Impacts of Mountaintop Removal Coal Mining on the Mud River, West Virginia: Selenium Accumulation, Trophic Transfer, and Toxicity in Fish

Selenium (Se) is a micronutrient necessary for the function of a variety of important enzymes; Se also exhibits a narrow range in concentrations between essentiality and toxicity. Oviparous vertebrates such as birds and fish are especially sensitive to Se toxicity, which causes reproductive impairment and defects in embryo development. Selenium occurs naturally in the Earth's crust, but it can be mobilized by a variety of anthropogenic activities, including agricultural practices, coal burning, and mining.

Mountaintop removal/valley fill (MTR/VF) coal mining is a form of surface mining found throughout central Appalachia in the United States that involves blasting off the tops of mountains to access underlying coal seams. Spoil rock from the mountain is placed into adjacent valleys, forming valley fills, which bury stream headwaters and negatively impact surface water quality. This research focused on the biological impacts of Se leached from MTR/VF coal mining operations located around the Mud River, West Virginia.

In order to assess the status of Se in a lotic (flowing) system such as the Mud River, surface water, insects, and fish samples including creek chub (Semotilus atromaculatus) and green sunfish (Lepomis cyanellus) were collected from a mining impacted site as well as from a reference site not impacted by mining. Analysis of samples from the mined site showed increased conductivity and Se in the surface waters compared to the reference site in addition to increased concentrations of Se in insects and fish. Histological analysis of mined site fish gills showed a lack of normal parasites, suggesting parasite populations may be disrupted due to poor water quality. X-ray absorption near edge spectroscopy techniques were used to determine the speciation of Se in insect and creek chub samples. Insects contained approximately 40-50% inorganic Se (selenate and selenite) and 50-60% organic Se (Se-methionine and Se-cystine) while fish tissues contained lower proportions of inorganic Se than insects, instead having higher proportions of organic Se in the forms of methyl-Se-cysteine, Se-cystine, and Se-methionine.

Otoliths, calcified inner ear structures, were also collected from Mud River creek chubs and green sunfish and analyzed for Se content using laser ablation inductively couple mass spectrometry (LA-ICP-MS). Significant differences were found between the two species of fish, based on the concentrations of otolith Se. Green sunfish otoliths from all sites contained background or low concentrations of otolith Se (< 1 µg/g) that were not significantly different between mined and unmined sites. In contrast creek chub otoliths from the historically mined site contained much higher (≥ 5 µg/g, up to approximately 68 µg/g) concentrations of Se than for the same species in the unmined site or for the green sunfish. Otolith Se concentrations were related to muscle Se concentrations for creek chubs (R2 = 0.54, p = 0.0002 for the last 20% of the otolith Se versus muscle Se) while no relationship was observed for green sunfish.

Additional experiments using biofilms grown in the Mud River showed increased Se in mined site biofilms compared to the reference site. When we fed fathead minnows (Pimephales promelas) on these biofilms in the laboratory they accumulated higher concentrations of Se in liver and ovary tissues compared to fathead minnows fed on reference site biofilms. No differences in Se accumulation were found in muscle from either treatment group. Biofilms were also centrifuged and separated into filamentous green algae and the remaining diatom fraction. The majority of Se was found in the diatom fraction with only about 1/3rd of total biofilm Se concentration present in the filamentous green algae fraction

Finally, zebrafish (Danio rerio) embryos were exposed to aqueous Se in the form of selenate, selenite, and L-selenomethionine in an attempt to determine if oxidative stress plays a role in selenium embryo toxicity. Selenate and selenite exposure did not induce embryo deformities (lordosis and craniofacial malformation). L-selenomethionine, however, induced significantly higher deformity rates at 100 µg/L compared to controls. Antioxidant rescue of L-selenomethionime induced deformities was attempted in embryos using N-acetylcysteine (NAC). Pretreatment with NAC significantly reduced deformities in the zebrafish embryos secondarily treated with L-selenomethionine, suggesting that oxidative stress may play a role in Se toxicity. Selenite exposure also induced a 6.6-fold increase in glutathione-S-transferase pi class 2 gene expression, which is involved in xenobiotic transformation. No changes in gene expression were observed for selenate or L-selenomethionine-exposed embryos.

The findings in this dissertation contribute to the understanding of how Se bioaccumulates in a lotic system and is transferred through a simulated foodweb in addition to further exploring oxidative stress as a potential mechanism for Se-induced embryo toxicity. Future studies should continue to pursue the role of oxidative stress and other mechanisms in Se toxicity and the biotransformation of Se in aquatic ecosystems.

Faunistic notes

Notes on dredging and trawling work during the latter half of 1895. On Doris maculata, a new species of Nudibranchiate mollusk found at Plymouth. On the changes in the pelagic fauna of Plymouth during September, 1893 and 1895. Notes on Nautilograpsus minutus, Milne Edwards.

English Channel towed sledge seabed images. Phase 2: Analysis of selected tow images

During the 1970s and 1980s, the late Dr Norman Holme undertook extensive towed sledge surveys in the English Channel and some in the Irish Sea. Only a minority of the resulting images were analysed and reported before his death in 1989 but logbooks, video and film material has been archived in the National Marine Biological Library (NMBL) in Plymouth. A study was therefore commissioned by the Joint Nature Conservation Committee and as a part of the Mapping European Seabed Habitats (MESH) project to identify the value of the material archived and the procedure and cost to undertake further work (Phase 1 of the study reported here: Oakley & Hiscock, 2005). Some image analysis was undertaken as a part of Phase 1. Phase 2 (this report) was to further analyse selected images. Having determined in Phase 1 that only the 35 mm photographic transparencies provided sufficient clarity to identify species and biotopes, the tows selected for analysis were ones where 35mm images had been taken. The tows selected for analysis of images were mainly in the vicinity of Plymouth and especially along the area between Rame Head and the region of the Eddystone. The 35 mm films were viewed under a binocular microscope and the taxa that could be recognised recorded in note form. Twenty-five images were selected for inclusion in the report. Almost all of the images were of level sediment seabed. Where rocks were included, it was usually unplanned and the sled was hauled before being caught or damaged. The main biotopes or biotope complexes identified were: SS.SMU.CSaMu. Circalittoral sandy mud. Extensively present between the shore and the Eddystone Reef complex and at depths of about 48 to 52 m. At one site offshore of Plymouth Sound, the turret shell Turritella communis was abundant. In some areas, this biotope had dense anemones, Mesacmaea mitchelli and (more rarely) Cerianthus lloydii. Queen scallops, Aequipecten opercularis and king scallops, Pecten maximus, were sometimes present in small numbers. Hard substratum species such as hydroids, dead mens fingers Alcyonium digitatum and the cup coral Caryophyllia smithii occurred in a few places, probably attached to shells or stones beneath the surface. South of the spoil ground off Hilsea Point at 57m depth, the sediment was muddier but is still assigned to this biotope complex. It is notable that three small sea pens, most likely Virgularia mirabilis, were seen here. SS.SMx.CMx. Circalittoral mixed sediment. Further offshore but at about the same depth as SS.SMU.CSaMu occurred, coarse gravel with some silt was present. The sediment was characterised must conspicuously by small queen scallops, Aequipecten opercularis. Peculiarly, there were ‘bundles’ of the branching bryozoan Cellaria sp. – a species normally found attached to rock. It could not be seen whether these bundles of Cellaria had been brought-together by terebellid worms but it is notable that Cellaria is recorded in historical surveys. As with many other sediments, there were occasional brittle stars, Ophiocomina nigra and Ophiura ophiura. Where sediments were muddy, the burrowing anemone Mesacmaea mitchelli was common. Where pebbles or cobbles occurred, there were attached species such as Alcyonium digitatum, Caryophyllia smithii and the fleshy bryozoan Alcyonidium diaphanum. Undescribed biotope. Although most likely a part of SS.SMx.CMx, the biotope visually dominated by a terebellid worm believed to be Thelepus cincinnatua, is worth special attention as it may be an undescribed biotope. The biotope occurred about 22 nautical miles south of the latitude of the Eddystone and in depths in excess of 70 m. SS.SCS.CCS.Blan. Branchiostoma lanceolatum in circalittoral coarse sand with shell gravel at about 48m depth and less. This habitat was the ‘classic’ ‘Eddystone Shell Gravel’ which is sampled for Branchiostoma lanceolatum. However, no Branchiostoma lanceolatum could be seen. The gravel was almost entirely bare of epibiota. There were occasional rock outcrops or cobbles which had epibiota including encrusting calcareous algae, the sea fan Eunicella verrucosa, cup corals, Caryophyllia smithii, hydroids and a sea urchin Echinus esculentus. The variety of species visible on the surface is small and therefore identification to biotope not usually possible. Historical records from sampling surveys that used grabs and dredges at the end of the 19th century and early 20th century suggest similar species present then. Illustrations of some of the infaunal communities from work in the 1920’s is included in this report to provide a context to the epifaunal photographs.

Assessing the sensitivity of seagrass bed biotopes to pressures associated with marine activities.

This project was commissioned to generate an improved understanding of the sensitivities of seagrass habitats to pressures associated with human activities in the marine environment - to provide an evidence base to facilitate and support management advice for Marine Protected Areas; development of UK marine monitoring and assessment, and conservation advice to offshore marine industries. Seagrass bed habitats are identified as a Priority Marine Feature (PMF) under the Marine (Scotland) Act 2010, they are also included on the OSPAR list of threatened and declining species and habitats, and are a Habitat of Principle Importance (HPI) under the Natural Environment and Rural Communities (NERC) Act 2006, in England and Wales. The purpose of this project was to produce sensitivity assessments with supporting evidence for the HPI, OSPAR and PMF seagrass/Zostera bed habitat definitions, clearly documenting the evidence behind the assessments and any differences between assessments. Nineteen pressures, falling in five categories - biological, hydrological, physical damage, physical loss, and pollution and other chemical changes - were assessed in this report. Assessments were based on the three British seagrasses Zostera marina, Z. noltei and Ruppia maritima. Z. marina var. angustifolia was considered to be a subspecies of Z. marina but it was specified where studies had considered it as a species in its own rights. Where possible other components of the community were investigated but the basis of the assessment focused on seagrass species. To develop each sensitivity assessment, the resistance and resilience of the key elements were assessed against the pressure benchmark using the available evidence. The benchmarks were designed to provide a ‘standard’ level of pressure against which to assess sensitivity. Overall, seagrass beds were highly sensitive to a number of human activities: • penetration or disturbance of the substratum below the surface; • habitat structure changes – removal of substratum; • physical change to another sediment type; • physical loss of habitat; • siltation rate changes including and smothering; and • changes in suspended solids. High sensitivity was recorded for pressures which directly impacted the factors that limit seagrass growth and health such as light availability. Physical pressures that caused mechanical modification of the sediment, and hence damage to roots and leaves, also resulted in high sensitivity. Seagrass beds were assessed as ‘not sensitive’ to microbial pathogens or ‘removal of target species’. These assessments were based on the benchmarks used. Z. marina is known to be sensitive to Labyrinthula zosterae but this was not included in the benchmark used. Similarly, ‘removal of target species’ addresses only the biological effects of removal and not the physical effects of the process used. For example, seagrass beds are probably not sensitive to the removal of scallops found within the bed but are highly sensitive to the effects of dredging for scallops, as assessed under the pressure penetration or disturbance of the substratum below the surface‘. This is also an example of a synergistic effect Assessing the sensitivity of seagrass bed biotopes to pressures associated with marine activities between pressures. Where possible, synergistic effects were highlighted but synergistic and cumulative effects are outside the scope off this study. The report found that no distinct differences in sensitivity exist between the HPI, PMF and OSPAR definitions. Individual biotopes do however have different sensitivities to pressures. These differences were determined by the species affected, the position of the habitat on the shore and the sediment type. For instance evidence showed that beds growing in soft and muddy sand were more vulnerable to physical damage than beds on harder, more compact substratum. Temporal effects can also influence the sensitivity of seagrass beds. On a seasonal time frame, physical damage to roots and leaves occurring in the reproductive season (summer months) will have a greater impact than damage in winter. On a daily basis, the tidal regime could accentuate or attenuate the effects of pressures depending on high and low tide. A variety of factors must therefore be taken into account in order to assess the sensitivity of a particular seagrass habitat at any location. No clear difference in resilience was established across the three seagrass definitions assessed in this report. The resilience of seagrass beds and the ability to recover from human induced pressures is a combination of the environmental conditions of the site, growth rates of the seagrass, the frequency and the intensity of the disturbance. This highlights the importance of considering the species affected as well as the ecology of the seagrass bed, the environmental conditions and the types and nature of activities giving rise to the pressure and the effects of that pressure. For example, pressures that result in sediment modification (e.g. pitting or erosion), sediment change or removal, prolong recovery. Therefore, the resilience of each biotope and habitat definitions is discussed for each pressure. Using a clearly documented, evidence based approach to create sensitivity assessments allows the assessment and any subsequent decision making or management plans to be readily communicated, transparent and justifiable. The assessments can be replicated and updated where new evidence becomes available ensuring the longevity of the sensitivity assessment tool. The evidence review has reduced the uncertainty around assessments previously undertaken in the MB0102 project (Tillin et al 2010) by assigning a single sensitivity score to the pressures as opposed to a range. Finally, as seagrass habitats may also contribute to ecosystem function and the delivery of ecosystem services, understanding the sensitivity of these biotopes may also support assessment and management in regard to these. Whatever objective measures are applied to data to assess sensitivity, the final sensitivity assessment is indicative. The evidence, the benchmarks, the confidence in the assessments and the limitations of the process, require a sense-check by experienced marine ecologists before the outcome is used in management decisions.

Tillin, H. & Tyler-Walters, H., 2014. Assessing the sensitivity of subtidal sedimentary habitats to pressures associated with marine activities. Phase 2 Report – Literature review and sensitivity assessments for ecological groups for circalittoral and offshore Level 5 biotopes. JNCC Report No. 512B, 260 pp.

Human activities within the marine environment give rise to a number of pressures on seabed habitats. Improved understanding of the sensitivity of subtidal sedimentary habitats is required to underpin the management advice provided for Marine Protected Areas, as well as supporting other UK marine monitoring and assessment work. The sensitivity of marine sedimentary habitats to a range of pressures induced by human activities has previously been systematically assessed using approaches based on expert judgement for Defra Project MB0102 (Tillin et al. 2010). This previous work assessed sensitivity at the level of the broadscale habitat and therefore the scores were typically expressed as a range due to underlying variation in the sensitivity of the constituent biotopes. The objective of this project was to reduce the uncertainty around identifying the sensitivity of selected subtidal sedimentary habitats by assessing sensitivity, at a finer scale and incorporating information on the biological assemblage, for 33 Level 5 circalittoral and offshore biotopes taken from the Marine Habitat Classification of Britain and Ireland (Connor et al. 2004). Two Level 6 sub-biotopes were also included in this project as these contain distinctive characterising species that differentiate them from the Level 5 parent biotope. Littoral, infralittoral, reduced and variable salinity sedimentary habitats were excluded from this project as the scope was set for assessment of circalittoral and offshore sedimentary communities. This project consisted of three Phases. • Phase 1 - define ecological groups based on similarities in the sensitivity of characterising species from the Level 5 and two Level 6 biotopes described above. • Phase 2 - produce a literature review of information on the resilience and resistance of characterising species of the ecological groups to pressures associated with activities in the marine environment. • Phase 3 - to produce sensitivity assessment ‘proformas’ based on the findings of Phase 2 for each ecological group. This report outlines results of Phase 2. The Tillin et al., (2010) sensitivity assessment methodology was modified to use the best available scientific evidence that could be collated within the project timescale. An extensive literature review was compiled, for peer reviewed and grey literature, to examine current understanding about the effects of pressures from human activities on circalittoral and offshore sedimentary communities in UK continental shelf waters, together with information on factors that contribute to resilience (recovery) of marine species. This review formed the basis of an assessment of the sensitivity of the 16 ecological groups identified in Phase 1 of the project (Tillin & Tyler-Walters 2014). As a result: • the state of knowledge on the effects of each pressure on circalittoral and offshore benthos was reviewed; • the resistance, resilience and, hence, sensitivity of sixteen ecological groups, representing 96 characteristic species, were assessed for eight separate pressures; • each assessment was accompanied by a detailed review of the relevant evidence; Assessing the sensitivity of subtidal sedimentary habitats to pressures associated with human activities • knowledge gaps and sources of uncertainty were identified for each group; • each assessment was accompanied by an assessment of the quality of the evidence, its applicability to the assessment and the degree of concordance (agreement) between the evidence, to highlight sources of uncertainty as an assessment of the overall confidence in the sensitivity assessment, and finally • limitations in the methodology and the application of sensitivity assessments were outlined. This process demonstrated that the ecological groups identified in Phase 1 (Tillin & Tyler-Walters 2014) were viable groups for sensitivity assessment, and could be used to represent the 33 circalittoral and offshore sediments biotopes identified at the beginning of the project. The results of the sensitivity assessments show: • the majority of species and hence ecological groups in sedimentary habitats are sensitive to physical change, especially loss of habitat and sediment extraction, and change in sediment type; • most sedimentary species are sensitive to physical damage, e.g. abrasion and penetration, although deep burrowing species (e.g. the Dublin Bay prawn - Nephrops norvegicus and the sea cucumber - Neopentadactyla mixta) are able to avoid damaging effects to varying degrees, depending on the depth of penetration and time of year; • changes in hydrography (wave climate, tidal streams and currents) can significantly affect sedimentary communities, depending on whether they are dominated by deposit, infaunal feeders or suspension feeders, and dependant on the nature of the sediment, which is itself modified by hydrography and depth; • sedentary species and ecological groups that dominate the top-layer of the sediment (either shallow burrowing or epifaunal) remain the most sensitive to physical damage; • mobile species (e.g. interstitial and burrowing amphipods, and perhaps cumaceans) are the least sensitive to physical change or damage, and hydrological change as they are already adapted to unstable, mobile substrata; • sensitivity to changes in organic enrichment and hence oxygen levels, is variable between species and ecological groups, depending on the exact habitat preferences of the species in question, although most species have at least a medium sensitivity to acute deoxygenation; • there is considerable evidence on the effects of bottom-contact fishing practices and aggregate dredging on sedimentary communities, although not all evidence is directly applicable to every ecological group; • there is lack of detailed information on the physiological tolerances (e.g. to oxygenation, salinity, and temperature), habitat preferences, life history and population dynamics of many species, so that inferences has been made from related species, families, or even the same phylum; • there was inadequate evidence to assess the effects of non-indigenous species on most ecological groups, and Assessing the sensitivity of subtidal sedimentary habitats to pressures associated with human activities • there was inadequate evidence to assess the effects of electromagnetic fields and litter on any ecological group. The resultant report provides an up-to-date review of current knowledge about the effects of pressures resulting from human activities of circalittoral and offshore sedimentary communities. It provides an evidence base to facilitate and support the provision of management advice for Marine Protected Areas, development of UK marine monitoring and assessment, and conservation advice to offshore marine industries. However, such a review will require at least annual updates to take advantage of new evidence and new research as it becomes available. Also further work is required to test how ecological group assessments are best combined in practice to advise on the sensitivity of a range of sedimentary biotopes, including the 33 that were originally examined.

Investigation of benthic community change over a century-wide scale in the western English Channel

Since the early part of the 20th Century the impact of a range of anthropogenic activities in our coastal seas has steadily increased. The effect of such activities is a major cause for concern but in the benthic environment few studies exist that date back more than a few decades. Hence understanding long term changes is a challenge. Within this study we utilized a historic benthic dataset and resurveyed an area west of Eddystone reef in the English Channel previously investigated 112 years ago. The aim of the present work was to describe the current benthic community structure and investigate potential differences between 1895 and 2007. For each of the four major phyla investigated (Polychaeta, Crustacea, Mollusca and Echinodermata), multivariate community analysis showed significant differences between the historic and contemporary surveys. Echinoderm diversity showed a clear reduction between 1895 and 2007. The sea urchins Echinus esculentus, Spatangus purpureus, and Psammechinus miliaris and large star-fish Marthasterias glacialis showed reductions in abundance, in some cases being entirely absent from the survey area in 2007. Polychaetes showed a shift from tubiculous species to small errant and predatory species such as Glycera, Nephtys, and Lumbrineris spp. Within the group Mollusca large species such as Pecten maximus and Laevicardium crassum decreased in abundance while small species increased. Crustaceans in 1895 were dominated by crab species which were present in similar abundances in 2007, but, the order Amphipoda appeared to show a significant increase. While some of the differences observed could stem from differences in methodologies between the surveys, in particular increases of small cryptic species, the loss of large conspicuous species was judged to be genuine. The study area is an important beam trawling and scallop dredging ground; the differences observed are concomitant with changes generally associated with disturbance from demersal fishing activities such as these.

Sediment contaminant surveillance in Milford Haven Waterway

Sediment contaminants were monitored in Milford Haven Waterway (MHW) since 1978 (hydrocarbons) and 1982 (metals), with the aim of providing surveillance of environmental quality in one of the UK’s busiest oil and gas ports. This aim is particularly important during and after large-scale investment in liquefied natural gas (LNG) facilities. However, methods inevitably have changed over the years, compounding the difficulties of coordinating sampling and analytical programmes. After a review by the MHW Environmental Surveillance Group (MHWESG), sediment hydrocarbon chemistry was investigated in detail in 2010. Natural Resources Wales (NRW) contributed their MHW data for 2007 and 2012, collected to assess the condition of the Special Area of Conservation (SAC) designated under the European Union Habitats Directive. Datasets during 2007-2012 have thus been more comparable. The results showed conclusively that a MHW-wide peak in concentrations of sediment polycyclic aromatic hydrocarbons (PAHs), metals and other contaminants occurred in late 2007. This was corroborated by independent annual monitoring at one centrally-located station with peaks in early 2008 and 2011. The spatial and temporal patterns of recovery from the 2007 peak, shown by MHW-wide surveys in 2010 and 2012, indicate several probable causes of contaminant trends, as follows: atmospheric deposition, catchment runoff, sediment resuspension from dredging, and construction of two LNG terminals and a power station. Adverse biological effects predictable in 2007 using international sediment quality guidelines, were independently tested by data from monitoring schemes of more than a decade duration in MHW (starfish, limpets), and in the wider SAC (grey seals). Although not proving cause and effect, many of these potential biological receptors showed a simultaneous negative response to the elevated 2007 contamination following intense dredging activity in 2006. Wetland bird counts were typically at a peak in the winter of 2005-2006 previous to peak dredging. In the following winter 2006-2007, shelduck in Pembroke River showed their lowest winter count, and spring 2007 was the largest ever drop in numbers of broods across MHW between successive breeding seasons. Wigeon counts in Pembroke River were again low in late 2012 after further dredging nearby. These results are strongly supported by PAH data reported previously from invertebrate bioaccumulation studies in MHW 2007-2010, themselves closely reflecting sediment

Analyses of sublittoral macrobenthic community change in a marine nature reserve using similarity profiles (SIMPROF).

Sublittoral macrobenthic communities in the Skomer Marine Nature Reserve (SMNR), Pembrokeshire, Wales, were sampled at 10 stations in 1993, 1996, 1998, 2003, 2007 and 2009 using a Day grab and a 0.5 mm mesh. The time series is analysed using Similarities Profiles (SIMPROF) tests and associated methods. Q-mode analysis using clustering with Type 1 SIMPROF addresses multivariate structure among samples, showing that there is clear structure associated with differences among years. Inverse (r-mode) analysis using Type 2 SIMPROF decisively rejects a hypothesis that species are not associated with each other. Clustering of the variables (species) with Type 3 SIMPROF identifies groups of species which covary coherently through the time-series. The time-series is characterised by a dramatic decline in abundances and diversity between the 1993 and 1996 surveys. By 1998 there had been a shift in community composition from the 1993 situation, with different species dominating. Communities had recovered in terms of abundance and species richness, but different species dominated the community. No single factor could be identified which unequivocally explained the dramatic changes observed in the SMNR. Possible causes were the effects of dispersed oil and dispersants from the Sea Empress oil spill in February 1996 and the cessation of dredge-spoil disposal off St Anne’s Head in 1995, but the most likely cause was severe weather. With many species, and a demonstrable recovery from an impact, communities within the SMNR appear to be diverse and resilient. If attributable to natural storms, the changes observed here indicate that natural variability may be much more important than is generally taken into account in the design of monitoring programmes.

The Secret Life of Oilbirds: New Insights into the Movement Ecology of a Unique Avian Frugivore

Background: Steatornis caripensis (the oilbird) is a very unusual bird. It supposedly never sees daylight, roosting in huge aggregations in caves during the day and bringing back fruit to the cave at night. As a consequence a large number of the seeds from the fruit they feed upon germinate in the cave and spoil.

Field fluxes and speciation of arsines emanating from soils

The biogeochemical cycle of arsenic (As) has been extensively studied over the past decades because As is an environmentally ubiquitous, nonthreshold carcinogen, which is often elevated in drinking water and food. It has been known for over a century that micro-organisms can volatilize inorganic As salts to arsines (arsine AsH(3), mono-, di-, and trimethylarsines, MeAsH(2), Me(2)AsH, and TMAs, respectively), but this part of the As cycle, with the exception of geothermal environs, has been almost entirely neglected because of a lack of suited field measurement approaches. Here, a validated, robust, and low-level field-deployable method employing arsine chemotrapping was used to quantify and qualify arsines emanating from soil surfaces in the field. Up to 240 mg/ha/y arsines was released from low-level polluted paddy soils (11.3 ± 0.9 mg/kg As), primarily as TMAs, whereas arsine flux below method detection limit was measured from a highly contaminated mine spoil (1359 ± 212 mg/kg As), indicating that soil chemistry is vital in understanding this phenomenon. In microcosm studies, we could show that under reducing conditions, induced by organic matter (OM) amendment, a range of soils varied in their properties, from natural upland peats to highly impacted mine-spoils, could all volatilize arsines. Volatilization rates from 0.5 to 70 µg/kg/y were measured, and AsH(3), MeAsH(2), Me(2)AsH, and TMAs were all identified. Addition of methylated oxidated pentavalent As, namely monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA), to soil resulted in elevated yearly rates of volatilization with up to 3.5% of the total As volatilized, suggesting that the initial conversion of inorganic As to MMAA limits the rate of arsine and methylarsines production by soils. The nature of OM amendment altered volatilization quantitatively and qualitatively, and total arsines release from soil showed correlation between the quantity of As and the concentration of dissolved organic carbon (DOC) in the soil porewater. The global flux of arsines emanating from soils was estimated and placed in the context of As atmospheric inputs, with arsines contributing from 0.9 to 2.6% of the global budget.

Arsenic speciation in the earthworms Lumbricus rubellus and Dendrodrilus rubidus

Two species of earthworm, Lumbricus rubellus Hoffmeister and Dendrodrilus rubidus (Savigny) collected from an arsenic-contaminated mine spoil site and an uncontaminated site were investigated for total tissue arsenic concentrations and for arsenic compounds by liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). For L. rubellus, whole-body total tissue arsenic concentrations were 7.0 to 17.0 mg arsenic/ kg dry weight in uncontaminated soil and 162 to 566 mg arsenic/kg dry weight in contaminated soil. For D. rubidus, whole-body tissue concentrations were 2.0 to 5.0 mg arsenic/kg dry weight and 97 to 321 mg arsenic/kg dry weight, respectively. Arsenobetaine was the only organic arsenic species detected in both species of earthworms, with the remainder of the extractable arsenic being arsenate and arsenite. There was an increase in the proportion of arsenic present as arsenobetaine in the total arsenic burden. Lumbricus rubellus and D. rubidus have similar life styles, both being surface living and litter feeding. Arsenic speciation was found to be similar in both species for both uncontaminated and contaminated sites, with dose-dependent formation of arsenobetaine. When L. rubellus and D. rabidus from contaminated sites were incubated in arsenic-free soils, the total tissue burden of arsenic diminished. Initially, L. rubellus from the tolerant populations (from the contaminated site) eliminated arsenic in the first 7 d of exposure before accumulating arsenic in tissues, whereas nontolerant populations (from the uncontaminated site) accumulated arsenic linearly. The tolerant and nontolerant L. rubellus eliminated tissue arsenic linearly over 21 d when incubated in uncontaminated soil.

Interactions between earthworms and arsenic in the soil environment:a review

Chemical pollution of the environment has become a major source of concern. In particular, many studies have investigated the impact of pollution on biota in the environment. Studies on metalliferous contaminated mine spoil wastes have shown that some soil organisms have the capability to become resistant to metal/metalloid toxicity. Earthworms are known to inhabit arsenic-rich metalliferous soils and, due to their intimate contact with the soil, in both the solid and aqueous phases, are likely to accumulate contaminants present in mine spoil. Earthworms that inhabit metalliferous contaminated soils must have developed mechanisms of resistance to the toxins found in these soils. The mechanisms of resistance are not fully understood; they may involve physiological adaptation (acclimation) or be genetic. This review discusses the relationships between earthworms and arsenic-rich mine spoil wastes, looking critically at resistance and possible mechanisms of resistance, in relation to soil edaphic factors and possible trophic transfer routes.

Arsenic-speciation in arsenate-resistant and non-resistant populations of the earthworm, Lumbricus rubellus

Arsenic speciation was determined in Lumbricus rubellus Hoffmeister from arsenic-contaminated mine spoil sites and an uncontaminated site using HPLC-MS, HPLC-ICP-MS and XAS. It was previously demonstrated that L. rubellus from mine soils were more arsenate resistant than from the uncontaminated site and we wished to investigate if arsenic speciation had a role in this resistance. Earthworms from contaminated sites had considerably higher arsenic body burdens (maximum 1,358 mg As kg-1) compared to the uncontaminated site (maximum 13 mg As kg-1). The only organo-arsenic species found in methanol/water extracts for all earthworm populations was arsenobetaine, quantified using both HPLC-MS and HPLC-ICP-MS. Arsenobetaine concentrations were high in L. rubellus from the uncontaminated site when concentrations were expressed as a percentage of the total arsenic burden (23% mean), but earthworms from the contaminated sites with relatively low arsenic burdens also had these high levels of arsenobetaine (17% mean). As arsenic body burden increased, the percentage of arsenobetaine present decreased in a dose dependent manner, although its absolute concentration rose with increasing arsenic burden. The origin of this arsenobetaine is discussed. XAS analysis of arsenic mine L. rubellus showed that arsenic was primarily present as As(III) co-ordinated with sulfur (30% approx.), with some As(v) with oxygen (5%). Spectra for As(III) complexed with glutathione gave a very good fit to the spectra obtained for the earthworms, suggesting a role for sulfur co-ordination in arsenic metabolism at higher earthworm arsenic burdens. It is also possible that the disintegration of As(III)-S complexes may have taken place due to (a) processing of the sample, (b) storage of the extract or (c) HPLC anion exchange. HPLC-ICP-MS analysis of methanol extracts showed the presence of arsenite and arsenate, suggesting that these sulfur complexes disintegrate on extraction. The role of arsenic speciation in the resistance of L. rubellus to arsenate is considered.

Resistance to copper toxicity in populations of the earthworms Lumbricus rubellus and Dendrodrilus rubidus from contaminated mine wastes

Two arsenic and heavy metal-contaminated mine spoil sites, at Carrock Fell, Cumbria, United Kingdom, and Devon Great Consols Mine, Devon, United Kingdom, have been found to support populations of the earthworms Lumbricus rubellus Hoffmeister and Dendrodrilus rubidus (Savigny). Lumbricus rubellus and D. rubidus collected from the Devon site and an uncontaminated site were kept for 28 d in uncontaminated soil and in soil containing 750 mg/kg CuCl2, the state of the specimens being recorded using a semiquantitative assessment of earthworm health (condition index). The condition index remained high for all specimens except those of L. rubellus and D. rubidus from uncontaminated sites, which displayed 100% mortality. Bioavailability of Cu in the soils from one uncontaminated and two contaminated sites and in the uncontaminated soil treated with CuCl2 was determined using sequential extraction. Soils from Devon Great Consols had the greatest availability of Cu, Carrock Fell the lowest. Total tissue Cu for L. rubellus and D. rubidus from the contaminated sites did not change significantly for each species during the experiment. Total tissue concentrations of Cu for L. rubellus and D. rubidus from uncontaminated sites increased significantly during the first 7 d, after which mortality was 90%, making it impossible to continue the analysis.

Soil conditioning for EPBMs

Nowadays, the Earth Pressure Balanced (EPB) shields are used more and more caused by both the mechanical development of the machines and the more effective use of additives such as foams and polymers. These additives are used to condition spoil in the screw conveyor. A crucial component of EPB tunnelling applications is ground conditioning. Thus, in order to permit quantitative measurements of the conditioned ground behavior, it is necessary to develop and use a test able to simulate the extraction of earth from the chamber with a screw conveyor. This reliable test procedure can provide quantitative parameters that can really describe the conditioned soil behavior.