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Human toenails as a biomarker of exposure to elevated environmental arsenic

A pilot study was conducted to determine the applicability of toenails as a biomarker of exposure to elevated environmental arsenic (As) levels. A total of 17 individuals were recruited for the pilot study: 8 residents living near to a former As mine, Devon, UK, forming the exposed group, plus 9 residents from Nottinghamshire, UK, with no anticipated As exposure who were used for comparison as a control group. All toenail samples were thoroughly washed prior to analysis and the wash solutions retained for As determination via ICP-MS to provide an indication of the background environmental As levels for each group. Total As was determined in washed toenail samples via ICP-MS following microwave assisted acid digestion. Concentrations of total As in the toenails of the exposed group were elevated, ranging from 858 to 25 981 microg kg(-1) (geometric mean = 5406 microg kg(-1)), compared to the control group whose toenail As concentrations ranged from 73 to 273 microg kg(-1) (geometric mean = 122 microg kg(-1)). Higher levels of exogenous As contamination were present on the toenails of the exposed group (geometric mean = 506 microg kg(-1)) compared to the control group (geometric mean = 4.0 microg kg(-1)) providing evidence of higher environmental As levels in the exposed group. Total As concentrations in toenail samples were positively correlated to environmental As levels (r = 0.60, p < 0.001). HPLC-ICP-MS analysis of aqueous toenail extracts revealed inorganic arsenite (As(III)) to be the dominant species extracted ( approximately 83%) with lesser amounts of inorganic arsenate (As(V)) and organic dimethylarsinate (DMA(V)) at approximately 13% and approximately 8.5%, respectively. Arsenic speciation in analysed toenail extracts from the two groups was comparable. The only notable difference between groups was the presence of small amounts (<1%) of organic methylarsonate (MA(V)) in two toenail samples from the exposed group. Toenails are presented as a viable biomarker of exposure at sites with elevated environmental As, such as the former mining sites found throughout Devon and Cornwall, UK.

DNA damage in earthworms from highly contaminated soils : assessing resistance to arsenic toxicity by use of the Comet assay

Earthworms native to the former mine site of Devon Great Consols (DGC), UK reside in soils highly contaminated with arsenic (As). These earthworms are considered to have developed a resistance to As toxicity. The mechanisms underlying this resistance however, remain unclear. In the present study, non-resistant, commercially sourced Lumbricus terrestris were exposed to a typical DGC soil in laboratory mesocosms. The earthworms bio-accumulated As from the soil and incurred DNA-damage levels significantly above those observed in the control mesocosm (assessed using the Comet assay). A dose response was observed between DNA damage (% tail DNA) and As concentration in soil (control, 98, 183, 236, 324 and 436mgkg(-1)). As-resistant earthworms (Lumbricus rubellus, Dendrodrilus rubidus and L. terrestris) collected from contaminated soils at DGC (203 to 9025mgkg(-1) As) had also bio-accumulated high levels of As from their host soils, yet demonstrated low levels of DNA damage compared with earthworms from uncontaminated sites. The results demonstrate that the As-contaminated soils at DGC are genotoxic to non-native earthworms and much less so to earthworms native to DGC, thus providing further evidence of an acquired resistance to As toxicity in the native earthworms.

Arsenic speciation in field-collected and laboratory-exposed earthworms Lumbricus terrestris.

Mature Lumbricus terrestris were host soils and leaf litter were collected from a former arsenic mine in Devon, UK (Devon Great Consols), a former gold mine in Ontario, Canada (Deloro), and an uncontaminated residential garden in Nottingham, UK. Arsenic concentrations determined by inductively coupled plasma-mass spectrometry (ICP-MS) in soils were 16-348 mg kg(-1), 6.0-239 mg kg(-1) in the earthworms and 8.6 mg kg(-1) in leaf litter sampled at Deloro (all dry weight). High performance liquid chromatography (HPLC-ICP-MS) analysis revealed arsenite (As(III)), arsenate (As(V)) and five organoarsenic species; arsenobetaine (AB), methylarsonate (MA(V)), dimethylarsinate (DMA(V)), arsenosugar 1 (glycerol sugar), arsenosugar 2 (phosphate sugar), and trimethylarsineoxide (TMAO) in field-collected L. terrestris. Differences were observed in the variety of organoarsenic species present between field sites. Several organoarsenic species were observed in the leaf litter (DMA(V), arsenosugar 2 and TMAO) but not AB. Depuration resulted in higher concentrations of inorganic As being detected in the earthworm whereas the concentration or variety of organoarsenic species was unchanged. Commercially sourced L. terrestris were exposed to As contaminated soil in laboratory mesocosms (1.0, 98, 183, 236, 324 and 436 mg kg(-1)) without leaf litter and were additionally analyzed using X-ray absorption near edge structure (XANES). Only inorganic As(III) and As(V) was observed. It is proposed that ingestion of leaf litter and symbiotic processes in the natural soil environment are likely sources of organoarsenic compounds in field-collected L. terrestris.

Functionality of microbial communities in constructed wetlands used for pesticide remediation: Influence of system design and sampling strategy

The objective of this study was to compare the microbial community metabolic function from both unsaturated and saturated constructed wetland mesocosms (CWs) when treating the pesticide tebuconazole. The comparison was performed for both interstitial water and substrate biofilm by community level physiological profiling (CLPP) via BIOLOG™ EcoPlates. For each CW design (saturated or unsaturated), six mesocosms were established including one unplanted and five planted individually with either Juncus effusus, Typha latifolia, Berula erecta, Phragmites australis or Iris pseudacorus. Microbial activity and metabolic richness of interstitial water from unsaturated CWs were significantly lower than that from saturated CWs. However, in general, the opposite result was observed for biofilm samples. Wetland plants promoted significantly higher biofilm microbial activity and metabolic richness than unplanted CWs in both CW designs. Differences in the microbial community functional profiles between plant species were only found for saturated CWs. Biofilm microbial metabolic richness was generally statistically higher than that of interstitial water in both unsaturated (1.4-24 times higher) and saturated (1.2-1.7 times higher) CWs. Carbon source (guild) utilization patterns were generally different between interstitial water and biofilm samples. Functionality of the biofilm microbial community was positively correlated to the removal of all pollutants (TN, NH4+-N, TP, TOC and tebuconazole) for both unsaturated and saturated CWs, suggesting the biofilm plays a more important role in pollutant removal than the interstitial water microbial community. Thus, merely observing the interstitial water microbial communities may underestimate the role of the microbial community in CW performance. Interestingly, the ability for the biofilm microbial community to utilize amino acids and amines/amides was positively correlated with tebuconazole removal in all system types.

Arsenic resistance and cycling in earthworms residing at a former gold mine in Canada.

Earthworms (Lumbricus castaneous and Dendrodrilus rubidus), their host soils and leaf litter were collected from a former gold mine with widespread arsenic (As) contamination in Nova Scotia, Canada and determined for total and speciated As. Resistance to As toxicity was investigated by measurement of DNA damage in exposed earthworm populations using the comet assay. Arsenobetaine (AB) was observed at low concentration in the earthworms but not in the host soil or leaf litter. Several different organoarsenic species were observed in the leaf litter and only inorganic As was found in the host soils. The results suggest that 1) adaptation to As toxicity in earthworms is widespread and not particular to a single species, 2) AB originates in the earthworm and not the consumed soil or leaf litter and 3) as previously hypothesised (Button et al., 2010), biotransformation of inorganic As to AB is not likely involved in the adaptation.

Community-Level Physiological Profiling of Microbial Communities in Constructed Wetlands: Effects of Sample Preparation

Community-level physiological profiling (CLPP) using BIOLOG® EcoPlates™ has become a popular method for characterizing and comparing the functional diversity, functional potential, and metabolic activity of heterotrophic microbial communities. The method was originally developed for profiling soil communities; however, its usage has expanded into the fields of ecotoxicology, agronomy, and the monitoring and profiling of microbial communities in various wastewater treatment systems, including constructed wetlands for water pollution control. When performing CLPP on aqueous samples from constructed wetlands, a wide variety of sample characteristics can be encountered and challenges may arise due to excessive solids, color, or turbidity. The aim of this study was to investigate the impacts of different sample preparation methods on CLPP performed on a variety of aqueous samples covering a broad range of physical and chemical characteristics. The results show that using filter paper, centrifugation, or settling helped clarify samples for subsequent CLPP analysis, however did not do so as effectively as dilution for the darkest samples. Dilution was able to provide suitable clarity for the darkest samples; however, 100-fold dilution significantly affected the carbon source utilization patterns (CSUPs), particularly with samples that were already partially or fully clear. Ten-fold dilution also had some effect on the CSUPs of samples which were originally clear; however, the effect was minimal. Based on these findings, for this specific set of samples, a 10-fold dilution provided a good balance between ease of use, sufficient clarity (for dark samples), and limited effect on CSUPs. The process and findings outlined here can hopefully serve future studies looking to utilize CLPP for functional analysis of microbial communities and also assist in comparing data from studies where different sample preparation methods were utilized.

Impacts of design configuration and plants on the functionality of the microbial community of mesocosm-scale constructed wetlands treating ibuprofen

Microbial degradation is an important pathway during the removal of pharmaceuticals in constructed wetlands (CWs). However, the effects of CW design, plant presence, and different plant species on the microbial community in CWs have not been fully explored. This study aims to investigate the microbial community metabolic function of different types of CWs used to treat ibuprofen via community-level physiological profiling (CLPP) analysis. We studied the interactions between three CW designs (unsaturated, saturated and aerated) and six types of mesocosms (one unplanted and five planted, with Juncus, Typha, Berula, Phragmites and Iris) treating synthetic wastewater. Results show that the microbial activity and metabolic richness found in the interstitial water and biofilm of the unsaturated designs were lower than those of the saturated and aerated designs. Compared to other CW designs, the aerated mesocosms had the highest microbial activity and metabolic richness in the interstitial water, but similar levels of biofilm microbial activity and metabolic richness to the saturated mesocosms. In all three designs, biofilm microbial metabolic richness was significantly higher (p < .05) than that of interstitial water. Both the interstitial water and biofilm microbial community metabolic function were influenced by CW design, plant presence and species, but design had a greater influence than plants. Moreover, canonical correlation analysis indicated that biofilm microbial communities in the three designs played a key role in ibuprofen degradation. The important factors identified as influencing ibuprofen removal were microbial AWCD (average well color development), microbial metabolic richness, and the utilization of amino acids and amine/amides. The enzymes associated with co-metabolism of l-arginine, l-phenyloalanine and putrescine may be linked to ibuprofen transformations. These results provide useful information for optimizing the operational parameters of CWs to improve ibuprofen removal.

Transcriptomic Responses During Early Development Following Arsenic Exposure in Western Clawed Frogs, Silurana tropicalis.

Arsenic compounds are widespread environmental contaminants and exposure elicits serious health issues, including early developmental anomalies. Depending on the oxidation state, the intermediates of arsenic metabolism interfere with a range of subcellular events, but the fundamental molecular events that lead to speciation-dependent arsenic toxicity are not fully elucidated. This study therefore assesses the impact of arsenic exposure on early development by measuring speciation and gene expression profiles in the developing Western clawed frog (Silurana tropicalis) larvae following the environmental relevant 0.5 and 1 ppm arsenate exposure. Using HPLC-ICP-MS, arsenate, dimethylarsenic acid, arsenobetaine, arsenocholine, and tetramethylarsonium ion were detected. Microarray and pathway analyses were utilized to characterize the comprehensive transcriptomic responses to arsenic exposure. Clustering analysis of expression data showed distinct gene expression patterns in arsenate treated groups when compared with the control. Pathway enrichment revealed common biological themes enriched in both treatments, including cell signal transduction, cell survival, and developmental pathways. Moreover, the 0.5 ppm exposure led to the enrichment of pathways and biological processes involved in arsenic intake or efflux, as well as histone remodeling. These compensatory responses are hypothesized to be responsible for maintaining an in-body arsenic level comparable to control animals. With no appreciable changes observed in malformation and mortality between control and exposed larvae, this is the first study to suggest that the underlying transcriptomic regulations related to signal transduction, cell survival, developmental pathways, and histone remodeling may contribute to maintaining ongoing development while coping with the potential arsenic toxicity in S. tropicalis during early development.

Impacts and fate of triclosan and sulfamethoxazole in intensified re-circulating vertical flow constructed wetlands

The impacts to microbial function, overall performance and eventual fate were assessed for triclosan (TCL) and sulfamethoxazole (SMX) in intensified (re-circulating) vertical subsurface flow (VSSF) constructed wetlands (CWs). The potential toxicity of each pharmaceutical to the intrinsic microbial communities was first assessed over a wide exposure range (0-1000 μg/l) via an ex-situ dose-response assay to estimate the concentration at which adverse effects were likely to occur. Based on these results an acute (7 day) in-situ exposures (500 μg/l) were then performed and impacts to the mesocosm systems monitored for 1 month via community-level physiological profiling (CLPP) alongside chemical oxygen demand (COD) removal rates and a range of water quality, and hydrological parameters. Despite the clear potential for negative impacts to microbial function from both compounds observed at 100 μg/l in the ex-situ dose-response test, no impacts were observed for the 500 μg/l in-situ exposure in the VSSF mesocosms. COD removal, water chemistry, plant health, and hydrological parameters did not significantly change in response to the in-situ exposure. In terms of fate, the removal efficiency for both TCL and SMX was high (>80%) after 1 h and complete removal (>99.7%) was observed after 168 h. Following the in-situ exposure, and subsequent one month effects-monitoring period, the mesocosms were decommissioned with the media biofilm spatially assessed for organic content as well as TCL and SMX concentrations. TCL and SMX were found to have persisted in the media and demonstrated spatial variation with an overall 2-20% and 5-6% recovered respectively. This suggests that biofilm bound TCL and SMX were biologically degraded in VSSF CWs, however may also accumulate in the biofilm if TCL and SMX are maintained in the influent. These results reinforce the robustness and potential of constructed wetlands for the treatment of pharmaceutical and personal care product (PPCP) contaminated wastewater.

Microbial community metabolic profiles in saturated constructed wetlands treating iohexol and ibuprofen

The aim of the present study was to elucidate the microbial community metabolic profiles in saturated constructed wetland (CW) mesocosms planted with five different wetland plant species fed with water individually spiked with 100 μg L-1 ibuprofen or iohexol. Community-level physiological profiling (CLPP) using Biolog Ecoplates was performed and coupled with the assessment of water quality parameters (water temperature, pH, DO and TOC, TN, NH4-N, PO4-P removal efficiency). The microbial community metabolic profiles (microbial activity, richness, and carbon source utilization), as well as the water quality parameters revealed similar trends among the control mesocosms and the mesocosms fed with water spiked with iohexol and ibuprofen. Significant differences were observed between the planted and unplanted mesocosms and between seasons (summer and winter) within each of the feeding lines (control, iohexol or ibuprofen). The microbial community metabolic profiles in the saturated CW were shaped by plant presence and plant species, while no negative impact of iohexol and ibuprofen presence was noticed at the 100 μg L-1. In addition, the microbial activity and richness were generally higher in planted mesocosms than in the unplanted systems in the summer. For the first time, a positive correlation between iohexol removal and the microbial community metabolic profiles (activity, richness and amines and amides utilization in summer, and carbohydrates utilization in winter) in the saturated mesocosms was observed. Putrescine utilization in the summer and d-cellobiose, d,l-alpha-glycerol phosphate in winter were linked with the metabolic processing of iohexol, while glycogen in summer and l-phenylalanine, Glycyl-l-glutamic acid in winter were linked with ibuprofen removal efficiency in the saturated CW.