Acute and subchronic toxicity of lead to the spotted Babylon, Babylonia areolata (neogastropoda, buccinidae).

ABSTRACT The acute and subchronic toxicity of lead was determined in the spotted babylon, Babylonia areolata. The 96-h static bioassay was conducted to estimate the median lethal concentration ([LC.sub.50). The snails were exposed to lead nitrate (Pb[[N[O.sub.3]].sub.2]). The [LC.sub.50] values for 24, 48, 72, and 96 h were 29.31, 14.64, 12.44, and 10.50 mg Pb/L, respectively. In the subchronic experiment, the snails were exposed to 0.5 mg Pb/L (10% MATC, the maximum acceptable toxicant concentration) of lead nitrate for 3 mo. Lead accumulation was found in different organs with the greatest accumulation in the stomach and lesser in the esophagus, gill, rectum, digestive gland, proboscis, and foot. The histopathologic alterations in the digestive system and gills of B. areolata were studied by light microscopy. The general tissue alterations were decrease in length of cilia, decrease in acidophilic granules, slight distension of nuclei, and the loss of heterochromation. There were increases of mucous vacuoles, damaged ciliated cells, and enlargement of vacuoles.

KEY WORDS: Babylonia areolata, lead, acute toxicity, subchronic toxicity, histopathology 

INTRODUCTION

The biologic impact of heavy metals in aquatic system has become a major problem in recent years. Lead is one of the most commonly encountered and used metals. Its widespread industrial usage and continual release into the environment as an exhaust emission had made lead one of the most interesting toxic heavy metal in environmental contamination in Thailand. At trace concentration, lead is essential for biologic growth; however, it can adversely affect all living organisms if present in excessive amounts (Venugopal & Luckey 1978). Its poison can affect growth, fecundity, and mortality of the organisms. Acute toxicity test has been widely used to obtain a rapid estimate of the concentration of toxic chemical that causes direct, irreversible harm to test organisms (Parrish 1985).

Lead is acutely toxic to aquatic invertebrate at concentration between 0.1 and >40 mg/L for freshwater organisms and between 2.5 and >500 mg/L for marine organisms (EPA 1999). It is a nonessential and accumulating metal. Gastropods and bivalves are excellent bioaccumulators of lead and a wide range of pollutants. They are filter feeders, herbivores, or carnivores and have the potential to bioconcentrate contaminants that would normally be present in the water or within sediments at concentration too low for detection by routine monitoring techniques. Lead was reported to be accumulated in the soft tissue, kidney, digestive gland, alimentary tracts, mantle, foot, and shell of bivalves and gastropods (Bolognani et al. 1982, Ireland 1984, Richmonds & Dutta 1989, Newman et al. 1994). However, to the best of our knowledge, there is virtually no information on the acute and subchronic toxicity of lead on histopathology of marine gastropods.

Hence the aims of this study are to determine the median lethal concentration ([LC.sub.50]) of lead and to study the histopathologic alterations in the digestive system and gills of the snails, Babylonia areolata Link 1807, in acute and subchronic lead exposure.

MATERIALS AND METHODS

Experimental Snails

The spotted babylon, B. areolata, were obtained from the Institute of Marine Science, Burapha University, Chonburi province, Thailand. Juvenile snails with the size of 1.5-2.0 cm (SL) and the age -3 mo were used in this study. Snails were acclimatized in a glass aquarium containing 50 L of artificial seawater and aerated for 7-10 days prior to use. Table 1 shows the physical and chemical characteristics of seawater used in the experiment. The ranges of salinity and temperature were 30-32 ppt and 29[degrees]C to 32[degrees]C, respectively. The snails were fed daily with small shrimp.

Chemical

The lead solution used in the experiment was prepared by dissolving lead nitrate (Pb[[N[O.sub.3]].sub.2]) of 99.5% purity in distilled water. Concentration of the solution was prepared as 1,000 mg/L.

Experimental Procedures

Acute Toxicity of Lead

The median lethal concentration ([LC.sub.50]) values of spotted babylon, B. areolata was determined by using the 4-day (96-h) static bioassay experiment.

The snails were not fed during 24 h prior to the experiment and 96 h during the experiment. The behavior and general condition of the snails were observed, and dead snails were removed immediately to protect the fouling of the test solution. Abnormalities and mortalities were recorded every 24 h.

A total of 105 acclimatized snails were transferred from the stock tank and distributed into 21 small aquaria (5 snails/ aquarium). The test consisted of a control and 6 concentrations of lead (10, 12, 14, 16, 18, and 20 mg/L). Each experiment consisted of 3 replicates, with 5 snails in each replicate. Hence, 15 snails were tested in each lead concentration. The snails were exposed for 96 h. The numbers of dead snails were recorded at 24, 48, 72, and 96 h. The [LC.sub.50] and 95% confidence limits were computed using the probit analysis computer program (Finney 1971).

Subchronic Toxicity Test

Subchronic toxicity test was performed to determine the possible adverse effects of the chemical under a long-term exposure condition at sublethal concentration. The threshold concentration that produces statistically significant deleterious effects is expressed as the maximum acceptable toxicant concentration (MATC). The MATC is hypothetical concentration and is in a range between the no-observed-effect concentration (NOEC) and the lowest-observed-effect concentration (LOEC). The test concentration is estimated by the use of the application factor (AF) concept (Thophon et al. 2003).

For the subchronic study, B. areolata were exposed to 10% of 96-h [LC.sub.50] of lead concentration for 3 mo. The experiments were carried out in glass aquaria containing 50 L of the artificial seawater with continuous aeration. A total of 90 snails were transferred from the stock tank and distributed into glass aquaria (15 snails/aquarium). The test consisted of a control (not exposed to lead) and 19.17 mg/L of Pb, 10% of 96-h [LC.sub.50]. Three replications were performed for each group. The actual concentrations of lead was verified against known standard using methyl isobutyl ketone/ ammonium pyrrolidine dithiocarbamate (MIBK/APDC) extraction/nitric acid backextraction technique and measured by an atomic absorption spectrophotometer. The snails were fed with shrimp meat, and the water in all aquaria was renewed every 3 days.

Heavy Metal Analysis

At the end of each 2 wk, five snails from each aquarium were randomly collected. Dissection of snails was done after completing the external examination. Shell pieces were isolated, and the soft tissue was dissected out to isolate different organs (gill, rectum, stomach, esophagus, proboscis, digestive gland, foot). The dissected organs were rinsed in distilled water and dried at 80[degrees]C for 48 h for determination of heavy metals in the digested tissues. Digestion of dried tissues was performed according to the standard method for the examination of water and wastewater (APHA 1998). The digested tissues were diluted with double distilled water to 50 mL and analyzed for lead by a flame atomic absorption spectrophotometer (FAAS), GBC 932 plus.

Histopathologic Study

Histopathologic study was performed in snails from acute toxicity test (96-h of [LC.sub.50] lead) after 96 h and subchronic toxicity test (after 1, 2, and 3 mo). Ten snails of each treatment were randomly collected. They were removed from the shells by knocking gently with coarse forceps without previous anesthetization. Shell pieces were isolated, and the shell-free snails were immediately fixed in Bouin fluid. The soft tissue was then rapidly dissected out to isolate the different organs (gill, proboscis, esophagus, stomach, and rectum) under the fixative. They were fixed in Bouin fluid for 24 h, washed three times in 70% alcohol to remove the fixative. After dehydration in a graded series of ethanol, the specimens were infiltrated with dioxan overnight. Finally, they were embedded in paraplast, sectioned at 5-[micro]m thickness, stained with PAS (Periodic-Acid-Schiff). Sections were examined for abnormality under an OLYMPUS CH40 light microscope.

RESULTS

Visual Observation

All snails in the control group survived and appeared normal in both acute and subchronic exposure. At 10 mg/L of lead solution, 50% of the snail died within 96 h. The order of toxicological symptoms were production of a very large amount of mucus along the periphery of the mantle edge, closing of opercula, silt accumulation, and finally death.

Acute Toxicity Study

The median lethal concentrations ([LC.sub.50]) and 95% confidence limit of lead of B. areolata at 24, 48, 72, and 96 h were 22.21, 14.86 (13.95-15.76), 12.44 (11.52-13.25), and 10.50 (9.56-11.17) mg/L, respectively (Table 2). The [LC.sub.50] of lead of B. areolata showed a progressive decrease when the exposure time was increased (P [less than or equal to] 0.05).

Lead Accumulation Study

Figure 1 shows lead accumulation in various organs of the control and treated snails at 2-wk interval in 3-mo exposure. In control snails, very low lead content was found in the gill (1.39 mg/kg), esophagus (5.21 mg/kg), stomach (3.93 mg/kg), rectum (7.99 mg/kg), digestive gland (2.80 mg/kg), and foot (1.31 mg/kg).

[FIGURE 1 OMITTED]

In general, lead accumulation was increased when the exposure time was increased (P [less than or equal to] 0.05). It seems that most organs of the snails have a high affinity for lead accumulation. The main accumulation of lead was found in the stomach. Lead was significantly increased (P [less than or equal to] 0.05) from 3466.40 mg/kg in the first month to 8788.03 mg/kg in the third month (Fig. 1). The decreasing order of lead content in the organs was stomach > esophagus > gills > rectum > digestive gland > proboscis > foot.

Histopathologic Observation

Histopathologic alterations were observed in the gill and the organs of digestive system (proboscis, esophagus, stomach, and rectum).

Gill

Control Group

Gill is composed of gill axis and numerous gill filaments (Fig. 2A). The cells lining the gill filaments are simple columnar type with long cilia. The basal part of the gill contains skeletal rods and clusters of mucous cells. The middle part of gill filament shows numerous ciliated cells. They contain round to oval nuclei with dense heterochromatin (Fig. 2B).

[FIGURE 2 OMITTED]

Treatment Group

Acute Exposure: The gills of snails exposed to 10.5 mg/L of lead for 96 h showed several pathologic changes, and their frequency increased with increasing time of exposure. The general alterations in the middle part of gill filament were decrease in length of cilia and dilation of cells, which contained enlarged vacuoles (Fig. 2C). In addition, a slight distension of nuclei was observed, and they had lost the herterochromatin (Fig. 2C).

Subchronic Exposure: At the first month, the gill of treated snail showed a loss or decrease of cilia (Fig. 2D). Dilation of cells was also observed. At the second month, the gills showed decrease in length of cilia. There was dilation of cells, which contained more vacuoles than in the first month (Fig. 2E). At the third month, similar alterations were observed but they were more pronounced (Fig. 2F). In addition, the epithelial cells appeared to be broken down with no definite boundary, and most nuclei have been lost or some became pyknotic (Fig. 2F).

Proboscis

Control Group

The proboscis is long when extended and slender with thick muscle. It contains the buccal mass and radula inside. The proboscis sheath is composed of bundles of muscles. The epithelium of the proboscis sheath is a simple ciliated columnar type (Fig. 3A). It consists of mucus-secreting goblet cells and columnar cells. The nuclei are elliptical or oval in shape and lie in the middle of the cells (Fig. 3A).

[FIGURE 3 OMITTED]

Treatment Groups

Acute Exposure: The proboscis of snails exposed to 10.5 mg/L of lead for 96 h showed several pathologic changes, and their frequency increased with increasing time of exposure. The general alteration was an increase of mucous vacuoles (Fig. 3B). Slight distension and dilation of nuclei were observed (Fig. 3F).

Subchrouic Exposure: At the first month, the proboscis sheath of the treated snail showed little damage of epithelium. There was dilation of cells, which contained vacuoles (Fig. 3C). At the second month, the proboscis showed dilation of cells and increase of mucous vacuoles (Fig. 3D). There were more gaps in the muscle sheath (Fig. 3D). At the third month, the proboscis showed more epithelium damage and increase of mucous vacuoles (Fig. 3E). The epithelium showed fragmentation and gaps (Fig. 3E).

Esophagus

The esophagus is long and can be divided into three parts: anterior, mid, and posterior. The anterior esophagus is straight with short nonciliated cells, whereas the mid and posterior esophagus are highly folded and contained long, ciliated cells.

Control Group

No recognizable changes were observed in the esophagus of the control snails throughout the course of the experiment. The epithelial lining of the esophagus is mostly ciliated columnar type with scattered mucus-secreting goblet cells (Fig. 4A). The columnar cells contain abundant secretion. The nuclei are ellipsoidal in shape and rich in heterochromatin, and they lie in the center of the cells (Fig. 4A).

[FIGURE 4 OMITTED]

Treatment Groups

Acute Exposure: The esophagus of snails exposed to 10.5 mg/L of lead for 96 h showed several pathologic changes, and their frequency increased with increasing time of exposure. There was an increase of mucous vacuoles (Fig. 4B). Most cells were filled with secretion, and the nuclei were dilated with mostly euchromatin (Fig. 4F).

Subchronic Exposure: At the first month, the esophagus of treated snails appeared similar to that of the control (Fig. 4C). At the second month, the treated snails' esophagus showed an increase of mucous vacuoles (Fig. 4D). There were loss of cilia and dilation of cells. Fragmentation of muscle sheath was also observed. Similar lesions occurred at the third month of exposure (Fig. 4E), and several nuclei became dilated and lost their heterchromatin (Fig. 4F).

Stomach

Control Group

The stomach is a bulk-like structure and triangular in shape. The epithelium lining of the stomach is highly folded and contains simple columnar cells. The cells are tall with short cilia. There are columnar cells and numerous mucus-secreting goblet cells (Fig. 5A). The nuclei are ellipsoidal in shape and contain heterochromatin, and they lie at the base of the epithelial cells (Fig. 5A).

[FIGURE 5 OMITTED]

Treatment Groups

Acute Exposure: The stomach of snails exposed to 10.5 mg/L of lead for 96 h showed several pathologic changes, and their frequency increased with increasing time of exposure. There were a slight distension of nuclei and dilation of cells and nuclei. The mucous vacuoles were increased (Fig. 5B).

Subchronic Exposure: At the first month, the treated snail stomach showed similar lesions to those of acute exposure (Fig. 5B). At the second and third month of exposure, the stomach of treated snails showed decrease in length of cilia and dilation of cells, which contained more vacuoles (Figs. 5C, 5D). There were slight distension of nuclei and dilation of cells (Fig. 5D).

Rectum

Control Group

The rectum is a tube-like structure. The epithelial lining is a simple ciliated columnar type (Fig. 6A). The cells are tall with longer cilia than those in the stomach and esophagus. They are mostly columnar secretory cells with occasional mucus-secreting goblet cells. The epithelial cells contain dense acidophilic granules. The dense nuclei are ellipsoid in shape and lie in the center of the epithelial cells (Fig. 6A).

[FIGURE 6 OMITTED]

Treatment Groups

Acute Exposure: The rectum of snails exposed to 10.5 mg/L of lead for 96 h showed several pathologic changes, and their frequency increased with increasing time of exposure. There was a decrease in cilia length and an increase of mucous vacuoles (Fig. 6B). Some cells were filled with large vacuoles and gaps. The cell outline was rarely discernible. The nuclei were reduced in size with irregular shape (Fig. 6B).

Subehronie Exposure: At the first month, the rectum of the snails showed decreased length of cilia and an increase of mucous vacuoles, similar to those in acute exposure (Fig. 6B). At the second month, the rectum showed increased cilia length and increased mucous vacuoles (Fig. 6C). The cells were highly distended with numerous acidophilic granules (Fig. 6C). At the third month, the treated snails' rectum showed increased cilia length and an increase of mucous vacuoles. The epithelial cells had irregular shapes (Fig. 6D), some were filled with large vacuoles and gaps, and their outlines were no longer discernible. The nuclei had become reduced in size with irregular shape (Fig. 6D).

DISCUSSION

The acute toxicity of inorganic lead to marine and freshwater invertebrate is generally less than that of cadmium, copper, mercury, and zinc. Acute toxic effects have been recorded at concentrations between 0.5-5.0 mg/L range in both marine and freshwater species (Oladimigi & Offem 1989, Bodar et al. 1989). Based on these [LC.sub.50] values, B. areolata seemed to have a high tolerance to lead because their [LC.sub.50] values for 24, 48, 72, and 96 h were 29.31, 14.64, 12.44, and 10.50 mg/L, respectively. Another marine prosobranch, Nerita saxtilis, an efficient biologic monitor to heavy metal pollution in the Red Sea had much lower [LC.sub.50] values (300.35 [micro]g/L). Comparatively, the freshwater prosobranch, Filopludina martensi martensi also had a high 96-h [LC.sub.50] value (191.69 mg/L) (Jantataeme et al. 1996). Other species of freshwater pulmonates such as Lymnaea palustris and Physa integra could survive at lead concentration of 0.5-19 mg/L (Borgmann et al. 1978, Spehar et al. 1978). There were many factors, such as exposure time, form of pollutant used and water hardness that may have some influences on the [LC.sub.50] values (Sprague 1985). Generally prosobranch snails have opercula to protect themselves when surrounding water becomes hazardous to them. Hence, they are more tolerant to pollutants than opisthobranchs and pulmonates.

Abnormal behaviors of snails under pollutant stress have been reported in many species of snails such as F. martensi martensi (Jantataeme et al. 1996), Babylonia lutosa (Cheung & Wong 1999), Lymnaea stagnalis (Pyatt et al. 2002). When B. areolata were exposed to lead at various concentrations, they showed abnormal behavior. They moved slowly, then did not move, closed their opercula, and secreted large amount of mucus. These abnormal appearances were also observed in F. martensi martensi exposed to 300 mg/L of lead (Jantataeme et al. 1996). Cheung & Wong (1999) also reported the effect of copper on the activity and feeding of subtildal prosobranch, B. lutosa exposed to sublethal concentration for 4 wk. The snails showed high percentage of retraction (body extended, foot not attached, unable to move) of body within the shell. Pyatt et al. (2002) reported on the effects of lead (5 or 10 ppm) on the survival of the freshwater snail L. stagnalis collected from lead contaminated or uncontaminated environments. Acute (72 h) exposure to lead inhibited several behavioral activities including locomotion, feeding, tentacle extension, and emergence from the shell. Lead was bioaccumulated in the snail tissues, especially the buccal mass and stomach.

Mollusks are known to accumulate high concentration of lead in polluted area. Lead has been shown to be accumulated in different organs of invertebrates, especially at high levels in the gills and digestive gland (Anderson 1978). Bolognani et al. (1982) lound that in the freshwater gastropod, Viviparus viviparus, most of the lead was stored in the mantle. In closely related species, F. martensi martensi, lead was accumulated mostly in the intestine (Jantataeme et al. 1996). Ireland (1984) demonstrated that in the slug, Arion ater, most lead was deposited in the intestine and least in the foot. However, in the land snail, Helix aspersa, Beeby and Richmond 1989) reported that shell was a site of lead deposits. In the same species of snails, high contents of lead in the midgut gland of snails from contaminated sites were found (Beeby & Richmond 1989).

In marine mollusks, N. saxtilis, the bioaccumulation capability of lead was reduced as: digestive gland > headfoot > shell. The digestive glands were suggested to be the sites of detoxification (Abd Allah & Moustafa 2002). The sublethal exposure of B. areolata to lead showed that the main accumulation of lead was found in the stomach and lesser in esophagus, gill, rectum, digestive gland, proboscis, and foot. Compared to the oysters, the spotted babylon seems to have higher affinity to lead. Mature eastern oysters exposed to 200 [micro]g/L of lead for 49 days could accumulate 200 mg/kg of lead in their soft tissues. In B. areolata, the highest accumulation was found in the stomach (8788.03 mg/L) after exposure to 19.17 mg/L of lead for 90 days. It seems that the mechanisms of lead entry to the snail's body is probably via ingestion.

Uptake of lead into the body via a permeable surface might occur all over the body, particularly in small and/or soft-bodied invertebrates. Uptake from solution would also take place in the alimentary tract when any of the medium was swallowed during drinking or food ingestion. Katalin (1988) found that gill tissues of the mussel, Unio pictorum, were most effective at accumulation of all heavy metals. In this study, the gill also accumulated high concentration of lead. This is probably the mechanism of lead entry into the snail body.

Histologic alterations of gill of B. areolata showed the decrease in length of cilia, dilation of cells that contained vacuoles in the cytoplasm, and slight distension of nuclei that have lost the heterochromatin. The gills are the primary site for uptake of soluble metals from the aquatic environment. The general alterations presented were epithelial cell degeneration and reduced ciliation of gill filament. Slight distention of nuclei, which have lost the heterochromatin was similar to that described by Triebskorn and Kunast (1990) in slug, Deroceras reticulatum exposed to carbamate molluscicide. Hinch and Stephenson (1987) have suggested that the high concentrations of metal seen in gills may be overestimated due to adherence of extraneous material, which cannot be washed off, to mucus on the large surface areas of this organ. Moore et al. (1982) assumed that changes in membrane fluidity induced this altered rate of vesicle fusion. In addition, dilation of cells and nuclei of gill epithelium is probably caused by the loss of ability of cells to control the volume due to the reduction of membrane stability (Axiak et al. 1988)

Histologic alterations of digestive tract observed frequently in B. areolata was an increase of mucous vacuoles. Davies et al. (1989) believed that these activities of mucus production and extrusion were considered as a main mode of action of molluscicide, leading to a dehydration of snails. Triebskorn (1991) suggested that mucus may serve to dilute or detoxify the toxin. Furthermore, snails secrete mucus as a defense mechanism. However, intensified exudation of mucus can also kill them. The exudation of mucus secretion would finally lead to a desiccation of the snail, and loss of mucous cells would prevent production of the very mucus that protects the surface of the snails from desiccation (Triebskorn & Kunast 1990).

Other histologic alterations in digestive organs observed were the decrease in length of cilia and dilation of cells, which contained more vacuoles. Axiak et al. (1988) reported that the decrease in length of cilia or microvilli, the enlargement of vacuoles by the increase fusion of vesicles, and vacuoles in the cells might result from the interaction of lipophilic molluscicide or heavy metals with membrane. These reactions might induce changes in composition fluidity and stability of the membrane. The intensified fusion between small and large vacuoles result in the increase of large vacuoles after intoxication. Bourne et al. (1991) studied the effects of metaldehyde and methiocarb in the slug, D. reticulatum. They found that the methiocarb seemed to have a greater effect on the osmotic balance of the crop and the formation of large intracellular space within the cytoplasm of the crop epithelial cells. They suggested that it would seem probable that this increase in fluid content might be responsible for some of these symptoms of poisoning. Moore et al. (1982) assumed that changes in membrane fluidity induced this altered rate of vesicle fusion.

TABLE 1.
Reef change observed between 1870s and 1940s.

             2D Footprint               3D Surface Area

        1870s    1940s   Change    1870s    1940s   Change
Reef
 1      41093    18394       55    41098    18413       55
 2      14594    13315        9    14631    13335        9
 3      40840    72368      -77    40850    72375      -77
 4      73476    64615       12    73490    64636       12
 5      11521    41225     -258    11524    41234     -258
 6     141922   142965       -1   141938   142982       -1
 7      72318    42553       41    72344    42569       41
 8      29841     8662       71    29858     8670       71
 9     129672    10490       92   129775    10494       92
10     129672    94142       27   129775    94171       27
11      51963    36795       29    51976    36810       29
12      19629    53100     -171    19633    53106     -171
13     175605   161831        8   175671   161871        8
14      96657    93865        3    96716    93908        3
15      45111    35589       21    45131    35601       21
16     279115   246323       12   279135   246338       12
17     355282   285655       20   355364   285700       20
18      74105    90664      -22    74128    90682      -22
19      37231    64931      -74    37239    64939      -74
20     230509   321112      -39   230572   321139      -39

                Volume

        1870s    1940s   Change
Reef
 1      17923     8723    -9200
 2      12640     9452    -3187
 3      18950    29680    10730
 4      46874    38638    -8236
 5       1887    17860    15973
 6      74938    59885   -15053
 7      46819    26726   -20093
 8      22420     3932   -18488
 9      99696     3141   -96555
10      99696    50411   -49286
11      32043    20444   -11599
12       6528    21673    15145
13     144779   113668   -31111
14      72887    60947   -11939
15      25266    17603    -7663
16     107960    89612   -18348
17     184992   136943   -48049
18      47629    43849    -3780
19      11944    22213    10269
20     175734   116072   -59662

TABLE 2.
Statistics of reef change between 1870s and 1940s.

                       1870s      1940s     Difference       P

2D Area ([m.sup.2])   102508      94930       7578       0.4390894
Volume (m.sup.3])      62580      44574      18007       0.0091378
Height (m)                -0.29      -0.76       0.47    0.0002479

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P. SUPANOPAS, (1) P. SRETARUGSA, (2), * M. KRUATRACHUE, (1) P. POKETHITIYOOK (1) AND E. S. UPATHAM (3)

(1) Departments of Biology and (2) Anatomy, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400, Thailand; (3) Faculty of Science, Burapha University, Chonburi 20130, Thailand

* Corresponding author. E-mail: [e-mail]; Fax: [phone]