A novel biomonitoring method to detect pyrethroid metabolites in saliva of occupationally exposed workers as a tool for risk assessment

Figures & data

Figure 1. Diagram of proposed mechanism of transport and metabolism for the fate of pyrethroid metabolites detected in saliva using permethrin as an example.

Figure 2. Six pyrethroid metabolites commonly used in biological monitoring studies as analytes of interest for detection in saliva.

Table 1. Recoveries (%) for liquid-liquid extraction (LLE) method development of 100 ng/mL six pyrethroid metabolite mixture spiked in saliva, comparing mixing speed and extraction solvent.

Analyte	Recoveries (%)
	Slow mixing		Fast mixing
	TOL	DCM	MTBE
c-DCCA^a	–	–	69
t-DCCA^a	–	–	77
t-CDCA^a	–	–	86
c-DBCA	92	87.8	49
FPBA	103.9	81.9	89
3PBA	85.2	72.5	103

^aData are not available for slow mixing because the instrument was unable to detect the compound at the time of analysis. TOL: toluene; DCM: dichloromethane; MTBE: methyl tert-butyl ether.

Figure 3. Two saliva collection devices tested for this study: (A) SalivaBio^® sample collection aide attached to 2 mL cryovial, and (B) salivette^® sample collection device showing cotton swab and cap separate from collection device.

Table 2. Retention time (RT), quantification (Q) ions and confirmation ions of six pyrethroid metabolites and three internal standards using gas chromatography – ion trap mass spectrometry in select ion storage mode.

Analyte	RT (min)	Q ion (m/z)	Confirmation ion (m/z)
D6-t-DCCA	15.700	329	–
c-DCCA	15.305	323	325
t-DCCA	15.724	323	325
t-CDCA	15.053	455	261
c-CDCA	16.197	455	261
DBCA	22.976	367	369
FPBA	33.099	382	251
3PBA	34.466	364	169
^13C6-3PBA	34.460	370	175

Table 3. Questionnaire data including demographic information, occupational history, occupational and non-occupational exposures, and metabolic interferences of the study population.

Variable	Average ± SD or % population (n = 9)
Demographics
Gender (% male)	100
Age (yr.)	38 ± 11
Weight (kg)	98 ± 20
Height (cm)	177 ± 10
Occupational history
Job length (yr.)	7.4 ± 6
Days/month active (%)
1–5 day(s)	11
6–10 days	22
11–15 days	33
15–20 days	22
20+ days	11
Hr./day active (%)
1–3 h	56
3–5 h	33
5–7 h	11
7–9 h	0
Occupational exposures
Application time (h)	1.0
Used pyrethroid product (%)	78
Gloves during mixing (%)	89
Gloves during application (Type – %)	None – 11
	Nitrile – 67
	Cloth – 11
PPE during application (Type – %)	None – 66
	Full suit – 11
	Goggles – 22
	Mask – 11
Non-occupational exposures
Vegetable intake (no.)	2.3
Fruit intake (no.)	1.4
Washes produce (%)	78
Pesticide use at home (no.)	0
Metabolism
Grapefruit consumption (%)	0
Medication with CYP interactions (%)	22

Data are presented as the average ± standard deviation (SD) or % population (n = 9).

Table 4. Intra-day (n = 7) and inter-day (n = 5) variation (%) of saliva methodology, limit of detection (LOD) (ng/mL) and detection frequencies (%) for six pyrethroid metabolites analyzed in pre-work (n = 9) and post-work (n = 8) saliva and pre-work (n = 9) and post-work urine (n = 9) of nine pest control operators before and after a full day of work.

Analyte	Intra-day variation (%) (n = 7)	Inter-day variation (%) (n = 5)	Urine LOD (ng/mL)	Saliva LOD (ng/mL)	Detection frequency (%)
					Urine		Saliva
					Pre-work (n = 9)	Post-work (n = 9)	Pre-work (n = 9)	Post-work (n = 8)
c-DCCA	26.7	12.1	0.23	0.04	33	33	0	0
t-DCCA	18.2	2.67	0.16	0.07	33	33	0	0
t-CDCA	14.5	16.7	0.34	0.07	0	0	0	0
c-DBCA	14.0	4.14	0.2	0.22	0	11	0	0
FPBA	10.7	6.64	0.25	0.05	0	0	0	0
3PBA	11.9	49.4	0.08	0.07	100	100	0	22^a

^a Analyte was confirmed to be present under the LOD but not quantifiable.

Table 5. Mean concentrations (ng/mL) of six pyrethroid metabolites in pre- and post-work urine samples of nine pest control operators.

Analyte	Urinary Concentration (ng/mL)
	Pre-work (n = 9)	Post-work (n = 9)
c-DCCA	0.09	0.18
t-DCCA	0.28	0.25
t-CDCA	0	0
c-DBCA	<0.2	<0.2
FPBA	0	0
3PBA	0.45	0.45

Figure 4. Comparison of mass spectra for 3PBA detected in (A) a pest control operator (PCO) saliva sample and (B) 10 ppb 3PBA-spiked saliva sample. Both spectra exhibit characteristic quantitation ions, 364 m/z and 169 m/z, and similar retention times.

Table 6. Biomonitoring studies assessing pesticide levels in saliva, population characteristics, methodology, and sample analysis.

References (location)	Population (study size)	Sample design				Sample type	No. Samples	Analyte	LOD (ng/mL)	DFs (%)	Conc. range (ng/mL)
		1	2	3	4
Present Study (USA)	Pesticide applicators (9)	X		X		Saliva/Urine	18/18	c-DCCA	0.06/0.2	0/22	0/0–0.7
								t-DCCA	0.08/0.2	0/22	0/0–2.4
								t-CDCA	0.3/0.2	0/0	0/0
								c-DBCA	0.3/0.2	0/6	0/0–0.2
								FPBA	0.2/0.2	0/0	0/0
								3PBA	0.2/0.6	<LOD/100	<LOD/0.1–1.6
Zhang et al. (2013) (China)	Pesticide manufacturing factory workers (5)			X	X	Saliva	10	TCP	0.47	100	∼LOD–9.5
Lu et al. (2006) (Nicaragua)	Agricultural applicators (10)	X	X	X	X	Saliva/Blood	62/37	Diazinon	0.02/0.02	54	0–0.15/0.05–0.28
	Applicator children (10)						62			41	0–0.11
Denovan et al. (2000) (USA)	Pesticide applicators (15)				X	Saliva	302	Atrazine	0.22	99	<LOD–149
Nigg et al. (1993) (USA)	Pesticide applicators (6)				X	Saliva	51/51	Ethion	NR	100	86–4,130
						Saliva/Urine		DEP, DETP, DEDTP	NR	NR/100	NR/0.3–7.0

LOD: limit of detection; DF: detection frequency; NR: not reported; DEP: diethyl phosphate; DETP: diethylthiophosphate; DEDTP: diethyl dithiophosphate; 1 = pre-work/pre-season, 2 = during work, 3 = directly after work, 4 = follow-up/next morning.

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