Influence of selected anti-cancer drugs on the induction of DNA double-strand breaks and changes in gene expression in human hepatoma HepG2 cells

In chemotherapy, various anti-cancer drugs with different mechanisms of action are used and may represent different risk of undesirable delayed side effects in treated patients as well as in occupationally exposed populations. The aim of the present study was to evaluate genotoxic potential of four widely used anti-cancer drugs with different mechanisms of action: 5-fluorouracil (5-FU), cisplatin (CDDP) and etoposide (ET) that cause cell death by targeting DNA function and imatinib mesylate (IM) that inhibits targeted protein kinases in cancer cells in an experimental model with human hepatoma HepG2 cells. After 24 h of exposure all four anti-cancer drugs at non-cytotoxic concentrations induced significant increase in formation of DNA double strand breaks (DSBs), with IM being the least effective. The analysis of the changes in the expression of genes involved in the response to DNA damage (CDKN1A, GADD45A, MDM2), apoptosis (BAX, BCL2) and oncogenesis (MYC, JUN) showed that 5-FU, CDDP and ET upregulated the genes involved in DNA damage response, while the anti-apoptotic gene BCL2 and oncogene MYC were downregulated. On the contrary, IM did not change the mRNA level of the studied genes, showing different mechanism of action that probably does not involve direct interaction with DNA processing. Genotoxic effects of the tested anti-cancer drugs were observed at their therapeutic concentrations that may consequently lead to increased risk for development of delayed adverse effects in patients. In addition, considering the genotoxic mechanism of action of 5-FU, CDDP and ET an increased risk can also not be excluded in occupationally exposed populations. The results also indicate that exposure to 5-FU, CDDP and ET represent a higher risk for delayed effects such as cancer, reproductive effects and heritable disease than exposure to IM.     

Prediction and assessment of ecogenotoxicity of antineoplastic drugs in binary mixtures

The combined genotoxic effects of four anticancer drugs (5-fluorouracil [5-FU], cisplatin [CDDP], etoposide [ET], and imatinib mesylate [IM]) were studied testing their binary mixtures in two crustaceans that are part of the freshwater food chain, namely Daphnia magna and Ceriodaphnia dubia. Genotoxicity was assessed using the in vivo comet assay. Assessment was based on two distinct effect sizes determined from dose-response experiments. Doses for single and combined exposures expected to result in these effect sizes were computed based on Bliss independence as reference model. Statistical comparison by analysis of variance of single and combined toxicities allowed accepting or rejecting the independency hypothesis. The results obtained for D. magna showed independent action for all mixtures except for IM+5-FU that showed an antagonistic interaction. In C. dubia, most mixtures had antagonist interactions except IM+5-FU and IM+CDDP that showed Bliss independence. Despite the antagonistic interactions, our results demonstrated that combinations of anticancer drugs could be of environmental concern because effects occur at very low concentrations that are in the range of concentrations encountered in aquatic systems.     

Toxicity of the mixture of selected antineoplastic drugs against aquatic primary producers

The residues of antineoplastic drugs are considered as new and emerging pollutants in aquatic environments. Recent experiments showed relatively high toxicity of 5-fluorouracil (5-FU), imatinib mesylate (IM), etoposide (ET) and cisplatin (CP) that are currently among most widely used antineoplastic drugs, against phytoplankton species. In this study, we investigated the toxic potential of the mixture of 5-FU?+?IM?+?ET against green alga Pseudokirchneriella subcapitata and cyanobacterium Synechococcus leopoliensis, and the stability and sorption of these drugs to algal cells. Toxic potential of the mixture was predicted by the concepts of ‘concentration addition’ and ‘independent action’ and compared to the experimentally determined toxicity. In both test species, the measured toxicity of the mixture was at effects concentrations EC₁₀–EC₅₀ higher than the predicted, whereas at higher effect concentration (EC₉₀), it was lower. In general, P. subcapitata was more sensitive than S. leopoliensis. The stability studies of the tested drugs during the experiment showed that 5-FU, IM and CP are relatively stable, whereas in the cultures exposed to ET, two transformation products with the same mass as ET but different retention time were detected. The measurements of the cell-linked concentrations of the tested compounds after 72 h exposure indicated that except for CP (1.9 % of the initial concentration), these drugs are not adsorbed or absorbed by algal cells. The results of this study showed that in alga and cyanobacteria exposure to the mixture of 5-FU?+?ET?+?IM, in particular at low effect concentration range, caused additive or synergistic effect on growth inhibition, and they suggest that single compound toxicity data are not sufficient for the proper toxicity prediction for aquatic primary producers.     

Heavy metal and trace elements in riparian vegetation and macrophytes associated with lacustrine systems in Northern Patagonia Andean Range

Vegetation associated with lacustrine systems in Northern Patagonia was studied for heavy metal and trace element contents, regarding their elemental contribution to these aquatic ecosystems. The research focused on native species and exotic vascular plant Salix spp. potential for absorbing heavy metals and trace elements. The native species studied were riparian Amomyrtus luma, Austrocedrus chilensis, Chusquea culeou, Desfontainia fulgens, Escallonia rubra, Gaultheria mucronata, Lomatia hirsuta, Luma apiculata, Maytenus boaria, Myrceugenia exsucca, Nothofagus antarctica, Nothofagus dombeyi, Schinus patagonicus, and Weinmannia trichosperma, and macrophytes Hydrocotyle chamaemorus, Isöetes chubutiana, Galium sp., Myriophyllum quitense, Nitella sp. (algae), Potamogeton linguatus, Ranunculus sp., and Schoenoplectus californicus. Fresh leaves were analyzed as well as leaves decomposing within the aquatic bodies, collected from lakes Futalaufquen and Rivadavia (Los Alerces National Park), and lakes Moreno and Nahuel Huapi (Nahuel Huapi National Park). The elements studied were heavy metals Ag, As, Cd, Hg, and U, major elements Ca, K, and Fe, and trace elements Ba, Br, Co, Cr, Cs, Hf, Na, Rb, Se, Sr, and Zn. Geochemical tracers La and Sm were also determined to evaluate contamination of the biological tissues by geological particulate (sediment, soil, dust) and to implement concentration corrections.     

Fate of antimicrobial resistance genes in response to application of poultry and swine manure in simulated manure-soil microcosms and manure-pond microcosms

This study aimed to determine the occurrence, abundance, and fate of nine important antimicrobial resistance genes (ARGs) (sul1, sul2, tetB, tetM, ermB, ermF, fexA, cfr, and Intl1) in the simulated soil and pond microcosms following poultry and swine manure application. Absolute quantitative PCR method was used to determine the gene copies. The results were modeled as a logarithmic regression (N?=?mlnt?+?b) to explore the fate of target genes. Genes sul1, Intl1, sul2, and tetM had the highest abundance following the application of the two manure types. The logarithmic regression model fitted the results well (R ² values up to 0.99). The reduction rate of all genes (except for the genes fexA and cfr) in manure-pond microcosms was faster than those in manure-soil microcosms. Importantly, sul1, intl1, sul2, and tetM had the lowest reduction rates in all the samples and the low reduction rates of tetM was the first time to be reported. These results indicated that ARG management should focus on using technologies for the ARG elimination before the manure applications rather than waiting for subsequent attenuation in soil or water, particularly the ARGs (such as sul1, intl1, sul2, and tetM investigated in this study) that had high abundance and low reduction rate in the soil and water after application of manure.     

Illumina sequencing-based analysis of a microbial community enriched under anaerobic methane oxidation condition coupled to denitrification revealed coexistence of aerobic and anaerobic methanotrophs

Methane is produced in anaerobic environments, such as reactors used to treat wastewaters, and can be consumed by methanotrophs. The composition and structure of a microbial community enriched from anaerobic sewage sludge under methane-oxidation condition coupled to denitrification were investigated. Denaturing gradient gel electrophoresis (DGGE) analysis retrieved sequences of Methylocaldum and Chloroflexi. Deep sequencing analysis revealed a complex community that changed over time and was affected by methane concentration. Methylocaldum (8.2%), Methylosinus (2.3%), Methylomonas (0.02%), Methylacidiphilales (0.45%), Nitrospirales (0.18%), and Methanosarcinales (0.3%) were detected. Despite denitrifying conditions provided, Nitrospirales and Methanosarcinales, known to perform anaerobic methane oxidation coupled to denitrification (DAMO) process, were in very low abundance. Results demonstrated that aerobic and anaerobic methanotrophs coexisted in the reactor together with heterotrophic microorganisms, suggesting that a diverse microbial community was important to sustain methanotrophic activity. The methanogenic sludge was a good inoculum to enrich methanotrophs, and cultivation conditions play a selective role in determining community composition.     

2,3,7,8-TCDD-mediated toxicity in peripheral blood mononuclear cells is alleviated by the antioxidants present in <Emphasis Type="Italic">Gelidiella acerosa</Emphasis>: an in vitro study

Seaweeds have been used as a source of traditional medicine worldwide for the treatment of various ailments, mainly due to their ability to quench the free radicals. The present study aims at evaluating the protective effect of methanolic extract of Gelidiella acerosa, an edible red seaweed against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity in peripheral blood mononuclear cells (PBMC). For evaluating the protective effect of G. acerosa, PBMC were divided into four groups: vehicle control, TCDD (10 nM), TCDD?+?G. acerosa (300 μg/ml), and G. acerosa alone treated. Scavenging of intracellular reactive oxygen species (ROS) induced by TCDD was assessed by the dichloro-dihydro-fluorescein diacetate (DCFH-DA) method. Alterations at macromolecular level were quantified through lipid peroxidation (LPO) level, protein carbonyl content (PCC) level, and comet assay. The cellular morphology upon TCDD toxicity and G. acerosa treatment was obtained by light microscopy and histopathological studies. The chemical composition present in the methanolic extract of G. acerosa was determined by gas chromatography-mass spectrometry (GC-MS) analysis. The results reveal that 10 nM TCDD caused significant (P?<?0.05) reduction in cell viability (94.10?±?0.99), and treatment with 300 μg/ml extract increased the cell viability (99.24?±?0.69). TCDD treatment resulted in a significant increase in the production of ROS, LPO (114?±?0.09), and PCC (15.13?±?1.53) compared to the control, whereas co-treatment with G. acerosa significantly (P?<?0.05) mitigated the effects. Further, G. acerosa significantly (P?<?0.05) prevented TCDD-induced genotoxicity and cell damage. GC-MS analysis showed the presence of n-hexadecanoic acid (retention time (RT) 13.15), cholesterol (RT 28.80), α-d-glucopyranose, 4-O-α-d-galactopyranosyl (RT 20.01), and azulene (RT 4.20). The findings suggest that G. acerosa has a strong protective ability against TCDD-induced cytotoxicity, oxidative stress, and DNA damage.     

Acute and sub-chronic toxicity of four cytostatic drugs in zebrafish

The acute and sub-chronic effects of four cytostatic drugs—5-fluorouracil (5-FU), cisplatin (CisPt), etoposide (ET) and imatinib mesylate (IM)—on zebrafish (Danio rerio) were investigated. Acute tests were carried out in a static system in accordance with the OECD guideline 203 for adult fish and the draft guideline for fish embryos (FET test) in order to find the LC50 values of the four cytostatic drugs. Early-life stage toxicity test on zebrafish was conducted according the OECD guideline 210 using the cytostatic drugs 5-FU and IM in a semistatic system with the objective of investigating the sub-chronic effects of the cytostatic drugs on fish. In adult fish, the cytostatic drugs 5-FU and ET did not pass the limit test, thus, are considered non-toxic. In case of cisplatin, LC50 was calculated at 64.5 mg L^?1, whereas in case of IM, LC50 was at 70.8 mg L^?1. In the FET test, LC50 of 5-FU at 72-h post fertilization (hpf) was 2441.6 mg L^?1. In case of CisPt, LC50 was 349.9 mg L^?1 at 48 hpf and it progressively decreased to 81.3 mg L^?1 at 120 hpf. In addition, CisPt caused a significant delay in the hatch of larvae. In case of ET, LC50 values were not calculable as they were higher than 300 mg L^?1 at which concentration the substance crystallized in the solution. LC50 values of IM were 48 hpf; 158.3 mg L^?1 , 72 hpf; 141.6 mg L^?1, 96 hpf; 118.0 mg L^?1, and 120 hpf; 65.9 mg L^?1. In the Early-life Stage Test with 5-FU, embryonic deformities were not detected during the tests. Regarding mortalities, the 10 mg L^?1 concentration can be considered as LOEC, as statistically significant difference in mortalities was detected in this group alone. Concerning dry body weight and standard length, 1 mg L^?1 is the LOEC. In case of IM, the highest tested concentration (10 mg L^?1) can be considered LOEC for mortalities, however, the treatment did not have an effect on the other investigated parameters (dry and wet weight, standard length). All four cytostatic drugs were characterized by low toxicity in zebrafish in acute and sub-chronic tests.     

Phenological development stages variation versus mercury tolerance,accumulation, and allocation in salt marsh macrophytes <Emphasis Type="Italic">Triglochin maritima</Emphasis> and <Emphasis Type="Italic">Scirpus maritimus</Emphasis> prevalent in Ria de Aveiro coastal lagoon (Portugal)

Efficient and sustainable management of rapidly mounting environmental issues has been the focus of current intensive research. The present study aimed to investigate the impact of plant phenological development stage variation on mercury (Hg) tolerance, accumulation, and allocation in two salt marsh macrophytes Triglochin maritima and Scirpus maritimus prevalent in historically Hg-contaminated Ria de Aveiro coastal lagoon (Portugal). Both plant samples and the sediments vegetated by monospecific stands of T. maritima and S. maritimus were collected from reference (R) and sites with moderate (M) and high (H) Hg contamination in Laranjo bay within Ria de Aveiro lagoon. Hg tolerance, uptake, and allocation in T. maritima and S. maritimus, physico-chemical traits (pH, redox potential, and organic matter content) and Hg concentrations in sediments vegetated by these species were impacted differentially by phenological development stages variation irrespective of the Hg contamination level. In T. maritima, Hg concentration increased with increase in Hg contamination gradient where root displayed significantly higher Hg followed by rhizome and leaf maximally at H. However, in S. maritimus, the highest Hg concentration was perceptible in rhizome followed by root maximally at M. Between the two studied plant species, S. maritimus displayed higher Hg tolerance index (depicted by higher plant dry mass allocated to reproductive stage) and higher available Hg at M (during all growth stages) and H (during senescent stage) when compared to T. maritimus. Both plant species proved to be Hg excluder (low root/rhizome–leaf Hg translocation). Additionally, T. maritima also acted as Hg stabilizer while, S. maritimus as Hg accumulator. It can be inferred from the study that (a) the plant phenological development stage variations significantly influenced plant Hg sensitivity by impacting sediment chemistry, plant growth (in terms of plant dry mass), Hg accumulation, and its subsequent allocation capacity, contingent to Hg contamination gradient; (b) S. maritimus accumulated higher Hg but restricted its translocation to above-ground part using exclusion process at both M and H due to its accelerated growth during Hg-tolerant reproductive/metabolically active phenological development stage greater than its counterpart T. maritima; and (c) the studied salt marsh plants although hailed from the same C3 and monocot group did not necessarily display similar phenotypic plasticity and behavior towards Hg-contaminated scenario during their life cycle.     

Fate and transport of perfluoro- and polyfluoroalkyl substances including perfluorooctane sulfonamides in a managed urban water body

Transport and fate of perfluoro- and polyfluoroalkyl substances (PFASs) in an urban water body that receives mainly urban runoff was investigated. Water, suspended solids, and sediment samples were collected during the monsoon (wet) and inter-monsoon (dry) season at different sites and depths. Samples were analyzed for C7 to C12 perfluoroalkyl carboxylate homologues (PFCAs) (PFHpA, PFOA, PFNA, PFDA, PFUnA, PFDoA), perfluorohexane, perfluorooctane, and 6:2-fluorotelomer sulfonate (PFHxS, PFOS, and 6:2FtS, respectively), perfluorooctane sulfonamide (FOSA), N-ethyl FOSA (sulfluramid), N-ethyl sulfonamidoethanol (N-EtFOSE), and N-methyl and N-ethyl sulfonamidoacetic acid (N-EtFOSAA and N-MeFOSAA, respectively). Concentrations in wet samples were only slightly higher. The sum total PFAS (ΣPFAS) concentrations dissolved in the aqueous phase and sorbed to suspended solids (SS) ranged from 107 to 253 ng/L and 11 to 158 ng/L, respectively. PFOA, PFOS, PFNA, PFHxS, and PFDA contributed most (approximately 90 %) to the dissolved ΣPFASs. N-EtFOSA dominated the particulate PFAS burden in wet samples. K _D values of PFOA and PFOS calculated from paired SS and water concentrations varied widely (1.4 to 13.7 and 1.9 to 98.9 for PFOA and PFOS, respectively). Field derived K _D was significantly higher than laboratory K _D suggesting hydrophobic PFASs sorbed to SS resist desorption. The ΣPFAS concentrations in the top sedimentary layer ranged from 8 to 42 μg/kg and indicated preferential accumulation of the strongly sorbing long-chain PFASs. The occurrence of the metabolites N-MeFOSAA, N-EtFOSAA and FOSA in the water column and sediments may have resulted from biological or photochemical transformations of perfluorooctane sulfonamide precursors while the absence of FOSA, N-EtFOSA and 6:2FtS in sediments was consistent with biotransformation.     

Enhanced UV-B and Elevated CO<Subscript>2</Subscript> Impacts Sub-Arctic Shrub Berry Abundance,Quality and Seed Germination

This study investigated the effects of long-term-enhanced UV-B, and combined UV-B with elevated CO₂ on dwarf shrub berry characteristics in a sub-arctic heath community. Germination of Vaccinium myrtillus was enhanced in seeds produced at elevated UV-B, but seed numbers and berry size were unaffected. Elevated UV-B and CO₂ stimulated the abundance of V. myrtillus berries, whilst UV-B alone stimulated the berry abundance of V. vitis-idaea and Empetrum hermaphroditum. Enhanced UV-B reduced concentrations of several polyphenolics in V. myrtillus berries, whilst elevated CO₂ increased quercetin glycosides in V. myrtillus, and syringetin glycosides and anthocyanins in E. hermaphroditum berries. UV-B × CO₂ interactions were found for total anthocyanins, delphinidin-3-hexoside and peonidin-3-pentosidein in V. myrtillus berries but not E. hermaphroditum. Results suggest positive impacts of UV-B on the germination of V. myrtillus and species-specific impacts of UV-B × elevated CO₂ on berry abundance and quality. The findings have relevance and implications for human and animal consumers plus seed dispersal and seedling establishment.     

Toxicity of Cu (II) to the green alga <Emphasis Type="Italic">Chlorella vulgaris</Emphasis>: a perspective of photosynthesis and oxidant stress

The toxic effects of Cu (II) on the freshwater green algae Chlorella vulgaris and its chloroplast were investigated by detecting the responses of photosynthesis and oxidant stress. The results showed that Cu (II) arrested the growth of C. vulgaris and presented in a concentration- and time-dependent trend and the SRichards 2 model fitted the inhibition curve best. The chlorophyll fluorescence parameters, including qP, Y (II), ETR, F _v /F _m , and F _v /F ₀, were stimulated at low concentration of Cu (II) but declined at high concentration, indicating the photosystem II (PSII) of C. vulgaris was destroyed by Cu (II). The chloroplasts were extracted, and the Hill reaction activity (HRA) of chloroplast was significantly decreased with the increasing Cu (II) concentration under both illuminating and dark condition, and faster decline speed was observed under dark condition. Activities of superoxide dismutase (SOD) and catalase (CAT) and malondialdehyde (MDA) content were also significantly decreased at high concentration Cu (II), companied with a large number of reactive oxygen species (ROS) production. All these results indicated a severe oxidative stress on algal cells occurred as well as the effect on photosynthesis, thus inhibiting the growth of algae, which providing sights to evaluate the phytotoxicity of Cu (II).     

Evaluation of air pollution tolerance index and anticipated performance index of plants and their application in development of green space along the urban areas

Air pollution due to vehicular emissions has become one of the most serious problems in the whole world and has resulted in huge threat to both the environment and the health of living organisms (plants, humans, animals, microorganisms). Plants growing along the roadsides get affected at the maximum as they are the primary recipients to different air pollutants and show varied levels of tolerance and sensitivity. Taking this into account, the present work was based on assessment of seasonal variation in air pollution tolerance index (APTI) and anticipated performance index (API) of four roadside plants, namely, Alstonia scholaris, Nerium oleander, Tabernaemontana coronaria, and Thevetia peruviana belonging to family Apocynaceae. APTI was calculated by the determination of four important biochemical parameters, viz., pH, relative water content (RWC), total chlorophyll (TChl), and ascorbic acid (AsA) content of leaves. The leaf samples were collected from plants growing at seven different sites of Amritsar (Punjab), India, for pre-monsoon and post-monsoon seasons. Highest APTI (82.14) was reported in N. oleander during the pre-monsoon season while the lowest was recorded in T. coronaria (18.59) in the post-monsoon season. On the basis of API score, A. scholaris was anticipated to be an excellent performer during the pre-monsoon and post-monsoon seasons followed by N. oleander, T. coronaria, and T. peruviana. Linear regression analysis and Pearson’s correlation coefficient depicted significant positive correlation between APTI and ascorbic acid content during the pre-monsoon and post-monsoon seasons.     

<Emphasis Type="Italic">Ailanthus Altissima</Emphasis> and <Emphasis Type="Italic">Phragmites Australis</Emphasis> for chromium removal from a contaminated soil

The comparative effectiveness for hexavalent chromium removal from irrigation water, using two selected plant species (Phragmites australis and Ailanthus altissima) planted in soil contaminated with hexavalent chromium, has been studied in the present work. Total chromium removal from water was ranging from 55 % (Phragmites) to 61 % (Ailanthus). After 360 days, the contaminated soil dropped from 70 (initial) to 36 and 41 mg Cr/kg (dry soil), for Phragmites and Ailanthus, respectively. Phragmites accumulated the highest amount of chromium in the roots (1910 mg Cr/kg_{(dry tissue)}), compared with 358 mg Cr/kg_{(dry tissue)} for Ailanthus roots. Most of chromium was found in trivalent form in all plant tissues. Ailanthus had the lowest affinity for Cr^VI reduction in the root tissues. Phragmites indicated the highest chromium translocation potential, from roots to stems. Both plant species showed good potentialities to be used in phytoremediation installations for chromium removal.     

Metals and PCB levels in some edible marine organisms from the Ionian Sea: dietary intake evaluation and risk for consumers

Concentrations of some metals (Cd, Cu, As, Hg, Pb) and polychlorinated biphenyls (PCBs) were investigated in edible marine organisms from different trophic levels and feeding behaviour like bivalve molluscs (Mytilus galloprovincialis and Chlamys glabra), gastropod molluscs (Hexaplex trunculus) and some commercial species of fish (Trachurus trachurus, Boops boops, Sarpa salpa and Gobius niger). These species were collected in the first inlet of the Mar Piccolo of Taranto (Ionian Sea, Southern Italy), classified as ‘Site of National Interest’ established by National Law 426 (1998) and included in the ‘National Environmental Remediation and Restoration Projects’. The aim of this work was to investigate contamination levels and public health risks, associated with consuming seafood harvested from these areas. Moreover, in this study, was also estimated the weekly intake in children and adults, both for metals and PCBs. In comparison with the permissible limits set by EC Regulations, Cd and Pb levels were over the limit in the H. trunculus (in all sampling stations) and in the fish T. trachurus respectively. PCBs were over the legal limit in all sampled species with the exception of M. galloprovincialis (station 1), C. glabra and the herbivorous fish S. salpa. In the fish T. trachurus, for example, the concentration of six target PCBs was about five times higher than the EC limit. The estimated intakes of those trace elements included in this study through seafood consumption by the population exceed the provisional tolerable weekly intake recommended by the Joint FAO/WHO Expert Committee on Food Additives for Cd and Hg in the H. trunculus and T. trachurus, especially in children. Moreover, hazard quotience (HQ) for Hg and Cd was >1 in the children for T. trachurus and H. trunculus consumption. As regard non-dioxin-like PCB (NDL-PCB), the estimated intake were always above the ‘provisional guidance value’ (70 ng/kg body weight) Arnich et al. (Regul Toxicol Pharm 54: 287–2, 2009) for all sampled organism. Thus, health risks due to the dietary Hg, Cd and PCBs intake, especially for children, cannot be excluded. Therefore, an extended remediation programme is necessary to safeguard marine ecosystem, human health and, not less important, the economic activities, in the Taranto marine area.     

Total staphylococci as performance surrogate for greywater treatment

Faecal indicator bacteria (FIB) are commonly used as water quality indicators; implying faecal contamination and therefore the potential presence of pathogenic enteric bacteria, viruses, and protozoa. Hence in wastewater treatment, the most commonly used treatment process measures (surrogates) are total coliforms, faecal coliforms, Escherichia coli (E. coli), and enterococci. However, greywater potentially contains skin pathogens unrelated to faecal load, and E. coli and other FIB may grow within greywater unrelated to pathogens. Overall, FIB occurs at fluctuating and relatively low concentrations compared to other endogenous greywater bacteria affecting their ability as surrogates for pathogen reduction. Therefore, unlike municipal sewage, FIB provides a very limited and unreliable log-reduction surrogate measure for on-site greywater treatment systems. Based on our recent metagenomic study of laundry greywater, skin-associated bacteria such as Staphylococcus, Corynebacterium, and Propionibacterium spp. dominate and may result in more consistent treatment surrogates than traditional FIB. Here, we investigated various Staphylococcus spp. as potential surrogates to reliably assay over 4-log₁₀ reduction by the final-stage UV disinfection step commonly used for on-site greywater reuse, and compare them to various FIB/phage surrogates. A collimated UV beam was used to determine the efficacy of UV inactivation (255, 265 and 285 nm) against E. coli, Enterococcus faecalis, E. faecium, E. casseliflavus, Staphylococcus aureus, and S. epidermidis. Staphylococcus spp. was estimated by combining the bi-linear dose-response curves for S. aureus and S. epidermidis and was shown to be less resistant to UV irradiation than the other surrogates examined. Hence, a relative inactivation credit is suggested; whereas, the doses required to achieve a 4 and 5-log₁₀ reduction of Staphylococcus spp. (13.0 and 20.9 mJ cm^?2, respectively) were used to determine the relative inactivation of the other microorganisms investigated. The doses required to achieve a 4 and 5-log₁₀ reduction of Staphylococcus spp. resulted in a log₁₀ reduction of 1.4 and 4.1 for E. coli, 0.8 and 2.8 for E. faecalis, 0.8 and 3.6 for E. casseliflavus and 0.8 and 1.2 for MS2 coliphage, respectively. Given the concentration difference of Staphylococcus spp. and FIB (3 to 5-log₁₀ higher), we propose the use of Staphylococcus spp. as a novel endogenous performance surrogate to demonstrate greywater treatment performance given its relatively high and consistent concentration and therefore ability to demonstrate over 5-log₁₀ reductions.     

Amphibian recovery after a decrease in acidic precipitation

We here report the first sign of amphibian recovery after a strong decline due to acidic precipitation over many decades and peaking around 1980–90. In 2010, the pH level of ponds and small lakes in two heavily acidified areas in southwestern Scandinavia (Aust-Agder and Østfold in Norway) had risen significantly at an (arithmetic) average of 0.14 since 1988–89. Parallel with the general rise in pH, amphibians (Rana temporaria, R. arvalis, Bufo bufo, Lissotriton vulgaris, and Triturus cristatus) had become significantly more common: the frequency of amphibian localities rose from 33% to 49% (n = 115), and the average number of amphibian species per locality had risen from 0.51 to 0.88. In two other (reference) areas, one with better buffering capacity (Telemark, n = 21) and the other with much less input of acidic precipitation (Nord-Trøndelag, n = 106), there were no significant changes in pH or amphibians.     

Phytoextraction of rare earth elements in herbaceous plant species growing close to roads

The aim of study was to determine the phytoextraction of rare earth elements (REEs) to roots, stems and leaves of five herbaceous plant species (Achillea millefolium L., Artemisia vulgaris L., Papaver rhoeas L., Taraxacum officinale and Tripleurospermum inodorum), growing in four areas located in close proximity to a road with varied traffic intensity. Additionally, the relationship between road traffic intensity, REE concentration in soil and the content of these elements in plant organs was estimated. A. vulgaris and P. rhoeas were able to effectively transport REEs in their leaves, independently of area collection. The highest content of REEs was observed in P. rhoeas leaves and T. inodorum roots. Generally, HREEs were accumulated in P. rhoeas roots and leaves and also in the stems of T. inodorum and T. officinale, whereas LREEs were accumulated in T. inodorum roots and T. officinale stems. It is worth underlining that there was a clear relationship between road traffic intensity and REE, HREE and LREE concentration in soil. No positive correlation was found between the concentration of these elements in soil and their content in plants, with the exception of T. officinale. An effective transport of REEs from the root system to leaves was observed, what points to the possible ability of some of the tested plant species to remove REEs from soils near roads.     

Monitoring an outdoor smoking area by means of PM<Subscript>2.5</Subscript> measurement and vegetal biomonitoring

The extension of pollutant accumulation in plant leaves associated with its genotoxicity is a common approach to predict the quality of outdoor environments. However, this approach has not been used to evaluate the environmental quality of outdoor smoking areas. This study aims to evaluate the effects of environmental tobacco smoke (ETS) by assessing particulate matter 2.5 μm (PM_2.5) levels, the pollen abortion assay, and trace elements accumulated in plant leaves in an outdoor smoking area of a hospital. For this, PM_2.5 was measured by active monitoring with a real time aerosol monitor for 10 days. Eugenia uniflora trees were used for pollen abortion and accumulated element assays. Accumulated elements were also assessed in Tradescantia pallida leaves. The median concentration of PM_2.5 in the smoking area in all days of monitoring was 66 versus 34 μg/m³ in the control area (P?<?0.001). In addition, the elements Al, Cd, Cu, Ni, Pb, Rb, Sb, Se, and V in Tradescantia pallida and Al, Ba, Cr, Cu, Fe, Mg, Pb, and Zn in Eugenia uniflora were in higher concentration in the smoking area when compared to control area. Smoking area also showed higher rate of aborted grains (26.1?±?10.7 %) compared with control (17.6?±?4.5 %) (P?=?0.003). Under the study conditions, vegetal biomonitoring proved to be an effective tool for assessing ETS exposure in outdoor areas. Therefore, vegetal biomonitoring of ETS could be a complement to conventional analyses and also proved to be a cheap and easy-handling tool to assess the risk of ETS exposure in outdoor areas.     

Molecular characterization of microbial communities and quantification of <Emphasis Type="Italic">Mycobacterium immunogenum</Emphasis> in metal removal fluids and their associated biofilms

A number of human health effects have been associated with exposure to metal removal fluids (MRFs). Multiple lines of research suggest that a newly identified organism, Mycobacterium immunogenum (MI), appears to have an etiologic role in hypersensitivity pneumonitis (HP) in case of MRFs exposed workers. However, our knowledge of this organism, other possible causative agents (e.g., Pseudomonads), and the microbial ecology of MRFs in general, is limited. In this study, culture-based methods and small subunit ribosomal RNA gene clone library approach were used to characterize microbial communities in MRF bulk fluid and associated biofilm samples collected from fluid systems in an automobile engine plant. PCR amplification data using universal primers indicate that all samples had bacterial and fungal contaminated. Five among 15 samples formed colonies on the Mycobacteria agar 7H9 suggesting the likely presence of Mycobacteria in these five samples. This observation was confirmed with PCR amplification of 16S rRNA gene fragment using Mycobacteria specific primers. Two additional samples, Biofilm-1 and Biofilm-3, were positive in PCR amplification for Mycobacteria, yet no colonies formed on the 7H9 cultivation agar plates. Real-time PCR was used to quantify the abundance of M. immunogenum in these samples, and the data showed that the copies of M. immunogenum 16S rRNA gene in the samples ranges from 4.33?×?10⁴ copy/ml to 4.61?×?10⁷ copy/ml. Clone library analysis revealed that Paecilomyces sp. and Acremonium sp. and Acremonium-like were dominant fungi in MRF samples. Various bacterial species from the major phylum of proteobacteria were found and Pseudomonas is the dominant bacterial genus in these samples. Mycobacteria (more specifically MI) were found in all biofilm samples, including biofilms collected from inside the MRF systems and from adjacent environmental surfaces, suggesting that biofilms may play an important role in microbial ecology in MRFs. Biofilms may provide a shield or sheltered microenvironment for the growth and/or colonization of Mycobacteria in MRFs.