Bioavailability: Linking complex Environmental …Prof Graham A Mills (University of Portsmouth)...

Society of Environmental Toxicology and Chemistry UK

Royal Society of Chemistry Toxicology Group Joint Meeting

Bioavailability: Linking complex Environmental Chemistry with

Environmental Response

17-18th September, 2012, The RSC Chemistry Centre, Royal Society of Chemistry,

Burlington House, Piccadilly, London, W1J 0BA, UK.

SETAC-UK- RSC Joint Meeting 2012 Bioavailability: Linking Complex Environmental

Chemistry with Environmental Response 17-18th September 2012

Monday, 17 September 2012 10:00 Registration desk open 10:30 Welcome and Introduction: Dr Kirit Wadhia and Kate Jones Session 1: Fate and behaviour and influences on bioavailability Chair: Dr Kirit Wadhia 10:35 Prof Ian T Burke (School of Earth and Environment University of Leeds) Investigations of the mobility of toxic trace elements following the bauxite residue (red mud) spill at Ajka, Western Hungary. [S1.1] 11:05 Mr Scott Lambert (Food Environment Research Agency, University of York) Degradation of latex in freshwater environment [S1.2] 11:30 Miss Geraldine SC Turner (University of Portsmouth) Evaluating the diffusive gradients in thin-films technique for the measurement of dissolved uranium in natural waters [S1.3] 11:55 Miss Dokuboba Amachree (University of Plymouth) Comparison of Intermittent and Continuous Exposures to Mercury in the Marine Mussel, Mytilus edulis: Accumulation and Sub-lethal Effects [S1.4] 12:30 Lunch and Posters



Session 2: Metal bioavailability in aquatic environments Chair: Prof Richard Handy 13:30 Prof Christer Hogstrand (King's College London) Bioavailability of metals to fish: its molecular basis, toxicological consequences and how it can be assessed. [S1.5] 14:00 Dr Graham Merrington (Wca-environment) Metal Bioavailability: The State of the Art [S1.6] 14:25 Prof Richard D Handy (Ecotoxicology Research and Innovation Centre University of Plymouth) The Bioavailability of Nanomaterials: A Comparison of Nanometals Versus Dissolved Metal Ions [S1.7] 14:50 Tea/coffee sponsored by the University of Plymouth, Research and Innovation Centre 15:20 Dr Andrew Turner (University of Plymouth) The geochemistry, bioavailability and toxicity of thallium in the aquatic environment [S1.8] 15:45 Miss Helen M Wilkinson (Environment Agency) Incorporating bioavailability into regulatory practice: Experiences with metals in water [S1.9] 16:10-16:25 SETAC AGM - open to all 19:00 Conference social event, drinks and meal in Covent Garden



Tuesday, 18 September 2012 09:30 Registration desk open Session 3: Bioavailability of compounds in soil and water Chair: Mr Dean Leverett 09:40 Dr Stephen Lofts (NERC Centre for Ecology and Hydrology) Bioavailability of metals in soils: integrating knowledge into risk assessment [S1.10] 10:10 Miss Laura J Carter (University of York) Uptake of Pharmaceuticals in the Terrestrial Environment [S1.11] 10:35 Ms Monika D Jürgens (Centre for Ecology and Hydrology (CEH)) Using the UK Fish Tissue Archive to monitor EU priority substances [S1.12] 11:00 Mrs Clare E Askem (CEFAS) Growth inhibition test with Ceramium tenuicorne (ISO 10710:2010): our findings from using the OECD protocol with various chemicals, static and flow through systems. [S1.13] 11:25 Tea and coffee



Session 4: Can we use 'omics technologies to measure bioavailability? Chair: Dr Danielle Ashton 11:45 Prof Francesco Falciani (University of Liverpool) A Systems Biology approach to link chemical features, organism response and biodiversity [S1.14] 12:10 Dr Eduarda M Santos (University of Exeter) Exploring chemical impacts for selected pollutants in the three-spined stickleback (Gasterosteus aculeatus) derived from UK rivers. [S1.15] 12:35 Dr Nicolas R Bury (King's College London) Investigating the versatility of a primary fish gill cell culture system for environmental monitoring. [S1.16] 13:00 Prizes for best poster and presentation 13:05 Lunch and posters



Session 5: Human health risk assessment Chair: Kate Jones 14:05 Dr Mark R Cave (British Geological Survey) Measurement of the Bioavailability of Potentially Harmful Substances in soil - The "How" and the "Why" [S1.17] 14:35 Dr Camilla K Pease (ENVIRON UK Ltd) Experiences with Exposure Models for Estimating the Bioavailability of Lead in Children in the [S1.18] 15:00 Tea and coffee 15:20 Prof John R Dean (Northumbria University) Oral bioaccessibility of PTE and PAH contaminants: interpretation of results from a range of contaminated sites. [S1.19] 15:45 Dr Samuel O Asagba (Delta State University Abraka Nigeria) A Comparative evaluation of the biochemical effects of environmental cadmium-contaminated control diet and laboratory cadmium supplemented test diet. [S1.20] 16:10 End of meeting



Poster Session: (Posters can remain on the boards throughout the meeting) Mr Keishiro Horiuchi (Tokyo University of Pharmacy and Life sciences) Hydro-dynamics of coccolithophorid observed using fluorescence correlation spectroscopy with ordinary fluorescence configuration [S1.21] Ms Kate Jones (Health Safety Laboratory) Arsenic Speciation in Urine using micro liquid chromatography-ICP-MS [S1.22] Ms Kate Jones (Health Safety Laboratory) Human volunteer study to compare absorption and elimination kinetics of different sized titanium dioxide particles [S1.23] Ms Kana Inoue (School of Life Sciences Tokyo University of Pharmacy and Life Sciences) Denaturation of protein induced by transition metal ions under observation by Fluorescence Correlation Spectroscopy [S1.24] Mr David Megson (Plymouth University) Forensic techniques to distinguish past and current human exposure to PCBs [S1.25] Miss Lisa Tucker (Royal Veterinary College) Drug-Drug Interactions in Daphnia magna [S1.26] Mr Dean H Leverett (Wca-environment) Characterisation of Dissolved Organic Carbon in Effluents and the Assessment of its Overall Benefits in Mitigating Environmental Impacts from Metals [S1.27] Mr Graham Merrington (Wca-environment) Delivering a practical methodology to account for metal (bio)availability in the Water Framework Directive – Case Studies [S1.28]



Prof Kehinde O Olayinka (University of Lagos) Uptake of heavy metals by vegetable plants grown on contaminated soil and their potential bioavailability using sequential extraction [S1.29] Ms Tamsyn M Uren Webster (University of Exeter) Molecular mechanisms of adaptation to metals in a population of brown trout (Salmo trutta) inhabiting a contaminated river in the South West of England [S1.30] Miss Laurence MC BERTHOD (University of Portsmouth AstraZeneca) Solid phase extraction to model binding of active pharmaceutical ingredients to sewage sludge [S1.31] Prof Graham A Mills (University of Portsmouth) Potential of passive sampling devices to provide bio-available concentrations of pollutants in the aquatic environment [S1.32] Miss Alexandra Whicher (Department of Geography King's College London) The use of transgenic nematodes (Caenorhabditis elegans) as a novel bioassay for the assessment of urban river sediment toxicity [S1.33]



ABSTRACTS-ORAL PRESENTATIONS

S1.1 Investigations of the mobility of toxic trace elements following the bauxite residue (red mud) spill at Ajka, Western Hungary. Ian T Burke (School of Earth and Environment University of Leeds) In 2010, a tailings dam breach at Ajka, Hungary released up to 1 million tonnes of bauxite residue (red mud) into the environment. The magnitude of release was unprecedented and resulted in loss of life, property damage and contamination of agricultural land. Remediation is now largely complete. Acid and gypsum dosing of rivers and streams reduced pH and ‘dig and dump’ cleared waste deposits from the land. In order to prevent dust formation, red mud was ploughed into fields where the covering was < 5 cm. Some wetland areas also remain untreated. Red mud is the highly caustic fine fraction residue from aluminium extraction from bauxite. Water in contact with red mud is hyperalkaline (pH 13) has elevated concentrations of metals and metalloids such as Al (1000 ppm) As, V, and Mo (4 - 6 ppm) and the red mud itself has elevated concentrations of As, V, Cr, Co, Ni and U (100-1000 ppm). These contaminants persist in water and sediment samples for up to 100 km downstream of the source. Microcosm experiments using red mud mixed with uncontaminated local soils show a marked increase of As, V, Cr and Mo concentrations into the aqueous phase over 30 days. These results suggest that at the circumneutral pH, oxic conditions found, incorporation and sorption respectively will restrict the environmental mobility of Cr and As. V and Mo are inefficiently removed from solution by sorption reactions, therefore, the red mud may act as a source of mobile oxyanions. Email Address for correspondence: [email protected] 10:35 Monday 17th September 2012



S1.2 Degradation of latex in freshwater environment Scott Lambert (Food Environment Research Agency, University of York), Chris Sinclair (Food Environment Research Agency), Alistair Boxall (University of York) The aim of this work was to characterise the degradation of a latex film under environmentally relevant conditions. To do this latex samples were immersed in different aquatic solutions and exposed to natural cycles of sunlight and temperature. Material recovery after 200 days exposure in freshwater 53.54 % ± 11.79. Modelled data show the degradation kinetics to take part over three phases. The first is characterised by a period of weight gain, the second phase is characterised by a period of fast degradation driven by high levels of solar radiation. This is then followed by a third slower period of degradation characterised by lower levels of solar radiation. Nanoparticle tracking analysis (NTA) and GC-MS were used to characterise potential degradates. The results show that as the latex film disintegrates and starts to degrade particles in the nano range are formed with concentrations ranging from less than 1 x 108 to 4 x 108 nanoparticles/ml over the course to the exposure period. A screening approach was applied to the characterisation of chemical degradates. No good library matches were obtained for GC-MS and the chromatographic data represented that of an unresolved complex mixture. To this end principal component analysis (PCA) was applied to identify patterns in the data. A mass balance was also preformed and a shows that not all of the latex can be accounted for over the exposure period. It is proposed that as the latex macromolecules degrade volatilization processes come into effect with the evolution of low-molecular volatile products. Email Address for correspondence: [email protected] 11:05 Monday 17th September 2012 S1.3 Evaluating the diffusive gradients in thin-films technique for the measurement of dissolved uranium in natural waters Geraldine S C Turner (University of Portsmouth), Gary R Fones (University of Portsmouth), Graham A Mills (University of Portsmouth), Jonathan L Burnett (AWE), Sean Amos (AWE) The UK’s nuclear industry and associated infrastructure is committed to provide assurance that it is not impacting the aquatic environment. To achieve this, extensive monitoring



activities are implemented to detect actinides in a range of natural waters. Passive samplers have been identified as a technique applicable to the monitoring of dissolved phase radionuclides in the aquatic environment, and have several advantages over conventional sampling methods including - simplicity, provision of time-weighted average concentrations, and automatic in-situpre-concentration of analytes. Diffusive Gradient in Thin Films (DGT) device has been identified as one type of passive sampler that can effectively measure dissolved concentrations uranium, thereby providing information on the bioavailability and speciation of this actinide. In our work the binding phase shown to be most effective in accumulating uranium in alkaline freshwaters is a TiO2 based sorbent Metsorb™. Data is presented for week long deployments of Metsorb™ DGT devices over a six month period at two alkaline freshwater locations - the River Lambourn and the River Enbourne, UK. The sites were chosen for their differences in catchment management techniques, flow regimes and background water chemistry that may impact upon the utility of DGT devices used in the long-term monitoring campaigns. In addition to the uranium DGT measurements, samples for nutrients, dissolved organic carbon, chlorophyll and major anions and cations were taken in order to model (Visual Minteq programme) uranium speciation and to show any relationships that may exist between these variables and the results obtained using the DGT. Email Address for correspondence: [email protected] 11:30 Monday 17th September 2012 S1.4 Comparison of Intermittent and Continuous Exposures to Mercury in the Marine Mussel, Mytilus edulis: Accumulation and Sub-lethal Effects DOKUBOBA AMACHREE (UNIVERSITY OF PLYMOUTH), JOHN AMOODY (UNIVERSITY OF PLYMOUTH), RICHARD DHANDY (UNIVERSITY OF PLYMOUTH) Intermittent and variable exposure to pollution is the most likely way aquatic organisms are exposed to chemicals, including metals. However, the hazard from intermittent exposure may not simply be predicted from existing continuous exposure. This study aimed to compare the sub-lethal responses of aqueous exposure to HgCl2 in continuous or intermittent regime and to establish whether or not Hg accumulation was simply a function of equivalent dose. M. edulis were exposed using a semi-static and triplicated design to either control (no added Hg) or 50 µg/l Hg as HgCl2 in continuous or intermittent (2 day exposure, 2 days in clean seawater alternately) mode for 14 days. Hg tissue burden was significantly increased in the continuous compared to the intermittent for digestive gland (5 fold), gonad and carcass (>2 folds), but not gill and adductor mussel. There were no



differences in tissue thiobarbituric acid reactive substances (TBARS) content or haemolymph chemistry in terms of osmoregulation and immunology between the two exposure regimes. Tissue ions (Na+, K+, Ca2+ and Mg2+) were not different in both exposure regimes. Total glutathione was depleted in the digestive gland of the continuous but not the intermittent exposure. Histological examination of the digestive gland showed significantly less inflammation in the continuous compared to the intermittent exposure. Overall, results indicate that accumulation and responses of Hg during intermittent compared to continuous exposure are different. In risk assessment, the continuous exposure may not necessarily protect for intermittent events. Email Address for correspondence: [email protected] 11:55 Monday 17th September 2012 S1.5 Bioavailability of metals to fish: its molecular basis, toxicological consequences and how it can be assessed. Christer Hogstrand (King's College London) Bioavailability and toxicity of metals is critically dependent on speciation. This is partly explained by the fact that metals gain entry into epithelial tissues through ion channels and transporters, which have differing degrees of ion selectivity. Essential metals have dedicated transport systems, but metal uptake also occurs through channels for other ions by molecular mimicry. Metal bioavailability is dictated by presence in the medium of complexing agents, which compete with these ion channels and by competition between metals and other cations for uptake sites. Mechanisms of toxicity for transition metals include interfering with biological functions of minerals including those of essential metals. Water-breathing animals, such as fish, are typically very sensitive to transition metals that are dissolved in water because they take up metals and other ions across the gills. Mechanisms of acute toxicity of fish to many waterborne metals have been relatively well described even at the biochemical level. For example, cadmium and zinc block uptake of calcium across the gill, leading to acute hypocalcaemia whilst copper and silver inhibit sodium and chloride uptake, resulting in a loss of osmotic control. Thanks to the relatively direct link between bioavailability of metals at the gill and adverse effects occurring in the same tissue it has been possible to develop computational models, such as Biotic Ligand Models, which can predict acute toxicity of metals in different water chemistries. We have also devised a cell culture-based method to measure metal toxicity in natural waters and shown how it could be used in biomonitoring. This FIsh Gill Culture System (FIGCS) is



based on a primary culture of gill cells that is capable of withstanding exposure to metals in water. The dietary route of exposure was for a long time ignored for inorganic toxicants. However, recent studies show that dietary toxicity to inorganic substances can be of importance. For example a natural arsenic-contaminated diet was found to have negative effects on reproduction in fish.

Email Address for correspondence: [email protected] 13:30 Monday 17th September 2012 S1.6 Metal Bioavailability: The State of the Art Graham Merrington (Wca-environment), Adam Peters (Wca-environment) Statutory EQS for several metals were set in the mid 1980’s which considered a bioavailability modifying effect of water hardness on metal toxicity. These standards also largely recognised that dissolved metal concentrations were more toxicologically relevant than total concentrations. The BLM concept evolved from the combination of improved chemical speciation models, which were able to predict metal binding by natural organic matter, with a model of the mechanism of interaction between metals and fish gills. This heralded a new era in our understanding of the effects of metals on organisms on real systems. Since the development of the BLM concept numerous models of the acute toxicity of cationic metals to standard ecotoxicity test species have been developed. The regulatory requirement in Europe to consider chronic toxicity directly has also resulted in the development of chronic models for a limited selection of metals. There is a considerable degree of consistency between the different models, and in some cases it has also been demonstrated that the same models can also be applied to taxonomically dissimilar species, with the intrinsic sensitivity of the species being the only parameter which requires adjustment. Several issues still remain to be addressed about the effect of bioavailability in surface waters, including how to address those waters which fall outside the acceptable physiological ranges of the test species, and ensuring that the models developed can also be applied to organisms from remote parts of the world. Email Address for correspondence: [email protected] 14:00 Monday 17th September 2012



S1.7 The Bioavailability of Nanomaterials: A Comparison of Nanometals Versus Dissolved Metal Ions Richard D Handy (Ecotoxicology Research and Innovation Centre University of Plymouth) Metal-containing engineered nanoparticles (NPs) are now finding many applications, but there are concerns about their toxicity, and whether or not existing environmental regulations for dissolved metals can be used for nanomaterials. Nanometals are taken up across epithelia, but there are some fundamental differences in the bioavailability of dissolved versus particulate metals. There are concerns that traditional equilibrium models (e.g., binding and uptake rate constants) are inappropriate for nanometals which have dynamic behaviours better explained by colloid chemistry (DLVO theory). Some NPs, such as Cu-NPs, may also release free metal ions from the surface of the particle, but the abiotic factors controlling this dissolution (pH, water hardness, etc.,) are poorly understood for most metallic particles. Other NPs such as TiO2 do not appreciably dissolve in freshwater, but there are some novel effects of partial pressure of gases on the Ti metal uptake. TiO2 NPs also illustrate the complexity of measuring bioavailability of nanomaterials, with apparently different pathways for the uptake of different crystal structures. In certain conditions, the TiO2 particles may partially dissolve inside the cells. Alternatively, silver NPs that we might expect to dissolve inside cells, do not because of the chloride rich environment in biological fluids. The pharmacological mechanisms involve both solute transport and endocytosis-like pathways for metallic NPs. Overall, the net uptake rates and bioavailability of the nanometals tested so far are broadly similar to their dissolved metal counterparts, but the underlying properties controlling bioavailability can be very different. Email Address for correspondence: [email protected] 14:25 Monday 17th September 2012 S1.8 The geochemistry, bioavailability and toxicity of thallium in the aquatic environment Andrew Turner (University of Plymouth) Thallium is a highly toxic element that has been classified as an EPA priority pollutant. Despite its toxicity, however, very little information exists on the environmental behaviour of Tl and, in particular, its mobilisation, distribution, impacts and fate in aquatic systems.



This may, partly, be attributed to the relatively low economic value of the metal and, until recently, difficulty associated with its analysis in natural samples. This presentation reports on measurements of dissolved and particulate Tl in the hydrosphere of south west England, performed by the latest generation of quadrupole ICP-MS. It is shown that the principal sources of Tl to the region are from disturbed sulphidic metal deposits, where Tl occurs as discrete minerals or in conjunction with other minerals (e.g. chalcopyrite), and that concentrations are greatest in systems affected by acid mine drainage. Being a biogeochemical analogue of potassium, interactions with sediment are relatively low and conventional, industrial means of metal remediation (e.g. flocculation of hydroxides) are not applicable. The toxicity of Tl has been evaluated by determining its effect on the chlorophyll fluorescence quenching of the estuarine macroalga, Ulva lactuca. Toxicity is shown to be greater than equivalent concentrations of silver and it is suggested that Tl is able to pass through the cell membrane through Na-Cl-K porters. Implications for the monitoring, regulation and impacts of Tl are discussed. Email Address for correspondence: [email protected] 15:20 Monday 17th September 2012 S1.9 Incorporating bioavailability into regulatory practice: Experiences with metals in water Helen M Wilkinson (Environment Agency), Paul Whitehouse (Environment Agency), Lindsey A Sturdy (Environment Agency), Jon Gulson (Environment Agency), Graham Merrington (WCA Environment Ltd), Adam Peters (WCA Environment Ltd) Inputs of metals remain a significant pressure on water quality. From research carried out in recent years, we now know much more about the behaviour of metals in natural waters, and how physical and biological factors determine their toxicity to aquatic organisms. Crucially, only the ‘bioavailable’ fraction of metal is in a form that can cause toxicity. This understanding has led to the development of a suite of ‘Biotic Ligand Models’ (BLMs) that explicitly deal with bioavailability and predict the toxicity of several metals under a range of conditions. In our presentation, we explain the Environment Agency’s approach to incorporating this understanding into the way we address risks from metals. We describe: - simplified tools for predicting bioavailable concentrations of metals - show how incorporating bioavailability affects the classification of waterbodies under the Water Framework Directive, and - show how we can prioritise sites where action is needed to reduce risks from metal contamination. Finally, we highlight some steps that need addressing before we can fully implement



bioavailability-based approaches into chemical regulation. Email Address for correspondence: [email protected] 15:45 Monday 17th September 2012 S1.10 Bioavailability of metals in soils: integrating knowledge into risk assessment Stephen Lofts (NERC Centre for Ecology and Hydrology), David Spurgeon (NERC Centre for Ecology and Hydrology), Claus Svendsen (NERC Centre for Ecology and Hydrology), Ed Tipping (NERC Centre for Ecology and Hydrology) In recent years there have been considerable advances in integrating bioavailability into risk assessment for metals such as nickel, copper, zinc, cadmium, lead and mercury. This presentation will focus on two aspects of metal bioavailability in soils: firstly, the influence of soil chemistry on metal speciation and uptake, and secondly the influence of long-term ‘aging’ reactions of metals in soils on their bioavailability and toxicity. The ‘free ion approach’ may be used to derive critical limit concentrations of metals that take the influence of soil chemistry into account. It will be shown how such critical limit concentrations have been applied to assess the risks due to the atmospheric deposition of metals to UK soils, and how the use of critical limit concentrations that account for bioavailability influences the risk estimation. It will also be shown how the methodology can be extended to allow for the calculation of Potentially Affected Fractions (PAFs), and for probabilistic risk assessment of metals in soils. Aging of metals in soils is an important speciation process, since it controls the size of the ‘geochemically active’ or labile pool, which in turn controls the free ion concentration. Using models derived from laboratory data on aging, it will be shown how aging processes should act to attenuate the risks due to repeated additions of metals to soils such as might occur due to agricultural practices. Finally, potential approaches to account for bioavailability in the context of metal mixtures will be presented. Email Address for correspondence: [email protected] 09:40 Tuesday 18th September 2012



S1.11 Uptake of Pharmaceuticals in the Terrestrial Environment Laura J Carter (University of York), Jim J Ryan (GlaxoSmithKline), Alistair B A Boxall (University of York) Increasing amounts of pharmaceuticals are being detected in soils, primarily due to sewage sludge application to fields, of which contains high levels of pharmaceuticals. This study investigated the uptake and depuration of pharmaceuticals into earthworms (Eisenia fetida)with the ultimate aim to develop models to improve the risk assessment of pharmaceuticals in the terrestrial environment. Studies were based on the OECD Guideline 317 and involved a 21 day uptake phase followed by a 21 day depuration phase. Studies were performed using radiolabelled compounds to allow for lower limits of detection thus enabling environmentally relevant concentrations to be used.E. fetida, soil and porewater were extracted throughout the experiment and analysed by liquid scintillation counting. The results were modelled using OpenModel and based on a one compartment toxicokinetic model. For all pharmaceuticals, namely, carbamazepine, diclofenac, fluoxetine and orlistat, uptake was seen within the first 6 hours. The modelled uptake rate constants (Kin) show that fluoxetine had the highest uptake rate at 1.1079 nmol/kg d-1. In the depuration phase, carbamazepine was immediately eliminated from theE.fetida reaching complete elimination after day 7 of the depuration phase. In comparison, by the end of the depuration phase there were still large traces of both orlistat and diclofenac in the earthworms. Bioconcentration factors were estimated from the uptake and depuration rates and were then used to evaluate existing quantitative structure-activity relationships used in environmental risk assessment This research shows earthworms, E. fetida,can accumulate pharmaceuticals if they are present in soils at environmentally relevant concentrations. Email Address for correspondence: [email protected] 10:10 Tuesday 18th September 2012



S1.12 Using the UK Fish Tissue Archive to monitor EU priority substances Monika D Jürgens (Centre for Ecology and Hydrology (CEH)), Andrew C Johnson (Centre for Ecology and Hydrology (CEH)), Alan Lawlor (Centre for Ecology and Hydrology (CEH)), Dave Hughes (Lancaster University), Athanasios Katsoyiannis (Norwegian Institute for Air Research (NILU) formerly at Lancaster University), Askin Birgül (Uludag University formerly at Lancaster University), Kevin C Jones (Lancaster University) EU Directive 2008/105/EC includes an (optional) biota standard for 3 substances (article 3, 2a) requiring member states choosing this option to “apply, for mercury and its compounds, an EQS* of 20 μg/kg, and/or for hexachlorobenzene, an EQS of 10 μg/kg, and/or for hexachlorobutadiene, an EQS of 55 μg/kg, these EQS being for prey tissue (wet weight), choosing the most appropriate indicator from among fish, molluscs, crustaceans and other biota” A suggested revision (COM/2011/0876) has 11 (including the current 3) compulsory (fish) biota standards. Since 2007 CEH and the Environment Agency are building an archive of frozen fish samples from English rivers to provide a resource for future retrospective monitoring of pollutants. We analysed some of those archived fish and found: Mercury: About half the samples exceeded the EU EQS of 20 µg/kg (median: 23 յg/kg), but even the maximum of 68 µg/kg is almost a factor of 10 below the EU food limit for fish of 500 or 1000 յg/kg. Zebramussels had much lower concentrations than fish. Hexachlorobenzene, a fungicide which is no longer used in the EU, was below the EQS of 10 µg/kg in all fish analysed with a maximum of 6.4 յg/kg in an eel. Hexachlorobutadiene, which was used as a solvent in polymer production and as a fungicide and seed dressing, was not detectable in the majority of samples. PBDEs (flame retardants) are among the chemicals in the new proposal and were detected at much higher levels than the proposed 0.085 µg/Kg standard *Environmental Quality Standard Email Address for correspondence: [email protected] 10:35 Tuesday 18th September 2012



S1.13 Growth inhibition test with Ceramium tenuicorne (ISO 10710:2010): our findings from using the OECD protocol with various chemicals, static and flow through systems. Clare E Askem (CEFAS) Macroalgae constitute an important part of the costal ecosystem and are not usually examined in the traditional suite of toxicity tests in the UK. A recently developed OECD growth inhibition test with Ceramium tenuicorne (ISO 10710:2010) was used and adapted for conditions in the CEFAS Lowestoft laboratory. Ceramium tenuicorne clones are cultured in the lab and harvested prior to each assay creating a relatively simple and useful addition to the ‘toolbox’ of assays in Ecotoxicology. Growth inhibition tests were performed with Ciprofloxacin and Triclosan alongside more traditional test species such as Skeletonema costatum. Out of 5 species tested Ceramium tenuicorne was found to be the most sensitive to Ciprofloxacin with a 7 day EC50 of 10.67 mg/l (95% confidence limits 9.96-10.33 mg/l) which was more sensitive than single celled algae. The results of these tests are presented as well as our findings from culturing Ceramium tenuicorne. A flow through Ceramium assay has also been developed at Cefas for investigating certain effluent scenarios and working with rapidly metabolised or degraded chemicals. Issues raised and solved in the development of this assay will also be discussed. Email Address for correspondence: [email protected] 11:00 Tuesday 18th September 2012 S1.14 A Systems Biology approach to link chemical features, organism response and biodiversity Francesco Falciani (University of Liverpool), Philipp Antczak (University of Liverpool), Leona Scanlan (UC Berkeley), Chris Vulpe (UK Berkeley) Environmental protection agencies worldwide are presented with a huge challenge as a result of human innovation. Increasingly large numbers of chemicals are released into the environment through a number of sources. Unfortunately, the mechanisms of action and the long-term effects of these exposures are still largely unknown. Statistical modelling approaches, combined to “omics” measurements, have been effective in identifying molecular signatures predictive of toxicity outcome but failed to provide a comprehensive



tool for monitoring the effects of environmental pollution. Ideally, such a tool should be able to simultaneously predict the chemical composition of contaminated waters, toxicity outcome and provide hypothesis on toxicity mechanisms from the organism molecular response to exposure. Several computational methodologies that have been recently developed in the field of Systems Biology may be appropriate to face this challenge. To demonstrate the effectiveness of these techniques in the environmental biology arena, we applied a well validated reverse engineering algorithm to an mRNA expression dataset representing Daphnia Magna exposed to sub-lethal concentrations of a number of relevant chemicals as well as to contaminated water samples from the environment. Indeed, we could develop models that can identify chemicals in contaminated water, predict organism survival and provide an understanding of the mechanistic response to chemical exposure. Here we also show the results of a proof of concept study linking environmental stressors to biodiversity, which we propose is a first step to link organism to population effects. The implications for monitoring water quality and for risk assessment will be discussed. Email Address for correspondence: [email protected] 11:45 Tuesday 18th September 2012 S1.15 Exploring chemical impacts for selected pollutants in the three-spined stickleback (Gasterosteus aculeatus) derived from UK rivers. Eduarda M Santos (University of Exeter), Tim D Williams (The University of Birmingham), Tamsyn M Uren-Webster (University of Exeter), Huifeng F Wu (The University of Birmingham), Jon S Ball (University of Exeter), Patrick B Hamilton (University of Exeter), Toby S Coe (University of Exeter), Alastair C Cook (Centre for Environment Fisheries and Aquaculture Science), Ceri Lewis (University of Exeter), Anke Lange (University of Exeter), Jan Shears (University of Exeter), Ronny Van Aerle (University of Exeter), Margaret M Brown (Glasgow Caledonian University), Fernando Ortega (The University of Birmingham), Francesco Falciani (The University of Birmingham), John A Craft (Glasgow Caledonian University), Mark R Viant (The University of Birmingham), Ioanna Katsiadaki (Centre for Environment Fisheries and Aquaculture Science), J K Chipman (The University of Birmingham), Charles R Tyler (University of Exeter) We applied a combination of transcriptomics, metabolomics and specific biomarkers to characterise the mechanisms of toxicity of three model environmental chemicals in male three-spined stickleback and to show diagnostic response signatures for the different chemicals. This work revealed novel mechanisms of toxicity, including a dose-dependent



down-regulation of the cholesterol biosynthesis pathway in the liver following exposure to copper. We then determined the responsiveness to copper and EE2 in fish originating from sites with different histories of pollution. For fish derived from the River Aire (historically heavily polluted), there was no evidence of a differential response to these chemicals. However, differential transcriptomic responses to copper were observed in fish originating from the Sibblyback Lake (a location geologically rich in metals), indicating a potential adaptation to copper in this population. Further studies on F1 fish confirmed these findings. We also investigated the possible effects of pollution on genetic structure and reproductive fitness of wild stickleback populations. Populations from three historically polluted sites, but not from two relatively unpolluted sites, showed evidence of recent population bottlenecks. Nevertheless, males from polluted sites were able to reproduce successfully in competition with reference males, indicating that these bottlenecks have not resulted in a substantial loss of reproductive fitness in males, under our study conditions. Together, our studies identified mechanisms of toxicity of model pollutants in the stickleback and illustrated impacts of living in contaminated environments on genetic structure, reproductive fitness and responsiveness to pollutants in this fish species. Email Address for correspondence: [email protected] 12:10 Tuesday 18th September 2012 S1.16 Investigating the versatility of a primary fish gill cell culture system for environmental monitoring Nicolas R Bury (King's College London), Matteo Minghetti (King's College London), Sabine Schnell (King's College London), Lucy Stott (King's College London), Christer Hogstrand (King's College London) A primary fish gill culture system has been developed that when cultured on inserts is able to tolerate water on the apical surface. Previous studies showed that the response of the system to metals mimicked that of whole organisms. The current study aimed to ascertain if this in vitro system could be taken from the lab to the field as a potential site specific environmental monitoring tool. As an exemplar we investigate whether the culture could tolerate natural waters in the field and respond in a predictable way to a gradient of poly-metal contamination. Three independent studies were conducted using sites on the metal contaminated River Hayle, Cornwall. Water chemistry was determined, exposures lasted 24hrs, transepithelial resistance (TER) assessed and the expression of 11 target genes were measured to assess their suitability as surrogate end-points for metal-specific toxicity. In all 3 studies the expression of metal lothionein A and B correlated to the polymetal gradient indicating these genes are useful indicators of metal contamination.



Filtering of the water (0.45μm or 0.2μm) had no effect on the response. The cultures tolerated transport to the field and their response (MT-A and MT-B expression) to exposure in the field was the same as those exposed to the same water in the laboratory. Results show that this system has the potential to be used as an environmental monitoring tool which could form part of an investigative work packages to identify reasons why water bodies fail WFD good ecological status and direct assessment toxicity(DTA) of effluents. Email Address for correspondence: [email protected] 12:35 Tuesday 18th September 2012 S1.17 Measurement of the Bioavailability of Potentially Harmful Substances in soil - The "How" and the "Why" Mark R Cave (British Geological Survey) In terms of human health risk assessment there are three main exposure pathways for a given contaminant present in soil. The largest area of concern is the oral/ingestion pathway followed by the dermal and respiratory exposure routes (Paustenbach, 2000). Whether contaminated soils pose a human health risk depends on the potential of the contaminant to leave the soil and enter the human bloodstream. The use of total contaminant concentrations in soils provides a conservative approach as it assumes that all of the metal present in the soil can enter the bloodstream. Results from animal tests e.g. (Denys et al., 2012) suggest that contaminants in a soil matrix maybe absorbed to a lesser extent and show fewer toxic effects compared to the same concentration of soluble salts of the contaminants in a food or liquid matrix. This presentation will give an overview of the interdisciplinary inputs required to develop and validate methods for measuring the bioavailability /bioaccessibility of contaminants in soil, contrasting and comparing the requirements for inorganic and organic substances. The need for allied methodologies for understanding the processes which control the bioavailability /bioaccessibility of contaminants in soil will be discussed and examples of how both the properties of the contaminant and the physico-chemical properties of the soil can be used to provide an insight to bioavailability mechanisms. Finally, case studies of how bioaccessibility measurements have been applied on a local regional level will be presented. Email Address for correspondence: [email protected] 14:05 Tuesday 18th September 2012



S1.18 Experiences with Exposure Models for Estimating the Bioavailability of Lead in Children in the Camilla K Pease (ENVIRON UK Ltd), Susan Bullock (ENVIRON UK Ltd) The adverse health effects from lead (Pb) exposure can include neurobehavioural (intelligence quotient (IQ) lowering) effects in children, effects on the kidney and cardiovascular systems. The neurobehavioural effects in children are regarded as non-thresholded and can occur at low levels of exposure (<10 µg Pb/dL blood). The European Foods Safety Authority (EFSA, 2010) reported a benchmark dose level relating to a 1% increased risk of a 1 point reduction in IQ (BMDL1 ) = 1.2 յg Pb/dL and recommended that levels of Pb in foods be reduced to as low as reasonably practical. A human exposure scenario/risk/impact assessment can be modelled prospectively (e.g. when considering whether to take action to reduce Pb in environmental and consumer product contamination scenarios). It is necessary to estimate the bioavailable blood Pb level (using an appropriate model) and compare the internal exposure data with the effects data. Here, we report on experiences in using exposure models, such as the US EPA Integrated Exposure Uptake BioKinetic (IEUBK) model, to estimate the blood level of Pb in children from various exposure scenarios, and illustrate how exposure modelling can be used to influence policy decisions within the UK/EU. Email Address for correspondence: [email protected] 14:35 Tuesday 18th September 2012 S1.19 Oral bioaccessibility of PTE and PAH contaminants: interpretation of results from a range of contaminated sites. John R Dean (Northumbria University), Jane A Entwistle (Northumbria University) Exposure risk to humans depends in part on the potential of the contaminant to mobilise from its matrix in the human digestive system (bioaccessibility) and enter the blood stream (bioavailability). This paper evaluates the findings of bioaccessibility analyses undertaken on soils sampled from a range of contaminated sites including historic landfill and former industrial sites. Our bioaccessibility ranking of potentially toxic elements (PTEs) in soils is consistent with X-ray diffraction (XRD) determined mineral phases. The potential risk to human health through this principal (ingestion) exposure pathway is modelled. The role of



bioaccessibility protocols as an additional line of evidence and as a valuable decision-support tool for determining contaminated sites is also highlighted. Email Address for correspondence: [email protected] 15:20 Tuesday 18th September 2012 S1.20 A Comparative evaluation of the biochemical effects of environmental cadmium-contaminated control diet and laboratory cadmium supplemented test diet. Samuel O Asagba (Delta State University Abraka Nigeria), Fred O Obi (University of Benin Benin-City Nigeria) This study was designed to examine the effects of a diet contaminated by environmental cadmium on organ / body weight ratio and selected toxicological indices. It was also designed to permit a comparative analysis of the effects of the diet contaminated by environmental cadmium and that of cadmium supplemented diet based on the same parameters. Our results show that even though the cadmium content of the environmental cadmium contaminated diet was 300% less than that of the cadmium supplemented diet, the former caused statistically significant changes in superoxide dismutase (SOD) activity (P < 0.025), Na+ / K+ ATPase activity (P < 0.005) and Malondialdehyde (MDA) level (P < 0.025) when values at the end of one month exposure were compared to the values at the end of three months exposure. These parameters were altered in the same manner by the cadmium content of the supplemented diet in addition to significant reduction in the liver / bodyweight ratio (P < 0.005) within the exposure periods examined. By virtue of the very close nature of the values of these parameters in rats exposed to the two different diets, it appears that the background cadmium (cadmium from the environment) content of the diets is largely responsible for the observed changes, except in the case of liver / body weight ratio. KEY WORDS: cadmium; diet; environment; rat; toxicity Email Address for correspondence: [email protected] 15:45 Tuesday 18th September 2012



ABSTRACTS-POSTER PRESENTATIONS

S1.21 Hydro-dynamics of coccolithophorid observed using fluorescence correlation spectroscopy with ordinary fluorescence configuration Keishiro Horiuchi (Tokyo University of Pharmacy and Life sciences), Yasuhiro Hashidume (Tokyo University of Pharmacy and Life sciences), Motohide Aoki (Tokyo University of Pharmacy and Life sciences), Shoko Fujiwara (Tokyo University of Pharmacy and Life sciences), Kitao Fujiwara (Tokyo University of Pharmacy and Life sciences) The measurements of coccolithophorid with coccolith was compared with coccolithophorid without coccolith under turbulent flow, using the fluorescence correlation spectroscopy (FCS) under the ordinary fluorescence configuration. The measurement volume was 8 × 10-12 L which was adjusted by changing the diameter of the pin-hole attached to the photo-detector and that of a laser beam (light source: 488 nm). The coccolithophorid suspension was put in an ordinary fluorescence cuvette and stirred with a circular stirrer. The decalcification was performed twice by suspending coccolithophorid in EDTA (0.5 M) dissolved in MES buffer (0.5 M, pH 5.5) for a few minutes. The FCS was observed for the intact and decalcified cell suspension (1,000 cells/μL) in HEPES buffer (0.5 M at pH8.0). The diffusion times of the intact and decalcified cells were 460 and 250 ms, respectively (rotation speed of the stirrer = 100 rpm). Measuring the flow cytometry based on the front scattering of cells confirmed that the cell size itself was not different for the intact and decalcified cells. This fact shows that the cell with coccolith moved significantly faster, or the movement was synchronized with the flow of water than those without coccolith. The diffusion time of the cells with coccolith decreased along with increase in the rotation speed of the stirrer from 100 to 300 rpm, but those without coccolith were not affected by the speed of the stirrer. Email Address for correspondence: [email protected]



S1.22 Arsenic Speciation in Urine using micro liquid chromatography-ICP-MS Kate Jones (Health Safety Laboratory), Liz Leese (Health Safety Laboratory), Jackie Morton (Health Safety Laboratory), Vikki Carolan (Sheffield Hallam University) Introduction: There are many species of arsenic that are found in the environment, wildlife and humans. These species have differing bioavailability and toxicology. Inorganic arsenic species are carcinogenic, but to assess exposure most laboratories report a ‘total’ arsenic result. Separate quantitation of individual arsenic species allows differentiation of dietary arsenic from inorganic arsenic (and its metabolites) and would improve exposure assessment and risk management. Methods: Separation of five arsenic species (arsenobetaine (AB), arsenite (As3+), arsenate (As5+), dimethylarsinate (DMA), and monomethylarsonate (MMA)) in urine was achieved using a hyphenated µLC (micro liquid chromatography) system, coupled to an inductively coupled plasma – mass spectrometer (ICP-MS). In this study 236 urine samples from a total of 79 workers in the semi-conductor industry were analysed for arsenic. Each worker provided pre and post working week urine samples in spring and autumn. Results: The limits of detection for the analytes were all less than 0.03 յg/L. Overall, the arsenic exposure in semi-conductor workers was low. Both arsenite and arsenate were present at very low levels, all samples giving a 90th percentile value of <0.3 µg/L. Statistical analysis showed there was no significant difference between the pre and post working week samples for any of the five species. However, there were significant differences observed for AB and DMA (gender) and for As3+ (smoking status). Conclusions: A routine method to measure five species of arsenic in urine by յLC-ICP-MS has been established. More data is required to aid the interpretation of the results. Email Address for correspondence: [email protected] S1.23 Human volunteer study to compare absorption and elimination kinetics of different sized titanium dioxide particles Kate Jones (Health Safety Laboratory), Jackie Morton (Health Safety Laboratory) Background: The European Food Standards Agency has recommended that research should be directed at generating information on the toxicokinetic properties of engineered nanomaterials (ENM) after oral exposure as currently the bioavailability, toxicity and toxicokinetics of ENMs cannot be inferred by extrapolation from their non-nano forms.



This study aims to assess the toxicokinetics of nanomaterials following oral intake using titanium dioxide as a model compound. Methods: A human volunteer study (N=9) has been conducted using an oral dose of 5 mg/kg body weight of nanoparticulate titanium dioxide. All voided urine for 24 hours before and 72 hours after dosing was collected in timed samples. Blood samples were collected at 0, 2, 4, 24 and 48 hours. All samples were analysed for titanium using inductively coupled plasma – mass spectrometry. Blood samples were also underwent biochemistry testing. Results: There was no significant difference in 24-hour urinary output of titanium following dosing. Similar results were also obtained for titanium in both full blood and serum. Based on the amount of titanium excreted over three post-dosing days, the recovered dose in urine (converted to TiO2 ) was less than 0.1% for all volunteers. No significant effects on full blood count or liver function tests were observed. Conclusions: This volunteer study has shown no evidence of significant absorption of titanium dioxide nanoparticles. Levels of excreted titanium did not noticeably exceed naturally-occurring background levels, even after a substantial dose, and there were no observable biological effects. Email Address for correspondence: [email protected] S1.24 Denaturation of protein induced by transition metal ions under observation by Fluorescence Correlation Spectroscopy Kana Inoue (School of Life Sciences Tokyo University of Pharmacy and Life Sciences), Keishiro Horiuchi (School of Life Sciences Tokyo University of Pharmacy and Life Sciences), Motohide Aoki (School of Life Sciences Tokyo University of Pharmacy and Life Sciences), Kitao Fujiwara (School of Life Sciences Tokyo University of Pharmacy and Life Sciences) Denaturation of protein induced by transition metal ions (Pb(Ⅱ),Cr(Ⅲ),Ni(Ⅱ)) has been observed by means of the Fluorescence Correlation Spectroscopy(FCS). Optical blocks ® (Hamamatsu Photonics Co.) were combined with a single mode optical fiber laser (473 nm) to construct a con-focal system for the FCS observation. Phycoerythrin(PE)-conjugated mouse monoclonal anti-human VEGF R2/KDR (50 µg/mL) was purchased and used as the target protein. The protein solution was diluted (2000 fold) with MES buffer (10 mM at pH 6.0) at from 300 to 2000 times, and was filtrated with a filter of 0.20 µm pore size. After metal ions of 0~100 mM were added to the protein, the mixed solution was observed using the FCS. As the concentration of metal ions increased, the diffusion time in FCS increased, suggesting the coagulation of protein molecules was progressing.



The power of protein denaturation was in the order of Ni(Ⅱ)<Cr(Ⅲ)<Pb(Ⅱ). When the protein denaturation occurs, the protein molecules were coagulated and the large particles (> 1 µm) were observed under the fluorescence microscope. Email Address for correspondence: [email protected] S1.25 Forensic techniques to distinguish past and current human exposure to PCBs David Megson (Plymouth University), Gwen O'Sullivan (Plymouth University), Maeve Lohan (Plymouth University), Sean Comber (Plymouth University), Paul Worsfold (Plymouth University) In environmental and human exposure studies it may be necessary to establish when an individual was exposed to PCBs. Age dating PCB exposure is a complex task, however assessments may be made because different PCB congeners appear to have different residence times in the human body. Previous studies have shown that less chlorinated congeners tend to have shorter residence times because they are metabolised and excreted faster than the more chlorinated congeners. Currently most investigations assume that the presence of high proportions of less chlorinated congeners is indicative of recent exposure, although in reality this may simply reflect an exposure profile dominated by less chlorinated congeners. Multivariate statistical analysis of the NHANES data showed that less chlorinated congeners are not always less resistant to metabolism. For example, PCB 28 (a tri-chlorobiphenyl) appears to be much more resistant to metabolism than PCB 101 and 110 (penta-chlorobiphenyls). The results show that the prediction of residence times of individual congeners may be improved by considering the exact location of chlorine atoms on the biphenyl rather than by the degree of chlorination alone. Congeners with chlorine atoms in the 2,5- and 2,3,6- positions appear to be very susceptible to metabolism whereas congeners with chlorine bonds in the 2,3,4- 2,4,5- 3,4,5- and 2,3,4,5- positions appear to be more persistent. It may be concluded that future investigations estimating PCB exposure would benefit on identifying the proportions of key congeners selected by the exact position of the chlorine atoms and not just on the degree of chlorination. Email Address for correspondence: [email protected]



S1.26 Drug-Drug Interactions in Daphnia magna Lisa Tucker (Royal Veterinary College), Alistair Boxall (University of York) Pharmaceuticals enter the aquatic environment as complex mixtures from sewage treatment plant effluent. Drug-drug interactions occur in humans when a number of pharmaceuticals are taken together and cause an unwanted adverse effect. The aim of this study is to determine if these drug-drug interactions that can be serious and life threatening in humans also occur in the natural environment. First, by reviewing the information on drug-drug interactions and selecting binary mixtures for study. Second, from the selected compounds perform acute studies in Daphnia magna. Acute EC50 studies of the single compounds were performed on Daphnia magna. The EC50 values determined from these studies were used as the basis for a concentration range used in the mixture EC50 studies. The results show that two of the selected mixtures have no effect and two of the selected mixtures have an additive effect on Daphnia magna. From the results we can see that some mixtures increase the harmful effect in Daphnia magna and therefore the mixture of pharmaceuticals increases the potential damaging effect in aquatic organisms. Further studies include chronic studies looking at the long term effects of the mixtures in Daphnia magna, acute studies in algae and chronic studies in fish. Email Address for correspondence: [email protected] S1.27 Characterisation of Dissolved Organic Carbon in Effluents and the Assessment of its Overall Benefits in Mitigating Environmental Impacts from Metals Dean H Leverett (Wca-environment), Adam Peters (Wca-environment), Anthony Gravell (NLS), Erik Smolders (Division Soil and Water Management Leuven University Belgium), Ronald Smernick (School of Agriculture Food and Wine University of Adelaide Australia), Brian Ellor (UKWIR) DOC is known to form complexes with metals which can markedly reduce the free ion concentration of the metal and mitigate toxicity. BLMs for metals include speciation calculations of DOC with the metal ions, with increasing DOC concentrations usually providing a significant reduction in the bioavailability of the metal. BLMs assume a composition for the DOC in terms of the ratio of humic and fulvic acids. These default DOC characteristics work well for natural waters with generally good agreement between predicted and observed no effect concentrations. Sewage effluent contains high concentrations of DOC but a significant fraction of this organic matter will not



be humic and fulvic acids but proteins, other natural acids, chelating agents such as EDTA, sugars etc. which may bind metals more strongly than predicted by BLMs and so provide greater protection against toxic effects of metals. The DOC in effluents will, therefore, almost certainly interact with metals in a different manner to that predicted by BLMs. Also, any advanced treatment options used to destroy or remove organic chemicals of concern such as oestrogens or pharmaceuticals are likely to either reduce the concentrations of effluent DOC or affect its composition. Therefore, it may be that infrastructure investment intended to remove some organic chemicals may make the receiving waters more sensitive to metals by reducing protective effects of DOC. This poster presents two practical elements designed to investigate the effects of advanced effluent treatment on DOC characterisation and metal speciation. Email Address for correspondence: [email protected] S1.28 Delivering a practical methodology to account for metal (bio)availability in the Water Framework Directive – Case Studies Graham Merrington (Wca-environment), Peter Simpson (Wca-environment), Chris Schlekat (NiPERA), Katrien Delbeke (ECI), Frank Van Assche (IZA), Frederik Verdonck (ARCHE) Significant scientific progress has been made for assessing the risk of metals in surface waters in recent years, both at a technical and regulatory level. The current state of the science has recently been incorporated into the new EU Technical Guidance for deriving Environmental Quality Standards (EQS) under the European Water Framework Directive (WFD). The guidance supports the use of a tiered compliance assessment regime for metals that incorporates bioavailability and recommends using Biotic Ligand Models (BLMs). This approach has been endorsed by the EU Scientific Committee on Health and Environmental Risks (SCHER). The bio-met project has developed an online resource (www.bio-met.net) that includes a user friendly BLM tool (the bio-met bioavailability tool) and associated guidance for simply determining the bioavailability of copper, nickel and zinc in European freshwaters and undertaking compliance assessment against a bioavailability based EQS. This poster describes a series of example case-studies conducted across EU Member States that compare the EQS compliance picture using the bio-met bioavailability tool compared to conventional (i.e. hardness-banded) EQS. Email Address for correspondence: [email protected]



S1.29 Uptake of heavy metals by vegetable plants grown on contaminated soil and their potential bioavailability using sequential extraction Kehinde O Olayinka (University of Lagos), Aderonke O. Oyeyiola (University of Lagos), Fausat A. Odujebe (University of Lagos), Bola O. Oboh (University of Lagos) Heavy metals in soils can bioaccumulate in plants and are transferred to the food chain where they raise human and animal health concern. Plant available fractions of metals were estimated in three vegetable samples -Amaranthus viridis, Celosea argentea and Corchorus olitoriusgrown on three different soil samples. Matured plants were harvested and analysed, soil samples were analysed before and after planting for their pseudototal metal concentration using the flame AAS. Metal accumulation of plants with bioavailability in soils were correlated using sequential extraction (SE) of modified BCR technique. A secondary soil reference material (GLAURM) was used for quality control. It was observed that each metal differed considerably in uptake. Of the plants investigated, Amaranthus viridis gave the highest tissue concentration of Cd, Cr, Pb and Zn from the soils with concentration of 1.4, 5.2 and 3.2 mgkg-1 of Cd, 76.5, 96 and 43.0 mgkg-1 of Cr, 49.5, 20.0 and 27.4 mgkg-1 of Pb and 78.1, 112 and 96.9 mgkg-1 of Zn in all the soils. Results of SE showed that Cd and Zn were mostly present in the acid exchangeable and reducible fractions, where these metals were mostly taken up. Soil–to-plant transfer factor values decreased in the order Zn > Cd > Cr > Pb. Email Address for correspondence: [email protected] S1.30 Molecular mechanisms of adaptation to metals in a population of brown trout (Salmo trutta) inhabiting a contaminated river in the South West of England Tamsyn M Uren Webster (University of Exeter), Nic R Bury (King's College London), Janice A Shears (University of Exeter), Ronny Van Aerle (University of Exeter), Eduarda M Santos (University of Exeter) Metal contamination originating from historical mining activity occurs in many rivers worldwide, yet some still support healthy populations of fish. The River Hayle in South West England is an example of this, where a population of brown trout is found in waters containing high levels of dissolved metals including copper, zinc, nickel and cadmium. Analysis of metal content in a number of tissues of fish originating from this river showed very high concentrations of metals indicating that this population is able to tolerate significant accumulation of metals within tissues. We are conducting global transcriptome



analysis to investigate the molecular mechanisms involved in this metal tolerance.In order to enable these studies, we have first sequenced and assembled the transcriptome of the brown trout. We obtained 147.5 million sequence reads from a variety of tissues (in an Illumina GAII), and assembled these reads into transcripts using a combined de novo and reference-guided approach. Subsequent annotation of these transcripts revealed over 25 thousand unique genes, with a mean length of 1464 bp, equating to approximately 78% coverage of the zebrafish transcriptome. Global gene expression profiling identified a number of genes that were differentially expressed between the metal-tolerant brown trout and trout from a control site (the River Teign). These include genes involved in metal binding and transport, in cellular repair mechanisms and redox cycling. These data will inform on potential tolerance mechanisms to metals in the brown trout and will contribute towards the management of locally adapted fish populations. Email Address for correspondence: [email protected] S1.31 Solid phase extraction to model binding of active pharmaceutical ingredients to sewage sludge Laurence MCBERTHOD (University of Portsmouth AstraZeneca), Gary C Roberts (Astrazeneca), Graham Mills (University of Portsmouth), Alan Sharpe (Astrazeneca), David Whitley (University of Portsmouth) Over the past decade there has been a growing concern over the fate of active pharmaceutical ingredients (APIs) that are released into the aquatic environment. Some APIs can be effectively biodegraded in waste water treatment plants but others can be sorbed onto sludges that are often subsequently used as fertilisers or disposed of as landfill. In order make an accurate risk assessment it is important to know how a given APIwill be distributed between the aqueous or solid phases within a treatment plant. A sorption test to determine the partitioning (determination of the Kd value) of an API in sewage sludge is already available (OPPTS 835.1110), but this method is long and fastidious. Predictions of Kd based on Log Kow have been attempted, but these are not very reliable, especially for ionisable compounds, such as many APIs of interest. Therefore, better predictive models are urgently needed that include binding mechanisms in addition to the hydrophobic interaction. As activated sewage sludge is a complex matrix and hard to fully characterise we used ten commercially available solid phase extraction (SPE) cartridges with different chemistries to try to reproduce these binding processes on three commonly used pharmaceuticals. Various eluents with different organic modifiers and different buffers at a range of pH were tested. The results of these experiments, where the sorption mechanism is known, were compared to results obtained using ‘in house’ prepared cartridges of sludge at different sludge/silica and sludge/silicon carbide ratios.



Email Address for correspondence: [email protected] S1.32 Potential of passive sampling devices to provide bio-available concentrations of pollutants in the aquatic environment Graham A Mills (University of Portsmouth), Richard Greenwood (University of Portsmouth), Janine Bruemmer (University of Portsmouth), Gary R Fones (University of Portsmouth), Geraldine S C Turner (University of Portsmouth), Anthony Gravell (Environment Agency) Passive sampling devices have been used for nearly 25 years in monitoring a wide range of organic and inorganic pollutants in the aquatic environment. A number of devices is available either commercially (e.g. SPMD, Chemcatcher, POCIS and DGT) or as research stage prototypes. Depending on their mode of use, passive samplers can provide either equilibrium or time-weighted average concentrations of pollutants in the water column. In addition, as some of these samplers effectively sequester large volumes of water over a deployment period, they can thereby lower analytical detection limits. Recently, there has been much interest by various end users in using passive samplers in regulatory (e.g. EU-WFD) monitoring activities. Most passive samplers use some type of diffusion limiting membrane or gel that is placed over the receiving phase. This has many functions e.g. limiting the rate of pollutant uptake and reducing biofouling. However, by varying the properties (e.g. pore size, thickness and surface chemistry) of the membrane used it is also possible to gain further information into the bio-availability of chemicals within the aquatic environment. This presentation will highlight the range of samplers that are commercially available and their key construction properties and discuss the how these devices can be used alongside other techniques to add to our understanding of the bio-available fraction of pollutants in water and give data to improve risk assessments and policy decisions. Email Address for correspondence: [email protected]



S1.33 The use of transgenic nematodes (Caenorhabditis elegans) as a novel bioassay for the assessment of urban river sediment toxicity Alexandra Whicher (Department of Geography King's College London), Chris Hopkins (KNUDRA Transgenics Salt Lake City Utah USA.), Florent Tarroux (UMR 5808 EPOC-OASU Universite Bordeaux), Stephen Sturzenbaum (Analytical and Environmental Sciences Division King's College London), Nic Bury (Diabetes and Nutritional Sciences Division King's College London) River sediment toxicity is not considered under the Water Framework Directive due to a limited understanding of the complex interactions that control contaminant toxicity in sediments, along with a lack of reliable toxicological data for sediment dwelling organisms. To address this issue we explored the use of transgenic nematodes (Caenorhabditis elegans) as a bioassay tool for the assessment of sediment toxicity from an urban river; the River Wandle, London. Sediment was collected from 6 sites, lyopholised and sieved to under 63 μm before extraction of organic pollutants with dichloromethane/hexane and dichloromethane/acetone. The extracts were dried and re-suspended in DMSO. Four strains of transgenicC. Elegans were exposed on agar plates to the equivalent of 0.5, 5 and 50 mg of dry sediment by mixing the extract with E. Coli bacteria (the food source). The nematode strains were genetically engineered to contain a stable genome-integrated copy of the target gene linked to a red fluorescent reporter (mCherry). In detail, biosensors includedcyp-35A2::mCherry (a cytochorme P450 homolog),mtl-2::mCherry (a metallothionein),hsp-4::mCherry (a heat shock protein) andgst-4::mCherry (a glutathione-s-transferase). Single worms were exposed for 48hrs and relative changes in mCherry fluorescence measured in vivo. These results were correlated to exposure induced changes in growth. Overall, we were able to demonstrate that gene expression is a more sensitive indicator of sediment pollutant load than growth. Furthermore, we were able to differentiate between sites via a ranking system and reproducibly pinpoint the most polluted sediment; notably without the need of a prior knowledge of the chemical composition or interactions within the sediment. Email Address for correspondence: [email protected]

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