Spatial and temporal distribution of toxic compounds in sediments and potential ecological effects on macrobenthic faunal species in Hangzhou Bay from 2003 to 2015.

The development of toxic compounds in sediment and macrobenthos species in Hangzhou bay (2003-2015) was evaluated. Concentrations were compared to Chinese sediment quality guidelines (CN-SQG) and risk assessed by the ecological risk index (ERI) and t-Distributed Stochastic Neighbour Embedding (t-SNE). To study seafood contamination, sediment and swimming crabs were collected. Chromium, copper, and arsenic exceeded CN-SQG. Organic contaminants did not exceed CN-SQG; however, t-SNE revealed a negative relationship with benthic species numbers. Since 2003, half of the benthic species have disappeared. Species sensitive to contamination were not observed after 2003-2007, while crustacea species are more tolerant: cadmium levels in crabs were 5-17 times those in the sediment, demonstrating strong bioaccumulation. These results suggest that metals and organic pollutants pose ecological and seafood risks. For good environmental management in HZB, it is important to analyze sediment, benthic biota, and seafood species for compounds known to pose toxic risks.

Dataset of liver proteins of eu- and hypothyroid rats affected in abundance by any of three factors: in vivo exposure to hexabromocyclododecane (HBCD), thyroid status, gender differences.

Male Wistar rats with different thyroid status (eu-, hypothyroid) were exposed to 0, 3 or 30 mg/kg body weight of the flame retardant HBCD for 7 days and obtained data compared with a previous study in females, "Hexabromocyclododecane (HBCD) induced changes in the liver proteome of eu- and hypothyroid female rats" (Miller et al., 2016) [1]. Specifically, proteomic investigation of liver protein patterns obtained by 2D-DIGE was performed and differences between animals groups recorded, based on the factors exposure, thyroid status and gender. All proteins with significantly changed abundance in any of these comparisons were identified by mass spectrometry. General, hormone and proteomic data of both the present and the previous studies are discussed in Miller et al. (2016) [1] and in "Gender specific differences in the liver proteome of rats exposed to hexabromocyclododecane (HBCD)" Miller et al. (2016) [2].

Dataset of liver proteins changed in eu- and hypothyroid female rats upon in vivo exposure to hexabromocyclododecane (HBCD).

Female Wistar rats with different thyroid status (eu-, hypothyroid) were exposed to 0, 3 or 30 mg/kg body weight of the flame retardant HBCD for 7 days. Changes in protein patterns obtained by 2D-DIGE were evaluated, and different animal groups compared taking into account their exposure and thyroid status. Proteins significantly altered in abundance in any of these comparisons were identified by mass spectrometry. These data, together with hormone data of the animals, are discussed in "Hexa-bromocyclododecane (HBCD) induced changes in the liver proteome of eu- and hypothyroid female rats" (Miller et al., 2016) [1].

Hexabromocyclododecane (HBCD) induced changes in the liver proteome of eu- and hypothyroid female rats.

Hexabromocyclododecane (HBCD) is a brominated flame retardant known for its low acute toxicity as observed in animal experiments. However, HBCD exposure can affect liver functioning and thyroid hormone (TH) status. As exact mechanisms are unknown and only limited toxicological data exists, a gel-based proteomic approach was undertaken. In a eu- and hypothyroid female rat model, rats were exposed to 3 and 30 mg/kg bw/day HBCD for 7 days via their diet, and exposure was related to a range of canonical endpoints (hormone status, body weight) available for these animals. Alterations in the liver proteome under HBCD exposure were determined in comparison with patterns of control animals, for both thyroid states. This revealed significantly changed abundance of proteins involved in metabolic processes (gluconeogenesis/glycolysis, amino acid metabolism, lipid metabolism), but also in oxidative stress responses, in both euthyroid and hypothyroid rats. The results provide a more detailed picture on the mechanisms involved in these alterations, e.g. at the protein level changes of the proposed influence of HBCD on the lipid metabolism. Present results show that proteomic approaches can provide further mechanistic insights in toxicological studies.

Gender specific differences in the liver proteome of rats exposed to short term and low-concentration hexabromocyclododecane (HBCD).

The influence of short term (7-day) exposure of male rats to the brominated flame retardant hexabromocyclododecane (HBCD) was studied by investigation of the liver proteome, both in euthyroid and hypothyroid rats and by comparing results with general data on animal physiology and thyroid hormone, leptin, insulin and gonadotropin concentrations determined in parallel. Proteome analysis of liver tissue by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) revealed that only small protein pattern changes were induced by exposure in males, on just a few proteins with different functions and not involved in pathways in common. This is in contrast to previous findings in similarly exposed eu- and hypothyroid female rats, where general metabolic pathways had been shown to be affected. The largest gender-dependent effects concerned basal concentrations of liver proteins already in control and hypothyroid animals, involving mainly the pathways which were also differently affected by HBCD exposure. Among them were differences in lipid metabolism, which - upon exposure to HBCD - may also be the reason for the considerably higher ratio of γ-HBCD accumulated in white adipose tissue of exposed female rats compared to males. The results further elucidate the already suggested different sensitivity of genders towards HBCD exposure on the protein level, and confirm the need for undertaking toxicological animal experiments in both genders.

Deconjugation of soy isoflavone glucuronides needed for estrogenic activity.

Soy isoflavones (SIF) are present in the systemic circulation as conjugated forms of which the estrogenic potency is not yet clear. The present study provides evidence that the major SIF glucuronide metabolites in blood, genistein-7-O-glucuronide (GG) and daidzein-7-O-glucuronide (DG), only become estrogenic after deconjugation. The estrogenic potencies of genistein (Ge), daidzein (Da), GG and DG were determined using stably transfected U2OS-ERα, U2OS-ERß reporter gene cells and proliferation was tested in T47D-ERß cells mimicking the ERα/ERß ratio of healthy breast cells and inT47D breast cancer cells. In all assays applied, the estrogenic potency of the aglycones was significantly higher than that of their corresponding glucuronides. UPLC analysis revealed that in U2OS and T47D cells, 0.2-1.6% of the glucuronides were deconjugated to their corresponding aglycones. The resulting aglycone concentrations can account for the estrogenicity observed upon glucuronide exposure. Interestingly, under similar experimental conditions, rat breast tissue S9 fraction was about 30 times more potent in deconjugating these glucuronides than human breast tissue S9 fraction. Our study confirms that SIF glucuronides are not estrogenic as such, and that the small % of deconjugation in the cell is enough to explain the slight bioactivity observed for the SIF-glucuronides. Species differences in deconjugation capacity should be taken into account when basing risk-benefit assessment of these SIF for the human population on animal data.

How much do PCB toxic equivalents account for PHAH toxicity in predatory birds?

Various diffuse polyhalogenated aromatic hydrocarbons (PHAHs) exert common toxicity through the aryl hydrocarbon receptor (AhR). Apex predators spatially and temporally integrate diffuse contamination and simultaneous exposure can cause additive toxicity. We investigated the extent to which PCBs, still amongst the most prevalent PHAHs accumulated by predators, accounted for total PHAH toxicity in raptors and fish eating birds from Britain. We analysed egg or liver extracts from six species and compared chemically determined ΣPCB-TEQs concentrations with total AhR-mediated toxicity determined using the chemical-activated luciferase gene expression bioassay (CALUX-TEQ). Dioxin-like PCB profiles in eggs and livers were dominated by congeners 118, 105 and 167. ΣPCB-TEQ and CALUX-TEQ concentrations were positively associated but not in a 1:1 relationship. ΣPCB-TEQ were broadly similar to CALUX-TEQ concentrations in eggs and livers with CALUX-TEQ concentrations >50-80 and 160-320 pg g(-1) lipid respectively, but were lower than CALUX-TEQ concentrations in less contaminated samples.

The added value of proteomics for toxicological studies.

Proteomics has the potential to elucidate complex patterns of toxic action attributed to its unique holistic a posteriori approach. In the case of toxic compounds for which the mechanism of action is not completely understood, a proteomic approach may provide valuable mechanistic insight. This review provides an overview of currently available proteomic techniques, including examples of their application in toxicological in vivo and in vitro studies. Future perspectives for a wider application of state-of-the-art proteomic techniques in the field of toxicology are discussed. The examples concern experiments with dioxins, polychlorinated biphenyls, and polybrominated diphenyl ethers as model compounds, as they exhibit a plethora of sublethal effects, of which some mechanisms were revealed via successful proteomic studies. Generally, this review shows the added value of including proteomics in a modern tool box for toxicological studies.

Human T47D-ERß breast cancer cells with tetracycline-dependent ERß expression reflect ERα/ERß ratios in rat and human breast tissue.

T47D-ERß breast cancer cells with tetracycline-dependent ERß expression and constant ERα expression can be used to investigate effects of varying ERα/ERß ratios on estrogen-induced cellular responses. This study defines conditions at which ERα/ERß ratios in T47D-ERß cells best mimic ERα/ERß ratios in breast and other estrogen-sensitive tissues in vivo in rat as well as in human. Protein and mRNA levels of ERα and ERß were analyzed in T47D-ERß cells exposed to a range of tetracycline concentrations and compared to ERα and ERß levels found in breast, prostate, and uterus from rat and human origin. The ERα/ERß ratio in T47D-ERß cells exposed to >150ng/ml tetracycline is comparable to the ratio found in rat mammary gland and in human breast tissue. The ERα/ERß ratio of other estrogen-sensitive rat and human tissues can also be mimicked in T47D-ERß cells. The ERα/ERß ratio found in MCF-7 and native T47D breast cancer cell lines did not reflect ratios in analyzed rat and human tissues, which further supports the use of T47D-ERß cells as model for estrogen-responsive tissues. Using 17ß-estradiol and the T47D-ERß cells under the conditions defined to mimic various tissues it could be demonstrated how these different tissues vary in their proliferative response.

Toxic concentrations in fish early life stages peak at a critical moment.

During the development of an embryo into a juvenile, the physiology and behavior of a fish change greatly, affecting exposure to and uptake of environmental pollutants. Based on experimental data with sole (Solea solea), an existing bioaccumulation model was adapted and validated to calculate the development of concentrations of persistent organic pollutants in the tissue of developing fish. Simulation revealed that toxic tissue concentrations of pollutants with log octanol-water partition ratio (K(OW)) > 5 peak at the moment when the larvae become free-feeding, when the lipid reserves are depleted. This may explain the delayed effects observed in fish early-life-stage experiments with exposed eggs. In the field, eggs can be exposed through maternal transfer to adult pollutant tissue concentrations, which will increase in the larva to peak tissue concentrations, exceeding those of the adult fish. The results demonstrate the risk of underestimating the effects of lipophilic persistent organic pollutants with log K(OW) > 5 in short-term, early-life-stage fish tests and underscore the importance of maternal transfer as an exposure route in the field situation.

Effects of mixtures of persistent organic pollutants (POPs) derived from cod liver oil on H295R steroidogenesis.

Crude cod liver oil and liver oil supplements are consumed as a source of vitamin A, D and polyunsaturated fatty acids; during winter and early pregnancy. Crude cod liver oil however constitutes a considerable source of persistent organic pollutants (POPs). This paper aimed at characterizing and quantifying the influence of POP mixtures extracted from three different steps in the cod liver oil industrial process on hormone production and the expression of steroidogenesis-related genes in H295R cells. Exposure to extracts from crude cod liver oil and from its industrial waste increased progesterone (P4), cortisol (Cort), testosterone (T) and estradiol (E2) production; and among others, the expression of MC2R, CYP11B1 and HSD3B2 genes. Observed effects after exposure to pharmaceutical cod liver oil extract were considerably lower. The type of effects on gene expression and hormone production were similar to those induced by forskolin and PCBs, the latter being the major contaminants within the extracts. Additional research is required to further unveil the mechanisms behind the observed steroidogenic effects and to assess whether the potential risk might outweigh the potential benefits of crude and processed cod liver oil consumption.

Application of bioassays in toxicological hazard, risk and impact assessments of dredged sediments.

Given the potential environmental consequences of dumped dredged harbour sediments it is vital to establish the potential risks from exposure before disposal at sea. Currently, European legislation for disposal of contaminated sediments at sea is based on chemical analysis of a limited number of well-known contaminants for which maximum acceptable concentrations, action levels (ALs), have been set. The present paper addresses the issue of the applicability of in vitro and in vivo bioassays for hazard, risk and local impact assessment of dredged polluted sediments to be disposed of at sea. It discusses how and to what extent selected bioassays can fill in the gaps left open by chemical analysis and the way in which the bioassays may contribute to the present licensing system for disposal. Three different purposes for application were distinguished: the most basic application (A) is a rapid determination of the hazard (potential toxicity) of dredged sediments which is then compared to ALs in a licensing system. As with chemical analysis on whole sediment extracts, the bioavailability of the chemicals is not taken into account. As in vitro assays with sediment extracts are not sensitive to matrix effects, a selection of specific in vitro bioassays can be suitable fast and standardized additions for the licensing system. When the outcome of (A) does not convincingly demonstrate whether the sediment is clean enough or too polluted, further bioanalysis can help the decision making process (B). More aspects of the mostly unknown complex chemical mixtures are taken into account, including the bioavailability and chronic toxicity focusing on ecologically relevant endpoints. The ecotoxicological pressure imposed by the dredged sediments can be quantified as the potentially affected fraction (PAF) based on chemical or biological analysis of levels of contaminants in sediment or biota. To validate the predicted risk, the actual impact of dumped harbour sediments on local ecosystems (C) can be determined using a dedicated set of in vitro and in vivo bioassays as well as bio-indicators selected based on the information obtained from (A) and (B) and on the characteristics of the local ecosystem. Conversely, the local sediment impact assessment (C) can direct fine-tuning of the selection of chemical and bioassay analyses and for setting safe levels in the licensing system. It is concluded that in vitro and in vivo bioassays and biological indicators are useful tools in the process of hazard, ecotoxicological risk and impact assessment of dredged harbour sediments, provided they are consciously chosen and quality criteria for assay performance are defined.

Superinduction of estrogen receptor mediated gene expression in luciferase based reporter gene assays is mediated by a post-transcriptional mechanism.

Several estrogenic compounds including the isoflavonoid genistein have been reported to induce a higher maximal response than the natural estrogen 17ß-estradiol in in vitro luciferase based reporter gene bioassays for testing estrogenicity. The phenomenon has been referred to as superinduction. The mechanism underlying this effect and thus also its biological relevance remain to be elucidated. In the present study several hypotheses for the possible mechanisms underlying this superinduction were investigated using genistein as the model compound. These hypotheses included (i) a non-estrogen receptor (ER)-mediated mechanism, (ii) a role for an ER activating genistein metabolite with higher ER inducing activity than genistein itself, and (iii) a post-transcriptional mechanism that is not biologically relevant but specific for the luciferase based reporter gene assays. The data presented in this study indicate that induction and also superinduction of the reporter gene is ER-mediated, and that superinduction by genistein could be ascribed to stabilization of the firefly luciferase reporter enzyme increasing the bioluminescent signal during the cell-based assay. This indicates that the phenomenon of superinduction may not be biologically relevant but may rather represent a post-transcriptional effect on enzyme stability.

Phytoestrogen-mediated inhibition of proliferation of the human T47D breast cancer cells depends on the ERalpha/ERbeta ratio.

This study investigates the importance of the intracellular ratio of the two estrogen receptors ERalpha and ERbeta for the ultimate potential of the phytoestrogens genistein and quercetin to stimulate or inhibit cancer cell proliferation. This is of importance because (i) ERbeta has been postulated to play a role in modulating ERalpha-mediated cell proliferation, (ii) genistein and quercetin may be agonists for both receptor types and (iii) the ratio of ERalpha to ERbeta is known to vary between tissues. Using human osteosarcoma (U2OS) ERalpha or ERbeta reporter cells it was shown that compared to estradiol (E2), genistein and quercetin have not only a relatively greater preference for ERbeta but also a higher maximal potential for activating ERbeta-mediated gene expression. Using the human T47D breast cancer cell line with tetracycline-dependent ERbeta expression (T47D-ERbeta), the effect of a varying intracellular ERalpha/ERbeta ratio on E2- or pythoestrogen-induced cell proliferation was characterised. E2-induced proliferation of cells in which ERbeta expression was inhibited was similar to that of the T47D wild type cells, whereas this E2-induced cell proliferation was no longer observed when ERbeta expression was increased. With increased expression of ERbeta the phytoestrogen-induced cell proliferation was also reduced. These results point at the importance of the cellular ERalpha/ERbeta ratio for the ultimate effect of (phyto)estrogens on cell proliferation.

In vivo and in vitro Ah-receptor activation by commercial and fractionated pentabromodiphenylether using zebrafish (Danio rerio) and the DR-CALUX assay.

The present study addresses the toxicity of a commercial pentabrominated diphenylether (PeBDE) flame retardant mixture, DE-71, in a model aquatic vertebrate. Four weeks' exposure of juvenile zebrafish (Danio rerio) to water-borne DE-71 resulted in dose-dependent induction of CYP1A immunoreactivity, predominantly in the endocardium and the endothelium of larger blood vessels, such as ventral aorta and branchial arteries, as well as the larger hepatic and pancreatic blood vessels. To investigate the impact of possible contaminating PBDD/Fs in the DE-71 product, the study was repeated after DE-71 had been fractionated into a non-planar (cleaned PBDEs) and a planar fraction (PBDD/Fs). Zebrafish were exposed under similar conditions to the planar and cleaned DE-71 fractions, and to uncleaned DE-71. In addition, the above fractions were chemically analyzed and tested in a reporter gene assay (DR-CALUX) for their aromatic hydrocarbon-receptor (AhR) stimulating potencies. A relatively strong CALUX response was detected from the planar DE-71 fraction (19.7ng TCDD equivalent (TEQ)/g DE-71), coinciding with a strong induction of CYP1A immunoreactivity in zebrafish. CYP1A immunoreactivity in zebrafish exposed to uncleaned DE-71 was intense, although the CALUX response was 10-fold less compared to the planar fraction. Only weak CYP1A immunoreactivity was found in fish exposed to cleaned DE-71, and none in control animals; no CALUX response was detected in cleaned DE-71. The present findings indicate that chemical impurities of the commercial PeBDE product account for AhR-mediated effects. Analytical isolation of a planar fraction from the commercial product increased the in vitro (DR-CALUX) signal 10 times. Immunohistochemistry showed a strong tissue specific reaction to DE-71 in vivo at these relatively low TEQ levels regardless of chemical pretreatment of the mix, reflecting the sensitivity of CYP1A induction in juvenile zebrafish to AhR agonists.

Behavioural responses of Gammarus pulex (Crustacea, Amphipoda) to low concentrations of pharmaceuticals.

The continuous discharge of pharmaceuticals and personal care products into the environment results in a chronic exposure of aquatic organisms to these substances and their metabolites. As concentrations in surface waters are in the ng/L range, and sometimes in the low microg/L range, they are not likely to result in lethal toxicity. However, prolonged exposure to low concentrations of anthropogenic chemicals may lead to sublethal effects, including changes in behaviour. The aim of this study was to assess the effect of three pharmaceuticals, the antidepressant fluoxetine, the analgesic ibuprofen and the anti-epileptic carbamazepine, and one cationic surfactant, cetyltrimethylammonium bromide (CTAB), on the activity of the benthic invertebrate Gammarus pulex (Crustacea: Amphipoda: Gammaridae). We used the multispecies freshwater biomonitor to assess changes in activity of G. pulex in a quantitative manner. Exposure to low concentrations (10-100ng/L) of fluoxetine and ibuprofen resulted in a significant decrease in activity, whereas the activity of G. pulex at higher concentrations (1microg/L-1mg/L) was similar to the control. Response to carbamazepine showed a similar pattern, however, differences were not significant. The tested surfactant CTAB evoked a decrease in activity at increasing concentration. These behavioural effect concentrations were 10(4) to 10(7) times lower than previously reported LOECs and in the range of environmentally occurring concentrations. The potential consequences of this decreased activity for G. pulex population growth and benthic community structure and the exposure to mixtures of pharmaceuticals deserves further attention.

Are dioxin-like contaminants responsible for the eel (Anguilla anguilla) drama?

Eel populations worldwide are dangerously close to collapsing. Our study is the first to show that current levels of dioxin-like contaminants are strong candidates because of their devastating effects on development and survival of eel embryos. Female and male silver eels were artificially stimulated to maturation and reproduction by treatment with carp pituitary extracts and hCG, respectively. During maturation of female European silver eels, about 60 g fat per kg eel is incorporated in the oocytes. Together with the fat, however, persistent organic pollutants such as dioxin-like polychlorinated biphenyls (PCBs) are incorporated too. The total dioxin-like toxic potency of the individual gonad batches was determined as 2,3,7,8-tetrachlorodibenzo-p-dioxine equivalents (TEQs), using an in vitro reporter gene assay. The observed differences in development and survival showed a significant negative correlation with the TEQ levels in the gonads, already at levels far below the maximal allowable level for fish consumption, i.e., 4 ng TEQ/kg fish. The clear inverse relationship between the TEQ level and the survival period of the fertilised eggs strongly suggests that the current levels of dioxin-like compounds seriously impair the reproduction of the European eel. The peak of the environmental levels of dioxin-like PCBs and the decline of eel coincide worldwide, further suggesting that, in addition to other threats, these contaminants contributed significantly to the current collapse in eel populations.

In vitro biomonitoring in polar extracts of solid phase matrices reveals the presence of unknown compounds with estrogenic activity.

Determination of estrogenic activity has so far mainly concentrated on the assessment of compounds in surface water and effluent. This study is one of the first to biomonitor (xeno-)estrogens in sediment, suspended particulate matter and aquatic organisms. The relatively polar acetone extracts from these solid phase matrices do not contain the well-known estrogenic compounds such as hormones, alkylphenols and phthalates. An in vitro 'estrogen receptor-mediated chemical activated luciferase gene expression' (ER-CALUX) assay was applied to samples from various locations in the Netherlands. Estrogenic activity measured in polar fractions of particulate matter and sediment extracts ranged from below detection limit to up to 4.5 pmol estradiol equivalents (EEQ)/g dry weight. Estrogenic activity in freshwater river sediments was up to five times higher compared to sediments from large lakes and coastal locations. Tissue extracts EEQs were determined in bream (Abramis brama), flounder (Platichthysflesus), freshwater mussels (Dreissena polymorpha) and marine mussels (Mytilus edulis). The highest biota EEQ levels were found in the freshwater zebra mussel (30 pmol EEQ/g lipid). One sample site showed greatly elevated EEQs in sediment and biota, which correlated with effects found in the wild populations of bream. The EEQ activity of the unknown compounds in the polar fraction mostly was much higher than the calculated EEQ levels based on known estrogens in the non-polar fraction (previously published data).

Environmental impact and recovery at two dumping sites for dredged material in the North Sea.

The environmental impact and recovery associated with the long and uninterrupted disposal of large volumes of moderately contaminated dredged material from the port of Rotterdam was studied at nearby dumping sites in the North Sea. Observations were made on sediment contamination, ecotoxicity, biomarker responses and benthic community changes shortly after dumping at the 'North' site had ceased and at the start of disposal at the new dumping site 'Northwest'. During the period of dumping, very few benthic invertebrates were found at the North site. Concentrations of cadmium, mercury, polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs) and tributyltin (TBT) in the fine sediment fraction (<63 microm) from this site were 2-3 times higher than at the reference site. In four different bioassays with marine invertebrates the sediments showed no acute toxic effects. In tissue (pyloric caeca) of resident starfish Asterias rubens, residual levels of mercury, zinc, PCBs and dioxin-like activity were never more than twice those at the reference site. Four different biomarkers (DNA integrity, cytochrome P450 content, benzo[a]pyrene hydroxylase activity and acetylcholinesterase inhibition) were used on the starfish tissues, but no significant differences were found between North and the reference site. Minor pathological effects were observed in resident dab Limanda limanda. One year after dumping had ceased at the North site, a significant increase in the species richness and abundance of benthic invertebrates and a concomitant decrease in the fine sediment fraction of the seabed were observed. After 8.2 million m3 of moderately contaminated dredged material had been dumped at the new dumping site Northwest, the species richness and abundance of benthic invertebrates declined over an area extending about 1-2 km eastwards. This correlated with a shift in sediment texture from sand to silt. The contamination of the fine sediment fraction at the Northwest location doubled. It is concluded that marine benthic resources at and around the dumping sites have been adversely affected by physical disturbance (burial, smothering). However, no causal link could be established with sediment-associated contaminants from the dredged spoils.

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.