocrine active pharmaceuticals with a focus on estrogenic pharmaceuticals in environmental waters is discussed.

Assessment of hormonal activity of UV filters in tadpoles of frog Xenopus laevis at environmental concentrations.

Residues of UV filters used as ultraviolet absorbers in sunscreens, cosmetics and in light protection have been found in surface water and fish. Recently some of them were shown bearing estrogenic activity, however, very little is known about possible adverse effects to aquatic life. Here we investigate whether two UV filters, 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor (3-BC), interfere with the thyroid and sex hormone system during frog metamorphosis. Xenopus laevis tadpoles were exposed to 1, 5 and 50 microg/L of 4-MBC and 3-BC, respectively, for 35 days (NF stage 52-66). The rate of metamorphosis was not affected, and no obvious differences in body and tail length compared to controls were observed. Neither 4-MBC, nor 3-BC led to effects on the sex ratio or gross gonad morphology of X. laevis at stage 66. Our results indicate that these UV filters do not negatively affect the thyroid system and sex ratio of frogs at environmental concentrations.

Release and environmental impact of silver nanoparticles and conventional organic biocides from coated wooden façades.

This study represents for the first time a comprehensive assessment of functionality and environmental impacts of metallic silver nanoparticles (Ag-NP) compared to conventional organic biocides. Four different transparent, hydrophobic coatings of wooden outdoor façades were tested during one year outdoor weathering. The total silver release from products with Ag-NP was proportional to the overall erosion of the coating. The results indicate that the Ag-NPs are likely transformed to silver complexes, which are considerably less toxic than ionic silver. The protective effect of the silver containing coatings against mold, blue stain and algae was insufficient, even in immaculate and non-weathered conditions. The release of organic biocides from conventional coatings was dependent on the weather conditions, the type of biocide and the use in the base or top coat. The conventional coating showed a good overall performance free from mold, blue stain and algae until the end of the test period.

A tentative environmental risk assessment of the UV-filters 3-(4-methylbenzylidene-camphor), 2-ethyl-hexyl-4-trimethoxycinnamate, benzophenone-3, benzophenone-4 and 3-benzylidene camphor.

UV-filters are increasingly used in cosmetics and in the protection of materials against UV-irradiation. The widespread occurrence of UV-filter residues in aquatic systems has been reported, but still little is known about their environmental effects. Some of these compounds negatively interact with the hormone system of fish, resulting in decreased fecundity and reproduction. Here we report on acute and chronic effects of UV-filters 3-(4-methylbenzylidene-camphor) (4MBC), 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC), benzophenone-3 (BP3) and benzophenone-4 (BP4) on Daphnia magna. The acute toxicity increased with log Pow of the compound. The LC50 values (48h) of 4MBC, EHMC, BP3 and BP4 were 0.56, 0.29, 1.9 and 50mg/L, respectively. A tentative preliminary environmental risk assessment (ERA) based on a limited set of data indicates that individual UV-filters should undergo further ecotoxicological analysis, as an environmental risk cannot be ruled out. Consequently new data on the environmental occurrence and the effects of UV-filters are needed for a more accurate ERA. When regarded as a mixture occurring in surface waters they may pose a risk for sensitive aquatic organisms.

Effects of the UV filter benzophenone-2 on reproduction in fish.

The UV filter benzophenone-2 (BP-2) is largely used in personal care products such as cosmetics and in numerous other materials for UV protection. Like other UV filters, BP-2 has been found to be estrogenic in vitro and in vivo, but potential effects on reproduction of fish are unknown. In this study, we evaluate whether BP-2 affects important reproductive parameters such as fecundity, gametogenesis and secondary sex characteristics. After a pre-exposure period of 19 days, reproductively mature fathead minnows (Pimephales promelas) were exposed to 0.002, 0.1, 1.2, 5.0 and 9.7 mg/L BP-2 for 15 days. BP-2 was accumulated in fish up to 3.1 microg/g body weight. In males, a dose-dependent vitellogenin induction and decrease in the number of nuptial tubercles occurred. Moreover, significant dose-related effects on gonads of male and female fish were observed. At concentrations of 1.2 mg/L and higher, spermatocyte and oocyte development was significantly inhibited in male and female fish, respectively. Testes of exposed males had much fewer spermatocytes and ovaries of exposed females had much fewer mature and more atretic follicles. Reproduction was negatively affected in a dose-dependent manner with a decrease in egg production at 5.0 mg/L and a complete cessation of spawning activity at 9.7 mg/L BP-2. Our findings show significant estrogenic effects of the common UV filter BP-2 on vitellogenin induction, secondary sex characteristics, gonadal development, and reproduction in fish.

Estrogenic activity of UV filter mixtures.

UV-absorbing chemicals (UV filters) are widely used for protection against UV radiation in sunscreens and in a variety of cosmetic products and materials. Depending on the breadth and factor of UV protection, they are added as single compounds or as a combination thereof. Some UV filters have estrogenic activity, but their activity and interactions in mixtures are largely unknown. In this work, we analyzed 8 commonly used UV filters, which are pure or partial hERalpha agonists, for their estrogenic activity in equieffective mixtures in a recombinant yeast assay carrying the human estrogen receptor alpha (hERalpha). Mixtures of two, four and eight UV filters alone, or in combination with 17 beta estradiol (E2), were assessed at different effect levels and no-observed-effect-concentrations (NOEC). Predictions of the joint effects of these mixtures were calculated by employing the concentration addition (CA) and independent action (IA) model. Most binary mixtures comprising of pure hERalpha agonists showed a synergistic activity at all mixture combinations. Only in combination with benzophenone-1, antagonistic activity was observed at some effect levels. All mixtures of four or eight, pure or pure and partial hERalpha agonists, alone or including E2, showed synergistic activity at concentrations giving an increase of 10% of basal activity (BC10). This occurred even at concentrations that were at the NOEC level of each single compound. Hence, there were substantial mixture effects even though each UV filter was present at its NOEC level. These results show that significant interactions occur in UV filter mixtures, which is important for the hazard and risk assessments of these personal care products.