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Biological monitoring of pyrethroids in blood and pyrethroid metabolites in urine: applications and limitations
ELSEVIER
The Science
of the Total
Environment
199 (1997)
173-181
Biological monitoring of pyrethroids in blood and pyrethroid metabolites in urine: applications and limitations Gabriele Leng” , Karl-Heinz Kiihn, Helga Idel Institute
of Hyg.ene,
Heinrich-Heine
University
Diisseldorf;
Moorenstr.
5, D-40225
Diisseldovf;
Germany
Abstract The objective of this study was to perform biological monitoring of subjects who are occupationally exposed to pyrethroids. The study group consisted of 30 pest control operators exposed to cyfluthrin, cypermethrin or permethrin. After exposure, 24-h urine samples were collected and 20 ml of blood was drawn. The pyrethroid metabolites cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethyl~clopropanecarbo~lic acid, 3-phenoxybenzoic acid and fluorophenoxybenzoic acid were determined in the urine samples (limit of detection: 0.5 pg/l) by GC MS and the pyrethroids in plasma (limit of detection: 5 pg/l) by GC-ECD. The concentrations of metabolites in the urine of the pest control operators ranged between < 0.5 @g/l and 277 pg/l urine. The concentrations of cyIkrthrin, cypermethrin and permethrin in the plasma were below the limits of detection (< 5 pg/l). To test if the metabolites are specific for pyrethroid exposure, they were determined in the urine of non-exposed subjects (n = 40). In no case could pyrethroid metabolites be detected. A cylhrthrin elimination experiment showed that cyfluthrin metabolites are eliminated following first-order kinetics (t,,, = 6.4 h). Storage experiments demonstrate that frozen urine samples ( - 21°C) show no significant losses of metabolites within a year. In contrast, pyrethroids stored in plasma are susceptible to further biodegeneration. 0 1997 Elsevier Science B.V. Keywords:
Pyrethroids in blood; Metabolites in urine; Biological monitoring; Pest control operators; Cyfluthrin
elimination
1. Introduction
Pyrethroid cypermethrin,
insecticides such as cyfluthrin, permethrin and deltamethrin are
*Corresponding author. Tel: +49 211 8112607; 211 8112619; e-mail: [email protected] 0048-9697/97/$17.00 PII SOO48-9697(97)
0 1997 Elsevier 05493-4
Science
fax:
+49
B.V. All rights
used increasingly in, for example, plant and storage protection, wood preservation, impregnation of wool carpets and textiles, disinfection and mosquito control. The neurotoxic effect is determined by a prolonged opening of the Na channel which evokes a repetitive nerve action associated with hyperactivity, tremor, ataxia, convulsions and possible paralysis Wdridge, 1990; reserved.
174
G. Leng et al. / The Science of the Total Environment 199 (1997) 173-181
Narahashi, 1992; Appel and Gericke, 1993). Due to the nature of the different toxicological effects and symptoms caused in animal studies, pyrethroids are divided in two classes: Type I pyrethroids without a cyano group such as permethrin and Type II pyrethroids bearing a cyano group such as cyfluthrin (Aldridge, 1990; Narahashi, 1992). For humans, pyrethroids are much less toxic than other insecticides. The AD1 values (acceptable daily intake) vary between 0.01 (deltamethrin) and 0.05 (permethrin) mg/kg body weight per day (Hilbig et al., 1994). Nevertheless, after exposure, a variety of reversible symptoms such as headache, dizziness, nausea, irritation of the skin and nose and paraesthesia are reported by He et al. (1988, 1989). As demonstrated in Fig. 1, halogenated ester pyrethroids such as cyfluthrin, cypermethrin and permethrin are metabolised rapidly by hydrolytic cleavage of the ester bond, followed by oxidation
(1 S. tram,
yielding cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (DCCA), 3phenoxybenzoic acid (3-PBA) and fluorophenoxybenzoic acid (FPBAJ (Casida et al., 1983). These metabolites are partly conjugated to, for example, glucuronic acid and finally eliminated renally (Eadsforth and Baldwin, 1983; Eadsforth et al., 1988). In this study, biological monitoring of 30 pestcontrol operators exposed to cypermethrin, cyfluthrin and permethrin was performed. The analytical method used is described elsewhere (Kuhn et al., 1996; Leng et al., 1996). For meaningful biological monitoring, information about the specificity of the analysed substances, their storage stability and the appropriate point of time of sample collection is essential. To test the specificity of pyrethroid metabolites in urine, 40 nonexposed subjects were investigated. For minimising pyrethroid exposure at the workplace,
a-RS) Cyfluthrin
(1 s. trans. a-RS)
(1 S, trans) Permethrin
Fig. 1. Some pyrethroids and their metabolites. FIBA
Cypemethfin
G. Leng et al. /The
Science of the Total Environment
knowledge of the route of uptake (dermal, inhalative, oral) plays an important role. Therefore, the isomeric ratio of truns-DCCA to cWDCCA was calculated (Eadsforth and Baldwin, 1983; Eadsforth et al., 1988; Woollen et al., 1992). Since human toxicokinetic studies to date only provide data for cypermethrin (Eadsforth and Baldwin, 1983), this study investigates the elimination profile of cyfluthrin in man. 2. Material
and methods
2.1. Subjects
The study was performed in the region of North Rhine-Westphalia, Germany. The study group consisted of 30 male pest-control operators (PCOs), 22-58 years old with 8 months to 22 years of employment, and a control group of 40 subjects, 20 male and 20 female, 22-60 years old. The exposure was assessed by questionnaire. The weekly working time of the PCOs ranged between 40 and 85 h. The control group was not occupationally exposed to pyrethroids. Moreover, there were no hints of non-occupational exposure. A cylluthrin elimination profile was established with the help of one healthy male volunteer who took a single oral dose of cyfluthrin (0.03 mg/kg body wt>. The AD1 of cyfluthrin is 0.02 mg/kg body weight/day (Hilbig et al., 1994). All subjects gave their informed consent after they had received both verbal and written information with respect to the aim of the study, and its execution. The study design was approved by an independent ethical committee. 24-h urine samples were collected in polyethylene bottles during an exposure-free period. Urine volume and creatinine levels were determined in all samples. The samples were stored for up to 4 weeks (-- 21°C). The pyrethroid metabolites cisand trans-DCCA, 3-PBA and FPBA were determined in each urine sample (Fig. 1). Moreover, 20 ml venous blood was drawn from each subject (4-12 h after exposure) for the determination of cytluthrin, cypermethrin and permethrin. Plasma was obtained by centrifugation (3 g, 25°C). The samples were analysed immediately.
199 (1997) 173-181
2.2. Determination
ofpyrethroid
175
metabolites in urine
For the quantification of pyrethroid metabolites, the urine samples (5 ml) were subjected to an acid-induced hydrolytic cleavage of the conjugates, followed by liquid-liquid extraction and methylation of the free acid metabolites (Kuhn et al., 1996). The prepared derivatives were separated by diastereoselective capillary gas chromatography using a Hewlett-Packard MS Engine with a GC 5890, an autoinjector 7673 and a 5989 A mass-selective detector. External as well as internal calibration (Zphenoxybenzoic acid) was applied. The limits of detection were 0.5 hg/l for all metabolites concerned. The within run coefficient of variation as well as the between run coefficient of variation was 12 + 4%. Due to the lack of commercially available methylated acid metabolites, no additional calculation of the recovery could be performed. 2.3. Determination
of pyrethroids
in plasma
Plasma samples (1 ml) were subjected to precipitation of proteins, followed by liquid-liquid extraction. Detection was performed by GC-ECD (Perkin Elmer Autosystem). The pyrethroid bifenthrin served as the internal standard for quantification. The limit of detection was 5 pg/l (Kuhn, 19961. 2.4. Chemicals
Bifenthrin, cytluthrin, cyhalothrin, cypermethrin, deltamethrin, fenvalerate and permethrin were obtained from Promochem (Wesel, Germany). The cholinesterase activity was measured with a kit from Boehringer (Mannheim, Germany) and creatinine with a kit from Merck (Darmstadt, Germany). The metabolites 2- and 3-PBA (purity: 98%) are commercially available from Aldrich (Steinheim, Germany). Cis- and truns-DCCA and FPBA were from Bayer (Leverkusen, Germany). The purity of each substance was > 99%.
G. Leng et al. / The Science of the Total Enuironment 199 (1947) 173-181
1’76
2.5 Storage stat&t), of pyrethroid metabolites in wine
methrin (starting concentration: 60 pg,/lj. Two aliquots were stored at + 3°C and two at - 21°C for 8 days. At each temperature level, formic acid was added to one aliquot, the other aliquot served as a control (no ChE-inhibition).
A real urine sample from a PC0 exposed to cyfluthrin was stored over 30 days at +4”C and a urine sample from a PC0 exposed to cypermethrin was stored over 1 year at - 21°C.
3. Results
2.6. Storage stability of pyrethroids in plasma
3.1. Storage stability of pyrethroid metabolites in urine
Since it is known that pyrethroids are hydrolysed by esterases, different potential esterase inhibitors were tested to improve storage stability. Among those, formic acid cl%, v/v> turned out to be the most suitable inhibitor, because a total inhibition of cholinesterase could be achieved. Cholinesterase (ChE) was chosen as an indicator enzyme for esterases in general. To investigate the influence of ChE-inhibition on storage stability, plasma was spiked with standard solutions of cyfluthrin, cyhalothrin, cyperdeltamethrin, fenvalerate and permethrin,
Cont. MN-)
The mean decrease in the concentrations of the metabolites cis-/trans-DCCA and FPBA (Fig. 2) and cis-/&ans-DCCA and 3-PBA (Fig. 3) was 11 + 3%, which is within the between-run coefficient of variation of 12 f 4%. The limit of detection was 0.5 pg/l for all metabolites. 3.2. Storage stability of pyrethroids in plasma Fig. 4 exemplarily shows the decrease in the concentrations of permethrin, cypermethrin and
80 60
20 0
0
5
10
15
20
25
30
‘% h, ‘Y
cis-DCCA trans-DCCA FPBA
time (days) Fig. 2. Storage
stability
(30 days at + 4°C) of cyfluthrin
metabolites
in urine
from
a pest control
operator.
G. Leng et al. /The
Cont. W/L)
Science of the Total Environment
177
199 (1997) 173-181
7oo 600 500 400 300 200’
0
2
4
6
a
10
12
‘4 h, \
cis-DCCA trans-DCCA 3-PEA
time (months) Fig. 3. Storage stability (1 year at -21°C) of cypermethrin metabolites in urine from a pest control operator. Continuous lines = linear regression; dotted lines = 95% confidence level.
Cont. hJsu Permethrin Cypermethrin Cyfluthrin
IOf.-----------0 12
.~'~'.~'-~'~~~~~~--~~-~~-~~ 4 6 7 3 5
8
9
time (days) Fig. 4. Storage stability (8 days at +4”C) of pyrethroids in plasma (spiked). Non-linear estimation with exponential regression cc, = C,tP).
17x
G. Leng et al. / The Science of the Total Environment 199 (1997) 173-181
Table 1 Effect of formic acid (cholinesterase inhibitor) on storage stability (8 days at -21°C) of pyrethroids in plasma (values given rn x i S.D.. n = 2) ~~~ -_~__...~_____ .__... __ _ ___Pyrethroids Starting cone Without formic acid With formic acid (1% v/v) -__--~~~_( PM) Recovery Cont. after Recovery Cont. after 8 days (%) 8 days (%I ( /G/l) ( Pkvl) ___---Permethrin 60.0 37.5 f 3.8 62.5 f 6.3 60.7 k 6.1 101.2 f 1.0 60.0 15.0 + 1.5 25.0 k 2.5 27.0 & 2.7 45.0 + 4.5 Cypermethrin Cyfluthrin 60.0 20.8 + 2.1 37.4 * 3.5 33.0 + 3.3 55.0 + 5.5 60.0 15.2 f 1.5 33.4 + 3.3 55.6 & 5.6 Cyhalothrin 25.3 + 2.5 Fenvalerate 60.0 17.1 * 1.7 28.5 rt 2.9 38.9 * 3.9 64.8 I 6.5 60.0 19.3 + 1.9 41.3 * 4.1 68.8 i 6.9 Deitamethrin 32.2 + 3.2 .-.
cyfluthrin for a storage time of 8 days ( + 4°C). As an approach non-linear estimation with exponential regression (c, = toe ?was chosen. The halflife time for permethrin was 16 h, for cypermethrin 5 h and for cyhuthrin 7 h. For the other pyrethroids investigated (cyhalothrin, fenvalerate and deltamethrin) the mean half-life time was 6.4 h. At +4”C the addition of formic acid did not improve storage stability (data not shown here). In Table 1 the effect of formic acid (total cholinesterase inhibition) on storage stability (storage time: 8 days at -21°C) is shown. By adding formic acid, storage stability could be improved significantly. There was no decrease in concentration in the case of permethrin. In the case of the other pyrethroids, twice as much was Table 2 Total metabolite concentration in urine of 19 pest control operators (PCOs), daily (Monday-Friday) exposed to pyrethroids before urine collection started Total metabolite concn ( pg/l24 h urine)
Frequency (absolute number of PCOS)
< 0.5 0.5-10 10-20 20-30 30-40 40-50 50-60 > 60
3 0 3 6 1 2 2 2
recovered compared to when there cholinesterase inhibition (Table 1). 3.3. Biological
monitoring
was no
of non-exposed subjects
Urine samples of 40 non-exposed subjects were determined over 1 year (7 times on the whole) with the result that the concentrations of metabolites were below the limit of detection ( < 0.5 /Lg/l). The concentrations of pyrethroids in the blood samples were also below the limit of detection ( < 5 pug/l). 3.4. Biological monitoring
of pest control operators
The pyrethroid metabolites cis-/tram-DCCA, 3-PBA and FPBA were determined in 24-h urine samples from 30 PCOs. During the week of investigation, 19 PCOs were exposed daily (Monday-Friday) to the pyrethroids cyhuthrin, permethrin and cypermethrin. Twenty-four-hour urine samples were taken over the weekend (exposure-free time). Total metabolite concentration varied between < 0.5 and 277 pg/l urine, the median being 30 pg/l (Table 2). The isomeric ratio (tram-DCCAxisDCCA) ranged from 1.5 to 3.2. Seven PCOs were exposed only 1-3 days (1 PC0 Monday-Wednesday, 2 PCOs Wednesday and Thursday, 3 PCOs Tuesday and 1 PC0 Thursday). Four PCOs were not exposed to pyrethroids at all. In these subjects metabolite concentrations were < 0.5 pg/l.
G. Leng et al. /The Science of the Total Environment 199 (1997) 173-181
Pyrethroid concentrations kg/l in all 30 cases.
in plasma were < 5
119
In comparison with the intake of 2.6 mg, a total of 1 mg cyfluthrin equivalent was recovered in the urine (40% of intake). Moreover, Fig. 5 shows an isomeric ratio of 2.3 for trans-DCCA:cis-DCCA. Furthermore, the total amount of FPBA was twice as much as the total amount of cis-/trans-DCCA.
3.5. Urinary excretion rate of CyJluthrin metabolites in one male volunteer Fig. 5 shows the urinary excretion rate (AM/At) of the cyfluthrin metabolites cis/frans-DCCA and FPBA after oral intake of 2.6 mg cyfluthrin (0.03 mg/kg body wt). Metabolite concentrations were measured at 12-h intervals for 2 days. It was assumed that elimination follows a first-order kinetics (represented by lines drawn in Fig. 5). Linear regression analysis was performed on urinary excretion rate vs. mid-point time of each collection interval. The half-life time was estimated from the expression 0.693/(slope x 2.303) (Woollen et al., 1992). The mean half-life time of metabolites was 6.44 f 0.64 h (c&DCCA: 6.66 h; trans-DCCA: 6.54 h; FPBA: 6.13 h). The regression coefficient r2 was > 0.96.
4. Discussion This study demonstrates that the determination of the pyrethroid metabolites cls-/pans-DCCA, 3-PBA and FPBA in urine with the analytical method developed (Kiihn et al., 1996) is suitable for biological monitoring of subjects who are occupationally exposed to cyfluthrin, cypermethrin and permethrin. It was shown that there are several advantages to preferring the determination of metabolites in urine to the analysis of pyrethroids in plasma. Data obtained from the biological monitoring of 30 PCOs regularly exposed to cyfluthrin, cypermethrin and permethrin showed that pyrethroid
8
0 r
AM/At (Wg crea t-0
. 0
IO
15
20
25
30
35
40
45
h,
\ -i h, 50 ‘s
cis-DCCA bans-DCCA
FPBA
t mid (hours) Fig. 5. Urinary excretion rate (AM/At) of cyfluthrin metabolites in one male volunteer after oral intake of cytluthrin (0.03 mg/kg body weight) vs. mid point time of each collection interval (t,,,J on a semi-logarithmic scale.
1x0
G. Leng et al. /The
Science of the Total Environment
metabolites were only found in the urine of the PCOs exposed daily to the pyrethroids before urine collection started ( < 0.5-277 pg/l urine). For PCOs exposed for only l-3 days and in no case the day before urine collection started, no metabolites could be found. Moreover, the metabolites turned out to be specific for pyrethroid exposure. In the case of 40 nonexposed subjects metabolite concentrations were below the limit of detection. For the risk assessment of pyrethroids, it is of prime interest to obtain information about the elimination kinetics in man. So far, these data are only available for cypermethrin, showing a mean half-life of 16.5 h after oral and 13 h after dermal application (Eadsforth and Baldwin, 1983; Woollen et al., 1992). In this study, the urinary excretion of cyfluthrin metabolites in man after oral intake followed first-order kinetics with a half-life of 6.4 h (Fig. 51, showing that 94% of the metabolites were eliminated renally over 48 h. Studies in rats showed that 98% of the cyfluthrin dose was eliminated over 48 h after intake (Tomlin, 1994). Thus, cyfluthrin is eliminated more rapidly than cypermethrin. Therefore, urine samples should be taken during the first 24 h after exposure for biological monitoring. In the case of cylluthrin, FPBA is related specifically only to this compound, whereas 3-PBA can be obtained from different pyrethroids (permethrin, cypermethrin, deltamethrin). This information can be used for an estimation of the extent of cyfluthrin exposure in the likely event of an application of formulations containing pyrethroid mixtures. The effectiveness of the personal means of protection (e.g. breathing mask, overalls) can be proved by quantification of the urinary metabolites cis-/trans-DCCA. Considering the oral exposure studies to cypermethrin mentioned above, approximately twice as much trans-DCCA was excreted in urine compared to cis-DCCA. This concurs with the results found in this study for cyfluthrin (containing 42% cis- and 58% trans-cyfluthrin). As no cis- to trans-conversion can be observed during acid hydrolysis and chromatography, a large excretion of trans-DCCA is a clear sign of a significant oral/inhalative uptake
199 (1997)
173~IKI
during pesticide application (Eadsforth and Baldwin, 1983; Woollen et al., 1992), whereas the ratio is approximately 1 after dermal contact (Woollen et al., 1992). For the PCOs investigated in this study, the most likely route of pyrethroid exposure seemed to be oral/inhalative as the isomeric ratio was > 1 in each case. The storage stability of the substance is essential for biological monitoring. Storage experiments demonstrated that pyrethroid metabolites are stable for at least 30 days at +4”C and for more than a year at -21°C. In contrast, pyrethroids in plasma are less stable (t,,z: 6-16 h at +4”C). However, it was possible to improve storage stability at -21°C by using formic acid as an inhibitor of cholinesterase. Acknowledgements This study was supported by Bundesministerium fiir Bildung, Wissenschaft, Forschung und Technologie (BMBF), Grant No. 07 INR 30. We thank Dr. Lewalter (Bayer AG, Leverkusen, FRG) for the donation of the metabolites, Mrs. Geicke and Mrs. Marsetz for their excellent technical assistance and Dr. A. Leng for fruitful discussions. References Aldridge, W.N. (1990) An assessment of the toxicological properties of pyrethroids and their neurotoxicity. Toxicology 21, 89-104. Appel, K.E. and Gericke, S. (1993) Zur Neurotoxizitst und Toxikokinetik von Pyrethroiden. Bundesgesundheitsblatt 6, 219-252. Casida, J.E., Gammon, D.W., Clickman, A.H. and Lawrence, L.J. (1983) Mechanism of selective action of pyrethroid insecticides. Toxicology 23, 413-438. Eadsforth, C.V. and Baldwin, M.K. (1983) Human dose-excretion studies with the pyrethroid insecticide cypermethrin. Xenobiotica 13, 67-72. Eadsforth, C.V., Bragt, P.C. and van Sitter& N.J. (1988) Human dose-excretion studies with pyrethroid insecticides cypermethrin and alphacypennethrin: relevance for biological monitoring. Xenobiotica 18, 603-614. He, F., Sun, J., Han, K., Wu, Y. and Yao, P. (1988) Effects of pyrethroid insecticides on subjects engaged in packaging pyrethroids. Br. J. Ind. Med. 45, 548-551. He, F., Wang, S., Liu, L., Chen, S., Zhang, Z. and Sun, J. (1989) Clinical manifestation and diagnosis of acute pyrethroid poisoning. Arch. Toxicol. 63, 54-58.
G. Leng et al. / The Science of the Total Environment
Hilbig, V., Pfeil, R., Schellschmidt, B. (1994) ADI-Werte und DTA-Werte fiir Pfanzenschutzmittel-Wirkstoffe. Bundesgesundheitsblatt 4, 182-184. Kuhn, K.-H., Leng, G., Bucholski, K.A., Duneman, L. and Idel, H. (1996) Determination of pyrethroid metabolites in human urine by capillary gas chromatography-mass spectrometry. Chromatographia 43, 285-292. Kuhn, K.-H. (1996) PhD thesis. University of Clausthal, Germany. Leng, G., Kuhn, K.-H., Dunemann, L. and Idel, H. (1996) Gaschromatographische und massenspektrometrische
199 (1997) 173-181
181
Methode zum Nachweis ausgewahlter Pyrethroidmetabolite. Urin. Zbl. Hyg. 198, 443-4.51. Narahashi, T. (1992) Nerve membrane Na channels as targets of insecticides. TIPS 13, 236-241. Tomlin, C. (1994) The Pesticide Manual, 10th ed. Crop Protection Publication, The Royal Sot. of Chem., Cambridge, 249 pp. Woollen, B.H., Marsh, J.R., Laird, W.J.D. and Lesser, J.E. (1992) The metabolism of cypermethrin in man: differences in urinary metabolite profiles following oral and dermal administration. Xenobiotica 22. 983-991.
The Science
of the Total
Environment
199 (1997)
173-181
Biological monitoring of pyrethroids in blood and pyrethroid metabolites in urine: applications and limitations Gabriele Leng” , Karl-Heinz Kiihn, Helga Idel Institute
of Hyg.ene,
Heinrich-Heine
University
Diisseldorf;
Moorenstr.
5, D-40225
Diisseldovf;
Germany
Abstract The objective of this study was to perform biological monitoring of subjects who are occupationally exposed to pyrethroids. The study group consisted of 30 pest control operators exposed to cyfluthrin, cypermethrin or permethrin. After exposure, 24-h urine samples were collected and 20 ml of blood was drawn. The pyrethroid metabolites cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethyl~clopropanecarbo~lic acid, 3-phenoxybenzoic acid and fluorophenoxybenzoic acid were determined in the urine samples (limit of detection: 0.5 pg/l) by GC MS and the pyrethroids in plasma (limit of detection: 5 pg/l) by GC-ECD. The concentrations of metabolites in the urine of the pest control operators ranged between < 0.5 @g/l and 277 pg/l urine. The concentrations of cyIkrthrin, cypermethrin and permethrin in the plasma were below the limits of detection (< 5 pg/l). To test if the metabolites are specific for pyrethroid exposure, they were determined in the urine of non-exposed subjects (n = 40). In no case could pyrethroid metabolites be detected. A cylhrthrin elimination experiment showed that cyfluthrin metabolites are eliminated following first-order kinetics (t,,, = 6.4 h). Storage experiments demonstrate that frozen urine samples ( - 21°C) show no significant losses of metabolites within a year. In contrast, pyrethroids stored in plasma are susceptible to further biodegeneration. 0 1997 Elsevier Science B.V. Keywords:
Pyrethroids in blood; Metabolites in urine; Biological monitoring; Pest control operators; Cyfluthrin
elimination
1. Introduction
Pyrethroid cypermethrin,
insecticides such as cyfluthrin, permethrin and deltamethrin are
*Corresponding author. Tel: +49 211 8112607; 211 8112619; e-mail: [email protected] 0048-9697/97/$17.00 PII SOO48-9697(97)
0 1997 Elsevier 05493-4
Science
fax:
+49
B.V. All rights
used increasingly in, for example, plant and storage protection, wood preservation, impregnation of wool carpets and textiles, disinfection and mosquito control. The neurotoxic effect is determined by a prolonged opening of the Na channel which evokes a repetitive nerve action associated with hyperactivity, tremor, ataxia, convulsions and possible paralysis Wdridge, 1990; reserved.
174
G. Leng et al. / The Science of the Total Environment 199 (1997) 173-181
Narahashi, 1992; Appel and Gericke, 1993). Due to the nature of the different toxicological effects and symptoms caused in animal studies, pyrethroids are divided in two classes: Type I pyrethroids without a cyano group such as permethrin and Type II pyrethroids bearing a cyano group such as cyfluthrin (Aldridge, 1990; Narahashi, 1992). For humans, pyrethroids are much less toxic than other insecticides. The AD1 values (acceptable daily intake) vary between 0.01 (deltamethrin) and 0.05 (permethrin) mg/kg body weight per day (Hilbig et al., 1994). Nevertheless, after exposure, a variety of reversible symptoms such as headache, dizziness, nausea, irritation of the skin and nose and paraesthesia are reported by He et al. (1988, 1989). As demonstrated in Fig. 1, halogenated ester pyrethroids such as cyfluthrin, cypermethrin and permethrin are metabolised rapidly by hydrolytic cleavage of the ester bond, followed by oxidation
(1 S. tram,
yielding cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (DCCA), 3phenoxybenzoic acid (3-PBA) and fluorophenoxybenzoic acid (FPBAJ (Casida et al., 1983). These metabolites are partly conjugated to, for example, glucuronic acid and finally eliminated renally (Eadsforth and Baldwin, 1983; Eadsforth et al., 1988). In this study, biological monitoring of 30 pestcontrol operators exposed to cypermethrin, cyfluthrin and permethrin was performed. The analytical method used is described elsewhere (Kuhn et al., 1996; Leng et al., 1996). For meaningful biological monitoring, information about the specificity of the analysed substances, their storage stability and the appropriate point of time of sample collection is essential. To test the specificity of pyrethroid metabolites in urine, 40 nonexposed subjects were investigated. For minimising pyrethroid exposure at the workplace,
a-RS) Cyfluthrin
(1 s. trans. a-RS)
(1 S, trans) Permethrin
Fig. 1. Some pyrethroids and their metabolites. FIBA
Cypemethfin
G. Leng et al. /The
Science of the Total Environment
knowledge of the route of uptake (dermal, inhalative, oral) plays an important role. Therefore, the isomeric ratio of truns-DCCA to cWDCCA was calculated (Eadsforth and Baldwin, 1983; Eadsforth et al., 1988; Woollen et al., 1992). Since human toxicokinetic studies to date only provide data for cypermethrin (Eadsforth and Baldwin, 1983), this study investigates the elimination profile of cyfluthrin in man. 2. Material
and methods
2.1. Subjects
The study was performed in the region of North Rhine-Westphalia, Germany. The study group consisted of 30 male pest-control operators (PCOs), 22-58 years old with 8 months to 22 years of employment, and a control group of 40 subjects, 20 male and 20 female, 22-60 years old. The exposure was assessed by questionnaire. The weekly working time of the PCOs ranged between 40 and 85 h. The control group was not occupationally exposed to pyrethroids. Moreover, there were no hints of non-occupational exposure. A cylluthrin elimination profile was established with the help of one healthy male volunteer who took a single oral dose of cyfluthrin (0.03 mg/kg body wt>. The AD1 of cyfluthrin is 0.02 mg/kg body weight/day (Hilbig et al., 1994). All subjects gave their informed consent after they had received both verbal and written information with respect to the aim of the study, and its execution. The study design was approved by an independent ethical committee. 24-h urine samples were collected in polyethylene bottles during an exposure-free period. Urine volume and creatinine levels were determined in all samples. The samples were stored for up to 4 weeks (-- 21°C). The pyrethroid metabolites cisand trans-DCCA, 3-PBA and FPBA were determined in each urine sample (Fig. 1). Moreover, 20 ml venous blood was drawn from each subject (4-12 h after exposure) for the determination of cytluthrin, cypermethrin and permethrin. Plasma was obtained by centrifugation (3 g, 25°C). The samples were analysed immediately.
199 (1997) 173-181
2.2. Determination
ofpyrethroid
175
metabolites in urine
For the quantification of pyrethroid metabolites, the urine samples (5 ml) were subjected to an acid-induced hydrolytic cleavage of the conjugates, followed by liquid-liquid extraction and methylation of the free acid metabolites (Kuhn et al., 1996). The prepared derivatives were separated by diastereoselective capillary gas chromatography using a Hewlett-Packard MS Engine with a GC 5890, an autoinjector 7673 and a 5989 A mass-selective detector. External as well as internal calibration (Zphenoxybenzoic acid) was applied. The limits of detection were 0.5 hg/l for all metabolites concerned. The within run coefficient of variation as well as the between run coefficient of variation was 12 + 4%. Due to the lack of commercially available methylated acid metabolites, no additional calculation of the recovery could be performed. 2.3. Determination
of pyrethroids
in plasma
Plasma samples (1 ml) were subjected to precipitation of proteins, followed by liquid-liquid extraction. Detection was performed by GC-ECD (Perkin Elmer Autosystem). The pyrethroid bifenthrin served as the internal standard for quantification. The limit of detection was 5 pg/l (Kuhn, 19961. 2.4. Chemicals
Bifenthrin, cytluthrin, cyhalothrin, cypermethrin, deltamethrin, fenvalerate and permethrin were obtained from Promochem (Wesel, Germany). The cholinesterase activity was measured with a kit from Boehringer (Mannheim, Germany) and creatinine with a kit from Merck (Darmstadt, Germany). The metabolites 2- and 3-PBA (purity: 98%) are commercially available from Aldrich (Steinheim, Germany). Cis- and truns-DCCA and FPBA were from Bayer (Leverkusen, Germany). The purity of each substance was > 99%.
G. Leng et al. / The Science of the Total Enuironment 199 (1947) 173-181
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2.5 Storage stat&t), of pyrethroid metabolites in wine
methrin (starting concentration: 60 pg,/lj. Two aliquots were stored at + 3°C and two at - 21°C for 8 days. At each temperature level, formic acid was added to one aliquot, the other aliquot served as a control (no ChE-inhibition).
A real urine sample from a PC0 exposed to cyfluthrin was stored over 30 days at +4”C and a urine sample from a PC0 exposed to cypermethrin was stored over 1 year at - 21°C.
3. Results
2.6. Storage stability of pyrethroids in plasma
3.1. Storage stability of pyrethroid metabolites in urine
Since it is known that pyrethroids are hydrolysed by esterases, different potential esterase inhibitors were tested to improve storage stability. Among those, formic acid cl%, v/v> turned out to be the most suitable inhibitor, because a total inhibition of cholinesterase could be achieved. Cholinesterase (ChE) was chosen as an indicator enzyme for esterases in general. To investigate the influence of ChE-inhibition on storage stability, plasma was spiked with standard solutions of cyfluthrin, cyhalothrin, cyperdeltamethrin, fenvalerate and permethrin,
Cont. MN-)
The mean decrease in the concentrations of the metabolites cis-/trans-DCCA and FPBA (Fig. 2) and cis-/&ans-DCCA and 3-PBA (Fig. 3) was 11 + 3%, which is within the between-run coefficient of variation of 12 f 4%. The limit of detection was 0.5 pg/l for all metabolites. 3.2. Storage stability of pyrethroids in plasma Fig. 4 exemplarily shows the decrease in the concentrations of permethrin, cypermethrin and
80 60
20 0
0
5
10
15
20
25
30
‘% h, ‘Y
cis-DCCA trans-DCCA FPBA
time (days) Fig. 2. Storage
stability
(30 days at + 4°C) of cyfluthrin
metabolites
in urine
from
a pest control
operator.
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199 (1997) 173-181
7oo 600 500 400 300 200’
0
2
4
6
a
10
12
‘4 h, \
cis-DCCA trans-DCCA 3-PEA
time (months) Fig. 3. Storage stability (1 year at -21°C) of cypermethrin metabolites in urine from a pest control operator. Continuous lines = linear regression; dotted lines = 95% confidence level.
Cont. hJsu Permethrin Cypermethrin Cyfluthrin
IOf.-----------0 12
.~'~'.~'-~'~~~~~~--~~-~~-~~ 4 6 7 3 5
8
9
time (days) Fig. 4. Storage stability (8 days at +4”C) of pyrethroids in plasma (spiked). Non-linear estimation with exponential regression cc, = C,tP).
17x
G. Leng et al. / The Science of the Total Environment 199 (1997) 173-181
Table 1 Effect of formic acid (cholinesterase inhibitor) on storage stability (8 days at -21°C) of pyrethroids in plasma (values given rn x i S.D.. n = 2) ~~~ -_~__...~_____ .__... __ _ ___Pyrethroids Starting cone Without formic acid With formic acid (1% v/v) -__--~~~_( PM) Recovery Cont. after Recovery Cont. after 8 days (%) 8 days (%I ( /G/l) ( Pkvl) ___---Permethrin 60.0 37.5 f 3.8 62.5 f 6.3 60.7 k 6.1 101.2 f 1.0 60.0 15.0 + 1.5 25.0 k 2.5 27.0 & 2.7 45.0 + 4.5 Cypermethrin Cyfluthrin 60.0 20.8 + 2.1 37.4 * 3.5 33.0 + 3.3 55.0 + 5.5 60.0 15.2 f 1.5 33.4 + 3.3 55.6 & 5.6 Cyhalothrin 25.3 + 2.5 Fenvalerate 60.0 17.1 * 1.7 28.5 rt 2.9 38.9 * 3.9 64.8 I 6.5 60.0 19.3 + 1.9 41.3 * 4.1 68.8 i 6.9 Deitamethrin 32.2 + 3.2 .-.
cyfluthrin for a storage time of 8 days ( + 4°C). As an approach non-linear estimation with exponential regression (c, = toe ?was chosen. The halflife time for permethrin was 16 h, for cypermethrin 5 h and for cyhuthrin 7 h. For the other pyrethroids investigated (cyhalothrin, fenvalerate and deltamethrin) the mean half-life time was 6.4 h. At +4”C the addition of formic acid did not improve storage stability (data not shown here). In Table 1 the effect of formic acid (total cholinesterase inhibition) on storage stability (storage time: 8 days at -21°C) is shown. By adding formic acid, storage stability could be improved significantly. There was no decrease in concentration in the case of permethrin. In the case of the other pyrethroids, twice as much was Table 2 Total metabolite concentration in urine of 19 pest control operators (PCOs), daily (Monday-Friday) exposed to pyrethroids before urine collection started Total metabolite concn ( pg/l24 h urine)
Frequency (absolute number of PCOS)
< 0.5 0.5-10 10-20 20-30 30-40 40-50 50-60 > 60
3 0 3 6 1 2 2 2
recovered compared to when there cholinesterase inhibition (Table 1). 3.3. Biological
monitoring
was no
of non-exposed subjects
Urine samples of 40 non-exposed subjects were determined over 1 year (7 times on the whole) with the result that the concentrations of metabolites were below the limit of detection ( < 0.5 /Lg/l). The concentrations of pyrethroids in the blood samples were also below the limit of detection ( < 5 pug/l). 3.4. Biological monitoring
of pest control operators
The pyrethroid metabolites cis-/tram-DCCA, 3-PBA and FPBA were determined in 24-h urine samples from 30 PCOs. During the week of investigation, 19 PCOs were exposed daily (Monday-Friday) to the pyrethroids cyhuthrin, permethrin and cypermethrin. Twenty-four-hour urine samples were taken over the weekend (exposure-free time). Total metabolite concentration varied between < 0.5 and 277 pg/l urine, the median being 30 pg/l (Table 2). The isomeric ratio (tram-DCCAxisDCCA) ranged from 1.5 to 3.2. Seven PCOs were exposed only 1-3 days (1 PC0 Monday-Wednesday, 2 PCOs Wednesday and Thursday, 3 PCOs Tuesday and 1 PC0 Thursday). Four PCOs were not exposed to pyrethroids at all. In these subjects metabolite concentrations were < 0.5 pg/l.
G. Leng et al. /The Science of the Total Environment 199 (1997) 173-181
Pyrethroid concentrations kg/l in all 30 cases.
in plasma were < 5
119
In comparison with the intake of 2.6 mg, a total of 1 mg cyfluthrin equivalent was recovered in the urine (40% of intake). Moreover, Fig. 5 shows an isomeric ratio of 2.3 for trans-DCCA:cis-DCCA. Furthermore, the total amount of FPBA was twice as much as the total amount of cis-/trans-DCCA.
3.5. Urinary excretion rate of CyJluthrin metabolites in one male volunteer Fig. 5 shows the urinary excretion rate (AM/At) of the cyfluthrin metabolites cis/frans-DCCA and FPBA after oral intake of 2.6 mg cyfluthrin (0.03 mg/kg body wt). Metabolite concentrations were measured at 12-h intervals for 2 days. It was assumed that elimination follows a first-order kinetics (represented by lines drawn in Fig. 5). Linear regression analysis was performed on urinary excretion rate vs. mid-point time of each collection interval. The half-life time was estimated from the expression 0.693/(slope x 2.303) (Woollen et al., 1992). The mean half-life time of metabolites was 6.44 f 0.64 h (c&DCCA: 6.66 h; trans-DCCA: 6.54 h; FPBA: 6.13 h). The regression coefficient r2 was > 0.96.
4. Discussion This study demonstrates that the determination of the pyrethroid metabolites cls-/pans-DCCA, 3-PBA and FPBA in urine with the analytical method developed (Kiihn et al., 1996) is suitable for biological monitoring of subjects who are occupationally exposed to cyfluthrin, cypermethrin and permethrin. It was shown that there are several advantages to preferring the determination of metabolites in urine to the analysis of pyrethroids in plasma. Data obtained from the biological monitoring of 30 PCOs regularly exposed to cyfluthrin, cypermethrin and permethrin showed that pyrethroid
8
0 r
AM/At (Wg crea t-0
. 0
IO
15
20
25
30
35
40
45
h,
\ -i h, 50 ‘s
cis-DCCA bans-DCCA
FPBA
t mid (hours) Fig. 5. Urinary excretion rate (AM/At) of cyfluthrin metabolites in one male volunteer after oral intake of cytluthrin (0.03 mg/kg body weight) vs. mid point time of each collection interval (t,,,J on a semi-logarithmic scale.
1x0
G. Leng et al. /The
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metabolites were only found in the urine of the PCOs exposed daily to the pyrethroids before urine collection started ( < 0.5-277 pg/l urine). For PCOs exposed for only l-3 days and in no case the day before urine collection started, no metabolites could be found. Moreover, the metabolites turned out to be specific for pyrethroid exposure. In the case of 40 nonexposed subjects metabolite concentrations were below the limit of detection. For the risk assessment of pyrethroids, it is of prime interest to obtain information about the elimination kinetics in man. So far, these data are only available for cypermethrin, showing a mean half-life of 16.5 h after oral and 13 h after dermal application (Eadsforth and Baldwin, 1983; Woollen et al., 1992). In this study, the urinary excretion of cyfluthrin metabolites in man after oral intake followed first-order kinetics with a half-life of 6.4 h (Fig. 51, showing that 94% of the metabolites were eliminated renally over 48 h. Studies in rats showed that 98% of the cyfluthrin dose was eliminated over 48 h after intake (Tomlin, 1994). Thus, cyfluthrin is eliminated more rapidly than cypermethrin. Therefore, urine samples should be taken during the first 24 h after exposure for biological monitoring. In the case of cylluthrin, FPBA is related specifically only to this compound, whereas 3-PBA can be obtained from different pyrethroids (permethrin, cypermethrin, deltamethrin). This information can be used for an estimation of the extent of cyfluthrin exposure in the likely event of an application of formulations containing pyrethroid mixtures. The effectiveness of the personal means of protection (e.g. breathing mask, overalls) can be proved by quantification of the urinary metabolites cis-/trans-DCCA. Considering the oral exposure studies to cypermethrin mentioned above, approximately twice as much trans-DCCA was excreted in urine compared to cis-DCCA. This concurs with the results found in this study for cyfluthrin (containing 42% cis- and 58% trans-cyfluthrin). As no cis- to trans-conversion can be observed during acid hydrolysis and chromatography, a large excretion of trans-DCCA is a clear sign of a significant oral/inhalative uptake
199 (1997)
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during pesticide application (Eadsforth and Baldwin, 1983; Woollen et al., 1992), whereas the ratio is approximately 1 after dermal contact (Woollen et al., 1992). For the PCOs investigated in this study, the most likely route of pyrethroid exposure seemed to be oral/inhalative as the isomeric ratio was > 1 in each case. The storage stability of the substance is essential for biological monitoring. Storage experiments demonstrated that pyrethroid metabolites are stable for at least 30 days at +4”C and for more than a year at -21°C. In contrast, pyrethroids in plasma are less stable (t,,z: 6-16 h at +4”C). However, it was possible to improve storage stability at -21°C by using formic acid as an inhibitor of cholinesterase. Acknowledgements This study was supported by Bundesministerium fiir Bildung, Wissenschaft, Forschung und Technologie (BMBF), Grant No. 07 INR 30. We thank Dr. Lewalter (Bayer AG, Leverkusen, FRG) for the donation of the metabolites, Mrs. Geicke and Mrs. Marsetz for their excellent technical assistance and Dr. A. Leng for fruitful discussions. References Aldridge, W.N. (1990) An assessment of the toxicological properties of pyrethroids and their neurotoxicity. Toxicology 21, 89-104. Appel, K.E. and Gericke, S. (1993) Zur Neurotoxizitst und Toxikokinetik von Pyrethroiden. Bundesgesundheitsblatt 6, 219-252. Casida, J.E., Gammon, D.W., Clickman, A.H. and Lawrence, L.J. (1983) Mechanism of selective action of pyrethroid insecticides. Toxicology 23, 413-438. Eadsforth, C.V. and Baldwin, M.K. (1983) Human dose-excretion studies with the pyrethroid insecticide cypermethrin. Xenobiotica 13, 67-72. Eadsforth, C.V., Bragt, P.C. and van Sitter& N.J. (1988) Human dose-excretion studies with pyrethroid insecticides cypermethrin and alphacypennethrin: relevance for biological monitoring. Xenobiotica 18, 603-614. He, F., Sun, J., Han, K., Wu, Y. and Yao, P. (1988) Effects of pyrethroid insecticides on subjects engaged in packaging pyrethroids. Br. J. Ind. Med. 45, 548-551. He, F., Wang, S., Liu, L., Chen, S., Zhang, Z. and Sun, J. (1989) Clinical manifestation and diagnosis of acute pyrethroid poisoning. Arch. Toxicol. 63, 54-58.
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Hilbig, V., Pfeil, R., Schellschmidt, B. (1994) ADI-Werte und DTA-Werte fiir Pfanzenschutzmittel-Wirkstoffe. Bundesgesundheitsblatt 4, 182-184. Kuhn, K.-H., Leng, G., Bucholski, K.A., Duneman, L. and Idel, H. (1996) Determination of pyrethroid metabolites in human urine by capillary gas chromatography-mass spectrometry. Chromatographia 43, 285-292. Kuhn, K.-H. (1996) PhD thesis. University of Clausthal, Germany. Leng, G., Kuhn, K.-H., Dunemann, L. and Idel, H. (1996) Gaschromatographische und massenspektrometrische
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Methode zum Nachweis ausgewahlter Pyrethroidmetabolite. Urin. Zbl. Hyg. 198, 443-4.51. Narahashi, T. (1992) Nerve membrane Na channels as targets of insecticides. TIPS 13, 236-241. Tomlin, C. (1994) The Pesticide Manual, 10th ed. Crop Protection Publication, The Royal Sot. of Chem., Cambridge, 249 pp. Woollen, B.H., Marsh, J.R., Laird, W.J.D. and Lesser, J.E. (1992) The metabolism of cypermethrin in man: differences in urinary metabolite profiles following oral and dermal administration. Xenobiotica 22. 983-991.