Background, aim and scope A newly developed fine bubble aeration system, by which air is transferred under supercavitation conditions, shows a clearly better performance than traditional, well-known aerators that rely on the jet-pump principle and its performance can be compared to oxygen transfer rates achieved in membrane and foil plate aerators. Materials and method A prototype supercavitation aerator installed at a sewage treatment plant revealed an air input rate, which was about one third lower than that of the jet-pump system, which it replaced. Results In spite of this low air input rate, the daily demand of pure oxygen for the additionally installed membrane aeration system went down by approximately 49%, from the original level of about 1,200 m³/day to about 600 m³/day—and this over a test period of more than 7 months. Conclusions and discussion The observed high oxygen transfer rates cannot be explained by traditional mass transfer mechanisms. It is assumed that a large amount of water being transferred into the gas phase by supercavitation contacting directly oxygen also in the gas phase and thereby overcoming mass transfer hindrances which might be favoured by hydroxyl radicals. With this new aerator, during the first 3 months of test phase, already more than 10,000 Euros had been saved because of the reduced pure oxygen demand.

Background, aim, and scope Soil remediation with ethylenediamine tetraacetic acid (EDTA) leaching is capable of removing only part of the total metal concentration in the soil, mostly the labile, bioavailable metal species (metal bioavailability stripping). However, reintroduction of remediated soil in the environment exposes the soil to various environmental factors, which could potentially shift nonlabile residual metals back to labile bioavailable forms. We studied the effect of autochthonous earthworm species as model biotic environmental factor on the fractionation and bioavailability of Cu residual in soil after remediation. Materials and methods We used soil from a 50-year-old vineyard regularly managed and treated with CuSO₄•5H₂O (Bordeaux mixture) as fungicide. Soil containing 400 mg kg⁻¹ of Cu was leached with total 15 mmol kg⁻¹ EDTA. Remediated and nonremediated soil was processed by fully clitellated adult specimens of Lumbricus terrestris L., a prevailing autochthonous soil earthworm species. Cu fractionation, phytoavailability, and oral-bioavailability in processed and nonprocessed soil were determined using six-step sequential extraction, extraction with diethylenediamine pentaacetic acid, and in vitro physiologically based extraction test, respectively. Results EDTA leaching removed 41% of the pseudototal Cu, mostly from the soil Fe- and Mn-oxides, carbonates, and organic matter. A 2.7-fold decrease in Cu phytoavailability and a 4.4- and 2.8-fold decrease in Cu oral-bioavailability in the stomach and small intestine fractions, respectively, were achieved after remediation. In nonremediated soil, earthworms increased the share of nonlabile Cu in residual soil fraction, while in remediated soil they increased the share of Cu bound to carbonates. A statistically significant 1.1- and 1.7-fold increase in Cu phytoavailability and intestinal oral-bioavailability, respectively, was observed in earthworm processed remediated soil. Discussion Cu occurs in various soil “pools” of different solubilities with different chemical characteristics and consequently different functions. By removing the labile part of the metals from the soil during remediation, we disrupt the chemical equilibrium; the nonlabile residual metals left in soil after remediation might become more labile in time in tendency to re-establish that equilibrium. Earthworms alter the physical and chemical properties of soil affecting consequently the fractionation of metals. The increase in earthworm's gut pH due to the excretion of ammonia and/or calcium carbonate into the intestine could lead to the transbounding of metals into the carbonate fraction. However, their activity in remediated soil increased Cu phytoavailability and intestinal oral-bioavailability, and it would, therefore, be improper to generalize the influence of earthworms on metal availability in soil. Conclusions The results presented here show that residual Cu in remediated soil is affected by environmental factors such as earthworms, which should be considered in evaluating the effect of Cu polluted soil remediation. Recommendations and perspectives Information on the behavior of residual metals in soil after its remediation is surprisingly scarce. The development of new effective remediation techniques should imply also the evaluation of postremediation effects on remediated soil. The results presented in this work indicate a possible tool for assessing the effect of biotic environmental factors on residual metals left in soil after its remediation.

Background, aim, and scope Arthropods, with over a million species described, are ubiquitous throughout different environments. Knowledge of their responses to human impact is crucial for understanding and predicting changes in ecosystem structure and functions. Our aim was to investigate the general patterns and to identify sources of variation in changes of the diversity, abundance and fitness of terrestrial arthropods (including Arachnida, Collembola and Insecta) in habitats affected by point polluters. Main features We found 134 suitable studies which were published between 1965 and 2007. These data came from impact zones of 74 polluters in 20 countries with the largest representation from Russia (28 polluters), Poland (12 polluters) and the USA (six polluters). The database allowed calculation of 448 effect sizes (i.e. relative differences between measurements taken from polluted and control sites) on the effects of various point polluters like non-ferrous industries, aluminium plants, cement, magnezite, fertilising and chemical plants, power plants, iron- and steel-producing factories. We used meta-analysis to search for general effects and to compare between polluter types and arthropod groups, and linear regression to describe the latitudinal gradient and to quantify relationships between pollution and arthropod responses. Results The overall effect of pollution on arthropod diversity did not differ from zero. However, species richness of soil arthropods (both living on the soil surface and within the soil) tended to decrease, and species richness of herbivores to increase, near point polluters. Abundance of terrestrial arthropods near point polluters decreased in general. This decrease resulted from strong adverse effects on soil arthropods, especially on decomposers and predators. Densities of herbivores increased, but a number of research biases that we discovered in published data may have led to overestimation of the latter effect. The dome-shaped density pattern along pollution gradients was discovered only in 5% of data sets. Among herbivores, only free-living defoliators and sap-feeders demonstrated higher densities in polluted sites; the effects of pollution on other guilds were not significant. Near the polluters, conifers suffered higher increase in damage from herbivores than deciduous woody plants and herbs. Overall effect of pollution on arthropod performance was negative; in particular, individuals from polluted sites were generally smaller than individuals from control sites. This negative effect weakened with increase in duration of the pollution impact, hinting evolution of pollution resistance in populations inhabiting polluted sites. Stepwise regression analysis demonstrated that pollution-induced changes in both the density and performance of arthropods depended on climate of the locality. Negative effects on soil fauna increased with increase in annual precipitation; positive effects on herbivore population density increased with increases in both mean July temperature and annual precipitation. Discussion We detected effects of research methodology on the outcome of published studies. Many of them suffer from research bias—the tendency to collect data on organisms or under conditions in which one has an expectation of detecting significant effects. Pseudoreplicated studies (one polluted site contrasted to one control site) frequently reported larger effects than replicated studies (several polluted sites contrasted with several control sites). These methodological flaws especially influenced herbivory studies; we conclude that increase in herbivory in both heavily and moderately polluted habitats is not as frequent as it was earlier suggested. In contrast, the decrease in abundance of predators is likely to be a widespread phenomenon. Thus, our analysis supports the hypothesis that pollution may favour herbivore populations by creating an enemy-free space. Consistent declines in abundance of soil arthropods in impact zones of different polluters suggest that this group can potentially be used in bioindication of pollution-induced changes in terrestrial ecosystems. Conclusions Main effects of pollution on arthropod communities (decreased abundance of decomposers and predators and increased herbivory) may have negative consequences for structure and services of entire ecosystems. Responses of arthropods to pollution depend on both temperature and precipitation in such a way that ecosystem-wide adverse effects are likely to increase under predicted climate change. Recommendations and perspectives Our analysis confirmed that local severe impacts of industrial enterprises on biota are well-suited to reveal the direction and magnitude of the biotic effects of aerial pollution, as well as to explore the sources of variation in responses of organisms and communities. Although we analysed the effects of point polluters, our conclusions can be applied to predict consequences of pollution impacts on regional and even global scales. We argue that possible interactions between pollution and climate should be accounted for in the analyses of global change impacts on biota.

Background, aim and scope The importance of groundwater for human life cannot be overemphasised. Besides fulfilling essential ecological functions, it is a major source of drinking water. However, in the industrial area of Bitterfeld, it is contaminated with a multitude of harmful chemicals, including genotoxicants. Therefore, recently developed methodologies including preparative capillary gas chromatography (pcGC), MOLGEN-MS structure generation and mutagenicity prediction were applied within effect-directed analysis (EDA) to reduce sample complexity and to identify candidate mutagens in the samples. A major focus was put on the added value of these tools compared to conventional EDA combining reversed-phase liquid chromatography (RP-LC) followed by GC/MS analysis and MS library search. Materials and methods We combined genotoxicity testing with umuC and RP-LC with pcGC fractionation to isolate genotoxic compounds from a contaminated groundwater sample. Spectral library information from the NIST05 database was combined with a computer-based structure generation tool called MOLGEN-MS for structure elucidation of unknowns. Finally, we applied a computer model for mutagenicity prediction (ChemProp) to identify candidate mutagens and genotoxicants. Results and discussion A total of 62 components were tentatively identified in genotoxic fractions. Ten of these components were predicted to be potentially mutagenic, whilst 2,4,6-trichlorophenol, 2,4-dichloro-6-methylphenol and 4-chlorobenzoic acid were confirmed as genotoxicants. Conclusions and perspectives The results suggest pcGC as a high-resolution fractionation tool and MOLGEN-MS to improve structure elucidation, whilst mutagenicity prediction failed in our study to predict identified genotoxicants. Genotoxicity, mutagenicity and carcinogenicity caused by chemicals are complex processes, and prediction from chemical structure still appears to be quite difficult. Progress in this field would significantly support EDA and risk assessment of environmental mixtures.

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.

Background, aim, and scope Mutagenic nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) have been known to arise in the environment through direct emissions from combustion sources and nitration of PAHs, primarily in the atmosphere. In the marine environment, PAHs are one of the classic anthropogenic organic pollutants, while nitrite (NO ₂ ⁻ ) is produced naturally via various biological processes like imbalance in nitrification/denitrification or eutrophication and subsequent oxygen depletion from an oversupply of nutrients. In this paper, we report the formation of PAH-DNA adducts in fish contaminated with PAHs and exposed to NO ₂ ⁻ in the ambient water. Electrospray ionization tandem mass spectrometric (ESI-MS/MS) analysis of the bile of the euryhaline fish Oreochromis mossambicus exposed simultaneously to field relevant sublethal concentrations of phenanthrene and NO ₂ ⁻ and collision-induced dissociation of selected ions revealed the presence of DNA-PAH adducts. The present study indicates that, although several high sensitivity techniques have been developed for the analysis of PAH derived DNA adducts, MS/MS has emerged as a powerful tool in the detection and structure elucidation of DNA adducts. Materials and methods Juvenile O. mossambicus from a local estuarine fish farm were used with increasing frequency for carcinogenicity testing and comparative cancer research. The fish were exposed to the alkylating agent phenanthrene in the presence of NO ₂ ⁻ . Composite untreated bile samples after dilution with methanol: water (1:1; v/v) were analyzed by ESI-MS. Results Several adducts could be evidenced in the bile by MS/MS. Deoxyadenosine/deoxyguanosine having a mass in the range of 450-650 amu is detected. In addition, a segment of modified dinucleotide with a mass that corresponds to a dimer consisting of a modified guanosine and a normal guanosine has also been identified in the bile. Discussion The formation of certain types of DNA adducts is a crucial step in the induction of cancer and a primary stage in mutagenesis. Phenanthrene injected by i.p. route led to the transformation of phenanthrene to N-formyl amino phenanthrene-N ⁶-deoxyadenosine adduct, whereas the fish co-exposed to phenanthrene and ambient nitrite metabolizes PAH to mono-, di- as well as trinitro derivatives, which then react with DNA leading to the formation of mainly modified guanosine and adenosine adducts. In the present investigation, dinitrophenanthrene diol epoxide (DNPDE) adduct with guanosine (m/z 587) seems to be the dominant adduct in the mixture, and its presence is shown first as a comparatively less stable adduct, which decomposes to give a more stable N² adduct (m/z 567). Conclusions MS/MS has proved to be useful in the rapid determination and discrimination of structurally different phenanthrene/derivatives DNA adducts in a complex mixture of fish bile co-exposed to phenanthrene and nitrite. However, the nature of metabolites formed is likely determined by the route of PAH administration, and there is a need to further define the early biochemical events of carcinogenesis in these species. Recommendations and perspectives DNA adduct analysis in fish bile offers a promising approach to study the risk of potentiation of anthropogenic chemicals into genotoxic compounds in the presence of nitrite in the marine environment. We believe this is the first report on the formation of DNA-phenanthrene adducts on co-exposure of the fish to PAH and nitrite.

Background, aim and scope The ergosterol biosynthesis-inhibiting (EBI) fungicide prochloraz can enhance the effect of other pesticides in a range of animal species. Approximately 50% of the fungicides used in Denmark are EBI fungicides. Hence, if they all have synergising potential, a risk assessment of pesticide mixtures based on additivity might not suffice. This study investigates the synergising potential of six different EBI fungicides representing the imidazoles (prochloraz), the triazoles (epoxiconazole, propiconazole and tebuconazole), the piperidines (fenpropidin) and the morpholines (fenpropimorph) together with the pyrethroid insecticide alpha-cypermethrin. Materials and methods Tests were made on the aquatic crustacean Daphnia magna. Mixtures of each of the fungicides were tested together with the insecticide both at a 50:50% effect mixture ratio and, subsequently, in a ray design including five mixture ratios. The results were tested against the concentration addition reference model using dose-response surface analyses. Results The results of the binary dose-response surface studies showed that mixtures with prochloraz increased toxicity up to 12-fold compared with what was expected using the reference model concentration addition (CA). Epoxiconazole and propiconazole enhanced toxicity up to six and sevenfold, respectively. Fenpropimorph showed antagonism, whilst mixtures with tebuconazole and fenpropidin did not deviate statistically from CA. Conclusions Hence, it can be concluded that both imidazoles and some, but not all, triazoles can enhance the effect of a pyrethroid insecticide towards D. magna substantially. Epoxiconazole and propiconazole are often sprayed out together with pyrethroids in tank mixtures. The extent to which this might create unforeseen ecological problems is discussed.

Purpose Besides classical organic pollutants and pesticides, pharmaceuticals and their residues have nowadays become recognized as relevant environmental contaminants. The risks of these chemicals for aquatic ecosystems are well known, but information about the pharmaca-plant interactions and metabolic pathways is scarce. Therefore, we investigate the process of uptake of acetaminophen (N-Acetyl-4-aminophenol) by Brassica juncea, drug-induced defense responses and detoxification mechanisms in different plant parts. Material and methods Hydroponically grown Indian mustard (Brassica juncea L. Czern.) plants were treated with acetaminophen and root and leaf samples were collected after 24, 72, and 168 h of treatment. The uptake of acetaminophen and the formation of its metabolites were analyzed using LC-MS/MS technique and enzyme activities including glutathione S-transferases (GSTs) as well as several plant defense enzymes like catalase, ascorbat peroxidase, peroxidase, and glutathione reductase were assayed spectrophotometrically. Results We determined the uptake and the translocation of acetaminophen, and we tried to identify the steps of the detoxification process by assaying typical enzymes, supposing the involvement of the same- or similar enzymes and reactions as in the mammalian detoxification process. After 24-h exposure, effective uptake and translocation were observed to the upper part of plants followed by two independent conjugative detoxification pathways. Changes in antioxidant defense enzyme activities connected to the defense pathway towards reactive oxygen species indicate an additional oxidative stress response in the plants. Conclusions The major metabolic pathways in mammals are conjugation with activated sulfate and glucuronic acid, while a small amount of acetaminophen forms a chemically reactive and highly toxic, hydroxylated metabolite. We identified a glutathionyl and a glycoside conjugate, which refer to the similarities to mammalian detoxification. Increased GST activities in leaf tissues were observed correlated with the appearance of the acetaminophen-glutathione conjugate which shows the involvement of this enzyme group in the metabolism of acetaminophen in plants to organic pollutants and xenobiotics. High acetaminophen concentrations lead to oxidative stress and irreversible damages in the plants, which necessitates further investigations using lower drug concentrations for the deeper understanding of the induced detoxification—and defense processes.

Background, aim, and scope Olive oil mill wastewater (OOMW) environmental impacts minimization have been attempted by developing more effective processes, but no chemical or biological treatments were found to be totally effective to mitigate their impact on receiving systems. This work is the first that reports simultaneously the efficiency of three different approaches: biological treatment by two fungal species (Trametes versicolor or Pleurotus sajor caju), enzymatic treatment by laccase, and chemical treatment by photo-Fenton oxidation on phenols removal. Materials and methods Those treatments were performed on OOMW with or without phenol supplement (p-coumaric, vanillin, guaiacol, vanillic acid, or tyrosol). OOMW samples resulted from treatments were extracted for phenols using liquid-liquid extraction and analyzed by gas chromatography coupled to mass spectrometry. Results Treatment with T. versicolor or P. sajor caju were able to remove between 22% and 74% and between 8% and 76% of phenols, respectively. Treatment by laccase was able to reduce 4% to 70% of phenols whereas treatment by photo-Fenton oxidation was responsible for 100% phenols reduction. Discussion Range of phenol degradation was equivalent between T. versicolor, P. sajor caju and laccase for p-coumaric, guaiacol, caffeic acid, and tyrosol in supplemented OOMW, which enhances this enzyme role in the biological treatment promoted by these two species. Conclusions Phenols were removed more efficiently by photo-Fenton treatment than by biological or enzymatic treatments. Recommendations and perspectives Use of fungi, laccase, or photo-Fenton presents great potential for removing phenols from OOMW. This should be further assessed by increasing the application scale and the reactor configurations effect on the performance, besides a toxicity evaluation of treated wastewater in comparison to raw wastewater.

Background, aim, and scope This review deals with publications concerning the mode of action of Bt proteins and their potential synergism with extrinsic factors. The aim was to assess the impact of those factors especially regarding selectivity and efficacy of Bt toxins and to discuss possible gaps in current risk assessment of genetically engineered plants expressing Bt toxins. Main features The review shows that several extrinsic factors are able to influence the selectivity and efficacy of Bt toxins. The findings are seen as being relevant for risk assessment in Bt plants. This conclusion is derived by discussing current state of knowledge about the mode of action of Bt proteins, unexpected effects on non-target organism, and the way how modified Bt toxins are expressed in genetically engineered plants. Results Several publications have been identified that show that certain factors and synergism can impact efficacy and selectivity of Bt toxins. These extrinsic factors are various and include other Bt toxins or parts from the spore of Bacillus thuringiensis as well as certain enzymes, environmental stress, non-pathogenic microorganisms, and infectious diseases. Discussion Research on the underlying mechanism of observed synergism might help to explain some of the effects found in non-target organisms. In general, possible synergism of Bt toxins with extrinsic factors can be relevant for risk assessment of genetically engineered Bt plants since they expose a modified Bt toxin to the environment under various conditions and over a long period of time. Conclusions Risk assessment of genetically engineered plants should put into question the general assumption of a high selectivity and a linear dose-response relationship in the toxicity of Bt proteins. Both selectivity and efficacy can be influenced by synergism, which can provoke unexpected and undesired effects in non-target organisms. Perspectives It is suggested that systematic research be promoted on synergism between Bt toxins and potential extrinsic factors that could impact the spectrum of susceptible organisms. This research should become a prerequisite for risk assessment of Bt plants.