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Sukanya Boonchalermkit*, Janewit Wongsanoon* ,Munehiro Fukuda**
*»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 ‚∑√ “√. 0-2577-1138Environmental Research and Training Center, Department of Environmental Quality Promotion. Technopolis. Klong 5 Klong Luang, Pathumthani 12120 e-mail: [email protected]**ºŸâ‡™’ˬ«™“≠Õߧå°√§«“¡√à«¡¡◊Õ√–À«à“ߪ√–‡∑»≠’˪ÿÉπ (JICA Expert)
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ABSTRACTThis 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 fur ther
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 inf luence
of arsenic in the bay. Arsenic species-distri-
bution of mussel samples were also studied
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. IntroductionNatural 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
par t 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 mani-
festation 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 countr ies
including Taiwan. (5,6), India (7), and USA (8).
Arsenic accumulation in soil, plankton, aquatic
»Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡ °-13
animals, agricultural products etc. also havebeen reported in many literature (4, 9, 10, 11,12, 13).
Local extent of ground watercontamination by arsenic in Ron-Phibundistrict was evaluated by several studies (2,13).However there was no study investigatingthe influence of long-term arsenic run of todown stream environment. Surface waterfrom Ron-Phibun district are flowing, throughnumerous canals and Pak Pa-Nang river, intoPak Pa-Nang bay which is the importantarea for aqua culture. High level of arsenicrelease to the bay could have been occurredduring geological age, monitoring of currentsurface water alone may not be sufficient tostudy the influence of arsenic. The study ofthis paper intends the assessment of possibleinfluence of long term arsenic inputs from theupstream of Ron Phibun district to the PakPa-Nang bay using marine organisms.
Arsenic and its compounds in marineorganisms has been studied to identifythe chemical species and its toxicologicalimplication in recent research. Arsenicspecies study is very important because itstoxicity and possible carcinogenicity depend
on the chemical form. (14) For example, marineorganisms often contain arsenic in the rangeof 10 to 100mg/kg (15). This is considerablehigh compared with drinking water standardof arsenic at 0.01mg/l (16) In spite of suchhigh arsenic content, marine organism are nottoxic food to human because major arsenic isin the form of organic compounds which arelow in toxicity. Organic arsenic compoundsfound in marine organisms includemonomethylarsenic acid (MMA) anddimethylarsenic acid (DMA), arsenobetaine,arsenocholine and arsenosugar (17). Sean.et al. Reported that arsenosugars wereidentified as the major arsenic compoundspresent in marine algae and arsenobetainewas the dominant arsenic species in craband shrimp. His study also revealed thepresence of arsenosugars in addition toarsenobetaine in the bivalves. (18) Shibataet al. Concluded his researches that somebivalves contained not only arsenobetainebut also the arsenosugar derivatives (19).Arsenobetaine and arsenocholine, whenpresent in seawater , are ef f ic ient lyaccumulated by blue mussels and presentin the t issue as arsenobetaine. (20)
°-14 »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡
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 HPLC-
ICP-MS analysis.
2. Methods and Materials2.1 Sampling98 biological sample such as
shrimp (Penaeus merguiensis, P. monodon),
crab (Scylla serrata), fish, (Liza vaigiensis,
Arius truncatus, Plotosus anguillaris, Sillagomaculate , Cynoglossus macrolepidotus),mussel (Perna viridis) were collected fromPak Pa-Nang bay during 1994-1995 forscreening purpose. Shrimp, crab and fishare collected from fishermen who caughtthem in the bay.
After screening, Green musselsamples (Perna viridis) were collected at 48sampling station in and out of Pak Pa-Nangbay (39 in the bay and 9 out of the bay asshown in Figure 2) in November 1995.Each station, about 1 kilogram of musselsthat struck with the bamboo stick werecollected (40-80 mussels) and cleaned bywater at sampling site for 2-3 times to washmud and removed bornacles. Then sampleswere washed by distilled water for 2-3 timesand packed freshly in the plastic bags. Sampleswere then stored at temperature about 4 ÌCbefore analysis.
2.2 Sample preparationShrimp : Only meat of shrimps was
used for analysis. Shell, head, tail and stomachwere removed. Meat from 25-30 shrimps werecombined by using stainless steel cutter andground in glass blender.
Figure 2 Mussel sampling point in Pak Pa-Nang Bay Nakornsritamaraj, Thailand
Out Bay
WESTERN CENTRAL EASTERN
»Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡ °-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
fol lowed by hydride generat ion/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.
2.3 AnalysisTotal arsenic determination : Approx.
2 gm of grounded sample (wet weight)
were gently digested by HNO3 at room
temperature for over night and then at
130 ÌC. Then H2SO
4 and HClO
4 were added
and heated up to 290 ÌC until white fume
was observed. Attention was paid to avoid
overheating and boiling during digestion
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 tetramethylanmonium-
hydroxide, 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.
Figure 3 Arsenic Compounds inMarine environment
°-16 »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡
3. Result and Discussion3.1 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 concen-
tration 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 dif ficult 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
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 arseniclevel 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 con-
centration of this study and NOAA report.(23)
Table 1 Total arsenic concentration of biological samples
Sample type Average Minimum Maximum Number of
(mg/kg dry wt.) (mg/kg dry wt.) (mg/kg dry wt.) samples
Fish (surface) 0.97 ND 5.25 14
Fish (deep) 0.45 ND 11.0 27
Crab 6.40 (1.10) 0.38 32.3 6
Shrimp 2.23 0.11 12.8 9
Mussel 5.92 0.32 25.2 20
ND < 0.002 ppm
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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.
F = S12/ S22 = 4.63 / 3.54
= 1.31
S12 (Variance of in the bay group) = 2.152
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 - X
2 / Sp √ (1 /n1)+(1/n2)
X1(mean of out of the bay group) = 4.96
X2(mean of in the bay group) = 4.22
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.
Table 2 Arsenic concentration in Mussels
Average Standard Minimum Maximum Number ofDeviation samples
In the bay sample 4.22 2.15 0.74 9.20 39Out of the bay samples 4.96 1.88 2.37 7.56 9United states (23) 11.1 6.36 2.6 41.0 177
Unit in the table : mg/kg (dry wt.)
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.
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.
°-18 »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 ofarsenic 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 fur ther discussion, we f i rst
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
Figure 5 Spatial distribution of arsenic levelin 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 populat ion which supply arsenic
compounds to mussels.
»Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡ °-19
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 af fected by some act iv i t ies
from city which is quite close to the bay.
Second hypothesis may not be reasonable as
we could not f ind any evidence that
mussels in the bay has higher arsenic level
than that of out of the bay. In addition,
the concentrat ion 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 studyFigure 8 are HPLC-ICP/MS chroma-
togram 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
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 arsenobe-
taine (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.
Figure 6 Polynominal Trend Surface Map
Figure 7 Correlation of Arsenic concentrationvs. distance from the river mouth
°-20 »Ÿπ¬å«‘®—¬·≈–Ωñ°Õ∫√¡¥â“π ‘Ëß·«¥≈âÕ¡ °√¡ à߇ √‘¡§ÿ≥¿“æ ‘Ëß·«¥≈âÕ¡
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. ConclusionIn conclusion, the present study
revealed that the arsenic level in biological
samples in Pak Pa-Nang bay is not consi-
dered 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 relat ive
abundance rat io 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 fur ther study focused on
variation of biological activities, particularly of
planktion, inside the bay is important for
understanding arsenic distribution.
5. AcknowledgementsThe 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 prepara-
tion 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.
Figure 8 HPLC-ICP/MS Chromatograms(m/z=75) of Mussel Extracts
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