0

5

10

15

20

25

30

2 6 10 14 18

Conc.(ug/g)

Dep

t (cm

)

0

5

10

15

20

25

30

2 6 10 14 18

Conc.(ug/g)

Dep

t (cm

)

0

5

10

15

20

25

30

2 6 10 14 18

Conc.(ug/g)

Dep

t (cm

)

4 -8 u g /g

8 -1 2 u g /g

12 -1 6 u g /g

EVALUATION OF TOTAL AND SPECIES CONTAMINATION FROM ARSENIC IN THE EVALUATION OF TOTAL AND SPECIES CONTAMINATION FROM ARSENIC IN THE RIVERS AND BAY OF THE PAK PA-NANG CATCHMENT, SOUTHERN THAILANDRIVERS AND BAY OF THE PAK PA-NANG CATCHMENT, SOUTHERN THAILAND

M. E. FoulkesM. E. Foulkes11, S., S. RattanachongkiatRattanachongkiat33, G.E. Millward, G.E. Millward11, W. Utoomprurkporn, W. Utoomprurkporn22, M. Taiyaqupt, M. Taiyaqupt22,,P. ChongprasithP. Chongprasith33 and P. Tantichodok and P. Tantichodok44

11 School of E, O and Environmental Sciences, University of Plymouth, Plymouth PL4 8AA UK School of E, O and Environmental Sciences, University of Plymouth, Plymouth PL4 8AA UK22Faculty of Science, Chulalongkorn University, Bangkok; Faculty of Science, Chulalongkorn University, Bangkok; 33Pollution Control Department, MNRE Bangkok;Pollution Control Department, MNRE Bangkok;

44Walailak University, Nakhon Si Thammarat, Thailand Walailak University, Nakhon Si Thammarat, Thailand INTRODUCTIONINTRODUCTION

The Pak Pa-Nang Estuary is located in southern Thailand and its catchment comprises a 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 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 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 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 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 arsenic in this bay will help identify any current toxicity problem and also serve in formulating protection strategies for the future.formulating protection strategies for the future.

AIMS OF THE STUDYAIMS OF THE STUDY

Determination of arsenic speciation in fauna and sediment samples using Determination of arsenic speciation in fauna and sediment samples using High – 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 techniqueApplication of the technique to assess the impact of As contamination to to assess the impact of As contamination to the Pak the Pak Pa-Nang EstuaryPa-Nang Estuary

METHODOLOGYMETHODOLOGY

Sediments and commercial sea foods, [sardines (Sediments and commercial sea foods, [sardines (Escualosa thoracataEscualosa thoracata), croakers (), croakers (Johnius Johnius belangeriibelangerii), catfish (), catfish (Plotosus caniusPlotosus canius) and swimming crabs () and swimming crabs (Portunus pelagicusPortunus pelagicus)] were )] were collected from the Pak Pa-Nang Estuary in August 2001 and immediately freeze-dried. The 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 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 Teflon bomb by microwave digestion using nitric acid and hydrogen peroxide. Following the digestion total the digestion total As As was determined using Nwas determined using N22–– ICP-MS ICP-MS☻☻. .

Arsenic speciation studies in the samples were employed using HPLC coupled with ICP-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 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 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. 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 The combination of chlorine introduced via the sample with argon from the plasma can give rise to the formation of 4040ArAr3535ClCl++, which interferes with the monoisotopic , which interferes with the monoisotopic 7575AsAs++ ; the problem was solved by adding the molecular gas nitrogen ; 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[about 4.5 % (v/v) of total carrier gas] to the nebulizer gas of ICP-MS (N22 –– ICP-MS) ICP-MS) [1][1]

As species Toxicity Inorganic species

arsenite (AsIII), arsenate (AsV) extremely toxic, carcinogenic [2]

Organic species

monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) cancer promoter [3]

arsenobetaine (AsB) non-toxic [4-5]

THE STUDY AREA IS CLOSE TO FORMER TIN MINING AREAS AND THOUSANDS OF THE STUDY AREA IS CLOSE TO FORMER TIN MINING AREAS AND THOUSANDS OF PEOPLE IN THE FORMER MINING AREAS ARE SUFFERING FROM ARSENIC POISONINGPEOPLE IN THE FORMER MINING AREAS ARE SUFFERING FROM ARSENIC POISONING

Former tin mining area

Pak Pa-Nang Estuary

Black spot disease

Boat-launchingBoat-launching Long Tail Fishing Long Tail Fishing boatboat

Freeze-dried crabsFreeze-dried crabs

0.25 g dry sample0.25 g dry sample

4 ml HNO4 ml HNO33 + 1 ml H + 1 ml H22OO22

Microwave digestion Microwave digestion for 5 minfor 5 min

50 ml solution50 ml solution

+

NN22-ICP-MS Analysis-ICP-MS Analysis

0%

Inorganic As6%

AsB77%

DMA17% 0%

Inorganic As12%

AsB77%

DMA11% 0%

Inorganic As9%

AsB75%

DMA16%

Sardines (Escualosa thoracata)

REFERENCESREFERENCES

1 Hill, S.J, Ford, M.J., and Ebdon, L., 1 Hill, S.J, Ford, M.J., and Ebdon, L., J. Anal. At. SpectromJ. Anal. At. Spectrom., 1992, 7, 719.., 1992, 7, 719. 2 2 Penrose, W.R., Penrose, W.R., CRC Crit. Rev. Environ. ControlCRC Crit. Rev. Environ. Control, 1974, 4, 465. , 1974, 4, 465. 33 Brown, J., Kitchen, K.and George, M., Brown, J., Kitchen, K.and George, M., Teratog. Carcinog. MutagenTeratog. Carcinog. Mutagen, 1997, 17, 71., 1997, 17, 71. 44 Cullen, W.R.and Reimer, K.J., Cullen, W.R.and Reimer, K.J., Chem. Rev.Chem. Rev., 1989, 89, 713., 1989, 89, 713. 5 Neff, J.M., 5 Neff, J.M., Environ. Toxicol. Chem.Environ. Toxicol. Chem., 1997, 5, 917., 1997, 5, 917. 6 Francesconi, K.A. and Edmonds, J.S., Arsenic and Marine Organisms. 6 Francesconi, K.A. and Edmonds, J.S., Arsenic and Marine Organisms. Advances in Advances in Inorganic ChemistryInorganic Chemistry., 1997, 44, 147.., 1997, 44, 147. 7 7 Branch, S., Ebdon, L. and O’neill, P., Branch, S., Ebdon, L. and O’neill, P., J. Anal. At. SpectromJ. Anal. At. Spectrom., 1994, 9, 33.., 1994, 9, 33.

Catfish (Plotosus canius) Swimming crabs (Portunus pelagicus)Croakers (Johnius belangerii)

ARSENIC SPECIES IN THE FAUNA SAMPLESARSENIC SPECIES IN THE FAUNA SAMPLES

0%Inorganic As

5%

AsB80%

DMA15%

DISCUSSION AND CONCLUSIONSDISCUSSION AND CONCLUSIONS

Methods have been developed for the determination of As and its species in fauna and sediments from the 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 Pak Pa-Nang Estuary, using analytical procedures that gave near to full extraction efficiencies and recoveries.recoveries.  While the As concentration in the fauna and sediment samples is relatively low (not greater than 16 µg gWhile the As concentration in the fauna and sediment samples is relatively low (not greater than 16 µg g -1-1) ) the major species present and available for cycling are different for the two types of sample analysed. 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 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) 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 gconstituted some 5 to 12% of the total As in fauna. This converts to approximately 2 µg g -1-1 inorganic As for inorganic As for a consumable fish or crustacean, at the higher total As content found of 16 µg ga consumable fish or crustacean, at the higher total As content found of 16 µg g -1-1. ‘Advisable levels’ for As . ‘Advisable levels’ for As in foodstuffs suggest a 1 µg gin foodstuffs suggest a 1 µg g-1-1 limit on inorganic As particularly where the foodstuff constitutes a regular limit on inorganic As particularly where the foodstuff constitutes a regular or staple diet. or staple diet.   Only the highly toxic inorganic As species (AsIII and AsV) were found in the sediment samples. 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 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. 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. 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 TOXICITYARSENIC SPECIES AND THEIR TOXICITY

Sediment samplingSediment sampling This work sponsored byThis work sponsored by

The Royal Thai GovernmentThe Royal Thai Government The British CouncilThe British Council

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

The authors gratefully acknowledge University of Plymouth, Chulalongkorn and Walailak Universities and also their 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.staff for their very kind help in the laboratories and with the field sampling.

PAK PA-NANG SAMPLING (AUGUST 2001)PAK PA-NANG SAMPLING (AUGUST 2001)

Sediment core sampleSediment core sample

0.25 g dry fish0.25 g dry fish

0.1 g trypsin in 0.1 M NH0.1 g trypsin in 0.1 M NH44HCOHCO33

Extraction in a Extraction in a shaking bath (37shaking bath (37°°C) for C) for

12 hr12 hr

25 ml solution25 ml solution

+

HPLC-ICP-MS AnalysisHPLC-ICP-MS Analysis

0.5 g dry sediment0.5 g dry sediment

25 ml 1M H25 ml 1M H33POPO44

Extraction in a microwave Extraction in a microwave digester (45w) for 20 mindigester (45w) for 20 min

25 ml solution25 ml solution

+

HPLC-ICP-MS AnalysisHPLC-ICP-MS Analysis

HPLC System for As Species in Fish

Column Hamilton 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

* Adjusted with ammonia solution

HPLC System for As Species in Sediments

Column Hamilton 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

* Adjusted with ammonia solution

Chromatogram of Standards

Range MeanFinfish Australia (9)[6] 0.8-14 6.5

Plymouth (8)[7] 1-187 66Pak Pa-Nang (3) 3-8 6

Crustaceans Australia (5)[6] 7-91 27Pak Pa-Nang (1) 14-16 15

Sediment Thames[6] 4-16 -Pak Pa-Nang 5-16 12

Location (no. of species)TypeArsenic concentration (µg g-1 dry wt.)

TOTAL ARSENIC IN THE SAMPLES COMPARED WITH OTHER AREASTOTAL ARSENIC IN THE SAMPLES COMPARED WITH OTHER AREAS

 

DISTRIBUTION OF ‘TOTAL AVAILABLE’ ARSENIC IN THE SEDIMENT SAMPLESDISTRIBUTION OF ‘TOTAL AVAILABLE’ ARSENIC IN THE SEDIMENT SAMPLES

1

2

3

Station 1 Station 3 Station 4

ARSENIC SPECIES IN THE SEDIMENT SAMPLES ARSENIC SPECIES IN THE SEDIMENT SAMPLES (µg g(µg g-1-1))

45

6 7

8

9

Chromatogram of Standards

X Sediment sampling stationX 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%.

Available As AsIII* AsV*1 14.1 1.7 (11%) 13.7 (89%)2 11.7 1.2 (10%) 11.2 (90%)3 12.9 1.4 (11%) 11.3 (89%)4 14.0 1.4 (10%) 12.6 (90%)

Surface 5 6.1 0.7 (12%) 5.2 (88%)6 12.0 1.5 (12%) 11 (88%)

7 9.4 1.7 (20%) 6.9 (80%)8 6.8 1.0 (14%) 6.2 (86%)9 7.9 1.1 (12%) 7.8 (88%)1 11.5 0.8 (7%) 10.8 (93%)

Sub-surface 3 8.0 0.4 (5%) 7.2 (95%)4 13.8 0.8 (6%) 12.4 (94%)

StationDIGESTION PROCEDURE DIGESTION PROCEDURE FOR TOTAL As IN FISH AND FOR TOTAL As IN FISH AND

AVAILABLE As IN SEDIMENTAVAILABLE As IN SEDIMENT

EXTRACTION OF As SPECIES EXTRACTION OF As SPECIES FROM FISH FROM FISH

EXTRACTION OF As SPECIES EXTRACTION OF As SPECIES FROM SEDIMENT 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

200.00 400.00 600.00 800.00rt0

100

%

Inorganic As

MMADMA

AsB

200.00 400.00 600.00 800.00rt0

100

%

As(V)

MMA

DMA

As(III)

M. Foulkes1M. Foulkes1