S-Oxygenation of Thiobencarb in Tap Water Processed by Chlorination

Article abstract of DOI:10.1021/jf960552u

Thiobencarb, an herbicide, was incubated in tap water containing 0.7 mg/L residual chlorine at 30 °C, and the solution was analyzed by HPLC. Thiobencarb could not be detected after 3 h, and only a byproduct was detected. The increase in concentration of the byproduct correlated well with the decrease in that of thiobencarb. In a high free chlorine medium (more than 10 mg/L), the byproduct degraded to p-chlorobenzyl alcohol, p-chlorobenzaldehyde, and p-chlorobenzyl chloride, but not in a low free chlorine medium (0.1-10 mg/L). The byproduct was identified to be thiobencarb sulfoxide by LC/MS, infrared, and NMR spectra, and a reduction technique using 2-mercaptoethanol. Chiral HPLC analysis made it clear that the sulfoxide was a racemic compound. The sulfoxide was mutagenic regardless of the presence or absence of S9mix in the Ames Salmonella typhimurium TA100 assay. It was suggested that the management and control of thiobencarb in tap water processed by chlorination should include monitoring of thiobencarb sulfoxide, using HPLC.

Full text of DOI:10.1021/jf960552u

990
J. Agric. Food Chem. 1997, 45, 990−994
S-Oxygen a tion of Th ioben ca r b in Ta p Wa ter P r ocessed by
Ch lor in a tion
Shuji Kodama,* Atsushi Yamamoto, and Akinobu Matsunaga
Toyama Institute of Health, 17-1 Nakataikoyama, Kosugi-machi, Toyama 939-03, J apan
Thiobencarb, an herbicide, was incubated in tap water containing 0.7 mg/L residual chlorine at 30
°C, and the solution was analyzed by HPLC. Thiobencarb could not be detected after 3 h, and only
a byproduct was detected. The increase in concentration of the byproduct correlated well with the
decrease in that of thiobencarb. In a high free chlorine medium (more than 10 mg/L), the byproduct
degraded to p-chlorobenzyl alcohol, p-chlorobenzaldehyde, and p-chlorobenzyl chloride, but not in a
low free chlorine medium (0.1-10 mg/L). The byproduct was identified to be thiobencarb sulfoxide
by LC/MS, infrared, and NMR spectra, and a reduction technique using 2-mercaptoethanol. Chiral
HPLC analysis made it clear that the sulfoxide was a racemic compound. The sulfoxide was
mutagenic regardless of the presence or absence of S9mix in the Ames Salmonella typhimurium
TA100 assay. It was suggested that the management and control of thiobencarb in tap water
processed by chlorination should include monitoring of thiobencarb sulfoxide, using HPLC.
Keyw or d s: Thiobencarb; thiobencarb sulfoxide; chlorination; HPLC; tap water; herbicide
INTRODUCTION
(pesticide grade), acetonitrile (HPLC grade), and other chemi-
cals (reagent grade) were obtained from Wako Pure Chemical
Industries Ltd.
Stock solutions (2000 mg/L) of thiobencarb, thiobencarb
sulfoxide, p-chlorobenzoic acid, p-chlorobenzyl alcohol, p-
chlorobenzaldehyde, p-chlorotoluene, and p-chlorobenzyl chlo-
ride were separately prepared in acetonitrile or acetone.
Standard solutions were obtained by mixing together all of
their stock solutions in 10 mM phosphoric acid to final
concentrations of 1-100 µg/L each.
Thiobencarb (S-p-chlorobenzyl diethylthiocarbamate)
is a carbamate herbicide that has been widely used for
weed control in rice crops. It was moderately toxic to
aquatic invertebrates and fishes in acute toxicity tests
(Sanders and Hunn, 1982). The Integrated Risk Infor-
mation System has set up the toxicological constant of
thiobencarb at 0.01 mg/kg/d, and Smith (1996) has set
the risk-based concentration of thiobencarb in tap water
at 370 µg/L. In J apan, the level of thiobencarb remain-
ing in tap water is being regulated at less than 20 µg/L
by the Ministry of Health and Welfare. There are many
studies on the analysis of thiobencarb in river water,
soil, and tap water (Takahashi and Morita, 1988;
Redondo et al., 1994; Kodama et al., 1995).
It was reported that irradiation of thiobencarb at 300
nm in aqueous solution yielded 30 photoproducts con-
taining thiobencarb sulfoxide, N-desethyl and N-acetyl
derivatives of thiobencarb, and so on (Ruzo and Casida,
1985). Thus, it seems that thiobencarb is able to be
degraded under environmental conditions. It was re-
ported that thiobencarb was detected in raw water but
not in tap water processed by chlorination (Takahashi
and Morita, 1988). Takahashi and Morita (1993) and
Magara et al. (1994) have reported that thiobencarb was
degraded by chlorination to produce p-chlorobenzyl
alcohol, p-chlorobenzaldehyde, p-chlorobenzyl chloride,
p-chlorotoluene, and p-chlorobenzoic acid as chlorination
byproducts. They concluded that the management and
control of pesticides in drinking water should include
testing for chlorination byproducts.
HP LC Ap p a r a tu s. The HPLC system consisted of a
Hitachi autosampler model AS-4000, two Hitachi model
L-6300 pumps, a Rheodyne manual injector, a Shimadzu
photodidoe array detector model SPD-M10AV, a Shimadzu
column oven model CTO-10AC and a Senshu Scientific model
E1E010 switching valve. Separations were attained using
RSpak DE-613 column (6 mm i.d. × 150 mm, Showa Denko)
maintained at 40 °C, in conjunction with a 4 mm i.d. × 10
mm enrichment column packed with a polymethacrylate-based
gel. For the chiral resolution, Chiralcel OB column (4.6 mm
i.d. × 250 mm, Daicel Chemical Industries, Ltd.) and both a
Shimadzu photodiode array detector and a J asco model OR-
990 chiroptical detector were used. The system was controled
by a Hitachi HPLC manager model D-6100. Data acquisition
and processing were conducted with a Shimadzu LC model
CLASS-M10A workstation.
HP LC An alysis of Ch lor in ation Bypr odu cts of Th ioben -
ca r b. HPLC was performed by a fully-automated system
previously described (Kodama et al., 1995). That is, 3 mL of
the standard solution or the reaction mixture injected by using
an autosampler was applied onto an enrichment column
followed by washing. The switching valve was moved to the
injection position, and the analytes trapped on the enrichment
column were desorbed with a mobile phase and transferred
to the analytical column. The separated components were
measured with a photodiode array detector. The mobile phase
used was 53% (v/v) acetonitrile containing 10 mM phosphoric
acid. The flow rate was 1.5 mL/min, and the column temper-
ature was kept at 40 °C.
In this paper, we have found that thiobencarb disap-
peared by incubation with tap water containing 0.7
mg/L of residual chlorine and only a byproduct was
detected by high-performance liquid chromatography
(HPLC). We have also studied the identification and
the mutagenesis of the byproduct.
P r ep a r a tion of a Ch lor in a tion Byp r od u ct of Th ioben -
ca r b (Th ioben ca r b Su lfoxid e). Thiobencarb was added to
9 L of distilled water containing 3 mg/L free chlorine to a final
concentration of 4 mg/L. The solution was left to stand at 30
°C for 3 h and was dechlorinated with 2 mM sodium ascorbate.
The dechlorinated solution was applied onto a SEP PAK PLUS
PS-2 cartridge (Waters), and the adsorbed material was eluted
with 50% (v/v) acetonitrile. The eluate was applied to HPLC
EXPERIMENTAL PROCEDURES
Ch em ica ls. Distilled water, acetone, hexane, and methanol
were of HPLC grade from Kanto Chemical Co., Inc. S9mix
was purchased from Oriental Yeast Co., Ltd. Thiobencarb
S0021-8561(96)00552-3 CCC: $14.00
© 1997 American Chemical Society

S-Oxygenation of Thiobencarb
J. Agric. Food Chem., Vol. 45, No. 3, 1997 991
RESULTS AND DISCUSSION
Ch lor in a t ion of Th iob en ca r b in Ta p Wa t er .
Thiobencarb was added to tap water containing 0.7
mg/L residual chlorine to a final concentration of 100
µg/L, and left to stand at 30 °C for 0-5 h. After
dechlorination with 2 mM sodium ascorbate, 3 mL of
the solution was applied to HPLC. Figure 1 shows the
time course of the peak areas of thiobencarb and its
chlorination byproduct. Thiobencarb decreased with
incubation time, and only an unknown byproduct was
detected. The increase in concentration of the byproduct
correlated well with the decrease in that of thiobencarb
and within 3 h reached a plateau.
The byproduct was different from the compounds such
as p-chlorobenzyl alcohol, p-chlorobenzaldehyde, or p-
chlorobenzyl chloride previously reported (Takahashi
and Morita, 1993). The byproduct was stable in water
for at least 1 month. It seemed that the byproduct was
more hydrophilic, as the retention time of the byproduct
was significantly shorter than that of thiobencarb on
the reversed-phase column.
F igu r e 1. Time course of chlorination of thiobencarb in tap
water. O, Thiobencarb; b, chlorination byproduct of thioben-
carb.
using a manual injector, and the chlorination byproduct of
thiobencarb was fractionated.
Id en tifica tion of th e Ch lor in a tion Byp r od u ct of
Th ioben ca r b. The chlorination byproduct of thioben-
carb was applied to LC/MS, and the mass spectrum on
the top of its peak was obtained (Figure 2). The
spectrum of the byproduct was characterized by ions at
m/z 274/276 and 291/293. It was reported that a ratio
between the protonated molecular adduct [M + H]+ and
the ammonia-cationized molecular adduct [M + NH4]+
depended on the chcaracteristics of the compound (Chiu
et al., 1989). It was suggested that ions at m/z 274/276
and 291/293 corresponded to [M + H]+ and [M + NH4]+
species, respectively. Therefore, it was also suggested
that the molecular weight of the byproduct was 273/
275 and the byproduct contained a chlorine atom.
Figure 3 shows the infrared spectra of thiobencarb
and its chlorination byproduct. Both spectra seemed
to be similar except that strong 1060 cm-1 absorbance
in the byproduct appeared and the carbonyl band at
1641 cm-1 for thiobencarb shifted to absorbance at 1695
cm-1 for the byproduct. It was suggested that the
strong 1060 cm-1 absorbance resulted in sulfoxide
(Tseng et al., 1977). It seems that the production of
sulfoxide affected the environment near the carbonyl
Liqu id Ch r om atogr aph y (LC)/Mass Spectr om etr y (MS).
LC/MS was performed with a Shimadzu model QP1100EX
thermospray mass spectrometer. The mobile phase was 50%
(v/v) acetonitrile-50 mM ammonium acetate and the flow rate
was 1 mL/min. The vaporizer tip temperature was 212 °C.
Positive ions were sampled from a scanning range of m/z 150-
400. Mass spectra were obtained using the discharge mode.
The chlorination byproduct of thiobencarb was dissolved in
acetonitrile to obtain a concentration of 20 mg/L, and a 20 µL
aliquot of the solution was applied.
In fr a r ed (IR) Sp ectr om etr y. IR spectra were recorded
in neat with a 1600 series FTIR (Perkin Elmer).
1H-Nu clea r Ma gn etic Reson a n ce (NMR) Sp ectr oscop y.
The 1H-NMR spectra were obtained on a J OEL EX-400 in
CDCl3 with tetramethylsilane as an internal standard.
Op tica l Resolu tion of a Ch lor in a tion Byp r od u ct of
Th ioben ca r b (Th ioben ca r b Su lfoxid e) by Usin g Ch ir a l
HP LC. Thiobencarb sulfoxide was dissolved in ethanol-
hexane (5:95) to obtain a concentration of 100 mg/L. 10 µL of
the solution was applied onto a Chiralcel OB column at 40
°C, and absorbance at 220 nm was measured. The mobile
phase used was ethanol-hexane (5:95) at a flow rate of 1 mL/
min. For the chiroptical detection, 10 µL of 4000 mg/L
thiobencarb sulfoxide was used.
Mea su r em en t of Resid u a l Ch lor in e. Residual chlorine
was measured by the diethyl p-phenylenediamine method
(J apan Waterworks Association, 1993).
residue to shift the carbonyl band at 1695 cm-1
.
1H-NMR spectrum data of thiobencarb was as fol-
lows: σ 7.28 (d, J ) 8.6 Hz, 2H, aromatic H), σ 7.24 (d,
J ) 8.6 Hz, 2H, aromatic H), σ 4.10 (s, 2H, SCH2), σ
3.36 (br d, J ) 34.2 Hz, 4H, NCH2), σ 1.15 (t, J ) 6.8
Hz, 6H, NCH2CH3). The data of the chlorination
byproduct of thiobencarb were as follows: σ 7.34 (d, J
) 8.3 Hz, 2H, aromatic H), σ 7.26 (d, J ) 8.3 Hz, 2H,
aromatic H), σ 4.23 (d, J ) 12.7 Hz, 1H, SOCH2), σ 4.18
(d, J ) 12.7 Hz, 1H, SOCH2), σ 3.35 (q, J ) 7.1 Hz, 2H,
NCH2), σ 3.25 (m, 1H, NCH2), σ 3.12 (m, 1H, NCH2), σ
Mu ta gen esis Assa y of Th ioben ca r b a n d Its Ch lor in a -
t ion Byp r od u ct (Th iob en ca r b Su lfoxid e). Salmonella
typhimurium strains TA98 and TA100 were used for the Ames
test (Ames et al., 1975) using the preincubation technique
(Yahagi et al., 1977) either in the presence or absence of a rat
liver microsomal fraction containing cofactors (known as
S9mix). Thiobencarb and its sulfoxide samples were purified
by HPLC and then dissolved in dimethyl sulfoxide, and two
plates were used per dose.
F igu r e 2. Thermospray liquid chromatography mass spectrum of a chlorination byproduct of thiobencarb.

992 J. Agric. Food Chem., Vol. 45, No. 3, 1997
Kodama et al.
F igu r e 5. Optical resolution of thiobencarb sulfoxide.
resolution of the sulfoxide carried out by a Chiralcel OB
column. The former peak area was almost the same as
the latter one. The optical activities of the former and
the latter peaks were negative and positive, respectively.
Therefore, it is shown that the thiobencarb sulfoxide
produced by chlorination of thiobencarb is a racemic
compound.
Ch lor in a tion of Th ioben ca r b a n d Its Su lfoxid e
a t Va r iou s Con cen tr a tion s of F r ee Ch lor in e. So-
dium hypochlorite was added to distilled water to obtain
free chlorine concentration of 0-300 mg/L. Thiobencarb
was added to the chlorine solution to a final concentra-
tion of 100 µg/L. After incubation of the solution at 40
°C for 3 h, 60 mM sodium ascorbate and 10 mM
phosphoric acid were added. 3 mL of the mixture was
applied to HPLC, and the byproducts were determined
(Table 1). As free chlorine increased, thiobencarb
decreased and it could not be detected at 0.7 mg/L free
chlorine. On the other hand, production of thiobencarb
sulfoxide proceeded in a similar manner to parallel the
decrease of thiobencarb, and its sulfoxide decreased
gradually at more than 10 mg/L free chlorine. p-
Chlorobenzyl alcohol, p-chlorobenzaldehyde, and p-
chlorobenzyl chloride were detected when thiobencarb
was treated with more than 10 mg/L free chlorine.
However, p-chlorobenzoic acid and p-chlorotoluene could
not be detected. According to Takahashi and Morita
(1993), p-chlorobenzyl alcohol, p-chlorobenzaldehyde,
and p-chlorobenzyl chloride were mainly produced by
chlorination (140 mg/L free chlorine) of thiobencarb, and
these compounds accounted for 3-20% of the total
thiobencarb. Our result obtained by incubation of
thiobencarb and free chlorine (more than 100 mg/L) was
identical with their results.
To confirm whether the compounds such as p-chlo-
robenzyl alcohol resulted in a degradation of thiobencarb
sulfoxide, the sulfoxide was treated with various con-
centrations of free chlorine. As shown in Table 2, it was
confirmed that p-chlorobenzyl alcohol, p-chlorobenzal-
dehyde, and p-chlorobenzyl chloride were produced by
the chlorination of the sulfoxide, as well as the produc-
tion of these compounds in Table 1.
Mu t a gen ic Act ivit ies of Th iob en ca r b a n d It s
Su lfoxid e. Mutagenic activities of thiobencarb and its
sulfoxide were assayed by the Ames test using the
preincubation technique (Figure 6). In order to know
the effect of metabolic activation of these compounds,
the preincubation was carried out either in the presence
or absence of S9mix. Neither of the samples showed
any mutagenic activity in the presence or absence of
S9mix against strain TA98 at the concentrations of 30-
1000 µg/plate. In the case of strain TA100, however,
both the samples were mutagenic regardless of being
in the presence or absence of S9mix. It seemed that
the decrease of strain TA100 revertant colonies in
concentrations of 300-1000 µg/plate thiobencarb was
responsible for the toxicity. Cashman and Olsen (1990)
F igu r e 3. Infrared spectra of thiobencarb and its clorination
byproduct. (A) Thiobencarb; (B) chlorination byproduct of
thiobencarb. Arrows 1, 2, and 3 indicate wavenumbers at
1060, 1695, and 1641 cm-1, respectively.
F igu r e 4. Structure formulas of thiobencarb and its chlorina-
tion byproduct (thiobencarb sulfoxide). (A) Thiobencarb; (B)
thiobencarb sulfoxide.
1.14 (t, J ) 7.1 Hz, 3H, NCH2CH3), σ 0.94 (t, J ) 7.1
Hz, 3H, NCH2CH3). This was nearly the same as the
data of thiobencarb sulfoxide previously reported (Cash-
man et al., 1990) except for the methylene resonance of
NCH2. On the basis of the data obtained, it is proposed
that the structure of thiocarbamoyl group in thiobencarb
sulfoxide under the NMR measurement condition is
sS(fO)sC(sO-)dN+(C2H5)2, but not sS(fO)sC-
(dO)sN(C2H5)2.
J ori et al. (1968) reported that methionine sulfoxide
was reduced back to methionine by treatment with
2-mercaptoethanol. The chlorination byproduct of
thiobencarb was also reduced to thiobencarb by treat-
ment with 5% (v/v) 2-mercaptoethanol in distilled water
at 30 °C for 20 h. This result also supported the finding
that chlorination of thiobencarb produced thiobencarb
sulfoxide (Figure 4).
Therefore, it was shown that thiobencarb sulfoxide
was produced not only by photochemical reaction (Draper
et al., 1981; Ruzo et al., 1985) or enzyme reaction
(Cashman et al., 1990), but also by chlorination of
thiobencarb.
Op tica l Resolu tion of Th ioben ca r b Su lfoxid e by
Ch ir a l HP LC. Compounds containing sulfoxide may
be optically active. Thiobencarb sulfoxide obtained by
chlorination was applied to HPLC with a RSpak DE-
613 column and a chiroptical detector, but it did not
show any optical activity. Figure 5 shows the optical

S-Oxygenation of Thiobencarb
J. Agric. Food Chem., Vol. 45, No. 3, 1997 993
Ta ble 1. An a lysis of Ch lor in a tion Byp r od u cts of Th ioben ca r b a t Va r iou s Con cen tr a tion s of F r ee Ch lor in e
thiobencarb
[tR ) 22.9 min]
thiobencarb sulfoxide p-chlorobenzyl alcohol p-chlorobenzaldehyde p-chlorobenzyl chloride
[ta ) 10.2 min] [tR ) 10.9 min] [tR ) 13.52 min] [tR ) 21.1 min]
a
free chlorine
added (mg/L) (µg/L)
(nM)
(µg/L)
(nM)
(µg/L)
(nM)
(µg/L)
(nM)
(µg/L)
(nM)
total (nM)
0
100
65
9
389
252
35
ND
ND
ND
ND
ND
ND
ND
ND
33
89
98
99
96
81
55
18
ND
ND
121
326
359
363
352
297
201
66
ND
ND
ND
ND
ND
ND
1
3
5
7
ND
ND
ND
ND
ND
ND
7
21
35
49
ND
ND
ND
ND
ND
ND
ND
ND
2
ND
ND
ND
ND
ND
ND
ND
ND
14
ND
ND
ND
ND
ND
ND
ND
1
ND
ND
ND
ND
ND
ND
ND
6
389
373
361
359
363
352
304
228
127
97
0.1
0.3
0.7b
1
3
10
30
100
300
NDc
ND
ND
ND
ND
ND
ND
2
2
12
12
ND
5
36
a
b
Retention time. Chlorination in tap water containing 0.7 mg/L of resudal chlorine. c Not detected (less than 1 µg/L).
Ta ble 2. An a lysis of Ch lor in a tion Byp r od u cts of Th ioben ca r b Su lfoxid e a t Va r iou s Con cen tr a tion s of F r ee Ch lor in e
thiobencarb sulfoxide p-chlorobenzyl alcohol p-chlorobenzaldehyde p-chlorobenzylchloride
free chlorine added (mg/L)
(µg/L)
(nM)
(µg/L)
(nM)
(µg/L)
(nM)
(µg/L)
(nM)
total (nM)
0
1
10
100
300
100
100
86
31
3
366
366
315
113
11
NDa
ND
ND
4
ND
ND
ND
28
ND
ND
ND
1
ND
ND
ND
7
ND
ND
ND
1
ND
ND
ND
6
366
366
315
154
100
6
42
5
36
2
12
a
Not detected (less than 1 µg/L).
F igu r e 6. Dose-response curve of thiobencarb and its sulfoxide by the Ames TA98 and TA100 assay. Mutagenic activities of
thiobencarb (O) and its sulfoxide (b) were measured by the Ames TA98 (A, B) and TA100 (C, D) assay in the presence (B, D) or
absence (A, C) of S9mix.
CONCLUSION
reported that thiobencarb sulfoxide was the principal
metabolite of thiobencarb in the presence of hepatic
microsomes from striped bass, and accounted for 98%
of the total thiobencarb. They also reported that this
reaction was catalyzed largely by flavin-containing
monooxygenase and to a lesser extent by cytochromes
P-450. The mutation frequency (1.0 rev/µg) of thioben-
carb sulfoxide in the presence of S9mix was almost the
same as that (1.2 rev/µg) in the absence of S9mix. In
contrast, the mutation frequency (3.0 rev/µg) of thioben-
carb in the presence of S9mix was lower than that (14.3
rev/µg) in the absence of S9mix. This might suggest
that thiobencarb was S-oxygenated in the presence of
S9mix during the preincubation time, because S9mix
contained microsomal fraction. Therefore, it could be
considered that thiobencarb and its sulfoxide were
mutagenic and both their mutagenic types were base-
pair substitutions but were not frameshift.
It has been found that thiobencarb sulfoxide is
produced by a reaction of thiobencarb and residual
chlorine contained in tap water. In our experiments,
the reaction occurred at low residual chlorine concen-
tration in a range of 0.1-10 mg/L. In high residual
chlorine concentration (more than 10 mg/L), the pro-
duced sulfoxide degraded followed by yielding p-chlo-
robenzyl alcohol, p-chlorobenzaldehyde, and p-chlo-
robenzyl chloride. On the basis of the above results, it
seems that thiobencarb sulfoxide is a major byproduct
from thiobencarb in tap water processed by chlorination.
And it was found that thiobencarb sulfoxide was mu-
tagenic despite its mutagenic activity being lower than
that of thiobencarb. In J apan, the level of thiobencarb
remaining in tap water is being regulated at less than
20 µg/L by the Ministry of Health and Welfare. In our
preliminary study, thiobencarb was detected but its

994 J. Agric. Food Chem., Vol. 45, No. 3, 1997
Kodama et al.
J apan Waterworks Association. Standard method of drinking
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photo-oxidation of the methionyl residues in lysozyme. J .
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Kodama, S.; Matsunaga, A.; Ohto, M.; Yamamoto, A.; Mi-
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17 column (J &W Scientific), because the sulfoxide may
be thermally unstable (Draper and Crosby, 1981). By
using a fully-automated HPLC analysis system, linear-
ity (r > 0.999) was demonstrated in a range of 1-100
µg/L by standard curves of thiobencarb and its sulfoxide.
Therefore, it was suggested that the management and
control of the herbicide thiobencarb in tap water pro-
cessed by chlorination as in J apan should include
monitoring of thiobencarb sulfoxide, using HPLC.
Redondo, M. J .; Ruiz, M. J .; Boluda, R.; Font, G. Determination
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ACKNOWLEDGMENT
We are grateful to Drs. Y. Asano and Y. Kato,
Biotechnology Research Center, Faculty of Engineering,
Toyama Prefectural University, for performing the H-
NMR spectra and for useful discussions. We thank Dr.
K. Hayakawa and Mr. A. Nakamura, Faculty of Phar-
maceutical Science, Kanazawa University, for the Ames
test and for valuable discussions. We also thank Mr.
N. Takayama and Mr. S. Tanaka, Chemistry Section,
Forensic Science Laboratory, Ishikawa Pref. Police H.Q.,
for performing the LC/MS spectra.
1
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