Salivary cortisol and cortisone are stable after long-term storage.

Frozen saliva samples are often used for later determination of salivary glucocorticoids in research studies on stress and endocrine disorders. We studied the stability of cortisol and cortisone in saliva after six years of storage at -80 °C by repeated analysis of 153 stored aliquots, collected with Salivette®, using liquid chromatography tandem mass spectrometry. We found a very high agreement between the first and the repeated measurement after six years at -80 °C, for both cortisol and cortisone concentrations (rs= 0.96 and rs= 0.98, respectively). Passing-Bablok regression equations were y = 0.02 + 1.00x and y = 0.02 + 1.14x for cortisol and cortisone, respectively. We conclude that salivary cortisol and cortisone concentrations remain essentially unaltered after six years of storage at -80 °C.

Salivary Cortisol and Cortisone Can Circumvent Confounding Effects of Oral Contraceptives in the Short Synacthen Test.

CONTEXT: Adrenal insufficiency (AI) is usually diagnosed by low plasma cortisol levels following a short Synacthen test (SST). Most plasma cortisol is bound to corticosteroid-binding globulin, which is increased by estrogen in combined estrogen-progestin oral contraceptives (COCs). Women with AI using COCs are therefore at risk of having an apparently normal plasma cortisol level during SST, which would not adequately reflect AI. OBJECTIVE: This work aimed to test whether salivary cortisol or cortisone during SST is more robust against the COC effect and to calculate the lower reference limits (LRLs) for these to be used as tentative diagnostic cutoffs to exclude AI. METHODS: Forty-one healthy women on COCs and 46 healthy women without exogenous estrogens underwent an SST with collection of plasma and salivary samples at 0, 30, and 60 minutes after Synacthen injection. The groups were compared using regression analysis with age as covariate and the LRLs were calculated parametrically. RESULTS: SST-stimulated plasma cortisol levels were significantly higher in the COC group vs controls, while mean salivary cortisol and cortisone levels were slightly lower in the COC group. Importantly, COC use did not significantly alter LRLs for salivary cortisol or cortisone. The smallest LRL difference between groups was seen for salivary cortisone. CONCLUSION: Salivary cortisol and especially salivary cortisone are considerably less affected by COC use than plasma cortisol during SST. Due to similar LRLs, a common cutoff for salivary cortisol and cortisone during SST can be used to exclude AI in premenopausal women irrespective of COC use.

Salivary cortisol and cortisone in diagnosis of Cushing's syndrome - a comparison of six different analytical methods.

OBJECTIVES: Salivary cortisol and cortisone at late night and after dexamethasone suppression test (DST) are increasingly used for screening of Cushing's syndrome (CS). We aimed to establish reference intervals for salivary cortisol and cortisone with three liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques and for salivary cortisol with three immunoassays (IAs), and evaluate their diagnostic accuracy for CS. METHODS: Salivary samples at 08:00 h, 23:00 h and 08:00 h after a 1-mg DST were collected from a reference population (n=155) and patients with CS (n=22). Sample aliquots were analyzed by three LC-MS/MS and three IA methods. After establishing reference intervals, the upper reference limit (URL) for each method was used to calculate sensitivity and specificity for CS. Diagnostic accuracy was evaluated by comparing ROC curves. RESULTS: URLs for salivary cortisol at 23:00 h were similar for the LC-MS/MS methods (3.4-3.9 nmol/L), but varied between IAs: Roche (5.8 nmol/L), Salimetrics (4.3 nmol/L), Cisbio (21.6 nmol/L). Corresponding URLs after DST were 0.7-1.0, and 2.4, 4.0 and 5.4 nmol/L, respectively. Salivary cortisone URLs were 13.5-16.6 nmol/L at 23:00 h and 3.0-3.5 nmol/L at 08:00 h after DST. All methods had ROC AUCs ≥0.96. CONCLUSIONS: We present robust reference intervals for salivary cortisol and cortisone at 08:00 h, 23:00 h and 08:00 h after DST for several clinically used methods. The similarities between LC-MS/MS methods allows for direct comparison of absolute values. Diagnostic accuracy for CS was high for all salivary cortisol and cortisone LC-MS/MS methods and salivary cortisol IAs evaluated.

Confounding effects of liquorice, hydrocortisone, and blood contamination on salivary cortisol but not cortisone.

Objective: To determine the effects of liquorice consumption, topical hydrocortisone, and blood contamination on salivary cortisol and cortisone concentrations. Design and methods: Thirty healthy volunteers were randomized to a low, medium, or high dose of liquorice. Late-night saliva samples were collected using a Salivette® collection device at baseline, during 1 week of daily liquorice consumption, and during 4 weeks' washout. Saliva sampling was also performed before and after the application of topical hydrocortisone on the skin. Furthermore, in a subgroup (n = 16), saliva and venous blood were collected from each individual and mixed to achieve graded blood contamination in saliva. Salivary cortisol and cortisone were analyzed with liquid chromatography-tandem mass spectrometry. Results: Significant increases in salivary cortisol concentrations were observed during medium- (+49%) and high-dose (+97%) liquorice intake, which returned to baseline 4 days after liquorice withdrawal. Topical hydrocortisone on fingers holding the collection swab increased salivary cortisol concentrations >1000-fold with concomitant pronounced elevation of the cortisol:cortisone ratio. Salivary cortisol increased significantly after contamination with blood ≥0.5%. Visual examination could safely detect these samples. Salivary cortisone concentrations were unaffected by liquorice consumption and blood contamination, and only marginally affected by topical hydrocortisone. Conclusion: Liquorice, topical hydrocortisone, and blood contamination may all cause elevated salivary cortisol concentrations. Improved sampling instructions and visual examination of the sample may minimize these risks. Salivary cortisone is essentially unaffected by the different preanalytical confounders and may be used as a first-line screening test for Cushing's syndrome.

Polycyclic aromatic compounds in urban soils of Stockholm City: Occurrence, sources and human health risk assessment.

Polycyclic aromatic compounds (PACs) are ubiquitous pollutants that are found everywhere in our environment, including air, soil and water. The aim of this study was to determine concentrations, distribution, sources and potential health risk of 43 PACs in soils collected from 25 urban parks in Stockholm City, Sweden. These PACs included 21 PAHs, 11 oxygenated PAHs, 7 methylated PAHs, and 4 azaarenes whose concentrations ranged between 190 and 54 500, 30.5-5 300, 14.9-680, and 4.17-590 ng/g soil, respectively. Fluoranthene was found at the highest levels ranging between 17.7 and 9800 ng/g, benzo[a]pyrene between 9.64 and 4600 ng/g, and the highly potent carcinogen dibenzo[a,l]pyrene up to 740 ng/g. The most abundant oxy-PAH was 6H-benzo[cd]pyren-6-one (2.09-2300 ng/g). Primary sources of PAHs were identified by use of diagnostic ratios and Positive Matrix Factorization modelling and found to be pyrogenic including vehicle emissions and combustion of biomass. Estimating the incremental lifetime cancer risks (ILCRS) associated with exposure to PAHs in these soils indicated that the PAH levels in some parks constitute a considerable increased risk level for adults and children (total ILCR > 1 × 10-4). Compared to worldwide urban parks contamination, we conclude that the PAC soil levels in parks of Stockholm City in general are low, but that some parks are more heavily contaminated and should be considered for clean-up actions to limit human health risks.

Combining Leaching and Passive Sampling To Measure the Mobility and Distribution between Porewater, DOC, and Colloids of Native Oxy-PAHs, N-PACs, and PAHs in Historically Contaminated Soil.

Different methods to quantify soil porewater concentrations of contaminants will provide different types of information. Passive sampling measurements give freely dissolved porewater concentrations (Cpw,free), while leaching tests provide information on the mobile concentration (Cpw,leach), including contaminants associated with dissolved organic carbon (DOC) and particles/colloids in the porewater. This study presents a novel combination of these two measurements, to study the sorption and mobility of polycyclic aromatic compounds (PACs) to DOC and particulate organic carbon (POC) in 10 historically contaminated soils. The PACs investigated were polycyclic aromatic hydrocarbons (PAHs), oxygenated-PAHs, and nitrogen containing heterocyclic PACs. Observed Cpw,leach was up to 5 orders of magnitude higher than Cpw,free; implying large biases when Cpw,leach is used to assess bioavailability or soil partitioning. Sorption of PACs to DOC and POC was important for the mobility of compounds with log KOW > 4. Average DOC/water-partitioning coefficients (KDOC) correlated well with KOW (log KDOC = 0.89 × log KOW +1.03 (r2 = 0.89)). This relationship is likely more accurate for historically contaminated soils than previously published data, which suffer from artifacts caused by problems in measuring Cpw,free correctly or not using historically contaminated soils. POC/water-partitioning coefficients (KPOC) were orders of magnitude larger than corresponding KDOC, suggesting sorption to mobile particles/colloids is the dominant mechanism for PAC mobility.

14th congress of combustion by-products and their health effects-origin, fate, and health effects of combustion-related air pollutants in the coming era of bio-based energy sources.

The 14th International Congress on Combustion By-Products and Their Health Effects was held in Umeå, Sweden from June 14th to 17th, 2015. The Congress, mainly sponsored by the National Institute of Environmental Health Sciences Superfund Research Program and the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, focused on the "Origin, fate and health effects of combustion-related air pollutants in the coming era of bio-based energy sources". The international delegates included academic and government researchers, engineers, scientists, policymakers and representatives of industrial partners. The Congress provided a unique forum for the discussion of scientific advances in this research area since it addressed in combination the health-related issues and the environmental implications of combustion by-products. The scientific outcomes of the Congress included the consensus opinions that: (a) there is a correlation between human exposure to particulate matter and increased cardiac and respiratory morbidity and mortality; (b) because currently available data does not support the assessment of differences in health outcomes between biomass smoke and other particulates in outdoor air, the potential human health and environmental impacts of emerging air-pollution sources must be addressed. Assessment will require the development of new approaches to characterize combustion emissions through advanced sampling and analytical methods. The Congress also concluded the need for better and more sustainable e-waste management and improved policies, usage and disposal methods for materials containing flame retardants.

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.

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.

Determination of polyoxymethylene (POM)--water partition coefficients for oxy-PAHs and PAHs.

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are a class of ubiquitously occurring pollutants of which little is known. They can be co-emitted with PAHs or formed from PAHs in the environment. The environmental fate and risk of oxy-PAHs are difficult to assess due to a lack of methods to quantify their pore water concentrations. One sampler that can be used to determine freely dissolved concentrations of organic contaminants is polyoxymethylene (POM). In this study, POM - water partition coefficients (KPOM) were determined for 11 oxy-PAHs. KPOM values of 8 PAHs with similar hydrophobicities as the oxy-PAHs were determined for comparison. Results showed that logKPOM values ranged from 2.64 to 4.82 for the PAHs (2-4 rings), similar to previously determined values. LogKPOM values for investigated oxy-PAHs ranged from 0.96 to 5.36. The addition of carbonylic oxygen on a parent PAH generally lowered KPOM by 0.5 to 1.0 log units, which is attributable to the presence of carbonylic oxygens increasing water solubility. The KPOM values presented here will facilitate simultaneous assessments of freely dissolved water concentrations of oxy-PAHs and PAHs in environmental media.

In vitro mammalian mutagenicity of complex polycyclic aromatic hydrocarbon mixtures in contaminated soils.

This study employed an in vitro version of the lacZ transgenic rodent mutation assay to assess the mutagenicity of nonpolar neutral and semipolar aromatic soil fractions from 10 PAH-contaminated sites, and evaluated the assumption of dose additivity that is routinely employed to calculate the risk posed by PAH mixtures. Significant mutagenic activity was detected in all nonpolar neutral fractions, and 8 of 10 semipolar aromatic fractions (nonpolar > semipolar). Mutagenic activity of synthetic PAH mixtures that mimic the PAH content of the soils (i.e., 5-PAH or 16-PAH mix) were greater than that of the PAH-containing soil fractions, with 5-PAH mix >16-PAH-mix. Predictions of mutagenic activity, calculated as the sum of the contributions from the mutagenic mixture components, were all within 2-fold of the observed activity of the nonpolar neutral fractions, with one exception. Observed differences in mutagenic activity are likely the result of dynamic metabolic processes, involving a complex interplay of AhR agonsim and saturation of metabolic machinery by competitive inhibition of mixture components. The presence of hitherto unidentified polar compounds present in PAH-contaminated soils may also contribute to overall hazard; however, these compounds are generally not included in current contaminated site risk assessment protocols.

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.

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.

Leachability and desorption of PCBs from soil and their dependency on pH and dissolved organic matter.

pH affects both soil-water partitioning coefficient (Kd) of polychlorinated biphenyls (PCBs) and dissolved organic matter (DOM), thereby influencing PCBs' leachability from contaminated soils. To explore these incompletely understood interactions, the leachability of 11 selected PCBs in a naturally aged soil was investigated in pH static leaching tests spanning a wide pH range (2 to 9). The K(d) was calculated for each of the PCBs, based on their observed concentrations in the soil and leachates obtained from each test. The concentration and composition of DOM in each leachate were also determined, the latter using FTIR spectroscopy. Correlations between the DOM's FTIR spectra and K(d) values were investigated by orthogonal projections to latent structures. The log K(d)-values varied among the PCB congeners and were most variable at low pH, but the values for all studied congeners decreased with increasing pH, by up to 3 log units (for PCB 187). In the pH 5-7 interval, an abrupt decrease in log K(d) values with increases in pH was observed, although the total organic carbon content remained relatively stable. The FTIR data indicate that fulvic and humic acids in DOM partially deprotonate as the pH rises from 5 to 7.

The influence of soil composition on the leachability of selected hydrophobic organic compounds (HOCs) from soils using a batch leaching test.

The influence of soil composition (peat and clay content) on the leachability was investigated in batch leaching experiments for chemically diverse hydrophobic organic compounds (HOCs: PCP, PAHs, HCB, HCHs, PCBs, and TCDD/Fs). An experimental design was applied to generate 8 diverse soil matrices, and the results were evaluated by orthogonal projections to latent structures (OPLS), as well as compound specific response surface models. Overall, the distribution coefficients (logKd) of model HOCs were in the range of approx. 2.0-5.7. The Kd-values of HCHs, phenanthrene and PCP were positively correlated with the peat content. Kd-values of benzo(a)anthracene, HCB, and PCB 47 were positively correlated with both peat and clay content. The Kd-values of 1,3,6,8-TCDD and 1,3,6,8-TCDF were positively correlated with peat content but negatively correlated with clay content, while for PCB 153 and PCB 155 the correlations were reversed. The correlation between the Kd-values and the compounds' Kow-values was linearly for compounds with log Kow <6. For HOCs with log Kow>6, the Kd-values were leveling off, possibly due to small particles in the leachates. Our study demonstrated how complex interaction between both the organic matter and clay components influences the leachability of HOCs in a compound-specific manner.

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.

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.

Dioxin removal from contaminated soils by ethanol washing.

The aim of this study was to investigate the potential utility of ethanol washing for remediating soils contaminated with polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), as a cost-efficient alternative to conventional remediation methods of PCDD/F-contaminated soils. Initially, screening experiments were performed with a two-level full factorial design to examine the effects of temperature, extraction time and ethanol concentration on the removal efficiency. The screening experiments showed that the ethanol concentration was the most important parameter. In addition, repeated washing cycles considerably improved the results. Ethanol washing conditions were then selected (10 wash cycles with 75% ethanol at 60 degrees C), and applied to four soils with different soil characteristics and contamination levels to test the robustness of the selected method. Treatment efficiencies of 81% and 85% were obtained for a lightly contaminated sandy-silty soil and a highly contaminated clay soil rich in graphite particles, respectively. Even higher treatment efficiencies (> or = 97%) were obtained for two other highly contaminated soils, one of which contained high amounts of organic matter. PCDD/Fs were found to both dissolve in the solvent and migrate into it as species adsorbed to particles. The relative contributions of these mechanisms and the overall efficiency of the removal seem to depend on contaminant concentration, the types of carbon in the soil matrix and the particle size distribution. The study shows that ethanol washing has effective remediation potential for a variety of PCDD/F-contaminated soils.

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 gammaH2AX 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.

Mutagenicity of an aged gasworks soil during bioslurry treatment.

This study investigated changes in the mutagenic activity of organic fractions from soil contaminated with polycyclic aromatic hydrocarbons (PAHs) during pilot-scale bioslurry remediation. Slurry samples were previously analyzed for changes in PAH and polycyclic aromatic compound content, and this study examined the correspondence between the chemical and toxicological metrics. Nonpolar neutral and semipolar aromatic fractions of samples obtained on days 0, 3, 7, 24, and 29 of treatment were assayed for mutagenicity using the Salmonella mutation assay. Most samples elicited a significant positive response on Salmonella strains TA98, YG1041, and YG1042 with and without S9 metabolic activation; however, TA100 failed to detect mutagenicity in any sample. Changes in the mutagenic activity of the fractions across treatment time and metabolic activation conditions suggests a pattern of formation and transformation of mutagenic compounds that may include a wide range of PAH derivatives such as aromatic amines, oxygenated PAHs, and S-heterocyclic compounds. The prior chemical analyses documented the formation of oxygenated PAHs during the treatment (e.g., 4-oxapyrene-5-one), and the mutagenicity analyses showed high corresponding activity in the semipolar fraction with and without metabolic activation. However, it could not be verified that these specific compounds were the underlying cause of the observed changes in mutagenic activity. The results highlight the need for concurrent chemical and toxicological profiling of contaminated sites undergoing remediation to ensure elimination of priority contaminants as well as a reduction in toxicological hazard. Moreover, the results imply that remediation efficacy and utility be evaluated using both chemical and toxicological metrics.