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As [mg kg -1 ] Arsenic speciation in seaweeds using liquid chromatography hydride generation atomic fluorescence spectrometry (HPLC-HG-AFS) Liam Morrison 1, Bin Chen 2 and Warren T. Corns 2 1, Earth and Ocean Sciences, National University of Ireland, Galway, Ireland 2, P S Analytical Ltd, Kent, UK Background and Aims AcknowledgementsReferences Morrison et al., 2008. An assessment of metal contamination along the Irish coast using the seaweed Ascophyllum nodosum (Fucales, Phaeophyceae). Environmental Pollution 152, 293-303 Murcott, S. 2012. Arsenic Contamination in the World: An International Sourcebook. IWA Publishing, London, pp.282. Sirot et al., 2009. Dietary exposure and biomarkers of arsenic in consumers of fish and shellfish from France. Science of the Total Environment 407, 1875-1885. Villaescusa, I., Bollinger, J.-C. 2008. Arsenic in drinking water: sources, occurrence and health effects (a review). Reviews in Environment Science and Biotechnology 7, 307-323. Seaweeds accumulate metals to levels several orders of magnitude higher than those present in the surrounding seawater and have been widely used as biomonitors of water quality, avoiding the logistical difficulties associated with representative and comparative sampling of seawater and sediments (Morrison et al., 2008). Furthermore, they integrate short-term fluctuation in metal concentrations and reflect bioavailable concentrations in the water. Arsenic exists in both inorganic (i-As) or organic forms (o-As) and toxicity depends on the chemical form (Villaescusa and Bolloinger, 2008). Although elevated concentrations of arsenic have been reported in groundwater that affects an estimated 226 million people from 105 countries (Murcott, 2012), marine organisms exhibit much higher concentrations of arsenic. A recent European study has highlighted that algae, fish and shellfish remain a major sources of arsenic exposure for humans (Sirot et al., 2009). Consequently, the speciation analysis of arsenic in marine biota is important in terms of public health and ecotoxicology. The present study investigated arsenic speciation in three commercially important species of brown seaweed, Ascophyllum nodosum Linnaeus, Le Jolis, Fucus vesiculosus Linnaeus (Fucales, Phaeophyceae), and Laminaria digitata (Hudson) J. V. Lamouroux, (Laminariales, Phaeophyceae) collected in Galway Bay, Ireland (Fig. 1). In addition, metal concentrations were determined in different-aged tissue regions of A. nodosum up to a maximum age of ten years. Data analysis and graphing were performed using the statistical package R (http://www.R-project) and SigmaPlot® 12.http://www.R-project The study was funded by an Innovation Voucher (IV 2012 0026T) from Enterprise Ireland. This work includes Ordnance Survey Ireland data reproduced under OSi Licence number NUIG200803. Unauthorised reproduction infringes Ordnance Survey Ireland and Government of Ireland copyright. © Ordnance Survey Ireland, 2012. Conclusions and Future Research Methods Ireland Galway Bay Total Arsenic in Seaweeds Fig. 1. Map of sampling locations. Fig. 2. Inis an Ghainimh sampling site and Intertidal zone. Figs. 3. and 4. Ascophyllum nodosum. Fig. 5. Fucus vesiculosus. Fig. 6. Laminaria digitata. Fig. 1Fig. 2 Fig. 3 Fig. 4Fig. 5 Fig. 6 Year 1 Year 2 Ascophyllum nodosum, showing tissue regions differentiated by annual vesicle formation. As [mg kg -1 ] Fig. 7. Arsenic concentrations ranged from 27.1 – 53.4 mg kg -1 in A. nodosum, 28.9 – 53.6 mg Kg -1 in F. vesiculosus and 54.3 – 83.2 mg Kg -1 in L. digitata. Tukey’s HSD showed significant differences between the mean As level in L. digitata vs A. nodosum and F. vesiculosus vs L. digitata. Arsenic Speciation in L. digitata Fig. 8. Total As in different-aged tissue regions of A. nodosum. There was evidence of significant differences between the average concentrations of As in Years 5 & 6 versus Years 3 & 4.There was no evidence of a significant difference in As levels between the remaining years. The data from this study represent baseline levels of As in commercially important Irish seaweeds. Future work will focus on As speciation in different- aged seaweed tissue. Previously, inorganic arsenic concentrations in L. digitata were established using a selective hydride generation method (SHG) after sample was digested by HNO 3 and H 2 O 2, comparing well with the HPLC analysis. Four As species were found in L. digitata, As(III), As(V), sugar-1 and sugar-2 (S1 and S2). We presume these are arseno-sugars as it is common to find arseno-sugars in seaweed at high concentrations, and also as the retention time of these two species were different from the species tested. HPLC data represent ‘water extractable arsenic species’. The summation of the 4 species found by HPLC approach is far lower than the total arsenic data established by UV-HG-AFS after nitric/peroxide digestion. Therefore it is clear that the majority of the arsenic species (such as sugars, lipids etc.) were not extracted by hot water, while high extraction efficiencies were obtained for inorganic arsenic using water only. Total Inorganic Arsenic in Seaweeds Seaweed Sampling (n=20) Freeze Drying (- 50 °C) Total Analysis Hydride Generation - Atomic Fluorescence Spectrometry (HG-AFS) Pulverising and Homogenisation (Agate Ball Mill) Total As Extraction HNO 3, HCl and H 2 O 2 Speciation Analysis Liquid Chromatography - Hydride Generation - Atomic Fluorescence Spectrometry (HPLC-HG-AFS) Extraction for Speciation Water extraction in a water bath heated at 90C for 60 minutes (water extraction was used to avoid arseno-sugar breakdown) Total Inorganic As (iAs) [mg kg -1 ]% iAs Between Seaweeds Fig. 7Fig. 8Fig. 9Fig. 10 Fig. 9. Inorganic As concentration (mg kg -1 ) were significantly higher (p<0.001) in L. digitata when compared to A. nodosum and F. vesiculosus. Fig. 10. The mean % iAs level in A. nodosum was approximately 0.1% higher than in F. vesiculosus, while the mean % iAs level in L. digitata was 5% higher than the mean % iAs level in A. nodosum and approximately 5.1% higher than the mean % iAs level in F. vesiculosus (outlier included). Fig. 11. Chromatogram for arsenic species (water extracts) in L. digitata using HPLC coupled to HG-AFS. MMA and DMA was not detected in L. digitata. Fig. 12. Total As, total inorganic arsenic and concentrations of four species of As observed ( As(III), As(V), sugar-1 and sugar-2 (S1 and S2)) in L. digitata. Arsenic species in L. digitata [mg kg -1 ] Fig. 11 Fig. 12
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