1. EVALUATION OF TOTAL AND SPECIES CONTAMINATION FROM ARSENIC IN THE RIVERS AND BAY OF THE PAK PA-NANG CATCHMENT, SOUTHERN THAILAND EVALUATION OF TOTAL AND SPECIES CONTAMINATION FROM ARSENIC IN THE RIVERS AND BAY OF THE PAK PA-NANG CATCHMENT, SOUTHERN THAILAND M. E. Foulkes 1, S. Rattanachongkiat 3, G.E. Millward 1, W. Utoomprurkporn 2, M. Taiyaqupt 2, P. Chongprasith 3 and P. Tantichodok 4 1 School of E, O and Environmental Sciences, University of Plymouth, Plymouth PL4 8AA UK 2 Faculty of Science, Chulalongkorn University, Bangkok; 3 Pollution Control Department, MNRE Bangkok; 4 Walailak University, Nakhon Si Thammarat, Thailand INTRODUCTION The Pak Pa-Nang Estuary is located in southern Thailand and its catchment comprises a tin mining area. More than 1,000 inhabitants of the region are suffering from various stages of arsenic (As) poisoning. Drainage from the high concentration of As in spoil tips of the mined area could affect water and sediment quality in the bay, which is biologically productive, including substantial mussel aquaculture. Information on the speciation of arsenic in this bay will help identify any current toxicity problem and also serve in formulating protection strategies for the future. AIMS OF THE STUDY  Determination of arsenic speciation in fauna and sediment samples using High – Performance Liquid Chromatography (HPLC) coupled with Inductively Coupled Performance Liquid Chromatography (HPLC) coupled with Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Plasma Mass Spectrometry (ICP-MS)  Application of the technique to assess the impact of As contamination to the Pak Pa-Nang Estuary Pa-Nang EstuaryMETHODOLOGY Sediments and commercial sea foods, [sardines (Escualosa thoracata), croakers (Johnius belangerii), catfish (Plotosus canius) and swimming crabs (Portunus pelagicus)] were collected from the Pak Pa-Nang Estuary in August 2001 and immediately freeze-dried. The dried samples were ground, using an agate mortar and pestle, and digested for total As in a Teflon bomb by microwave digestion using nitric acid and hydrogen peroxide. Following the digestion total As was determined using N 2 – ICP-MS ☻. Arsenic speciation studies in the samples were employed using HPLC coupled with ICP- MS following a low power microwave extraction and an enzymatic extraction for sediment and fauna samples, respectively. Speciation studies of arsenic are necessary because the toxicity of arsenic depends on the nature of its species rather than total concentration. ☻ The combination of chlorine introduced via the sample with argon from the plasma can give rise to the formation of 40 Ar 35 Cl +, which interferes with the monoisotopic 75 As + ; the problem was solved by adding the molecular gas nitrogen 40 Ar 35 Cl +, which interferes with the monoisotopic 75 As + ; the problem was solved by adding the molecular gas nitrogen [about 4.5 % (v/v) of total carrier gas] to the nebulizer gas of ICP-MS (N 2 – ICP-MS) [1] [about 4.5 % (v/v) of total carrier gas] to the nebulizer gas of ICP-MS (N 2 – ICP-MS) [1] THE STUDY AREA IS CLOSE TO FORMER TIN MINING AREAS AND THOUSANDS OF PEOPLE IN THE FORMER MINING AREAS ARE SUFFERING FROM ARSENIC POISONING  Former tin mining area Pak Pa-Nang Estuary Black spot disease Boat-launching Long Tail Fishing boat Freeze-dried crabs 0.25 g dry sample 4 ml HNO 3 + 1 ml H 2 O 2 Microwave digestion for 5 min 50 ml solution 50 ml solution + N 2 -ICP-MS Analysis N 2 -ICP-MS Analysis Sardines (Escualosa thoracata) REFERENCES 1 Hill, S.J, Ford, M.J., and Ebdon, L., J. Anal. At. Spectrom., 1992, 7, 719. 1 Hill, S.J, Ford, M.J., and Ebdon, L., J. Anal. At. Spectrom., 1992, 7, 719. 2 Penrose, W.R., CRC Crit. Rev. Environ. Control, 1974, 4, 465. 2 Penrose, W.R., CRC Crit. Rev. Environ. Control, 1974, 4, 465. 3 Brown, J., Kitchen, K.and George, M., Teratog. Carcinog. Mutagen, 1997, 17, 71. 3 Brown, J., Kitchen, K.and George, M., Teratog. Carcinog. Mutagen, 1997, 17, 71. 4 Cullen, W.R.and Reimer, K.J., Chem. Rev., 1989, 89, 713. 4 Cullen, W.R.and Reimer, K.J., Chem. Rev., 1989, 89, 713. 5 Neff, J.M., Environ. Toxicol. Chem., 1997, 5, 917. 5 Neff, J.M., Environ. Toxicol. Chem., 1997, 5, 917. 6 Francesconi, K.A. and Edmonds, J.S., Arsenic and Marine Organisms. Advances in 6 Francesconi, K.A. and Edmonds, J.S., Arsenic and Marine Organisms. Advances in Inorganic Chemistry., 1997, 44, 147. Inorganic Chemistry., 1997, 44, 147. 7 Branch, S., Ebdon, L. and O’neill, P., J. Anal. At. Spectrom., 1994, 9, 33. Catfish (Plotosus canius)Swimming crabs (Portunus pelagicus) Croakers (Johnius belangerii) ARSENIC SPECIES IN THE FAUNA SAMPLES DISCUSSION AND CONCLUSIONS Methods have been developed for the determination of As and its species in fauna and sediments from the Pak Pa-Nang Estuary, using analytical procedures that gave near to full extraction efficiencies and recoveries. While the As concentration in the fauna and sediment samples is relatively low (not greater than 16 µg g -1 ) the major species present and available for cycling are different for the two types of sample analysed. In fish and crustaceans the major species present is the non-toxic arsenobetaine (75 to 80%) with smaller quantities of the mildly toxic DMA (11 to 17%). The highly toxic inorganic As species (AsIII and AsV) constituted some 5 to 12% of the total As in fauna. This converts to approximately 2 µg g -1 inorganic As for a consumable fish or crustacean, at the higher total As content found of 16 µg g -1. ‘Advisable levels’ for As in foodstuffs suggest a 1 µg g -1 limit on inorganic As particularly where the foodstuff constitutes a regular or staple diet. Only the highly toxic inorganic As species (AsIII and AsV) were found in the sediment samples. Considering the dynamic conditions found in the estuary together with the part that benthic organisms play in the estuarine food chain, the supply of these highly toxic As species to humans is likely to continue. This may be for many years, particularly when the levels of arsenic in sample cores are considered. ☺ August 2002 10 km ARSENIC SPECIES AND THEIR TOXICITY Sediment sampling This work sponsored by The Royal Thai Government The British Council ACKNOWLEDGEMENTS The authors gratefully acknowledge University of Plymouth, Chulalongkorn and Walailak Universities and also their staff for their very kind help in the laboratories and with the field sampling. PAK PA-NANG SAMPLING (AUGUST 2001) Sediment core sample 0.25 g dry fish 0.1 g trypsin in 0.1 M NH 4 HCO 3 Extraction in a shaking bath (37 ° C) for 12 hr 25 ml solution 25 ml solution + HPLC-ICP-MS Analysis HPLC-ICP-MS Analysis 0.5 g dry sediment 25 ml 1M H 3 PO 4 Extraction in a microwave digester (45w) for 20 min 25 ml solution 25 ml solution + HPLC-ICP-MS Analysis HPLC-ICP-MS Analysis HPLC System for As Species in Fish ColumnHamilton Resin PRP-X100 10µm i.c. ( 250 x 4.6 mm ) Injection loop/µl100 Flow rate/ml min -1 1.5 Mobile phasesa: 5 m mol l -1 Na 2 SO 4 pH 10-10.5 * b: 0.05 mol l -1 Na 2 SO 4 pH 10-10.5 * Standard solution200 ppb AsB, DMA, MMA and Inorganic As Retention time/min15 * Adjusted with ammonia solution HPLC System for As Species in Sediments ColumnHamilton Resin PRP-X100 10µm i.c. ( 250 x 4.6 mm ) Injection loop/µl20 Flow rate/ml min -1 1.2 Mobile phasesa: 2 m mol l -1 H 3 PO 4 pH 7.5 * b: 50 m mol l -1 H 3 PO 4 pH 6 * Standard solution100 ppb As III, DMA, MMA and As V Retention time/min15 * Adjusted with ammonia solution Chromatogram of Standards TOTAL ARSENIC IN THE SAMPLES COMPARED WITH OTHER AREAS DISTRIBUTION OF ‘TOTAL AVAILABLE’ ARSENIC IN THE SEDIMENT SAMPLES 1 2 3 Station 1Station 3Station 4 ARSENIC SPECIES IN THE SEDIMENT SAMPLES (µg g -1 ) 4 5 6 7 8 9 Chromatogram of Standards X Sediment sampling station X Sediment core sampling station *Extraction efficiency of arsenic using phosphoric acid compared with ‘total available’ arsenic in sediments ranged from 95 to 108 %. Recovery of species from spiking of sediments ranged from 90 to 100%. DIGESTION PROCEDURE FOR TOTAL As IN FISH AND AVAILABLE As IN SEDIMENT EXTRACTION OF As SPECIES FROM FISH EXTRACTION OF As SPECIES FROM SEDIMENT HPLC cycle -Isocratic elution –Step gradient –Re-equilibrate Mobile phase >a 5 min >b 3 min >a till finish HPLC cycle -Isocratic elution –Step gradient –Re-equilibrate Mobile phase >a 3 min >b 6 min >a till finish M. Foulkes1