INTRODUCTION

1. EVALUATION OF TOTAL AND SPECIES CONTAMINATION FROM ARSENIC IN THE RIVERS AND BAY OF THE PAK PA-NANG CATCHMENT, SOUTHERN THAILAND M. E. Foulkes1, S.Rattanachongkiat3, G.E. Millward1, W. Utoomprurkporn2, M. Taiyaqupt2, P. Chongprasith3 and P. Tantichodok4 1 School of E, O and Environmental Sciences, University of Plymouth, Plymouth PL4 8AA UK 2Faculty of Science, Chulalongkorn University, Bangkok; 3Pollution Control Department, MNRE Bangkok; 4Walailak 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 Plasma Mass Spectrometry (ICP-MS) • Application of the technique to assess the impact of As contamination to the Pak Pa-Nang Estuary METHODOLOGY • 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 N2– 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. ARSENIC SPECIES IN THE SEDIMENT SAMPLES (µg g-1) DIGESTION PROCEDURE FOR TOTAL As IN FISH AND AVAILABLE As IN SEDIMENT EXTRACTION OF As SPECIES FROM FISH EXTRACTION OF As SPECIES FROM SEDIMENT 0.25 g dry sample 0.25 g dry fish 0.5 g dry sediment + + + 4 ml HNO3 + 1 ml H2O2 0.1 g trypsin in 0.1 M NH4HCO3 25 ml 1M H3PO4 Microwave digestion for 5 min Extraction in a shaking bath (37°C) for 12 hr Extraction in a microwave digester (45w) for 20 min 50 ml solution 25 ml solution 25 ml solution N2-ICP-MS Analysis HPLC-ICP-MS Analysis HPLC-ICP-MS Analysis *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%. DISTRIBUTION OF ‘TOTAL AVAILABLE’ ARSENIC IN THE SEDIMENT SAMPLES HPLC System for As Species in Fish ColumnHamilton Resin PRP-X100 10µm i.c. ( 250 x 4.6 mm ) Injection loop/µl 100 Flow rate/ml min-1 1.5 Mobile phases a: 5 m mol l-1 Na2 SO4 pH 10-10.5* b: 0.05 mol l-1 Na2 SO4 pH 10-10.5* Standard solution 200 ppb AsB, DMA, MMA and Inorganic As Retention time/min 15 Chromatogram of Standards M. Foulkes1 HPLC cycle -Isocratic elution –Step gradient –Re-equilibrate Mobile phase >a 5 min >b 3 min >a till finish ARSENIC SPECIES AND THEIR TOXICITY Station 1 Station 3 Station 4 *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/µl 20 Flow rate/ml min-1 1.2 Mobile phases a: 2 m mol l-1 H3 PO4 pH 7.5* b: 50 m mol l-1 H3 PO4 pH 6* Standard solution 100 ppb AsIII, DMA, MMA and AsV Retention time/min 15 Chromatogram of Standards 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. THE STUDY AREA IS CLOSE TO FORMER TIN MINING AREAS AND THOUSANDS OF PEOPLE IN THE FORMER MINING AREAS ARE SUFFERING FROM ARSENIC POISONING 4 9 5 8 Pak Pa-Nang Estuary HPLC cycle -Isocratic elution –Step gradient –Re-equilibrate Mobile phase >a 3 min >b 6 min >a till finish 3 6 7 *Adjusted with ammonia solution 2 1 TOTAL ARSENIC IN THE SAMPLES COMPARED WITH OTHER AREAS X Sediment sampling station X Sediment core sampling station  Former tin mining area 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. 10 km Black spot disease ARSENIC SPECIES IN THE FAUNA SAMPLES PAK PA-NANG SAMPLING (AUGUST 2001) REFERENCES 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. 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. 5 Neff, J.M., Environ. Toxicol. Chem., 1997, 5, 917. 6 Francesconi, K.A. and Edmonds, J.S., Arsenic and Marine Organisms. Advances in Inorganic Chemistry., 1997, 44, 147. 7 Branch, S., Ebdon, L. and O’neill, P., J. Anal. At. Spectrom., 1994, 9, 33. Boat-launching Long Tail Fishing boat Sediment sampling Sediment core sample Freeze-dried crabs This work sponsored by ☻The combination of chlorine introduced via the sample with argon from the plasma can give rise to the formation of 40Ar35Cl+, which interferes with the monoisotopic 75As+ ; 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 (N2– ICP-MS) [1] Croakers (Johnius belangerii) Sardines (Escualosa thoracata) Catfish (Plotosus canius) Swimming crabs (Portunus pelagicus) The Royal Thai Government The British Council ☺ August 2002