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4
II.7.4 Carbamate pesticides
by Kiyoshi Ameno
Introduction
Among many carbamate pesticides commercially available in Japan, those with relatively high
toxicities are shown in > Table 4.1 [1]. Carbamate pesticides are generally classified into
N-methylcarbamate insecticides and N-allylcarbamate herbicides in view of their chemical
structures and biological actions. The number of fatalities due to poisoning by carbamate pes-
ticides is 50–100 every year in Japan; many of them are poisoned by methomyl [2]. According
to statics reported by National Research Institute of Police Science of Japan, the number of
fatalities is highest with paraquat plus diquat, followed by organophosphates and then carba-
mates among pesticides. The toxicity of carbamate pesticides is due to inhibition of acetylcho-
linesterase (AchE) by their binding with the active site of the enzyme; the inhibition of the
hydrolysis reaction of acetylcholine (Ach) results in the accumulation of Ach, provoking
poisoning symptoms, such as miosis, lacrimation, sweating, hypersalivation and convulsion of
extremities. The binding of carbamate pesticides to AchE is much weaker than that of organo-
phosphorus pesticides, and the former pesticides are easily decomposeda in mammalian bodies.
Therefore, the damages of organs by carbamate pesticides have not been reported.
For analysis of carbamate pesticides, methods by GC [3–7], GC/MS [5, 7, 8–10], HPLC
[11] and LC/MS [10] were reported. In this chapter, the extraction procedures and analytical
methods using the above 4 instruments are described for this group of pesticides.
• The authentic standards of compounds listed in > Table 4.1 and ethion can be purchased
from Wako Pure Chemical Industries, Ltd., Osaka, Japan. A standard mixture of seven
N-methylcarbamate pesticides can be obtained from Kanto Chemicals (Tokyo, Japan)b.
Carbofuran, carbaryl and ethion are separately dissolved in acetone to prepare 100 µg/mL
solutions as stock solutions for internal standards (ISs)c. Carbendazole (Aldrich, Milwau-
kee, WI, USA) is dissolved in distilled water to prepare 100 µg/mL solution. o-Methoxyl-
phenol can be also obtained from Aldrich.
• Sep-Pak C18 and Oasis MCX cartridges are purchased from Waters (Milford, MA, USA).
• The solutions to be prepared are: n-hexane/ethyl acetate (1:1, v/v); acetonitrile/distilled water
(1:1, v/v); methanol/distilled water (5:59, v/v); 0.1 M HCl solution; and 2 % NaCl solution.
• 0.1 M Phosphate buffer solution (pH 7.0): 6.81 g of KH2PO4 is dissolved in 400 mL distilled
water, and the pH of the solution is adjusted to 7.0 with 1.0 M NaOH solution; the final
volume is adjusted to 500 mL with distilled water.
• 5 % Ammonia/methanol solution: 2.5 mL of 3 M NH4OH solution is mixed with 47.5 mL
methanol.
• HPLC mobile phases: methanol/distilled water (65:35, v/v); acetonitrile/2 mM ammonium
formate (30:70 and 80:20, v/v, pH 3.0).
⊡ Table 4.1
Structures, properties and toxicities of carbamate pesticides
Name, MF, MW, property and structure Use Acute oral LD50
(rat, mg/kg)
bendiocarb insecticide 40 ~ 156
C11H13NO4
MW: 223.2
MP: 125 ~ 129
furathiocarb insecticide 53
C18H26N2O5S
MW: 382.5
BP: 250<
Carbamate pesticides 561
⊡ Table 4.1
(continued)
methomyl insecticide 17 24
C5H13N2O2S
MW: 162.2
MP: 78 ~ 79
oxamyl insecticide 5.4
C7H13N3O3S
MW: 219.3
MP: 100 ~ 102
pirimicarb insecticide 147
C11H18N4O2
MW: 238.3
MP: 90.5
propoxur insecticide 50
C11H15NO3
MW: 209.2
MP: 90
thiodicarb insecticide 66
C10H18N4O4S3
MW: 354.5
MP: 173 ~ 174
MF: molecular formula; MW: molecular weight; MP (°C): melting point; BP (°C): boiling point.
562 Carbamate pesticides
Extraction methods
Liquid-liquid extraction
i. A 2-g aliquot of specimens (blood, tissues and stomach contents d) is mixed with 10 mL
acetonitrile (containing an appropriate IS), homogenized with a Polytron homogenizer
and centrifuged at 3,000 rpm for 5 min to obtain clear supernatant solution.
ii. For the sediment, the above i) step of extraction is repeated two times.
iii. The three acetonitrile supernatant solutions thus obtained are combined and mixed with
80 mL of 2 % NaCl solution and 25 mL of n-hexane/ethyl acetate (1:1, v/v) in a 250-mL
volume separating funnele.
iv. The funnel is shaken for 10 min (with a shaking machine).
v. The n-hexane/ethyl acetate layer is obtained.
vi. The layer is evaporated to dryness under reduced pressure in a rotary evaporator at room
temperature.
vii. The residue is dissolved in 100 µL methanol.
viii. A 2-µL aliquot of it is injected into GC or GC/MS; a 20-µL aliquot into HPLC.
ix. For quantitation, each calibration curve is constructed using peak area ratios of a target
compound to IS. A ratio obtained from a specimen is applied to the curve to calculate its
concentration.
GC conditions
GC columnsm: DB-5 and DB-1 fused silica capillary columns (30 m × 0.25 mm i.d., film thick-
ness 0.25 µm, J&W Scientific, Folsom, CA, USA).
GC conditions; instrument: Shimadzu GC14B (Shimadzu Corp., Kyoto, Japan); detector:
flame thermionic ionization detector (FTD)n; column temperature: 50 °C → 20 °C/min →
120 °C → 5 °C/min → 260 °C(10 min); injection and detector temperature o: 230 °C; carrier gas:
He (13 kPa).
GC/MS conditions
GC column: a DB-5MS fused silica capillary column (30 m × 0.25 mm i.d., film thickness
0.25 µm, J&W Scientific).
GC/MS; instrument: Shimadzu GC-MS5000 (Shimadzu Corp.); column temperature:
120 °C (1 min) → 20 °C/min → 240 °C (8 min); injection and separator temperature: 230 °C;
carrier gas: He (2.0 mL/min); ionization mode: EI (70 eV).
⊡ Table 4.2
Retention times of carbamate pesticides obtained by GC*
⊡ Table 4.3
EI mass spectra of carbamate pesticides
times of carbamate pesticides, when a DB-5 fused silica capillary column was used for GC
analysis.
> Table 4.3 shows principal ions (5 peaks each including a molecular ion) observed in EI
mass spectra for carbamate pesticides obtained by GC/MS [4, 9]. The intensities of molecular
ions are generally low; there are compounds without appearance of molecular ionsp. Sufficient
qualitative analysis is achieved by measuring retention times and mass spectra of compounds
tested. The sensitive quantitation analysis can be made using the selected ion monitoring
(SIM).
Methomyl can be analyzed by GC (/MS) in its underivatized from; but a method using the
conversion of methomyl into methomyl-oxime by alkali, followed by silylation (TMS) for GC/
MS, was reported [8]. This method is aimed to overcome the thermolability of underivatized
methomyl, and a good mass spectrum including the molecular ion can be obtained for the
methomyl-oxime-TMS (M.W. 177); the mass spectrum shows ions at m/z 177 (molecular ion),
162, 130, 121 and 105. Chemical ionization (CI) mode is preferably used for quantitation,
because it gives the base peaks at m/z 178 and 197 of protonated ions of methomyl-oxime-TMS
and o-methoxyphenol-TMS (IS). The quantitation can be made with peak area ratios of the ion
at m/z 178 to that at m/z 197. The quantitative range is reported to be 0.1–6 µg/mL; the details
of the method can be obtained from the reference [8].
HPLC columnq: Finepak SIL C18 (25 cm × 4.6 mm i.d., particle size 5 µm, Nihonbunko, Tokyo,
Japan).
HPLC conditions; instrument: Shimadzu HPLC10A (Shimadzu Corp.); detector: UV
(wavelength, 254 nm); mobile phase: methanol/distilled water (65:35, v/v); its flow rate: 1 mL/
min.
The analysis of carbamate pesticides by HPLC is much inferior to that by capillary GC in reso-
lution ability, but does not suffer from decomposition of analytes by heat. In HPLC analysis of
566 Carbamate pesticides
⊡ Table 4.4
Retention times and detection limits (on-column) of carbamate pesticides obtained by HPLC
methomyl and methomyl-oxime, the peaks are frequently interfered with by a solvent peak; to
avoid the problem, the ratio of water content of the mobile phase is increased to about 50 %.
There are various detectors commercially available for HPLC; they are useful for improving
specificityr and sensitivitys.
> Table 4.4 shows retention times and detection limits for carbamate pesticides obtained
under the above HPLC-UV conditions.
The analysis by LC/MS has been established recently; it requires no concern about the
decomposition by heat and also give mass spectra. The method is very useful for qualitative
analysis and enables sensitive quantitation in the SIM mode. > Table 4.5 shows ions selected
⊡ Table 4.5
Selected ions for qualitative and quantitative LC/MS analysis of carbamate pesticides
for qualitative and quantitative analyses. The sensitivity obtained by SIM of LC/MS is as high
as that of GC/MS.
Although the number of fatalities due to carbamate pesticide poisoning is not small in the
world, the reports on their concentrations in blood and organs are not many. The concen-
trations reported in literature and measured in the author’s laboratories are summarized
in > Table 4.6.
The blood methomyl concentrations in fatalities by its poisoning is 0.6–57 µg/mL (mean
concentration in 13 cases, 20 µg/mL) [3, 8, 12–14]. When the blood methomyl concentrations
of survived subjects, after ingestion followed by treatments, are also taken into consideration,
the fatal blood concentration of methomyl is estimated not lower than 1.0 µg/mL.
The respective blood concentrations of carbofuran, benfuracarb and furathiocarb after
their ingestion for suicidal purposes are as follows. The blood carbofuran concentrations
in 7 fatality cases after ingestion of the pesticide only were 0.32–29.3 µg/mL (n = 7, average
9.2 µg/mL) [7, 10, 15, 16]. The blood benfuracarb concentrations after its ingestion were 0.30–
2.32 µg/mL (n = 3, average 0.98 µg/mL) and the blood concentrations of carbofuran, the
metabolite of benfuracarb, were not lower than 1.45 µg/mL [6], suggesting that benfuracarb is
rapidly metabolized into carbofuran in human bodies. After furathiocarb ingestion, its blood
concentrations were 0.1–21.6 µg/mL (n = 7, average 3.6 µg/mL); but carbofuran, also the meta-
bolite of furathiocarb, has never been analyzed simultaneouslyt [5]. As shown in > Table 4.1,
the acute toxicities of furathiocarb and benfuracarb in rats are 8–30 times less than that of
carbofuran; the both compounds are metabolized into a highly toxic metabolite carbofuran in
mammals. In furathiocarb and benfuracarb poisoning cases, carbofuran should be analyzed
simultaneously.
In fatal cases of propoxur poisoning, its blood concentrations were 0.3–41.1 µg/mL (n = 6,
average 10.7 µg/mL) [17] and ethanol was also detected from blood in many cases. This sug-
gests that ethanol enhances the toxicity of propoxur.
Notes
⊡ Table 4.6
Blood concentrations of carbamate pesticides in their fatal poisoning cases
aldicarb, propoxur, carbofuran, carbaryl and methiocarb can be analyzed at ppb levels
simultaneously.
t) Carbofuran as a metabolite of furathiocarb has never been analyzed. This does not mean
“not detectable”, but means “not determined”. If it is analyzed, an appreciable amount of
carbofuran seems to be detected.
References
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2) National Research Institute of Police Science (ed) (1997–2001) Annual Case Reports of Drug and Toxic Poison-
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3) Araki M, Yonemitsu K, Kambe T et al. (1982) Forensic toxicological investigations on fatal cases of carbamate
pesticide methomyl (Lannate®) poisoning. Jpn J Legal Med 36:584–588 (in Japanese with an English abstract)
4) Suzuki O, Hattori H, Liu J et al. (1990) Positive-and negative-ion mass spectrometry and rapid clean-up of some
carbamate pesticides. Forensic Sci Int 46:169–180
5) Lee SK, Ameno K, In SW et al. (1999) Acute fatal poisoning cases due to furathiocarb ingestion. Forensic Sci Int
101:65–70
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benfuracarb ingestion. Int J Legal Med 112:268–270
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Int 116:59–61
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suicide. Forensic Sci Int 42:263–270
9) Tsunoda N, Kishi T (1988) Mass spectra of pesticides 4. N-Methylcarbamate pesticides. Rep Natl Res Inst Police
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Forensic Toxicol News 3 (3):33–34 (in Japanese)
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rience. Sci Justice 36:35–39
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cholinesterase-inhibiting carbamate insecticide and its effects on cholinesterase activity in various biological
fluids. J Forensic Sci 37:337–344
17) Pfordt J, Magerl H, Vock R (1987) Tödliche Vergiftungen mit Propoxur®. Z Rechtsmed 98:43–48