Synthetic and natural rubber associated chemicals drive functional and structural changes as well as adaptations to antibiotics in in vitro marine microbiomes.

The leaching of additives from plastics and elastomers (rubbers) has raised concerns due to their potential negative impacts on the environment and the development of antibiotic resistance. In this study, we investigated the effects of chemicals extracted from two types of rubber on microbiomes derived from a benthic sea urchin and two pelagic fish species. Additionally, we examined whether bacterial communities preconditioned with rubber-associated chemicals displayed adaptations to antibiotics. At the highest tested concentrations of chemicals, we observed reduced maximum growth rates and yields, prolonged lag phases, and increased alpha diversity. While the effects on alpha and beta diversity were not always conclusive, several bacterial genera were significantly influenced by chemicals from the two rubber sources. Subsequent exposure of sea urchin microbiomes preconditioned with rubber chemicals to the antibiotic ciprofloxacin resulted in decreased maximum growth rates. This indicates a more sensitive microbiome to ciprofloxacin when preconditioned with rubber chemicals. Although no significant interaction effects between rubber chemicals and ciprofloxacin exposure were observed in bacterial alpha and beta diversity, we observed log-fold changes in two bacterial genera in response to ciprofloxacin exposure. These findings highlight the structural and functional alterations in microbiomes originating from various marine species when exposed to rubber-associated chemicals and underscore the potential risks posed to marine life.

A simple protocol for estimating the acute toxicity of unresolved polar compounds from field-weathered oils.

Crude oil spilled at sea is chemically altered through environmental processes such as dissolution, biodegradation, and photodegradation. Transformation of hydrocarbons to oxygenated species increases water-solubility. Metabolites and oxidation products largely remain uncharacterized by common analytical methods but may be more bioavailable to aquatic organisms. Studies have shown that unresolved (i.e. unidentified) polar compounds ('UPCs') may constitute > 90% of the water-accommodated fraction (WAF) of heavily weathered crude oils, but still there is a paucity of information characterizing their toxicological significance in relation to other oil-derived toxicants. In this study, low-energy WAFs (no droplets) were generated from two field-weathered oils (collected during the 2010 Deepwater Horizon incident) and their polar fractions were isolated through fractionation. To allow establishment of thresholds for acute toxicity (LC50) of the dissolved and polar fraction of field collected oils, we concentrated both WAFs and polar fractions to beyond field-documented concentrations, and the acute toxicity of both to the marine copepod Acartia tonsa was measured and compared to the toxicity of the native WAF (non-concentrated). The difference in toxic units (TUs) between the total of the mixture and of identified compounds of known toxicity (polycyclic aromatic hydrocarbons [PAHs] and alkyl phenols) in both WAF and polar fractions was used to estimate the contribution of the UPC to overall toxicity. This approach identified that UPC had a similar contribution to toxicity as identified compounds within the WAFs of the field-weathered oils. This signifies the relative importance of polar compounds when assessing environmental impacts of spilled and weathered oil.

Comparison of two field systems for determination of crude oil biodegradation in cold seawater.

Marine oil spills have devastating environmental impacts and extrapolation of experimental fate and impact data from the lab to the field remains challenging due to the lack of comparable field data. In this work we compared two field systems used to study in situ oil depletion with emphasis on biodegradation and associated microbial communities. The systems were based on (i) oil impregnated clay beads and (ii) hydrophobic Fluortex adsorbents coated with thin oil films. The bacterial communities associated with the two systems displayed similar compositions of dominant bacterial taxa. Initial abundances of Oceanospirillales were observed in both systems with later emergences of Flavobacteriales, Alteromonadales and Rhodobacterales. Depletion of oil compounds was significantly faster in the Fluortex system and most likely related to the greater bioavailability of oil compounds as compared to the clay bead system.

Comprehensive Two-Dimensional Gas Chromatography with Peak Tracking for Screening of Constituent Biodegradation in Petroleum UVCB Substances.

Petroleum substances, as archetypical UVCBs (substances of unknown or variable composition, complex reaction products, or biological substances), pose a challenge for chemical risk assessment as they contain hundreds to thousands of individual constituents. It is particularly challenging to determine the biodegradability of petroleum substances since each constituent behaves differently. Testing the whole substance provides an average biodegradation, but it would be effectively impossible to obtain all constituents and test them individually. To overcome this challenge, comprehensive two-dimensional gas chromatography (GC × GC) in combination with advanced data-handling algorithms was applied to track and calculate degradation half-times (DT50s) of individual constituents in two dispersed middle distillate gas oils in seawater. By tracking >1000 peaks (representing ∼53-54% of the total mass across the entire chromatographic area), known biodegradation patterns of oil constituents were confirmed and extended to include many hundreds not currently investigated by traditional one-dimensional GC methods. Approximately 95% of the total tracked peak mass biodegraded after 64 days. By tracking the microbial community evolution, a correlation between the presence of functional microbial communities and the observed progression of DT50s between chemical classes was demonstrated. This approach could be used to screen the persistence of GC × GC-amenable constituents of petroleum substance UVCBs.

Spawning time in adult polar cod (Boreogadus saida) altered by crude oil exposure, independent of food availability.

Fish early life stages are well known for their sensitivity to crude oil exposure. However, the effect of crude oil exposure on adults and their gametes during their spawning period is not well studied. Polar cod, a key arctic fish, may be at risk for crude oil exposure during this potentially sensitive life stage. Additionally, this species experiences lower food availability during their spawning season, with unknown combined consequences. In the present study, wild-caught polar cod were exposed to decreasing levels of a water-soluble fraction (WSF) of crude oil or control conditions and fed either at a low or high feed ration to assess the combined effect of both stressors. Samples were taken during late gonadal development, during active spawning (spawning window), and in the post-spawning period. Histology analysis of gonads from fish sampled during the spawning window showed that oil-exposed polar cod were more likely to have spawned compared to controls. Oil-exposed females had 947 differentially regulated hepatic genes, and their eggs had a higher polycyclic aromatic hydrocarbon body burden compared to controls. Feed ration did not consistently affect polar cod's response to oil exposure for the endpoints measured, however, did alone result in decreases in some sperm motility parameters. These results suggest that polar cod's spawning period is a sensitive life event to crude oil exposure, while feed limitation may play a minor role for this supposedly capital breeder. The effects of adult exposure to crude oil on gamete quality and the next generation warrant further investigation.

Organic chemicals associated with rubber are more toxic to marine algae and bacteria than those of thermoplastics.

The current study investigated the chemical complexity of fifty plastic (36) and elastomer/rubber (14) methanol extracts from consumer products, focusing on the association with toxicity in two screening assays (bacteria luminescence and marine microalgae). The chemical composition varied considerably between the products and polymers. The most complex sample (car tire rubber) contained 2456 chemical features and the least complex (disposable water bottle) only 39 features, with a median of 386 features across all products. Individual extract toxicity also varied significantly across the products and polymers, with the two toxicity assays showing comparable results in terms of defining low and high toxicity extracts, and correlation between medium toxicity extracts. Chemical complexity and abundance both correlated with toxicity in both assays. However, there were strong differences in toxicity between plastic and elastomer extracts. Overall, 86-93 % of the 14 elastomer extracts and only 33-36 % of other polymer extracts (n = 36) were more toxic than the median. A range of compounds were tentatively identified across the sample set, with several concerning compounds being identified, mostly in the elastomers. While the current focus on plastic chemicals is towards thermoplastics, we show that elastomers may be of more concern from an environmental and human health perspective.

In situ biomonitoring using caged lumpfish (Cyclopterus lumpus) eggs reveal plastic and rubber associated chemicals in a harbour area in Central Norway.

Plastics- and rubber-derived chemicals are given increasing focus due to their migration into the environment and potential for causing detrimental effects. The current study demonstrates the use of a novel biomonitoring platform using caged fertilized eggs of lumpfish (Cyclopterus lumpus) in combination with gas chromatography tandem mass spectrometry analysis of a selection of target chemicals extracted from the lumpfish eggs after deployment. A monitoring campaign in the Trondheim harbor and off the coast of Trøndelag in Norway was executed using the described system. Here we found accumulation of UV stabilizers (benzophenone and benzothiazoles), plasticizers (n-butylbenzenesulfonamide), reagents, and polymer synthesis precursors (bisphenol A, acetophenone, phthalide, and phthalimide) in deployed eggs. Several of the compounds were detected in concentrations above previously quantified legacy contaminants in the same study areas.

Effects of Exposure Timing on cyp1a Expression, PAH Elimination, and Lipid Utilization in Lumpfish Embryos Exposed to Produced Water.

Intentional discharges of produced water from oil production platforms to the marine environment contain a complex mixture of toxicants, including polycyclic aromatic hydrocarbons (PAHs). Early life stages of fish are highly sensitive to petrogenic exposure, and short-term exposure during critical periods of embryonic development may have detrimental effects on larvae health and survival. However, why different periods are more sensitive to exposure than others are not fully understood. Three identical exposure experiments (48 h, approx. 30 µg/L tPAH, sum 42 PAHs) on lumpfish (Cyclopterus lumpus) embryos were conducted where only exposure timing was varied: 0-48 h post fertilization (hpf, starting before chorion hardening), 36-84 hpf (starting after chorion hardening), and 240-288 hpf (during organogenesis). Total PAH (tPAH) uptake at the end of exposure was 5× higher when exposed during fertilization than when exposed late (during organogenesis). The first evidence of cyp1a induction in lumpfish during embryogenesis was observed after 84 hpf. Early exposure affected lipid droplet coagulation, indicating altered lipid utilization during embryogenesis. Although no significant impacts of exposure were observed on hatching success, hatching was delayed when exposed at the latest time point. This study shows that chorion properties, lipid content, biotransformation potential, and timing of produced water exposure during lumpfish embryogenesis affected PAH uptake and elimination.

No observed developmental effects in early life stages of capelin (Mallotus villosus) exposed to a water-soluble fraction of crude oil during embryonic development.

The rise in offshore oil and gas operations, maritime shipping, and tourism in northern latitudes enhances the risk of oil spills to sub-Arctic and Arctic coastal environments. Therefore, there is a need to understand the potential adverse effects of petroleum on key species in these areas. Here, we investigated the effects of oil exposure on the early life stages of capelin (Mallotus villosus), an ecologically and commercially important Barents Sea forage fish species that spawns along the coast of Northern Norway. Capelin embryos were exposed to five different concentrations (corresponding to 0.5-19 µg/L total PAHs) of water-soluble fraction (WSF) of crude oil from 6 days post fertilization (dpf) until hatch (25 dpf), and development of larvae in clean seawater was monitored until 52 dpf. None of the investigated endpoints (embryo development, larval length, heart rate, arrhythmia, and larval mortality) showed any effects. Our results suggest that the early life stages of capelin may be more robust to crude oil exposure than similar life stages of other fish species.

Physicochemical characterization and quantification of nanoplastics: applicability, limitations and complementarity of batch and fractionation methods.

A comprehensive physicochemical characterization of heterogeneous nanoplastic (NPL) samples remains an analytical challenge requiring a combination of orthogonal measurement techniques to improve the accuracy and robustness of the results. Here, batch methods, including dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM), as well as separation/fractionation methods such as centrifugal liquid sedimentation (CLS) and field-flow fractionation (FFF)-multi-angle light scattering (MALS) combined with pyrolysis gas chromatography mass spectrometry (pyGC-MS) or Raman microspectroscopy (RM) were evaluated for NPL size, shape, and chemical composition measurements and for quantification. A set of representative/test particles of different chemical natures, including (i) polydisperse polyethylene (PE), (ii) (doped) polystyrene (PS) NPLs, (iii) titanium dioxide, and (iv) iron oxide nanoparticles (spherical and elongated), was used to assess the applicability and limitations of the selected methodologies. Particle sizes and number-based concentrations obtained by orthogonal batch methods (DLS, NTA, TRPS) were comparable for monodisperse spherical samples, while higher deviations were observed for polydisperse, agglomerated samples and for non-spherical particles, especially for light scattering methods. CLS and TRPS offer further insight with increased size resolution, while detailed morphological information can be derived by electron microscopy (EM)-based approaches. Combined techniques such as FFF coupled to MALS and RM can provide complementary information on physical and chemical properties by online measurements, while pyGC-MS analysis of FFF fractions can be used for the identification of polymer particles (vs. inorganic particles) and for their offline (semi)quantification. However, NPL analysis in complex samples will continue to present a serious challenge for the evaluated techniques without significant improvements in sample preparation.

Crude oil exposure of early life stages of Atlantic haddock suggests threshold levels for developmental toxicity as low as 0.1 µg total polyaromatic hydrocarbon (TPAH)/L.

Atlantic haddock (Melanogrammus aeglefinus) embryos bind dispersed crude oil droplets to the eggshell and are consequently highly susceptible to toxicity from spilled oil. We established thresholds for developmental toxicity and identified any potential long-term or latent adverse effects that could impair the growth and survival of individuals. Embryos were exposed to oil for eight days (10, 80 and 300 µg oil/L, equivalent to 0.1, 0.8 and 3.0 µg TPAH/L). Acute and delayed mortality were observed at embryonic, larval, and juvenile stages with IC50 = 2.2, 0.39, and 0.27 µg TPAH/L, respectively. Exposure to 0.1 µg TPAH/L had no negative effect on growth or survival. However, yolk sac larvae showed significant reduction in the outgrowth (ballooning) of the cardiac ventricle in the absence of other extracardiac morphological defects. Due to this propensity for latent sublethal developmental toxicity, we recommend an effect threshold of 0.1 µg TPAH/L for risk assessment models.

Non-target and suspect screening reveal complex pattern of contamination in Arctic marine zooplankton.

Although increasing, there is still limited knowledge of the presence of 'contaminants of emerging concern' in Arctic marine biota, particularly in lower trophic species. In the present study, we have applied a novel pipeline to investigate the presence of contaminants in a variety of benthic and pelagic low-trophic organisms: amphipods, copepods, arrow worms and krill. Samples collected in Kongsfjorden in Svalbard in 2018 were subject to extraction and two-dimensional gas chromatography coupled to high-resolution mass spectrometry (GC×GC-HRMS). Tentatively identified compounds included plastic additives, antioxidants, antimicrobials, flame retardants, precursors, production solvents and chemicals, insecticides, and pharmaceuticals. Both legacy contaminants (PAHs, PCBs, PBDEs, hexachlorobenzene) as well as novel and emerging contaminants (triclosan, bisphenol A, and ibuprofen) were quantified in several species using target analysis by GC-MS/MS. The significance of these discoveries is discussed considering the potential for detrimental effects caused by these chemicals, as well as suggested local and distant sources of the components to the Arctic environment.

Monitoring ocean water quality by deployment of lumpfish (Cyclopterus lumpus) eggs: In situ bioaccumulation and toxicity in embryos.

Fish embryos can bioaccumulate and are particularly sensitive to a wide range of contaminants, which makes them suitable sentinels for environmental biomonitoring. However, fish embryos are very rarely utilized in environmental monitoring surveys, possibly due to their fragility and seasonality. In the present work, we assessed the applicability of caged lumpfish (Cyclopterus lumpus) eggs for in situ biomonitoring of exposure and effects of organic contaminants focusing on polyaromatic hydrocarbons and phenolic compounds. Fertilized eggs (1 dpf) were transplanted for 17-19 days at different locations that differed in terms of contaminant load, depths and weather conditions, namely at three stations close to the city of Trondheim (two harbour areas and a one in the Fjord) and three stations at a coastal aquaculture facility. High survival upon retrieval after deployment showed that lumpfish eggs are relatively robust and survive encaging in different environments. Bioaccumulation of organic contaminants (PAHs and phenolic compounds) was measured and potential effects on hatching, development, survival and larvae morphometry were determined. Chemical analyses showed that especially PAHs were effectively accumulated in eggs in contaminated sites, with concentrations of Æ©PAHs being 15 - 25 times higher in harbour areas compared to those at the aquaculture facility. A higher incidence of embryonic deformations was observed in the most polluted deployment location, but larvae morphometry revealed no evidence of toxicity related to pollutant body burden. In conclusion, the in-situ exposure method was proven to work well, making it attractive for implementations in environmental monitoring programs.

The fate of conventional and potentially degradable gillnets in a seawater-sediment system.

Abandoned gillnets in the marine environment represent a global environmental risk due to the ghost fishing caused by the nets. Degradation of conventional nylon gillnets was compared to that of nets made of polybutylene succinate co-adipate-co-terephthalate (PBSAT) that are designed to degrade more readily in the environment. Gillnet filaments were incubated in microcosms of natural seawater (SW) and marine sediments at 20 °C over a period of 36 months. Tensile strength tests and scanning electron microscopy analyses showed weakening and degradation of the PBSAT filaments over time, while nylon filaments remained unchanged. Pyrolysis-gas chromatography/mass spectrometry revealed potential PBSAT degradation products associated with the filament surfaces, while nylon degradation products were not detected by these analyses. Microbial communities differed significantly between the biofilms on the nylon and PBSAT filaments. The slow deterioration of the PBSAT gillnet filaments shown here may be beneficial and reduce the ghost fishing periods of these gillnets.

Atlantic cod (Gadus morhua) embryos are highly sensitive to short-term 3,4-dichloroaniline exposure.

3,4-dichloroaniline (3,4-DCA) is one of the most widely produced anilines world-wide, used in plastic packaging, fabrics, pharmaceuticals, pesticides, dyes and paints as well as being a degradation product of several pesticides. 3,4-DCA has been detected in freshwater, brackish and marine environments. Although freshwater toxicity thresholds exist, very little toxicological information is available on marine and cold-water species. In this study, we exposed Atlantic cod (Gadus morhua) embryos (3-7 days post fertilization) to 3,4-DCA concentrations ranging from 8-747 µg/L for 4 days followed by a recovery period in clean sea water until 14 days post fertilization (dpf). The cod embryos were significantly more sensitive to acute 3,4-DCA exposure compared to other species tested and reported in the literature. At the highest concentration (747 µg/L), no embryos survived until hatch, and even at the lowest concentration (8 µg/L), a small, but significant increase in mortality was observed at 14 dpf. Delayed and concentration-dependent effects on surviving yolk-sac larvae, manifested as cardiac, developmental and morphometric alterations, more than a week after exposure suggest potential long-term effects of transient embryonic exposure to low concentrations of 3,4-DCA.

The sub-lethal impact of plastic and tire rubber leachates on the Mediterranean mussel Mytilus galloprovincialis.

Ocean contamination by synthetic polymers can represent a risk for the fitness of marine species due to the leaching of chemical additives. This study evaluated the sub-lethal effects of plastic and rubber leachates on the mussel Mytilus galloprovincialis through a battery of biomarkers encompassing lysosomal endpoints, oxidative stress/detoxification parameters, and specific responses to metals/neurotoxicants. Mussels were exposed for 7 days to leachates from car tire rubber (CTR), polypropylene (PP), polyethylene terephthalate (PET), polystyrene (PS) and polyvinyl chloride (PVC), containing organic additives and metals in the ng-µg/L range. The leachate exposure affected general stress parameters, including the neutral lipid content (all leachates), the lysosomal membrane stability (PS, PP, PVC and CTR leachates) and lysosomal volume (PP, PVC and TR leachates). An increased content of the lipid peroxidation products malondialdehyde and lipofuscin was observed in mussels exposed to PET, PS and PP leachates, and PP, PVC and CTR leachates, respectively. PET and PP leachates increased the activity of the phase-II metabolism enzyme glutathione S-transferase, while a decreased acetylcholinesterase activity was induced by PVC leachates. Data were integrated in the mussel expert system (MES), which categorizes the organisms' health status based on biomarker responses. The MES assigned healthy status to mussels exposed to PET leachates, low stress to PS leachates, and moderate stress to PP, CTR and PVC leachates. This study shows that additives leached from selected plastic/rubber polymers cause sub-lethal effects in mussels and that the magnitude of these effects may be higher for CTR, PVC and PP due to a higher content and release of metals and organic compounds.

Combined effects of crude oil exposure and warming on eggs and larvae of an arctic forage fish.

Climate change, along with environmental pollution, can act synergistically on an organism to amplify adverse effects of exposure. The Arctic is undergoing profound climatic change and an increase in human activity, resulting in a heightened risk of accidental oil spills. Embryos and larvae of polar cod (Boreogadus saida), a key Arctic forage fish species, were exposed to low levels of crude oil concurrently with a 2.3 °C increase in water temperature. Here we show synergistic adverse effects of increased temperature and crude oil exposure on early life stages documented by an increased prevalence of malformations and mortality in exposed larvae. The combined effects of these stressors were most prevalent in the first feeding larval stages despite embryonic exposure, highlighting potential long-term consequences of exposure for survival, growth, and reproduction. Our findings suggest that a warmer Arctic with greater human activity will adversely impact early life stages of this circumpolar forage fish.

Microplastic fibres from synthetic textiles: Environmental degradation and additive chemical content.

Microplastic fibres (MPFs) often make up the largest fraction of microplastic pollution in aquatic environments, yet little is known about their degradative fate and persistence. This study investigates the environmentally relevant photodegradation of common MPFs: polyester (PET), polyamide (PA) and polyacrylonitrile (PAN), their respective additive chemical profile, together with their potential for additive leaching. MPFs were subject to ultraviolet (UV) exposure in seawater and freshwater media over 10 months. PET and PA MPFs showed significant fragmentation and surface changes following UV exposure, additionally PA showed evidence of chemical changes. PAN did not undergo significant photodegradation in the same exposure period. Chemicals tentatively identified in MPFs and aqueous leachates via non-target gas chromatography-mass spectrometry include monomers, UV stabilisers and degradation products. Characterisation of several bisphenols (BPs) and benzophenones (BzPs) was performed via ultraperformance liquid chromatography tandem mass spectrometry. Bisphenol A, bisphenol S and benzophenone-3 were quantified in all MPFs and wool at concentrations between 4.3 and 501 ng/g, with wool displaying the highest sum concentration of BPs and BzPs at 863 and 27 ng/g, respectively.

UV degradation of natural and synthetic microfibers causes fragmentation and release of polymer degradation products and chemical additives.

A high proportion of the total microplastic (MP) load in the marine environment has been identified as microfibers (MFs), with polyester (PET) and polyamide (PA) typically found in the highest abundance. The potential for negative environmental impacts from MPs may be dependent on their degree of degradation in the environment, which is influenced by both intrinsic properties (polymer type, density, size, additive chemicals) and extrinsic environmental parameters. Most polymer products break down slowly through a combination of environmental processes, but UV degradation can be a significant source of degradation. The current study aimed to investigate the effect of UV irradiance on the degradation of natural (wool) and synthetic (PET and PA) MFs. Degradation of MFs was conducted in seawater under environmentally relevant accelerated exposure conditions using simulated sunlight. After 56 days of UV exposure, PA primarily exhibited changes in surface morphology with no significant fragmentation observed. PET and wool fibers exhibited both changes in surface morphology and fragmentation into smaller particles. A range of molecular degradation products were identified in seawater leachates after UV exposure, with increasing abundance over the duration of the experiment. Furthermore, a variety of additive chemicals were shown to leach from the MFs into seawater. While some of these chemicals were also susceptible to UV degradation and some are expected to biodegrade rapidly, others may be persistent and contribute to the overall load of chemical pollution in the marine environment.

DNA damage and health effects in juvenile haddock (Melanogrammus aeglefinus) exposed to PAHs associated with oil-polluted sediment or produced water.

The research objective was to study the presence of DNA damages in haddock exposed to petrogenic or pyrogenic polyaromatic hydrocarbons (PAHs) from different sources: 1) extracts of oil produced water (PW), dominated by 2-ring PAHs; 2) distillation fractions of crude oil (representing oil-based drilling mud), dominated by 3-ring PAHs; 3) heavy pyrogenic PAHs, mixture of 4/5/6-ring PAHs. The biological effect of the different PAH sources was studied by feeding juvenile haddock with low doses of PAHs (0.3-0.7 mg PAH/kg fish/day) for two months, followed by a two-months recovery. In addition to the oral exposure, a group of fish was exposed to 12 single compounds of PAHs (4/5/6-ring) via intraperitoneal injection. The main endpoint was the analysis of hepatic and intestinal DNA adducts. In addition, PAH burden in liver, bile metabolites, gene and protein expression of CYP1A, GST activity, lipid peroxidation, skeletal deformities and histopathology of livers were evaluated. Juvenile haddock responded quickly to both intraperitoneal injection and oral exposure of 4/5/6-ring PAHs. High levels of DNA adducts were detected in livers three days after the dose of the single compound exposure. Fish had also high levels of DNA adducts in liver after being fed with extracts dominated by 2-ring PAHs (a PW exposure scenario) and 3-ring PAHs (simulating an oil exposure scenario). Elevated levels of DNA adducts were observed in the liver of all exposed groups after the 2 months of recovery. High levels of DNA adduct were found also in the intestines of individuals exposed to oil or heavy PAHs, but not in the PW or control groups. This suggests that the intestinal barrier is very important for detoxification of orally exposures of PAHs.