Passive sampler housing and sorbent type determine aquatic micropollutant adsorption and subsequent bioassay responses.

The combination of integrative passive sampling and bioassays is a promising approach for monitoring the toxicity of polar organic contaminants in aquatic environments. However, the design of integrative passive samplers can affect the accumulation of compounds and therewith the bioassay responses. The present study aimed to determine the effects of sampler housing and sorbent type on the number of chemical features accumulated in polar passive samplers and the subsequent bioassay responses to extracts of these samplers. To this end, four integrative passive sampler configurations, resulting from the combination of polar organic chemical integrative sampler (POCIS) and Speedisk housings with hydrophilic-lipophilic balance and hydrophilic divinylbenzene sorbents, were simultaneously exposed at reference and contaminated surface water locations. The passive sampler extracts were subjected to chemical non-target screening and a battery of five bioassays. Extracts from POCIS contained a higher number of chemical features and caused higher bioassay responses in 91% of cases, while the two sorbents accumulated similar numbers of features and caused equally frequent but different bioassay responses. Hence, the passive sampler design critically affected the number of accumulated polar organic contaminants as well as their toxicity, highlighting the importance of passive sampler design for effect-based water quality assessment.

The contribution of PFAS to thyroid hormone-displacing activity in Dutch waters: A comparison between two in vitro bioassays with chemical analysis.

Per- and polyfluoroalkyl substances (PFAS) are a group of xenobiotics that are widely distributed throughout the aquatic environment. Many PFAS are possible thyroid hormone (TH) system disrupting compounds, because they have the capacity to -amongst other- inhibit the TH thyroxine (T4) from binding to its transport protein transthyretin (TTR). This study investigated the occurrence of TH-displacing activity in the Dutch water cycle, and more specifically, the contribution of PFAS to this effect. Over one year of monitoring data of 29 PFAS (linear and branched) showed the continuous presence of PFAS in drinking waters and their surface water sources. Secondly, the FITC-T4 and TTR-TRß-CALUX bioassays were mutually compared using positive (HPLC-grade water spiked with PFOA) and negative control samples (HPLC-grade water), as well as relative potency factors (RPFs) of up to 20 PFAS congeners. Both assays were found to be suitable for measuring TH-displacing activity in water samples. As a third aim, a field study was performed in the Dutch water cycle that was comprised of samples from drinking water, surface water, PFAS contaminated sites, and 2 wastewater treatment plants. All samples were analyzed with 1. chemical analysis for 29 PFAS, 2. the FITC-T4 bioassay, and 3. the TTR-TRß-CALUX bioassay. The bioassays mutually showed good correlation (R2 0.85). Bioanalytical equivalent concentrations (BEQ) based on chemically-determined concentrations and RPFs (BEQchem) revealed that analyzed PFAS only explained ≤4.1 % of their activity in water extracts measured by both bioassays (BEQbio). This indicated that as yet unknown compounds contribute to the majority of the measured TH-displacing activity. Moreover, water treatment processes (e.g. DW production from SW) showed a larger contribution of target PFAS to the BEQbio. This could be a first lead to identify unknown compounds that contribute to this activity, and as such, enable the assessment of possible risks associated by the occurrence of TH-displacing activity in water.

Advancements in effect-based surface water quality assessment.

Legally-prescribed chemical monitoring is unfit for determining the pollution status of surface waters, and there is a need for improved assessment methods that consider the aggregated risk of all bioavailable micropollutants present in the aquatic environment. Therefore, the present study aimed to advance effect-based water quality assessment by implementing methodological improvements and to gain insight into contamination source-specific bioanalytical responses. Passive sampling of non-polar and polar organic compounds and metals was applied at 14 surface water locations that were characterized by two major anthropogenic contamination sources, agriculture and wastewater treatment plant (WWTP) effluent, as well as reference locations with a low expected impact from micropollutants. Departing from the experience gained in previous studies, a battery of 20 in vivo and in vitro bioassays was composed and subsequently exposed to the passive sampler extracts. Next, the bioanalytical responses were divided by their respective effect-based trigger values to obtain effect-based risk quotients, which were summed per location. These cumulative ecotoxicological risks were lowest for reference locations (4.3-10.9), followed by agriculture locations (11.3-27.2) and the highest for WWTP locations (12.8-47.7), and were mainly driven by polar organic contaminants. The bioanalytical assessment of the joint risks of metals and (non-)polar organic compounds resulted in the successful identification of pollution source-specific ecotoxicological risk profiles: none of the bioassays were significantly associated with reference locations nor with multiple location types, while horticulture locations were significantly characterized by anti-AR and anti-PR activity and cytotoxicity, and WWTP sites by ERα activity and toxicity in the in vivo bioassays. It is concluded that the presently employed advanced effect-based methods can readily be applied in surface water quality assessment and that the integration of chemical- and effect-based monitoring approaches will foster future-proof water quality assessment strategies on the road to a non-toxic environment.

Dioxins, PCBs, chlorinated pesticides and brominated flame retardants in free-range chicken eggs from peri-urban areas in Arusha, Tanzania: Levels and implications for human health.

The environment in the northern part of Tanzania is influenced by rapid population growth, and increased urbanization. Urban agriculture is common and of economic value for low income families. In Arusha, many households sell eggs from free-ranging backyard chicken. In 2011, 159 eggs from different households in five different locations in Arusha were collected, homogenized, pooled into 28 composite samples and analyzed for a wide selection of POPs. Levels of POPs varied widely within and between the locations. The levels of dieldrin and ΣDDT ranged between 2 and 98,791 and 2 and 324ng/g lipid weight (lw), respectively. EU MRLs of 0.02mg/kg dieldrin for eggs were exceeded in 4/28 samples. PCBs, HCHs, chlordanes, toxaphenes and endosulfanes were found at lower frequency and levels. Brominated flame retardants (BFRs), e.g polybrominated diphenylethers (PBDEs), hexabromocyclododecane (HBCD) and 1,2-bis(2,4,6-tribromphenoxy)ethane (BTBPE) were present in 100%, 60% and 46% of the composite samples, respectively. Octa-and deca-BDEs were the dominating PBDEs and BDE 209 levels ranged between

Programming of metabolic effects in C57BL/6JxFVB mice by exposure to bisphenol A during gestation and lactation.

The global rise in prevalence of obesity is not fully explained by genetics or life style factors. The developmental origins of health and disease paradigm suggests that environmental factors during early life could play a role. In this perspective, perinatal exposure to bisphenol A (BPA) has been indicated as a programming factor for obesity and related metabolic disorders later in life. Here we study early life programming by BPA using an experimental design that is relevant for human exposure. C57BL/6JxFVB hybrid mice were exposed during gestation and lactation via maternal feed to 8 non-toxic doses (0-3000 µg/kg body weight/day (µg/kg bw/d)) of BPA. After weaning, offspring were followed for 20 weeks without further exposure. Adult male offspring showed dose-dependent increases of body and liver weights, no effects on fat pad weights and a dose-dependent decrease in circulating glucagon. Female offspring showed a dose-dependent decrease in body weight, liver, muscle and fat pad weights, adipocyte size, serum lipids, serum leptin and adiponectin. Physical activity was decreased in exposed males and suggested to be increased in exposed females. Brown adipose tissue showed slightly increased lipid accumulation in males and lipid depletion in females, and ucp1 expression was dose-dependently increased in females. The effects in females were more reliable and robust than in males due to wide confidence intervals and potential confounding by litter size for male data. The lowest derived BMDL (lower bound of the (two-sided) 90%-confidence interval for the benchmark dose) of 233 µg/kg bw/d (for interscapular weight in females) was below the proposed BMDL of 3633 µg/kg bw/d as a basis for tolerable daily intake. Although these results suggest that BPA can program for an altered metabolic phenotype, the sexual dimorphism of effects and diversity of outcomes among studies similar in design as the present study do not mark BPA as a specific obesogen. The consistency within the complex of observed metabolic effects suggests that upstream key element(s) in energy homeostasis are modified. Sex-dependent factors contribute to the final phenotypic outcome.

Effects of endocrine disrupting chemicals on in vitro global DNA methylation and adipocyte differentiation.

Recent studies suggest that endocrine disrupting chemicals (EDCs) may form a risk factor for obesity by altering energy metabolism through epigenetic gene regulation. The goal of this study is to investigate the effects of a range of EDCs with putative obesogenic properties on global DNA methylation and adipocyte differentiation in vitro. Murine N2A and human SK-N-AS neuroblastoma cells and murine preadipocyte fibroblasts (3T3-L1) were exposed to tributyltin (TBT), diethylstilbestrol (DES), bisphenol A (BPA), 2,3,7,8-tetrachlorodibenzo-[p]-dioxin (TCDD), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153), hexachlorobenzene (HCB), hexabromocyclododecane (HBCD), 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47) , perfluorinated octyl acid (PFOA) and perfluorinated octyl sulfonate (PFOS). A modest decrease in global DNA methylation was observed in N2A cells exposed to 10 µM DES, BPA, TCDD, BDE-47, PCB-153 and 1 µM HCB, but no changes were found in the human SK-N-AS cells. We reveal for the first time that BDE-47 increases adipocyte differentiation in a dose-dependent manner (2.5-25 µM). Adipocyte differentiation was also enhanced by TBT (≥ 10 nM) and BPA (>10 µM) and inhibited by TCDD (≥ 0.1 nM). The other chemicals showed either modest or no effects on adipocyte differentiation at the concentrations tested (PFOA, PFOS and HBCD at 10 µM; PCB-153, 3.4 µM and HCB, 1 µM). This study demonstrates that selected EDCs can induce functional changes in murine adipocyte differentiation in vitro which are accompanied by decreased global DNA methylation.

Integrated chemical and biological analysis to explain estrogenic potency in bile extracts of red mullet (Mullus barbatus).

A biological screening was performed to establish the total exposure to estrogenic compounds of red mullet (Mullus barbatus) collected at several sites along the Spanish Mediterranean coast by testing male fish bile extracts using the in vitro ER-LUC reporter gene assay. In addition, major metabolites were identified and measurements of OH-PAHs (1-naphthol, 9-phenantrol, 9-fluorenol, 1-pyrenol, 1OH-BaP and 3OH-BaP) and alkylphenols (4-n-nonylphenol (4-n-NP) and 4-tertoctylphenol (4-tert-OP)) in the same fish bile extracts were taken by gas chromatography-mass spectrometry in electron ionization mode (GC-EI-MS). Relative in vitro estrogenic potencies of the chemically quantified compounds were also tested. The highest biliary concentrations of 1-pyrenol, 9-fluorenol and 4-n-NP were found in fish from Barcelona and from the Mar Menor coastal lagoon. However, these concentrations can be considered relatively low compared to values reported in red mullet from other polluted waters in the Mediterranean Sea. The contribution of 1-pyrenol, 4-n-NP and 4-tert-OP to the total estrogenic potency measured in male fish bile was found to be negligible, indicating the presence of other estrogenic compounds in red mullet bile. Estrogenic potency in bile from male fish was markedly elevated in Mar Menor lagoon (234.8±5.7 pg E2EQ/µl), and further research will be necessary to explain whether the presence of natural and synthetic-hormones in the lagoon contributed to this finding. Values of approximately 15-16E2EQ pg/mg bile can be regarded as preliminary baseline levels of bile estrogenicity in male red mullet from the western Mediterranean Sea.

Lack of a distinct gradient in biomarker responses in small mammals collected at different distances from a highway.

This study describes biomarker effects in small mammals exposed to traffic emissions. Animals were collected at 10-50 m (site 1), 150-200 m (site 2), and 5 km (site 3) from a very busy highway (A2). To distinguish between routes of exposure, strictly carnivorous common shrews ( Sorex araneus) and predominantly herbivorous bank voles ( Clethrionomys glareolus) were collected. As a measure of exposure to polycyclic aromatic hydrocarbons (PAHs), aromatic DNA adduct levels were determined by (32)P-postlabeling techniques in tissue from heart, lung, and liver. Lead (Pb), cadmium (Cd), and copper (Cu) levels were analyzed in kidney as a measure of exposure to heavy metals. EROD and PROD activity and retinoid levels were determined in liver as effect biomarkers for exposure to PAHs and polyhalogenated aromatic hydrocarbons (PHAHs). Relatively high Cd levels in S. araneus and in particular elevated DNA adduct levels in C. glareolus indicated that small mammals at site 3 were exposed to more compounds than at sites 1 and 2 (3 > or = 1 > 2). The latter effect is probably due to an incidental and actual input of airborne pollutants that is deposited on plant surfaces. By consumption of above-ground vegetation, voles are chronically exposed to this pollution. Relatively high background input of PAHs probably hinders that the traffic-related gradient of airborne PAH concentrations found in an earlier study is reflected in DNA adduct levels in small mammals in the present study. Moreover, historical biomarkers for exposure to traffic emissions, such as increased kidney Pb levels, increased hepatic EROD activity, and disturbed hepatic vitamin A homeostasis are no longer applicable to indicate differences in exposure. This is a result of the ban on addition of Pb and chlorinated scavengers to gasoline and of cleaner combustion techniques, which were enforced by law over the past decade. Finally, it is advisable to use only juvenile small mammals for in situ monitoring of diffuse pollution because DNA adduct levels increased with age.

Biological and chemical analysis of the toxic potency of pesticides in rainwater.

A newly developed method for measuring the integrated esterase inhibiting potency of rainwater samples was applied in practice, and the results are compared to the toxic potency calculated from concentrations of 31 organophosphate (OP) and carbamate pesticides, out of a total of 66 chemically analyzed pesticides. In addition, the general toxic potency of the rainwater samples was evaluated in a microtiter luminescence assay with Vibrio fischeri bacteria. Rainwater samples were collected over four consecutive 14-day periods in both open and wet-only samplers. The esterase inhibiting potency of the open rainwater samples (expressed as ng dichlorvos-equivalents/l) corresponded well with the chemical analyses of the rainwater samples collected by both types of samplers (r = 0.83-0.86). By far, the highest esterase inhibiting potency was found in a sample collected in an area with intense horticultural activities in June, and was attributed to high concentrations of dichlorvos, mevinphos, pirimiphos-methyl and methiocarb. The esterase inhibiting potency of this sample was equivalent to a dichlorvos concentration of 1380 ng/l in the rainwater, which is almost 2000 times higher than the maximum permissible concentration (MPC) of dichlorvos set for surface water in Netherlands. Maximum individual concentrations of dichlorvos and pirimiphos-methyl even exceeded the EC50 for Daphnia, suggesting that pesticides in rainwater pose a risk for aquatic organisms. Not all responses of the luminescence-assay for general toxicity could be explained by the analyzed pesticide concentrations. The bio-assays enable a direct assessment the toxic potency of all individual compounds present in the complex mixture of rainwater pollutants, even if they are unknown or present at concentrations below the detection limit. Therefore, they are valuable tools for prescreening and hazard characterization purposes.

The application of reporter gene assays for the determination of the toxic potency of diffuse air pollution.

Diffuse air pollution consists of a mixture of numerous compounds. It is emitted by many distributed sources and is omnipresent due to atmospheric transport. Risk assessment of the complex mixture of air pollutants on the basis of the toxicity of the individual compounds is not yet possible because the chemical identity and/or toxicity of the constituencies of a substantial fraction is unknown. In addition, no adequate procedures are available to integrate toxicity data of such complex mixtures, so that an individual risk assessment of the constituents of air pollution disregards possible combination effects. In the present study, an approach has been developed to assess the toxic potency by using in vitro bio-assay techniques. Genotoxicity was assessed in the umu-assay, a reporter gene assay using a strain of Salmonella typhimurium stably transfected with a plasmid (pSK1002) carrying the SOS-gene umuC fused to the reporter gene lacZ. Arylhydrocarbon-receptor activation was assessed in the DR-CALUX-assay, using a stably transfected H4IIE hepatoma cell line containing a plasmid for the luciferase gene under transcriptional control of dioxin-responsive elements. Samples of airborne particulate matter (APM) were collected with a high volume sampler next to a highway and in a natural conservation area. Both assays proved to be applicable to quantify genotoxicity and the presence of polycyclic aromatic hydrocarbons (PAHs) in small extracts from air-filter samples. Results indicate that PAHs from traffic exhausts seem to be largely responsible for an increased genotoxic activity of APM collected down-wind from the highway (western wind). APM collected at eastern wind directions seems to have a different composition of compounds, with a higher genotoxic activity that is less related to highway-emitted PAH-like compounds. At northern wind directions, APM is relatively less genotoxic and contains less PAHs than at other wind directions. Dioxin-like compounds contribute negligibly to the Ah-receptor agonistic potency of APM. Airborne pollutants with genotoxic and/or PAH-like characteristics form an undesired mutagenic risk, which will be evaluated in further in vivo studies.

A small-volume bioassay for quantification of the esterase inhibiting potency of mixtures of organophosphate and carbamate insecticides in rainwater: development and optimization.

The goal of this study was to develop a sensitive in vitro bioassay for quantification of the total esterase inhibiting potency of low concentrations of organophosphate and carbamate insecticides in relatively small rainwater samples. Purified acetylcholinesterase (AChE) from electric eel (Electrophorus electricus) and carboxylesterases from a homogenate of honeybee heads (Apis mellifera) were used as esterases, each having different affinities for the substrates S-acetylthiocholine-iodide (ATC) and N-methylindoxylacetate (MIA). MIA hydrolysis by honeybee homogenate was more sensitive to inhibition by organophosphate insecticides than ATC hydrolysis by purified AChE, although the latter parameter is often used for in vitro monitoring of esterase inhibitors. The higher sensitivity of carboxylesterases is attributed to the instant formation of a reversible Michaelis-Menten complex with the inhibitor, which competes with MIA for the active sites of the free enzymes. This dose-dependent instant inhibition can be quantified with kinetics for competitive inhibition at dichlorvos concentrations < 16 nM. At similar concentrations, purified AChE was not instantly inhibited, whereas both AChE and carboxylesterases were irreversibly and progressively inhibited at higher dichlorvos concentrations (IC50(10min) >/= 0.1 microM). Honeybee homogenate mediated MIA hydrolysis was applied as the most sensitive enzyme-substrate combination for experiments with fractionated extracts of 4 rainwater samples collected in a natural conservation area. Most esterase inhibiting potency was found in the polar methanol fraction, with recalculated concentrations equivalent to 12-125 ng dichlorvos per liter rainwater.

Predator-prey relationships in a two-species toxicity test system.

In a two-species toxicity test system survival and reproduction of both the predator Hypoaspis aculeifer (Gamasida) and the prey Folsomia fimetaria (Collembola) were studied after 21 days of residual exposure to a soil contamination of the insecticide dimethoate. Additional experiments were run to analyze which species-species and compound-species relationships determine the outcome of this two-species experiment. Number of adult F. fimetaria were reduced by both predation and dimethoate exposure, whereas mites preyed less efficiently on adults than on juveniles. At 0.357 mg dimethoate/kg soil, numbers of juvenile F. fimetaria were mainly reduced by predation on adults and juveniles. At 0.7 mg/kg, an additional dimethoate effect was found, which was attributed to an effect on the reproduction of F. fimetaria, mainly due to lethality of adults. It was reasoned that lethal effects on juvenile springtails are less important. Adult H. aculeifer was not affected by dimethoate exposure, whereas numbers of juvenile H. aculeifer demonstrated a decline only at the highest concentration of 0.7 mg/kg. It is hypothesized that this latter effect is possibly due to food depletion caused by a decreased availability of prey, rather than to the lethal effects of dimethoate on juvenile mites. Such a secondary effect of a pesticide application could not have been derived from a single-species toxicity experiment and demonstrates the additional value of a two-species toxicity test system.