Background, aim and scope Perfluorinated compounds (PFCs) are global environmental pollutants that bioaccumulate in wildlife and humans. Laboratory experiments have revealed toxic effects such as delayed development, humoral suppression, and hepatotoxicity. Although numerous human blood levels have been reported, little is known about distribution in the human body. Knowledge about PFC distribution and accumulation in the human body is crucial to understanding uptake and subsequent effects as well as to conduct risk assessments. The present study reports PFC levels in human liver and breast milk from a general population living in Catalonia, Spain. Liver and milk levels are compared to previously reported levels in blood from the same geographic area as well as to other existing reports on human liver and milk levels in other countries. Materials and methods Human liver (n = 12) and milk (n = 10) samples were collected in 2007 and 2008 in Catalonia, Spain. Liver samples were taken postmortem from six males and six females aged 27-79 years. Milk samples were from healthy primipara women (30-39 years old). Both liver and milk were analyzed by solid-phase extraction and ultra-performance liquid chromatography tandem mass spectrometry. Results Six PFCs were detected in liver, with perfluorooctanesulfonate (PFOS, 26.6 ng/g wet weight) being the chemical with the highest mean concentration. Other PFCs such as perfluorohexanesulfonate (PFHxS), perfluorooctanoic acid (PFOA), and acids with chain lengths up to C11 were also detected, with mean levels ranging between 0.50 and 1.45 ng/g wet weight. On the other hand, PFOS and PFHxS were the only PFCs detected in human milk, with mean concentrations of 0.12 and 0.04 ng/mL, respectively. Discussion While milk concentrations were similar to reported levels from other countries, liver samples contained more PFCs above quantification limits and higher PFOS concentrations compared to the only two other reports found in the literature. Differences between the results of the present study and those concerning previous investigations can be due to declining levels of some PFCs, which have been reported for the USA. The relationship between PFC concentrations in human liver, milk, and blood was assessed using blood concentrations previously determined in Catalonia. Those levels resulted in liver/serum ratios of 1.7:1, 1.4:1, and 2.1:1 for PFOS, perfluorodecanoic acid, and perfluoroundecanoic acid, respectively. Accumulation in liver is suggested for PFOS and the perfluorocarboxylic acids with carbon chain lengths C9, C10, and C11. For PFOA and PFHxS, fivefold and 14-fold higher concentrations, respectively, were seen in serum as compared to liver. The mean concentration of PFOS and PFHxS in milk was only 0.8% and 0.6% of the reported mean serum level, respectively. Conclusions The results of the present study show that several PFCs could be detected in human liver samples of subjects living in Tarragona. Concerning human milk, the mechanism by which PFCs are transferred from mother's blood to breast milk is still unclear. Considering that PFCs are strongly bound to the protein fraction in blood, the possibility of PFCs entering the milk and accumulating to levels observed in maternal plasma is limited. Recommendations and perspectives Interestingly, the potential accumulation difference for PFCs with different chain lengths might be of great importance for risk assessment. Continuing studies on the distribution of different PFCs in human tissue are therefore justified.

Background, aim, and scope Linear alkylbenzene sulfonate (LAS) is the most used anionic surfactant in a worldwide scale and is considered a high-priority pollutant. LAS is regarded as a readily biodegradable product under aerobic conditions in aqueous media and is mostly removed in wastewater treatment plants, but an important fraction (20-25%) is immobilized in sewage sludge and persists under anoxic conditions. Due to the application of the sludge as a fertilizer, LAS reaches agricultural soil, and therefore, microbial toxicity tests have been widely used to evaluate the influence of LAS on soil microbial ecology. However, molecular-based community-level analyses have been seldom applied in studies regarding the effects of LAS on natural or engineered systems, and, to our knowledge, there are no reports of their use for such appraisals in agricultural soil. In this study, a microcosm system is used to evaluate the effects of a commercial mixture of LAS on the community structure of Alphaproteobacteria, Actinobacteria, and Acidobacteria in an agricultural soil. Material and methods The microcosms consisted of agricultural soil columns (800 g) fed with sterile water (8 ml h⁻¹) added of different concentration of LAS (10 or 50 mg l⁻¹) for periods of time up to 21 days. Sterile water was added to control columns for comparison. The structures of Alphaproteobacteria, Actinobacteria, and Acidobacteria communities were analyzed by a cultivation independent method (temperature gradient gel electrophoresis (TGGE) separation of polymerase chain reaction (PCR)-amplified partial 16S rRNA genes). Relevant populations were identified by subsequent reamplification, DNA sequencing, and database comparisons. Results Cluster analysis of the TGGE fingerprints taking into consideration both the number of bands and their relative intensities revealed that the structure of the Alphaproteobacteria community was significantly changed in the presence of LAS, at both concentrations tested. The average number of bands was significantly lower in the microcosms receiving 50 mg l⁻¹ LAS and in the lower portion of soil cores. The clear differentiation of the samples of the upper portion of the soil columns amended with LAS was specifically related to the presence and intensity of a distinctive major band (named band class 7). There was a statistically significant positive correlation between the concentrations of LAS detected in soil portions taken from LAS 10 mg l⁻¹ and LAS 50 mg l⁻¹ microcosms and the relative intensity of band class 7 in the corresponding TGGE profiles. Prevalent Alphaproteobacteria populations in the soil microcosms had close similarity (>99%) to cultivated species affiliated to genera of the Rhizobiaceae, Methylocystaceae, Hyphomicrobiaceae, Rhodospirillaceae, Brucellaceae, Bradyrhizobiaceae, and Caulobacteraceae families. The population represented by band class 7 was found closely related to the genus Phenylobacterium (Caulobacteraceae). According to cluster analysis of TGGE profiles, the structure of both Actinobacteria and Acidobacteria communities in the soil microcosms was remarkably stable in the presence of LAS at the two concentrations tested, as most bands were universally present in all samples and displayed fairly similar relative intensities. Discussion Previous studies by others authors, based on biological and chemical tests, concluded that LAS toxicity was not an important microbial selection factor in sludge amended soil, while work based on the use of molecular fingerprinting to evaluate the impact of LAS in aqueous media and marine sediments showed that concentrations as low as 1 mg l⁻¹ significantly influence the development of the bacterial community structure. Although TGGE is not a strictly quantitative method due to the bias introduced by the PCR reaction, changes of band intensity through experiments are a consequence of a change in the relative abundance of the corresponding populations in the community and can be used as a semiquantitative measure of bacterial diversity. Our results evidence that the Phenylobacterium population represented by band class 7 was favored by the presence of increasing concentrations of LAS in the soil and turned into a dominant population, suggesting its possible ability to use LAS in soil as a source of nutrients. As studies with pure cultures are required to confirm the ability of this population to degrade LAS, isolation strategies are currently under development in our laboratory. The weak effect of LAS on the structure of Actinobacteria and Acidobacteria communities is particularly interesting, as to our knowledge, there are no previous reports regarding the effects of LAS on these bacterial groups in soil. Conclusions, recommendations, and perspectives The Phenylobacterium-related alphaproteobacterial population identified in this work was selectively enriched in LAS polluted soil and is a plausible candidate to play a relevant role in the biotransformation of the surfactant under the conditions tested. The surfactant had no remarkable effects on the Actinobacteria and Acidobacteria fingerprints in soil, even when present at concentrations widely exceeding those reached in soil immediately after sludge application. TGGE fingerprinting provides a reliable and low time-consuming method for the monitoring of the bacterial community structure and dynamics, and we recommend its integration with the biological and chemical analyses usually applied in risk assessment of LAS in the environment.

Purpose Dissolved humic acids abiotically reduced inorganic arsenic to varying degrees, depending on solution pH, ionic strength, and type of humate used. The functionalities of dissolved organic matter responsible for these redox reactions remained in question, but quinoid moieties undoubtedly played an important role. It is not fully understood whether the quinoids are solely responsible for arsenate reduction, and what the kinetics of the relevant processes are. Methods Electron spin resonance (ESR) spectroscopy was used to monitor the radical content of the humates, both as bulk material and as size fractions. Information on the redox status of the humates was obtained from fluorescence excitation-emission matrices and correlated with the observed spin count. Size data were obtained from fractionation and UV-Vis spectrometry. Arsenic speciation was carried out by ion chromatography. Results ESR spectroscopy showed a free radical content of 3.4 × 1,017-20 × 1,017 spins/g for bulk and fractionated aqueous humic acids. The number of electrons corresponding to these counts could not account for the entire charge transferred to arsenate during abiotic reduction. The rate constants of the reactions were found to be independent of the humic concentration. Leonardite humic acid separated on a XAD-8 resin yielded fractions that on the short time frame (0-5 h) had rate constants of 0.035 h⁻¹ for the hydrophobic fraction compared to 0.0052 h⁻¹ for the hydrophilic fraction. The rate constants for the hydrophobic and hydrophilic fractions over the longer time frame (100-200 h) were similar—7.3 × 10⁻⁴ and 7.2 × 10⁻⁴ h⁻¹, respectively. Fluorescence excitation-emission matrices of humates involved in arsenate reduction exhibited shifts typical of quinoid components undergoing redox transformations. These gradual shifts took place during the first 24 h of reduction process, after which the spectra no longer changed. The reduction of arsenate, however, continued after this period, indicating that species other than quinoids were involved. This was consistent with the fact that the rate constants for the later processes were smaller. Conclusions The existence of different redox pools within the humate was confirmed, with the quinoid-centered redox entities showing the fastest kinetics. The results pertained to all size and polarity fractions.

Purpose Experiments were conducted to examine the effects of mercury (Hg) on soil nitrification activities and the microbial communities of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Methods The soil samples spiked with different Hg concentrations were incubated for a period of 1, 2, 4, and 8 weeks in triplicate and the potential nitrification rate (PNR) of the samples was determined. The abundance of AOB and AOA was measured after an 8-week incubation by real-time polymerase chain reaction (PCR) assay of the amoA genes, while the community compositions by cloning and sequencing approaches. Results The soil PNR differed with different incubation periods. It tended to decrease with increasing soil Hg concentrations at week 1, basing on which the half-maximal effective concentration (EC50) was 1.59 mg kg⁻¹. There was no significant difference in the abundance of AOB or AOA among the treatments. The AOB community was dominated by Nitrosospira-like sequences and more than 70% of the obtained clones were affiliated with the cluster 3a.2. The percentage of cluster 3a.1 in AOB community appeared to a consistent trend of decreasing with ascending soil Hg concentrations. While all the AOA sequences in the clone libraries were grouped into cluster S (soil and sediment origin). Conclusions This study revealed that Hg could inhibit soil potential nitrification and the extent varied with incubation periods. Soil Hg pollution changed the composition of soil AOB to some extent. These findings will be helpful to recognize the effects of Hg on the activity and community composition of soil ammonia oxidizers.

Background, aim and scope The climatic characteristic is a major parameter affecting on the distribution variation of organic pollutants such as polycyclic aromatic hydrocarbons (PAHs). The Tumen River is located in Northeastern of China. The winter era lasts for more than 5 months in a year, and the river water was frozen and covered by ice phase. Coal combustion is an essential heating source in the Tumen River Basin. The objective of this research is to study ice phase effect on the seasonal variation of PAHs in the Tumen River environment. Materials and methods Samples were collected from 13 sites along the River in March, July, October, and December of 2008. In addition, the ice sample, under ice water and air particulate were also collected in winter. The samples were analyzed for 16 PAHs (naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, beazo[a]anthene, chrysene, beazo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno(1,2,3-cd)pyrene, dibenz(a,h)anthracene, and benzo(ghi)perylene). The compounds were extracted from the water samples and solid samples using LLE and Soxhlet extraction technique, respectively, and it is determined by gas chromatography-mass spectrometry. Results and discussion Among 16 PAHs, fluorene, phenanthrene, and pyrene were found to be present in high concentrations and at high detection frequencies. The total concentration of PAHs in the water, particulate, sediment and ice phase ranged from 35.1-1.05 × 10³ ng L⁻¹, 25.4-817 ng L⁻¹, 117-562 ng g⁻¹and 62.8-136 ng g⁻¹, respectively. The levels of PAHs were generally higher in spring than other seasons. The ice phase in winter acts like a major reservoir of the pollutants and it is major contributor on the seasonal variation of PAHs in Tumen River. The PAHs found in water, particulate, and sediment in the Tumen River were possibly derived from similar pollution sources a proposition based on the compositions and isomer ratios of PAHs. Conclusions The distribution of PAHs was showed clear seasonal variation in the Tumen River environment, the ice phase and air pollution look like an important factor affecting on the seasonal variation. Recommendations and perspectives The ice phase as an important factor affecting on the seasonal variation of PAHs in Tumen River environment. Further studies regarding the effects of air pollution on the river and the mechanisms of migration and transformation of them in the environment are currently being conducted in our laboratory.

Background, aim and scope Landfill gas (LFG) tends to escape from the landfill surface even when LFG collecting systems are installed. Since LFG leaks are generally a noticeable percentage of the total production of LFG, the optimisation of the collection system is a fundamental step for both energy recovery and environmental impact mitigation. In this work, we suggest to take into account the results of direct measurements of gas fluxes at the air-cover interface to achieve this goal. Materials and methods During the last 5 years (2004-2009), 11 soil gas emission surveys have been carried out at the Municipal Solid Waste landfill of Legoli (Peccioli municipality, Pisa Province, Italy) by means of the accumulation chamber method. Direct and simultaneous measurements of CH₄ and CO₂ fluxes from the landfill cover (about 140,000 m²) have been performed to estimate the total output of both gases discharged into the atmosphere. Three different data processing have been applied and compared: Arithmetic mean of raw data (AMRD), sequential Gaussian conditional simulations (SGCS) and turning bands conditional simulations (TBCS). The total amount of LFG (captured and not captured) obtained from processing of direct measurements has been compared with the corresponding outcomes of three different numerical models (LandGEM, IPCC waste model and GasSim). Results Measured fluxes vary from undetectable values (<0.05 mol m⁻² day⁻¹ for CH₄ and <0.02 mol m⁻² day⁻¹ for CO₂) to 246 mol m⁻² day⁻¹ for CH₄ and 275 mol m⁻² day⁻¹ for CO₂. The specific CH₄ and CO₂ fluxes (flux per surface unit) vary from 1.8 to 7.9 mol m⁻² day⁻¹ and from 2.4 to 7.8 mol m⁻² day⁻¹, respectively. Discussion The three different estimation methodologies (AMRD, SGCS and TBCS) used to evaluate the total output of diffused CO₂ and CH₄ fluxes from soil provide similar estimations, whereas there are some mismatches between these results and those of numerical LFG production models. Isoflux maps show a non-uniform spatial distribution, with high-flux zones not always corresponding with high-temperature areas shown by thermographic images. Conclusions The average value estimated over the 5-year period for the Legoli landfill is 245 mol min⁻¹ for CH₄ and 379 mol min⁻¹ for CO₂, whereas the volume percentage of CH₄ in the total gas discharged into the atmosphere varies from 29% to 51%, with a mean value of 39%. The estimated yearly emissions from the landfill cover is about 1.29 × 10⁸ mol annum⁻¹ (2,100 t year⁻¹) of CH₄ and 1.99 × 10⁸ mol annum⁻¹ (8,800 t year⁻¹) of CO₂. Considering that the CH₄ global warming potential is 63 times greater than that of CO₂ (20 a time horizon, Lashof and Ahuja 1990), the emission of methane corresponds to 130,000 t annum⁻¹ of CO₂. Recommendations and perspectives The importance of these studies is to provide data for the worldwide inventory of CH₄ and CO₂ emissions from landfills, with the ultimate aim of determining the contribution of waste disposal to global warming. This kind of studies could be extended to other gas species, like the volatile organic compounds.

Background, aim and scope The applicability of the Whole Effluent Assessment concept for the proof of compliance with the “best available techniques” has been analysed with paper mill wastewater from Germany by considering its persistency (P), potentially bio-accumulative substances (B) and toxicity (T). Materials and methods Twenty wastewater samples from 13 paper mills using different types of cellulose fibres as raw materials have been tested in DIN or ISO standardised bioassays: the algae, daphnia, luminescent bacteria, duckweed (Lemna), fish-egg and umu tests with lowest ineffective dilution (LID) as test result. The potentially bio-accumulative substances (PBS) were determined by solid-phase microextraction and referred to the reference compound 2,3-dimethylnaphthalene. Usually, a primary chemical-physical treatment of the wastewater was followed by a single or multi-stage biological treatment. One indirectly discharged wastewater sample was pre-treated biologically in the Zahn-Wellens test before determining its ecotoxicity. Results No toxicity or genotoxicity at all was detected in the acute daphnia and fish egg as well as the umu assay. In the luminescent bacteria test, moderate toxicity (up to LIDlb = 6) was observed. Wastewater of four paper mills demonstrated elevated or high algae toxicity (up to LIDA = 128), which was in line with the results of the Lemna test, which mostly was less sensitive than the algae test (up to LIDDW = 8). One indirectly discharged wastewater sample was biodegraded in the Zahn-Wellens test by 96% and was not toxic after this treatment. Low levels of PBS have been detected (median 3.27 mmol L⁻¹). The colouration of the wastewater samples in the visible band did not correlate with algae toxicity and thus is not considered as its primary origin. Further analysis with a partial wastewater stream from thermomechanically produced groundwood pulp (TMP) revealed no algae or luminescent bacteria toxicity after pre-treatment of the sample in the Zahn-Wellens test (chemical oxygen demand elimination 85% in 7 days). Thus, the algae toxicity of the respective paper mill cannot be explained with the TMP partial stream; presumably other raw materials such as biocides might be the source of algae toxicity. Discussion Comparative data from wastewater surveillance of authorities confirmed the range of ecotoxicity observed in the study. Wastewater from paper mills generally has no or a moderate ecotoxicity (median LID 1 and 2) while the maximum LID values, especially for the algae and daphnia tests, are considerably elevated (LIDA up to 128, LIDD up to 48). Conclusions Wastewater from paper mills generally is low to moderately ecotoxic to aquatic organisms in acute toxicity tests. Some samples show effects in the chronic algae growth inhibition test which cannot be explained exclusively with colouration of the samples. The origin of elevated algae ecotoxicity could not be determined. In the algae test, often flat dose-response relationships and growth promotion at higher dilution factors have been observed, indicating that several effects are overlapping. Recommendations and perspectives At least one bioassay should be included in routine wastewater control of paper mills because the paper manufacturing industry is among the most water consuming. Although the algae test was the most sensitive test, it might not be the most appropriate test because of the complex relationship of colouration and inhibition and the smooth dose-effect relationship or even promotion of algae growth often observed. The Lemna test would be a suitable method which also detects inhibitors of photosynthesis and is not disturbed by wastewater colouration.

Background, aim, and scope In response to concerns about chemical substances that can alter the function of endocrine systems and may result in adverse effects on human and ecosystem health, a number of in vitro tests have been developed to identify and assess the endocrine disrupting potential of chemicals and environmental samples. One endpoint that is frequently used in in vitro models for the assessment of chemical effects on the endocrine system is the alteration of aromatase activity (AA). Aromatase is the enzyme responsible for converting androgens to estrogens. Some commonly used aromatase assays, including the human microsomal assay that is a mandatory test in US-EPA's endocrine disruptor screening program (EDSP), detect only direct effects of chemicals on aromatase activity and not indirect effects, including changes in gene expression or transcription factors. This can be a problem for chemical screening initiatives such as the EDSP because chemicals can affect aromatase both indirectly and directly. Here we compare direct, indirect, and combined measurements of AA using the H295R cell line after exposure to seven model chemicals. Furthermore, we compare the predictability of the different types of AA measurements for 17β-estradiol (E2) and testosterone (T) production in vitro. Materials and methods H295R cells were exposed to forskolin, atrazine, letrozole, prochloraz, ketoconazole, aminoglutethimide, and prometon for 48 h. Direct, indirect, and combined effects on aromatase activity were measured using a tritiated water-release assay. Direct effects on aromatase activity were assessed by exposing cells only during the conduct of the tritium-release assay. Indirect effects were measured after exposing cells for 48 h to test chemicals, and then measuring AA without further chemical addition. Combined AA was measured by exposing cells prior and during the conduction of the tritium-release assay. Estradiol and testosterone were measured by ELISA. Results and discussion Exposure to the aromatase inhibitors letrozole, prochloraz, ketoconazole, and aminoglutethimide resulted in greater indirect aromatase activity after a 48-h exposure due to presumed compensatory mechanisms involved in aromatase activity regulation. Forskolin and atrazine caused similar changes in hormone production and enzyme profiles, and both chemicals resulted in a dose-dependent increase in E2, T, and indirect AA. Neither of these two chemicals directly affected AA. For most of the chemicals, direct and combined AA and E2 were good predictors of the mechanism of action of the chemical, with regard to AA. Indirect aromatase activity was a less precise predictor of effects at the hormone level because of presumed feedback loops that made it difficult to predict the chemicals' true effects, mostly seen with the aromatase inhibitors. Further, it was found that direct and indirect AA measurements were not reliable predictors of effects on E2 for general inducers and inhibitors, respectively. Conclusions Differential modulation of AA and hormone production was observed in H295R cells after exposure to seven model chemicals, illustrating the importance of measuring multiple endpoints when describing mechanisms of action in vitro. Recommendations and perspectives For future work with the H295R, it is recommended that a combination of direct and indirect aromatase measurements is used because it was best in predicting the effects of a chemical on E2 production and its mechanism of action. Further, it was shown that direct AA measurements, which are a common way to measure AA, must be used with caution in vitro.