Staffan Lundstedt

Sources, Fate, and Toxic Hazards of Oxygenated Polycyclic Aromatic Hydrocarbons (PAHs) at PAH- contaminated Sites

Abstract In this paper we show that oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are important cocontaminants that should be taken into account during risk assessment and remediation of sites with high levels of PAHs. The presented data, which have been collected both from our own research and the published literature, demonstrate that oxy-PAHs are abundant but neglected contaminants at these sites. The oxy-PAHs show relatively high persistency and because they are formed through transformation of PAHs, their concentrations in the environment may even increase as the sites are remediated by methods that promote PAH degradation. Furthermore, we show that oxy-PAHs are toxic to both humans and the environment, although the toxicity seems to be manifested through other effects than those known to be important for polycyclic aromatic compounds in general, that is, mutagenicity and carcinogenicity. Finally, we present data that support the hypothesis that oxy-PAHs are more mobile in the environment than PAHs, due to their polarity, and thus have a higher tendency to spread from contaminated sites via surface water and groundwater. We believe that oxy-PAHs should be included in monitoring programs at PAH-contaminated sites, even if a number of other toxicologically relevant compounds that may also be present, such as nitro-PAHs and azaarenes, are not monitored. This is because oxy-PAH levels are difficult to predict from the PAH levels, because their environmental behavior differs substantially from that of PAHs, and oxy-PAHs may be formed as PAHs are degraded.

Pressurised liquid extraction of polycyclic aromatic hydrocarbons from contaminated soils

The reliability and efficiency of the pressurised liquid extraction technique (PLE) for extracting polycyclic aromatic hydrocarbons (PAHs) from contaminated soil has been investigated. Experimental design was used to study the influence of seven extraction variables (sample load, solvents used, solvent ratios, pressure, temperature, extraction time, and rinse volume). The results show that large sample loads in combination with small solvent volumes may result in low extraction efficiency. They also indicate that the recovery of low-molecular-mass PAHs is reduced by low extraction temperatures. The exact settings of the other variables are, however, less significant for the extraction efficiency. Repeated extractions at optimised settings of the tested variables show that PLE is an exhaustive extraction technique that generally results in high yields. In addition, extraction of a certified reference material (CRM 103-100) revealed that the method is both accurate and precise. Another finding was that adding the internal standard on top of the soil in the extraction cell causes considerable over-estimation of the concentrations when large samples are extracted with small solvent volumes. This is because the PLE-cell resembles a chromatographic column, so compounds added to the top of the soil layer have a longer distance to travel through the soil compared to the average distance of the native compounds, which are distributed evenly throughout the column. We therefore recommend that the internal standard should be added to the extract immediately after the extraction or, alternatively, carefully mixed with the sample prior to extraction.

Native Oxy-PAHs, N-PACs, and PAHs in Historically Contaminated Soils from Sweden, Belgium, and France : Their Soil-Porewater Partitioning Behavior, Bioaccumulation in Enchytraeus crypticus, and Bioavailability

Soil quality standards are based on partitioning and toxicity data for laboratory-spiked reference soils, instead of real world, historically contaminated soils, which would be more representative. Here 21 diverse historically contaminated soils from Sweden, Belgium, and France were obtained, and the soil-porewater partitioning along with the bioaccumulation in exposed worms (Enchytraeus crypticus) of native polycyclic aromatic compounds (PACs) were quantified. The native PACs investigated were polycyclic aromatic hydrocarbons (PAHs) and, for the first time to be included in such a study, oxygenated-PAHs (oxy-PAHs) and nitrogen containing heterocyclic PACs (N-PACs). The passive sampler polyoxymethylene (POM) was used to measure the equilibrium freely dissolved porewater concentration, Cpw, of all PACs. The obtained organic carbon normalized partitioning coefficients, KTOC, show that sorption of these native PACs is much stronger than observed in laboratory-spiked soils (typically by factors 10 to 100), which has been reported previously for PAHs but here for the first time for oxy-PAHs and N-PACs. A recently developed KTOC model for historically contaminated sediments predicted the 597 unique, native KTOC values in this study within a factor 30 for 100% of the data and a factor 3 for 58% of the data, without calibration. This model assumes that TOC in pyrogenic-impacted areas sorbs similarly to coal tar, rather than octanol as typically assumed. Black carbon (BC) inclusive partitioning models exhibited substantially poorer performance. Regarding bioaccumulation, Cpw combined with liposome-water partition coefficients corresponded better with measured worm lipid concentrations, Clipid (within a factor 10 for 85% of all PACs and soils), than Cpw combined with octanol-water partition coefficients (within a factor 10 for 76% of all PACs and soils). E. crypticus mortality and reproducibility were also quantified. No enhanced mortality was observed in the 21 historically contaminated soils despite expectations from PAH spiked reference soils. Worm reproducibility weakly correlated to Clipid of PACs, though the contributing influence of metal concentrations and soil texture could not be taken into account. The good agreement of POM-derived Cpw with independent soil and lipid partitioning models further supports that soil risk assessments would improve by accounting for bioavailability. Strategies for including bioavailability in soil risk assessment are presented.

Mutagenic Hazards of Complex Polycyclic Aromatic Hydrocarbon Mixtures in Contaminated Soil.

The objective of the present study was to evaluate hazard/risk assessment methods for complex environmental mixtures that involve a targeted, priority chemical approach based on the cumulative hazard/risk of known mixture components or analyses of sufficiently similar mixtures. Ten polycyclic aromatic hydrocarbon (PAH)-contaminated soils were separated into nonpolar and semipolar fractions, and both fractions elicited positive responses on the Salmonella reverse mutation assay. Targeted and nontargeted methods of hazard prediction routinely overestimated mutagenic activities for the nonpolar soil fractions, suggesting nonadditive interactions of PAHs in complex mixtures. This suggests that current risk assessment methods for complex mixtures may provide conservative estimates regarding soils contaminated with priority PAHs alone. Significant underestimations of total risk, however, will be obtained if the soils also contain unidentified PAHs as well as polycyclic aromatic compounds and related compounds that contribute to the total mutagenic activity. Furthermore, estimates of excess lifetime cancer risk associated with the nondietary ingestion of the PAH-contaminated soils studied here indicate that a traditional risk assessment model based on identified priority PAHs and an assumption of additivity generally underestimates the risk associated with the nonpolar soil fractions (in comparison to bioassay-derived risk estimates). Additional cancer risk may be associated with the more polar compounds that also are found at these contaminated sites and that rarely are included in the standard risk assessment methodology.

Exposure of HepG2 Cells to Low Levels of PAH-Containing Extracts from Contaminated Soils Results in Unpredictable Genotoxic Stress Responses

Contaminated soil is a serious environmental problem, constituting a risk to humans and the environment. Polycyclic aromatic hydrocarbons (PAHs) are often present at contaminated sites. However, risk levels are difficult to estimate because of the complexity of contaminants present. Here, we compare cellular effects of extracts from contaminated soils collected at six industrial settings in Sweden. Chemical analysis showed that all soils contained complex mixtures of PAHs and oxy‐PAHs. Western blotting and immunocytochemistry were used to investigate DNA damage signaling in HepG2 cells exposed to extracts from these soils. The effects on phosphorylated Mdm2, p53, Erk, H2AX, 53BP1, and Chk2, cell cycle regulating proteins (cyclin D1 and p21), and cell proliferation were compared. We found that most soil extracts induced phosphorylation of Mdm2 at the 2A10 epitope at low concentrations. This is in line with previous studies suggesting that this endpoint reflects readily repaired DNA‐damage. However, we found concentration‐ and time‐dependent γH2AX and 53BP1 responses that were sustained for 48 hr. These endpoints may reflect the presence of different types of persistent DNA‐damage. High concentrations of soil extracts decreased cyclin D1 and increased p21 response, indicating cell cycle arrest. Phosphorylation of Mdm2 at Ser166, which attenuates the p53 response and is induced by many tumor promoters, was induced in a time‐dependent manner and was associated with Erk phosphorylation. Taken together, the PAH extracts elicited unpredictable signaling responses that differed between samples. More polar compounds, i.e., oxy‐PAHs, also contributed to the complexity. Environ. Mol. Mutagen. 2009.

Emissions of PBDD/Fs, PCDD/Fs and PBDEs from flame-retarded high-impact polystyrene under thermal stress

The emissions of polybrominated diphenyl ethers (PBDEs), polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) and their chlorinated analogues (PCDD/Fs) during the thermal treatment of a high impact polystyrene (HIPS) TV casing were investigated. The halogenated compounds were analyzed in the original material and in the gases emitted during its treatment at temperatures between 50 °C and 250 °C. DecaBDE was the primary PBDE in the TV casing, which also contained high levels of PBDFs (ppm range). At the lower treatment temperatures, non-modified PBDEs evaporated from the samples. Conversely, at 200 °C or above, debromination reactions led to the formation of additional tri- through nonaBDE. The formation of new PBDD/Fs was also detected in the gas phase when the plastic was heated to 200 °C or 250 °C, with higher yields of furans than dioxins. This appreciably increased the toxic equivalent (TEQ) levels of the gas phase relative to those seen in the untreated sample. In all cases, the levels and TEQ contributions from PCDD/Fs were negligible compared to those for brominated analogues.

Cancer Risk Assessment of Polycyclic Aromatic Hydrocarbon Contaminated Soils Determined Using Bioassay-Derived Levels of Benzo(a)pyrene Equivalents

Here we evaluate the excess lifetime cancer risk (ELCR) posed by 10 PAH-contaminated soils using (i) the currently advocated, targeted chemical-specific approach that assumes dose additivity for carcinogenic PAHs and (ii) a bioassay-based approach that employs the in vitro mutagenic activity of the soil fractions to determine levels of benzo[a]pyrene equivalents and, by extension, ELCR. Mutagenic activity results are presented in our companion paper.1 The results show that ELCR values for the PAH-containing fractions, determined using the chemical-specific approach, are generally (i.e., 8 out of 10) greater than those calculated using the bioassay-based approach; most are less than 5-fold greater. Only two chemical-specific ELCR estimates are less than their corresponding bioassay-derived values; differences are less than 10%. The bioassay-based approach, which permits estimation of ELCR without a priori knowledge of mixture composition, proved to be a useful tool to evaluate the chemical-specific approach. The results suggest that ELCR estimates for complex PAH mixtures determined using a targeted, chemical-specific approach are reasonable, albeit conservative. Calculated risk estimates still depend on contentious PEFs and cancer slope factors. Follow-up in vivo mutagenicity assessments will be required to validate the results and their relevance for human health risk assessment of PAH-contaminated soils.

Aryl Hydrocarbon Receptor Activation and Developmental Toxicity in Zebrafish in Response to Soil Extracts Containing Unsubstituted and Oxygenated PAHs

Many industrial sites are polluted by complex mixtures of polycyclic aromatic compounds (PACs). Besides polycyclic aromatic hydrocarbons (PAHs), these mixtures often contain significant amounts of more polar PACs including oxygenated PAHs (oxy-PAHs). The effects of oxy-PAHs are, however, poorly known. Here we used zebrafish embryos to examine toxicities and transcriptional changes induced by PAC containing soil extracts from three different industrial sites: a gasworks (GAS), a former wood preservation site (WOOD), and a coke oven (COKE), and to PAH and oxy-PAH containing fractions of these. All extracts induced aryl hydrocarbon receptor (Ahr)-regulated mRNAs, malformations, and mortality. The WOOD extract was most toxic and the GAS extract least toxic. The extracts induced glutathione transferases and heat shock protein 70, suggesting that the toxicity also involved oxidative stress. With all extracts, Ahr2-knock-down reduced the toxicity, indicating a significant Ahr2-dependence on the effects. Ahr2-knock-down was most effective with the PAH fraction of the WOOD extract and with the oxy-PAH fraction of the COKE extract. Our results indicate that oxy-PAH containing mixtures can be as potent Ahr activators and developmental toxicants as PAHs. In addition to Ahr activating potency, the profile of cytochrome P4501 inhibitors may also determine the toxic potency of the extracts.

γH2AX, pChk1, and Wip1 as potential markers of persistent DNA damage derived from dibenzo[a,l]pyrene and PAH-containing extracts from contaminated soils.

Polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete combustion of organic material and are ubiquitous environmental contaminants. High levels of PAHs are commonly found in soils at industrial sites, thereby constituting a risk for humans and the environment. However, this risk is often difficult to estimate due to the complexity of the contamination. In the present study, we investigated the cellular DNA damage response induced by extracts of PAH-contaminated soils collected at various industrial sites in Sweden. The results show that interactions of PAHs in the soil extracts caused activation of DNA damage signaling consistent with persistent DNA damage. Signaling in HepG2 cells exposed to soil PAH extracts corresponding to 1 μM benzo[a]pyrene was similar to that of 0.1 μM dibenzo[a,l]pyrene, a highly carcinogenic PAH known to produce persistent DNA damage. The response involved prolonged activation of DNA damage marker (H2AX), check point kinase (Chk1), and phosphatases (Wip1). Furthermore, blocking DNA damage signaling using specific inhibitors and siRNA showed the important role of signaling through Chk1 for the level of DNA damage. We conclude that the combination of prolonged Chk1 phosphorylation and induced expression of Wip1 might serve as potential markers for persistent DNA damage induced by complex mixtures of environmental PAHs. Discrepancies between mRNA and protein levels of Wip1 in response to soil extracts, in parallel with increased microRNA (miR)-16 levels, suggest a role of miR-16 in the regulation of DNA damage signaling in response to PAHs. Taken together, our data indicate that PAH extracts induce irreparable DNA damage and that this is consistent with the prolonged activation of DNA damage signaling.

Optimization of selective pressurized liquid extraction for extraction and in-cell clean-up of PCDD/Fs in soils and sediments.

This paper describes the development of methods for selective extraction of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) from soils and sediments, using pressurized liquid extraction with in-cell clean-up (SPLE). Two binary solvent mixtures, viz. dichloromethane/n-heptane (DCM/Hp), and diethylether/n-heptane (DEE/Hp), were evaluated. The SPLE extraction conditions were optimized using central composite face (CCF) design. Three factors were investigated: extraction temperature (60-160 °C), number of extraction cycles (1-3) and time per cycle (2-18 min). The results showed that DCM/Hp (1/1, v/v) and DEE/Hp (1/2, v/v) were the best extraction solvent compositions and that the extraction temperature was a critical factor that needed careful optimization to achieve high extraction efficiency without co-extraction of sulfuric acid. Under the optimal conditions, the SPLE methods provided results with good accuracy and precision. For the sandy soil certified reference material (CRM-529) the quantification results ended up in the range 82-110% as compared to the concentrations obtained by a reference method based on Soxhlet extraction and external column clean-up. Furthermore, for a clay soil (CRM-530) and a sediment reference material (WMS-01), the accuracy (trueness) of the TEQ values were +11% (DCM/Hp) and +8% (DEE/Hp) for CRM-530, +8% and -7% for WMS-01, respectively. The individual congener concentrations also agreed well with the certified values. These findings show that SPLE is a promising method for combined extraction and clean-up of PCDD/Fs in soil/sediment samples.