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*»Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡ ‡∑§‚π∏“π’ µ.§≈ÕßÀâ“ Õ.§≈ÕßÀ≈«ß ®.ª∑ÿ¡∏“π’ 12120 ‚∑√. 0-2577-1136 ‚∑√ “√. 0-2577-1138 Environmental Research and Training Center, Department of Environmental Quality Promotion. Technopolis. Klong 5 Klong Luang, Pathumthani 12120 e-mail: Sukanya@deqp.go.th **ºŸâ‡™’ˬ«™“≠Õߧå°√§«“¡√à«¡¡◊Õ√–À«à“ߪ√–‡∑»≠’˪ÿÉπ (JICA Expert)

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ABSTRACT This paper presents a study of Arsenic (As) contamination at Nakorn Sri Thammaraj province, which is known as the tin-mining area especially in Ronpibool District where several tin mines are still operational.

Arsenic from this area has been out flowing through Pak Pa-Nang river into Pak Pa-Nang bay in the southern part of Thailand which is the important area for aquatic organism culture. The study focuses on the marine environment of Pak Pa-Nang bay with regard to possible influence of long term arsenic input from the upstream of Pak Pa-Nang river.

The various biosample including mussel, fish, shrimp are collected and analyzed for total arsenic by acid digestion and AAS analysis.

Based on the relative arsenic concentration level, mussel was selected for further study. Mussel samples from the bay and from open sea representing background were systematically collected and analyzed for arsenic. The difference between two groups (inside and outside of the bay) were examined in order to understand the influence of arsenic in the bay. Arsenic species-distribution of mussel samples were also studied »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡

by MeOH/water-ultrasonic extraction followed

by HPLC-ICP-MS analysis. The data indicated arsenic composition of the mussel in the bay have no unique character compared with those reported in previous study.

1. Introduction Natural arsenic commonly enriches in gold and base metal mineralization.

Mining and mineral processing is often became a source of arsenic contamination to environment. The granites of the southern part of Peninsula Thailand occurred as isolated plutons associated with numerous tin and tungsten deposits. Hence the region is called çtin belté (1). Extensive mining of tin and associated minerals have been done throughout the region. Ron-Phibun district, Nakorn Sri-Tammaraj Province, which lies in the main range of the tin belt, approximately 800 km. south of Bangkok (Figure 1,2) is one of such mining area and mining has been active for almost 100 years. The occurrence of keratosis and hyperpig-mentation was known for long time in the region. The skin manifestation of chronic arsenic poisoning was first highlighted in 1987 in Ron Phibun district. (2) Clinical survey during 1987-1988 showed that more than 1000 people between age from 4 months to 85 years were affected.

In some school, over 80% of students showed high arsenic level in their hair and nails. (3).

According to the most recent survey (1992) of school children in this district, a 22% incidence of skin lesions and hyperkeratosis was recorded. (2) The cause of sickness was related to the consumption of contaminated surface and ground water that probably sourced from tin mining site. (4) Example of chronic arsenic poisoning associated with regionally contaminated water have been documented from numerous countries including Taiwan. (5,6), India (7), and USA (8).

Arsenic accumulation in soil, plankton, aquatic °-13

animals, agricultural products etc. also have been reported in many literature (4, 9, 10, 11, 12, 13).

Local extent of ground water contamination by arsenic in Ron-Phibun district was evaluated by several studies (2,13).

However there was no study investigating the influence of long-term arsenic run of to down stream environment. Surface water from Ron-Phibun district are flowing, through numerous canals and Pak Pa-Nang river, into Pak Pa-Nang bay which is the important area for aqua culture. High level of arsenic release to the bay could have been occurred during geological age, monitoring of current surface water alone may not be sufficient to study the influence of arsenic. The study of this paper intends the assessment of possible influence of long term arsenic inputs from the upstream of Ron Phibun district to the Pak Pa-Nang bay using marine organisms.

Arsenic and its compounds in marine organisms has been studied to identify the chemical species and its toxicological implication in recent research. Arsenic species study is very important because its toxicity and possible carcinogenicity depend

°-14

on the chemical form. (14) For example, marine organisms often contain arsenic in the range of 10 to 100mg/kg (15). This is considerable high compared with drinking water standard of arsenic at 0.01mg/l (16) In spite of such high arsenic content, marine organism are not toxic food to human because major arsenic is in the form of organic compounds which are low in toxicity. Organic arsenic compounds found in marine organisms include monomethylarsenic acid (MMA) and dimethylarsenic acid (DMA), arsenobetaine, arsenocholine and arsenosugar (17). Sean. et al. Reported that arsenosugars were identified as the major arsenic compounds present in marine algae and arsenobetaine was the dominant arsenic species in crab and shrimp. His study also revealed the presence of arsenosugars in addition to arsenobetaine in the bivalves. (18) Shibata et al. Concluded his researches that some bivalves contained not only arsenobetaine but also the arsenosugar derivatives (19).

Arsenobetaine and arsenocholine, when present in seawater, are efficiently accumulated by blue mussels and present in the tissue as arsenobetaine. (20)

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Figure 2 Mussel sampling point in Pak Pa-Nang Bay Nakornsritamaraj, Thailand Out Bay

WESTERN CENTRAL

However, the origin such organic arsenic compounds is still a matter of debate. On the other hand, as found in the data of mussel watch program, arsenic concentration is high in some of the sampling sites than other sites. It is implying some, but not direct, relation between arsenic level in mussels and environmental condition, not necessarily arsenic level but also other biological/chemical factors, specific to some sites. Therefore interpretation of arsenic content in marine organisms needs careful consideration. In this study, total arsenic level of mussels in and out of the Pak Pa-Nang bay would be statistically compared for objective judgment of arsenic influence. Then their spatial distribution in the bay would be investigated.

Further consideration would be given by the result of arsenic species study by HPLCICP-MS analysis.

EASTERN

Arius truncatus, Plotosus anguillaris, Sillago maculate, Cynoglossus macrolepidotus), mussel (Perna viridis) were collected from Pak Pa-Nang bay during 1994-1995 for screening purpose. Shrimp, crab and fish are collected from fishermen who caught them in the bay.

After screening, Green mussel samples (Perna viridis) were collected at 48 sampling station in and out of Pak Pa-Nang bay (39 in the bay and 9 out of the bay as shown in Figure 2) in November 1995.

Each station, about 1 kilogram of mussels that struck with the bamboo stick were collected (40-80 mussels) and cleaned by water at sampling site for 2-3 times to wash mud and removed bornacles. Then samples were washed by distilled water for 2-3 times and packed freshly in the plastic bags. Samples were then stored at temperature about 4 ÌC before analysis.

2.2 Sample preparation

2. Methods and Materials 2.1 Sampling 98 biological sample such as shrimp (Penaeus merguiensis, P. monodon), crab (Scylla serrata), fish, (Liza vaigiensis, »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡

Shrimp : Only meat of shrimps was used for analysis. Shell, head, tail and stomach were removed. Meat from 25-30 shrimps were combined by using stainless steel cutter and ground in glass blender. °-15

Fish : Only meat of fish was used.

Procedure is similar to that of shrimps. 5-10 fishes of same species were homogenized to prepare a sample.

Crab: Only meat of crab was used.

Procedure is similar to that of shrimps. 10-20 crabs were used to prepare a sample.

Mussel : In the laboratory, 25-30 mussels of the same size (6-9 cm. In length, 4-5.5 cm. In width) were selected from each sample. Whole and undeparated soft parts of the mussels were separated carefully from the shell to avoid contamination. Then samples at each station were grounded in a glass blender equipped with a stainless steel cutter. The samples were used for total arsenic determination by acid digestion followed by hydride generation/atomic adsorption spectrometry.

Another set of samples were prepared for qualitative arsenic species study of water soluble arsenic compounds. A mussel each from 5 sampling stations were used for preparation. After shells were removed, samples were weighed and homogenized into the centrifuge tube 20 ml of methanol/ water (1:1, v/v) was added to the tube and the tube was sonicated for 10 minutes.

After centrifugation (2000 rpm x 10 minutes), the extract was removed. Extraction process was repeated five times and the extracts were combined. The extract were heated to 40 ÌC in evaporator to dryness. Then sample dissolved in water were analyzed by HPLC-ICP-MS.

process. After digestion, HCl (1+1) was added and heated at 90 ÌC. After filtration by using filter paper (No.5B), Kl solution (200 g/l) was added. Continuous flow hydride generator using HCl and NaBH4 solution were connected to SHIMADZU AA-630 atomic adsorption spectrometer for analysis (21) NIES No.6 mussel standard reference material (SRM) was used for quality control of analytical procedure. Recovery of the arsenic from NIES No.6 SRM by the method was over 90%.

Arsenic species study : Qualitative arsenic species analysis was done by HPLC-ICP-MS (Perkin Elmer 410 Bio LC system combined with a Yokogawa Electric PMS100 ICP mass spectrometer). The analytical condition was as follows; column: Asahipak GS-220HQ 7.6x300 mm, buffer solution : 25 mM tetramethylanmoniumhydroxide, 25 mM maronic acid (pH = 6.8), flow rate 0.6 ml/minute. Arsenic compounds used as standards are that of Shibata et.al (19) and are shown in figure 3.

2.3 Analysis Total arsenic determination : Approx.

2 gm of grounded sample (wet weight) were gently digested by HNO 3 at room temperature for over night and then at 130 ÌC. Then H2SO4 and HClO4 were added and heated up to 290 ÌC until white fume was observed. Attention was paid to avoid overheating and boiling during digestion °-16

Figure 3 Arsenic Compounds in Marine environment

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Table 1 Total arsenic concentration of biological samples Sample type

Average (mg/kg dry wt.)

Fish (surface) Fish (deep) Crab Shrimp Mussel ND < 0.002 ppm

0.97 0.45 6.40 (1.10) 2.23 5.92

Minimum Maximum (mg/kg dry wt.) (mg/kg dry wt.)

ND ND 0.38 0.11 0.32

3. Result and Discussion 3.1 Preliminary investigation: Table above summarized arsenic concentration (mg/kg dry weight) level of various biological samples which were collected as preliminary investigation.

The average total arsenic concentration in surface fish samples (Liza vaigiensis) and deep fish sample (Sillago maculate, Plotosus anguillaris, Cynoglossus macrolepidotus, Arius truncatus) (categorized by their living habitat) were low and did not show much difference in arsenic level due to this different habit.

The arsenic level in crab and mussel were higher than the others. Their average concentration were 6.40 and 5.92 mg/kg (dry wt) respectively. Of all 6 collected crab samples, one sample was found arsenic concentration at level of 32.3 mg/kg. Which is big differently level than other crab samples.

If we removed this sample from the data, the average arsenic concentration in crab was only 1.1 mg/kg (dry wt). The sample matrix which has outlier is difficult for statistical treatment. For this reason, crab may not be the good sample matrix. The average level of arsenic in green mussel were 5.92 mg./kg (dry wt). Green mussels were selected for further investigation based on the reasons that the average arsenic concentration in green mussel sample was higher than the other kinds of sample. They stay stationary in this area while fish, shrimp »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡

5.25 11.0 32.3 12.8 25.2

Number of samples 14 27 6 9 20

and crab can easily move from one area to the other place. In addition data of mussel watch program from other location is available for comparison.

3.2 Comparison of arsenic level in Mussels Histograms showing arsenic level distribution by mg/kg (dry wt.) for the samples in the bay and out of the bay are shown in Figure 4. For the samples in the bay, the distribution of arsenic level seems to follow normal distribution curve. The histogram for the samples out of the bay seems somewhat different from normal distribution. This may be due to the small number of samples (n=9) for out of the bay group. For the statistical method discussed below, we assume out of the bay group also follow normal distribution.

Based on the statistical discussion, we could not find any evidence that indicate the significant difference in the two group, i.e., in the bay samples and out of the bay samples.

This implies that mussel in Pak Pa-Nang bay is not significantly influenced by arsenic.

Absolute arsenic concentration level of mussels in this study was compared with the data on mussel watch program in the United States. The data was obtained from NOAA (national Oceanic and Atmospheric Administration) progress report (1986-1988) of mussel watch project. Table 2 summarize the descriptive statistics for the arsenic concentration of this study and NOAA report.(23) °-17

Table 2 Arsenic concentration in Mussels Average In the bay sample Out of the bay samples United states (23)

4.22 4.96 11.1

Standard Deviation 2.15 1.88 6.36

Minimum

Maximum

0.74 2.37 2.6

9.20 7.56 41.0

Number of samples 39 9 177

Unit in the table : mg/kg (dry wt.)

When two independent sample groups are to be compared, statistical method commonly applied is F-test and t-test. As first step, we compare the variance of two group by F-test. F value for this study is as follows. = 4.63 / 3.54 F = S12/ S22 = 1.31 2 = 2.152 S1 (Variance of in the bay group) S22 (Variance of out of the bay group) = 1.882 Critical value of F at 5% level of significance for the given degree of freedom (for numerator = α denominator =8 ) is 3.08 (22). F value of 1.31 which is smaller than critical value of 3.08 means the null hypothesis station two groups have equal variance can not be rejected.

Small F value suggests two groups have same variance.

For the comparison of mean value of two groups when variance of each groups is equal, t-value can be obtained by following. t = X1 - X2 / Sp √ (1 /n1)+(1/n2) = 4.96 X1 (mean of out of the bay group) = 4.22 X2 (mean of in the bay group) n1 (number of out of the bay group) = 9 n2 (number of in the bay group) = 39 where Sp is the pooled estimate of the population standard deviation based on both groups and can be given by, Sp2 = (n1-1) S12 + (n2-1) S22 / (n1+n2-2) = 4.44 Sp = 2.11 Here we obtain t value = 0.98. Critical value of t value for 40 degree of freedom at 5% level of significance is 1.68. As computed t value of 0.98 is far lower than the critical value, again we can not reject the null hypothesis stating two groups means are equal.

NOAA report (23) represent statistics around whole countries of the United States of America while the data of this study represents relatively small area. Therefore data from NOAA report naturally has more variance in the data set than the data of this study. °-18

However, average value of this study, both in and out of the bay samples, are less than half of the NOAA report data. This fact also supports the previous statistical discussion that the mussel in the bay is not different from background level or that of open ocean. »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡

Figure 4 Comparison of Arsenic Level in Mussel Samples

3.3 Spatial distribution of arsenic level Figure 5 depict the spatial distribution of arsenic level in mussel samples. Black point and its size represents sampling location as well as arsenic level. When carefully examined we could see the samples in eastern side of the bay generally has lower arsenic level than others.

For further discussion, we first categorized sampling site inside the bay into 3 zones by their different geography.

The central zone of the bay is the deepest area where the Pak Pa-Nang river from upstream flow directly through this area.

The eastern zone is quite shallow, (The depth at low tide is around 1 m.) and quite static.

It is dominantly mangrove forest. The western zone locates on the western bank of the bay.

It is also shallower compared to the central zone and close to the city of Pak Pak-Nang district.

The concentration of total arsenic in ten (10) mussel samples from the eastern zone were average 2.8 mg/kg (dry wt.) which is lower than the other zones including outside bay (11 samples in western : 4.0 mg/kg, 18 samples in central : 5.1 mg/kg). Such large difference can not be considered as mere random distribution. Figure 6 is the polynomial regression surface applied to the data. Left map is second order polynomial and right »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡

Figure 5 Spatial distribution of arsenic level in mussel samples map is third order polynomial trend surface.

Polynomial trend surface map is, in some cases, very useful in recognizing distribution pattern from large scale perspective. Left map shows that arsenic level is decreasing if the point is more eastern side. Right map shows that arsenic level is high in central zone where Pak Pa-Nang river water directly flows. There are two alternate hypothesis at this moment.

1) Bio-activities may be different in eastern side where water is shallow and mangrove forest is well developed. Unknown factor may reduce the plankton activities and population which supply arsenic compounds to mussels. °-19

Figure 6 Polynominal Trend Surface Map 2) Central zone is influenced by the arsenic contaminated water from upstream because it directly receives water flowing from Ron Phibun district. Western zone can be affected by some activities from city which is quite close to the bay.

Second hypothesis may not be reasonable as we could not find any evidence that mussels in the bay has higher arsenic level than that of out of the bay. In addition, the concentration of arsenic in these mussel samples also did not show any correlation with the distance from the mouth of river as shown in figure 7. Therefore second hypothesis is unlikely, but we can not totally exclude the possibility at this moment.

However it is more reasonable to assume that the mussels in eastern side is low in arsenic while the mussel in central and western side is high, in view of comparison with background (out of bay samples at average 4.96 mg/kg dry wt.) The issue remains as future focus of the research.

3.4 Arsenic species study Figure 8 are HPLC-ICP/MS chromatogram of the five mussel samples in the bay.

As seen in the figure, two major peaks are common to all samples. A peak of retention °-20

Figure 7 Correlation of Arsenic concentration vs. distance from the river mouth time around 610 sec was identified as arsenosugar (XI). Another peak around 763 sec was identified as arsenobetaine (VIII) (refer to figure 3 for chemical form of each compound). Smaller peak before arsenobetaine (VIII) around 744 sec was tentatively assigned as arsenosugar (X). Other minor peaks could not be positively identified at this moments. The fact that major arsenic composition of mussels in this study are arsenobetaine and arsenosugar as commonly abserved in other studies suggests no influence to the mussel in Pak Pa-Nang Bay by the upstream arsenic contamination. »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡

Figure 8 HPLC-ICP/MS Chromatograms (m/z=75) of Mussel Extracts One interesting point is that ratio of arsenosugar (XI) and arsenobetaine (VIII) are similar to four samples, but ratio is reversed for sample No.3. For study of plankton which is considered as major food source of mussels, Shibata et al. found that phytoplankton contain arsenosugar as major arsenic compounds while some of zooplankton contain high arsenobetaine (24).

Therefore biological condition at sampling site No.3, specially character of plankton population might be different from other sites and it affected ratio of two arsenic compounds. As we discussed previously the possible variation of such biological condition in the bay is interesting topics for future study. Currently survey of plankton species distribution in the bay in under planning.

4. Conclusion In conclusion, the present study revealed that the arsenic level in biological

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samples in Pak Pa-Nang bay is not considered as seriously affected by upstream contamination at Ron Phibun. The conclusion was reached from three independent approach as follows.

4.1 Mussels in the bay and out of the bay does not have any significant difference in arsenic level and can be considered as same population.

4.2 Arsenic level of the mussels in the study is, in average, at (4.359 mg/kg dry wt).

Less than half of mussel watch program conducted by NOAA. 4.3 Arsenic species in the green mussels is similar to previous research.

Main arsenic composition is arsenobetain and arsenosugars.

However, the arsenic level in mussel samples collected from eastern zone of the bay (dominant as mangrove forest) were lower than other zones and outside bay (represented for natural background). The relative abundance ratio of arsenosugar and arsenobetaine found in mussel samples are not similar in all samples. One mussel sample showed the reversed ratio. These finding suggests that further study focused on variation of biological activities, particularly of planktion, inside the bay is important for understanding arsenic distribution.

5. Acknowledgements The authors wish to express thank to Dr. Y. Shibata for providing ICP-MS analysis, Mr. Suthaib Srilachai and Miss Lamyai Chaiyo for helping in sample preparation and technical assistance. We also acknowledge Dr. Monthip Sriratana Tabucanon, director of Environmental Research and Training Center (ERTC) and all Japanese experts in (ERTC) and all Japanese experts in ERTC for their thoughtful comments.

°-21

6. Reference 1. Ishihara Shunso, Sawata Hideho et.al.,(1980).

Granites and Sn-W deposits of Peninsular Thailand, Mining geology special issue, No 8, 223-241.

2. Fordyce F.M, Willian T.M., Paijitprapapan A, Charoenchaisri p, (1995), Hydrogeochemistry of arsenic in an area of chronic mining related arsenism, Ronphibun district, Nakorn si tammarat province, Thailand: Preliminary results, BGS technical report, WC/94/79R, 1-64.

3. Piamphongsant T, and Udom-nimitkul P, (1989), Arsenic levels in hair and nail samples of normal adolescents in Amphoe Ronphibul, Bull. Dept med Ser, 14, 225-229.

4. Na Chiangmai N.,(1990), Arsenic concentration in water, vegetables, fruits and hair of Amphoe Ronpibul, Nakorn Si Thammaratj province, Songklanakarin Journal of Sci. Tech, 13, 59-67 5. Chen S.L., Dzeng S.R., yang M.H., (1994), Arsenic species in ground-waters of the Blackfoot Disease area, Taiwan, Environ. Sci.Technol., 28,877-881.

6. Hsin S.Y.(1984), Blackfoot Disease and chronic arsenism in Southern Tiwan, International Journal of Dermatology, 23/4, 258-260.

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10. Bovornsachot P., (Feb 1888), Arsenic in water, Journal of geology, Yr 33, V2, 54-58 °-22

11. Davitiyananda D., (1983), Studies of arsenic residue in seafoods and pigûs organ Journal of environmental research, Yr.,30-39.

12. Davitiyananda D., and Panich-kriangkrai W., (1990), Arsenic residue determination in egg, Thai J., Hlth. Resch, 4(2), 109-116.

13. Arrykul S., kooptarnon K., Wittayawarawat W., (1996), Contamination of arsenic, cadmium, and lead in Pak-panang river basin, Nakorn Si Thammarat Thailand, International Symposium on Geology and Environment, 309-318.

14. Mok W.M., Shak W.K., Wai C.M., (1986), Extraction of arsenic (III) and arsenic (V) from natural waters for neutron activation analysis, Anal.Chem, 110-113 15. Craig P.J., (1982), Environmental aspects of organometallic chemistry, Pergamon Press, V2, 979.

16. WHO Environmental Health Criteria, Arsenic, No.18, 87-89.

17. Office of Food and Drug Adminsitration, ministry of Public Health., (1988), Arsenic toxicity and conclusion of arsenic poisoning problem in Ronpiblul district, Nakorn Sri-Tamamaraj province, Jan, 1-47 18. Le Sean X.C., Cullen William R., Reimer Kenneth J., (1994), Speciation of arsenic compounds in some marine organisms, Environ.Sci.technol., 28, 1598-1604 19. Shibata yasuyuki and Morita Masatoshi., (1992), Characterization of organic arsenic compounds in bivalves, Applied organometallic chemistry, 6, 343-349.

20. Gailer Jurgen, Francesconi Kevin A., Edmond John S. and Irgolic K.J., (1995) Metabolism of arsenic compounds by the blue mussel Mytilus edulis after accumulation from seawater spiked with arsenic compounds, Applied organometallic chemistry, 9, 341-355.

21. Jin Kazao, Oganwa Hiroshi, Taga Mitsuhiko., (1983), Study on wet digestion »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡

method for determination of total arsenic in marine organisms by continuous flow arsine generation and atomic absorption spectrometry using some model compound, The Japan society for analytical chemistry, 32, E171-E176.

22. Supunvanich Somchai, (1981), Principles of biostatistics, 3 rd.ed.Sammit Press, Thailand.

23. National Oceanic and Atmoshpheric Administration, (1989), A summary of data on tissue contamination from the first three year (1986 - 1988) of the mussel watch project, NOAA Technical memorandum, NOS OMA 49, 1-22.

24. Shibata Yasuyuki Personal communication.

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