Measurement of Organophosphate Metabolites in Postpartum Meconium as a Potential Biomarker of Prenatal Exposure: A Validation Study.

Experimental data have linked exposure to prenatal organophosphates to adverse neurocognitive sequalae. However, epidemiologic research has been hampered by lack of reliable dosimeters. Existing biomarkers reflect short-term exposure only. Measurements of pesticides in postpartum meconium may yield a longer-term dosimeter of prenatal exposure. As the initial step in biomarker validation, this research determined background levels, detection limits, and stabilities of six organophosphate metabolites in meconium: diethylphosphate (DEP), diethylthiophosphate (DETP), diethyldithiophosphate (DEDTP), dimethylphosphate (DMP), dimethylthiophosphate (DMTP), and dimethyldithiophosphate (DMDTP). Calibration curves were also constructed. The meconium was collected from 20 newborns at New York Presbyterian Hospital; analyses were undertaken at the Centers for Disease Control and Prevention (CDC). DEP was detected in 19/20 samples (range 0.8-3.2 [micro]g/g) and DETP was detected in 20/20 (range 2.0-5.6 [micro]g/g). DMP and DEDTP were each detected in 1/20 (at 16 and 1.8 [micro]g/g, respectively). DMTP and DMDTP were not detected. Detection limits were comparable to or lower than those in urine; levels were similar to those seen in adult urine in population-based research. Metabolites were stable at room temperature over 12 hr. Calibration curves were linear over the range tested (0.5-400 [micro]g/g); recoveries ranged from 18% to 66%. Using isotope dilution, recoveries of each analyte in individual samples can be corrected automatically based on the recovery of the respective stable isotope-labeled analogue, making this method fully quantitative. Results indicate that measurements of organophosphate metabolites in meconium have promise as biomarkers of prenatal exposure. Further research is needed to determine the time frame of exposure represented by pesticide levels in meconium and to evaluate the dose--response relationship. Key words: biomarkers, meconium, organophosphates, pesticides, prenatal exposures. Environ Health Perspect 109:417-420 (2001). [Online 29 March 2001]

http://ehpnet1.niehs.nih.gov/docs/2001/109p417-420whyatt/abstract.html

Residential use of organophosphate insecticides is widespread in the United States (1). Resultant exposures can be appreciable and have been shown to approach or even exceed health-based standards (2-6). Many organophosphate compounds are lipophilic and readily cross the placenta (7). Experimental evidence has linked organophosphate exposure during gestation or the early post-natal period to adverse neurodevelopmental sequelae in offspring (1,8). Exposures during the spurt in brain growth (which in humans begins during the third trimester) may be particularly deleterious (9-14). However, epidemiologic research on this relationship is limited and has been hampered partly because of uncertainties in exposure estimates. Although biologic markers can be useful in understanding the role of environmental contaminants during fetal development (15-17), research on the effects of prenatal organophosphate exposure has been limited by the lack of biomarkers reflecting cumulative exposures. Available biomarkers, including blood and urine measurements, provide short-term dosimeters only (half-lives range from 10 to 30 hr) (18-20). Residential pesticide exposures are episodic, with high peaks after application and decreasing levels over time (3). Thus use of available biomarkers as dosimeters can lead to exposure misclassification if sample collection is not timed to pesticide application. Although erythrocyte acetylcholinesterase is a good biomarker for acute organophosphate exposure, large intraindividual (13%-25%) and interindividual (10%-40%) variability makes it unreliable as a dosimeter in low-level exposure settings unless preexposure values have been determined on each subject (21-23).

Measurements of organophosphates in meconium may yield a longer-term dosimeter of prenatal exposure. In human fetuses, meconium begins to accumulate in the bowels at approximately 16 weeks gestation and is generally not excreted until after delivery (24). Meconium represents the intestinal contents of the fetus and is a complex matrix, consisting mainly of water but also containing mucopolysaccharides, lipids, proteins, bile acids and salts, epithelial cells, cholesterol and sterol precursors, blood-group substances, squamous cells, residual amniotic fluid, and enzymes (25). Prior research on a broad range of xenobiotics indicates that metabolites of compounds to which the fetus has been exposed can be detected in meconium. These include metabolites of illicit drugs (25-32) , nicotine (33), alcohol (34), analgesics, antihistamines, anesthetics, the food additive butylated hydroxytoluene (BHT), and heavy metals (26). One study has also measured pesticide levels in meconium (26). The xenobiotics appear to enter the meconium as a consequence of bile excretion into the intestines and/or of swallowing by the fetus of amniotic fluid (35). Other mechanisms may be operating as well; drugs injected directly into the amniotic fluid of pregnant ewes were detected in meconium in significant concentrations even after the fetuses had undergone esophageal ligation to prevent swallowing (36). The authors reasoned that the drugs reached the fetal circulation by absorption across the umbilical cord or diffusion across the placental surface. Evidence suggests that the half-life of xenobiotics in meconium can be protracted and that measured levels may reflect exposures from the second trimester of pregnancy through delivery (26,28,34,35,37).

Materials and Methods

After obtaining Institutional Review Board approval, we collected meconium samples from the diapers of 20 newborns without knowledge of prenatal pesticide use. Sample collection was conducted over a 3-week period by the postpartum staff at New York Presbyterian Hospital. Samples were transported to the Molecular Epidemiologic Laboratory at Columbia University and frozen within 8 hr of collection in all cases. At the end of the collection period, the samples were shipped on dry ice to the Centers for Disease Control and Prevention (CDC) for analysis.

Before analysis, samples were thawed and homogenized to ensure that the pesticides were distributed evenly throughout the meconium, and then lyophilized to remove residual water. Approximately 0.5-1 g dried meconium was suspended in 5 mL methanol. After the addition of a stable isotope-labeled internal standard, the suspension was mixed by rotation and centrifuged to separate the solids from the supernatant. The supernatant was removed, evaporated to dryness, and reconstituted in acetonitrile. The analytes in the acetonitrile were chemically derivatized to form their chloropropyl esters to make the analytes more suitable for analysis by isotope dilution gas chromatography--tandem mass spectrometry (ID GC-MS/MS). Analyses were undertaken by ID GC-MS/MS to evaluate background levels of six organophosphate metabolites: diethylphosphate (DEP), diethylthiophosphate (DETP), diethyldithiophosphate (DEDTP), dimethylphosphate (DMP), dimethylthiophosphate (DMTP), and dimethyldithiophosphate (DMDTP). These metabolites are common to 1 or more of 28 different organophosphates, as shown in Table 1, and have been measured extensively in biological, samples as specific indicators of both occupational and environmental exposure to organophosphate pesticides (38-43).

Table 1. Organophosphate pesticides, common metabolites,
and insecticidal uses.

                                    Metabolites

Pesticides            DMP   DMTP   DMDTP   DEP   DETP   DEDTP

Azinphos-methyl        X     X       X
Chlorethoxyphos                             X     X
Chlorpyrifos                                X     X

Chlorpyrifos-methyl    X     X
Coumafos                                    X     X
Oiazinon                                    X     X
Dichlorvos (DDVP)      X

Dicrotophos            X
Dimethoate             X     X       X
Disulfoton                                  X     X       X
Ethion                                      X     X       X
Fenitrothion           X     X
Fenthion               X     X
Isazofos-methyl        X     X
Malathion              X     X       X
Methidathion           X     X       X
Methyl parathion       X     X
Naled                  X

Oxydemeton-methyl      X     X
Parathion                                   X     X
Phorate                                     X     X       X
Phosmet                X     X       X
Pirimiphos-methyl      X     X
Sulfotepp                                   X     X
Temephos               X     X
Terbufos                                    X     X       X
Tetrachlorvinphos      X
Trichlorfon            X

Pesticides                    Insecticidal uses(a)

Azinphos-methyl       Crops, trees, ornamentals
Chlorethoxyphos       Crops (corn)
Chlorpyrifos          Crop, lawn/turf, residential, termiticide,
                        ornamentals, pet collars, pasture,
                        livestock(b)
Chlorpyrifos-methyl   Stored grain
Coumafos              Livestock
Oiazinon              Crop, lawn/turf, residential/commercial
Dichlorvos (DDVP)     Pest strips, residential, food, storage/
                        processing, livestock
Dicrotophos           Crops (cotton)
Dimethoate            Crops, ornamentals
Disulfoton            Crops, ornamentals
Ethion                Crops (citrus), livestock
Fenitrothion          Residential/commercial ant/roach bait
Fenthion              Livestock, mosquito control (Florida)
Isazofos-methyl       Registrations canceled
Malathion             Crops, livestock, lawn/turf, mosquito
Methidathion          Crops
Methyl parathion      Crops
Naled                 Crops, greenhouse, flea collars, mosquito,
                        fly
Oxydemeton-methyl     Crops
Parathion             Crops(c)
Phorate               Crops
Phosmet               Crops, ornamental, forestry, livestock
Pirimiphos-methyl     Stored corn, seed, grain, livestock, bulbs
Sulfotepp             Greenhouses, ornamentals
Temephos              Mosquito larva
Terbufos              Crops
Tetrachlorvinphos     Livestock, domestic animals (dogs/cats)
Trichlorfon           Ornamentals, turf, agricultural premises,
                        nurseries, ants

(a) Sources on insecticidal uses from U.S. EPA (47).

(b) Indoor uses being phased out.

(c) Crop uses being phased out.

To determine stability of the metabolites in meconium, aliquots of meconium from the 20 newborns were thawed, pooled, and kept at room temperature for 0-12 hr, with analyses performed every hour. For analyses to construct calibration curves and to determine recoveries, we spiked 0.5 g meconium with an appropriate concentration of standard and analyzed as described above. To evaluate the meconium matrix effects, we compared the calibration curve slopes and intercepts and the reconstructed ion chromatograms from the analysis of spiked meconium samples to those of pure standards analyzed using the same technique.

Results

Table 2 shows the levels of the six organophosphate metabolites in postpartum meconium samples from the 20 newborns. We verified that the measured metabolites were not present in the diapers themselves. DEP was detected in 19/20 (95%) of the samples (range 0.8-3.2 [micro]g/g), and [micro]g was detected in 20/20 (100%; range 2.0-5.6 [micro]g/g). DMP and DEDTP were each detected in 1/20 (5%) of the samples at levels of 16 [micro]g/g and 1.8 [micro]g/g, respectively. DMTP and DMDTP were not detected.

Table 2. Levels of six organophosphate metabolites in postpartum
meconium samples collected from 20 newborns ([micro]g/g).

SAMPLE   DEP    DETP   DEDTP    DMP    DMTP   DMDTP

1        1.90   2.00    ND      ND      ND     ND
2        1.40   3.80    ND      ND      ND     ND
3        1.70   4.30    ND      ND      ND     ND
4        2.00   2.30    ND      ND      ND     ND
5        3.20   3.50   1.80     ND      ND     ND
6        1.20   2.40    ND      ND      ND     ND
7        1.00   2.80    ND      ND      ND     ND
8        1.10   2.00    ND      ND      ND     ND
9        1.00   2.20    ND      ND      ND     ND
10       1.30   2.70    ND      ND      ND     ND
11       1.40   3.00    ND      ND      ND     ND
12       1.30   2.50    ND      ND      ND     ND
13       0.80   2.00    ND      ND      ND     ND
14       2.50   5.60    ND      ND      ND     ND
15       2.80   5.20    ND      ND      ND     ND
16       0.90   2.50    ND      ND      ND     ND
17       1.00   2.40    ND     16.00    ND     ND
18        ND    2.00    ND      ND      ND     ND
19       1.80   5.00    ND      ND      ND     ND
20       0.90   2.40    ND      ND      ND     ND

ND, not detected.

Table 3 shows the stability of the organophosphate metabolites in meconium at room temperature from 0 and 12 hr. Concentrations of DEP and DETP were stable over the entire period, with [is less than] 1.5% variability. Concentrations of DMP were more variable, but there was no trend with time. Levels of DEDTP were too low to determine stability.

Table 3. Concentrations of analytes in meconium
stored at room temperature.

Time (hr)   DEP    DETP    DMP

0           0.81   2.6     6.7
1           0.82   2.6     __(a)
2           0.82   2.6     6.4
3           0.82   2.6     4.0
4           0.83   2.6     6.9
5           0.82   2.6     7.4
6           0.83   2.6     5.8
7           0.83   2.7     8.7
8           0.83   2.6     5.5
9           0.83   2.6     7.2
10          0.81   2.7     4.4
11          0.82   2.6     6.4
12          0.83   2.6     8.2
Mean        0.82   2.6     6.5
RSD         0.9    1.4     22

RSD, relative standard deviation.

(a) Measurement not taken.

Figure 1 shows calibration curves for the six metabolites, and Table 4 shows the [R.sup.2] of the calibration lines and the detection limits and percent recovery of the pesticides in meconium. All calibrations were linear over the entire range tested (Table 4). All [R.sup.2] values were [is greater than] 0.99, and the standard error about the slope was [is less than] 4%. Minimal matrix effects were observed. Due to fewer interfering coextractants, limits of detection were comparable to or better than those observed previously in urine samples from population-based studies that have been analyzed at the CDC. As Table 4 shows, the recoveries of the dialkylphosphate metabolites from meconium range from 18% to 66%. Use of the isotope dilution technique allows complete and automatic correction for analyte recoveries for each sample, enabling a fully quantitative analysis of the meconium.

[GRAPH OMITTED]

Table 4. Specifications of the analytic method.

                          Percent
          [R.sup.2] of   error about     Percent         Limit of
          calibration    calibration     recovery        detection
Analyte      lines          slope      from meconium   ([micro]g/g)

DEP          0.9929          3.0            26             0.2
DETP         0.9908          3.4            55             0.09
DEDTP        0.9969          2.0            62             0.05
DMP          0.9963          2.2            18             0.51
DMTP         0.9998          0.5            63             0.18
DMDTP        0.9995          0.8            66             0.08

Discussion

Results from this initial validation study show that organophosphate metabolites can be detected in postpartum meconium. It is interesting that diethylphosphate and diethylthiophosphate were detected in 95%-100% of the samples. Both are metabolites of the organophosphates diazinon and chlorpyrifos as well as several additional organophosphates used primarily in agriculture (see Table 1), and our findings are consistent with the widespread residential use that has been reported for these two insecticides (1,2,44). These insecticides are also of concern because prenatal exposure to both chlorpyrifos and diazinon has been linked experimentally to adverse neurodevelopmental sequelae in the offspring (1,8). The other organophosphate metabolites were detected only once (dimethylphosphate and diethyldithiophosphate) or not at all (dimethylthiophosphate and dimethyldithiophosphate). As seen from Table 1, this may reflect the fact that they are metabolites of organophosphates with less frequent residential use.

Results also indicate that the measurement of organophosphate metabolites in meconium may have promise as a biomarker of prenatal exposure. Detection limits for the organophosphate metabolites in meconium are low and comparable to or better than those seen with adult urine (45). Further, metabolite levels in meconium are several orders of magnitude higher than those generally seen in umbilical cord blood samples (usually nanograms per liter) (46) and are similar to levels seen in adult urine in population-based studies (45). In addition, the pesticide metabolites appear stable in meconium over 12 hr at room temperature, which should facilitate ease of incorporation of meconium measurements into research protocols. Although recoveries of the metabolites in meconium varied, low or variable recoveries will not compromise analyses. Using isotope dilution, recoveries of each analyte in each individual sample can be corrected based on the recovery of its respective stable isotope-labeled analogue. Chemically, the isotopically labeled analogues behave identically to the analytes measured, but they can be distinguished according to their mass differences. Given these initial promising findings, further research is needed to determine the time frame of exposure represented by pesticide levels in meconium and to evaluate the dose-response relationship.

REFERENCES AND NOTES

(1.) Landrigan PJ, Claudio L, Markowitz SB, Berkowitz GS, Brenner BL, Romero H, Wetmur JG, Matte TD, Gore AC, Godbold JH, Wolff MS. Pesticides and inner-city children: exposures, risks, and prevention. Environ Health Perspect 107(suppl 3):431-437 (1999).

(2.) Whitmore RW, Immerman FW, Camann DE, Bond AE, Lewis RG, Schaum JL. Non-occupational exposure to pesticides for residents of two U.S. cities. Arch Environ Contam Toxicol 26:47-59 (1994).

(3.) Gurunathan S, Robson M, Freeman N, Buckley B, Roy A, Meyer A, Bukowski J, Lioy PJ. Accumulation of chlorpyrifos on residential surfaces and toys accessible to children. Environ Health Perspect 106:9-16 (1988).

(4.) Fenske RA, Black KG, Elkner KP, Lee CL, Methner MM. Potential exposure and health risk of infant from indoor residential pesticide application. Am J Public Health 80:689-693 (1990).

(5.) Davis DL, Ahmed AK. Exposures from indoor spraying of chlorpyrifos pose greater health risks to children than currently estimated. Environ Health Perspect 106:299-301 (1998).

(6.) Lemus R, Ahdelghani AA, Akers TG, Homer W. Potential health risks from exposure to indoor formaldehyde. Rev Environ Health 13:91-98 (1998).

(7.) Richardson R J. Assessment of the neurotoxic potential of chlorpyrifos relative to other organophosphorus compounds: a critical review of the literature. J Toxicol Environ Health 44:135-165 (1995).

(8.) Eskenazi B, Bradman A, Castorina R. Exposures of children to organophosphate pesticides and their potential adverse health effects. Environ Health Perspect 107(suppl 3):409-419 (1999).

(9.) Eriksson P. Developmental neurotoxicity of environmental agents in the neonate. Neurotoxicology 18:719-726 (1997).

(10.) Ahlbom J, Fredriksson A, Ericksson P. Exposure to an organophosphate (DFP) during a defined period in neonatal life induces permanent changes in brain muscarinic receptors and behavior in adult mice. Brain Res 677:13-19 (1995).

(11.) Whitney KD, Sielder FJ, Slotkin TA. Developmental neurotoxicity of chlorpyrifos: cellular mechanisms. Toxicol Appl Pharmacol 134:53-62 (1995).

(12.) Dam K, Seidler FJ, Slotkin TA. Developmental neurotoxicity of chlorpyrifos: delayed targeting of DNA synthesis after repeated administration. Brain Res Bev Brain Res 108:39-45 (1998).

(13.) Slotkin SM. Developmental cholinotoxicants: nicotine and chlorpyrifos. Environ Health Perspect 107(suppl 1):71-80 (1999).

(14.) Johnson DE, Seidler FJ, Slotkin TA. Early biochemical detection of delayed neurotoxicity resulting from developmental exposure to chloropyrifos. Brain Res Bull 45:143-147 (1998).

(15.) Whyatt RM, Perera FP. Application of biologic markers to studies of environmental risks in children and the developing fetus. Environ Health Perspect 103(suppl 6):105-110 (1995).

(16.) Perera FP, Whyatt RM, Jedrychowski W, Rauh V, Manchester D, Santella RM, Ottman R. Recent developments in molecular epidemiology: a study of the effects of environmental polycylic aromatic hyrdrocarbons on birth outcomes in Poland. Am J Epidemiol 147:309-314 (1998).

(17.) Whyatt RM, Santella RM, Jedrychowski W, Garte SJ, Bell DA, Ottman R, Gladek-Yarborough A, Cosma G, Young TL, Cooper TB, et al. Relationship between ambient air pollution and DNA damage in Polish mothers and newborns. Environ Health Perspect 106 (suppl 3):821-826 (1998).

(18.) Durham WF, Wolfe HR, Elliot JW. Absorption and excretion of parathion by spraymen. Arch Environ Health 24:381-387 (1972).

(19.) Nolan RJ, Rick DL, Freshour NL, Saunders JH. Chlorpyrifos: pharmacokinetics in human volunteers. Toxicol Appl Pharmacol 73:8-15 (1984).

(20.) van Sittert NJ, Dumas EP. Field study on exposure and health effects of an organophosphate pesticide for maintaining registration in the Philippines. Med Lav 81:483-473 (1990).

(21.) Lotti M. Cholinesterase inhibition: complexities in interpretation. Clin Chem 41:1814-1818 (1995).

(22.) He F. Biological monitoring of occupational pesticide exposure. Int Arch Occup Environ Health 65:S69-76 (1993).

(23.) Costa LG. Biochemical and molecular neurotoxicology: relevance to biomarker development, neurotoxicity testing and risk assessment. Toxicology Lett 102-103:417-421 (1998).

(24.) Moriya F, Chan KM, Noguchi TT, Wu PY. Testing for drugs of abuse in meconium of newborn infants. J Anal Toxicol 18:41-45 (1994).

(25.) Moore C, Negrusz A, Lewis D. Determination of drugs of abuse in meconium. J Chromatogr 713:137-146 (1998).

(26.) Ostrea EM. Testing for exposure to illicit drugs and other agents in the neonate: a review of laboratory methods and the role of meconium analysis. Curr Probl Pediatr 29:37-56 (1999).

(27.) Browne S, Moore C, Negrusz A, Tebbett I, Covert R, Dusick A. Detection of cocaine, norcocaine, and cocaethylene in the meconium of premature neonates. J Forensic Sci 39:1515-1519 (1994).

(28.) Lewis DE, Moore CM, Leikin JB, Koller A. Meconium analysis for cocaine: a validation study and comparison with paired urine analysis. J Anal Toxicol 19:148-150 (1995).

(29.) Ryan RM, Wagner CL, Schultz JM, Varley J, DiPreta J, Sherer DM, Phelps DL, Kwong T. Meconium analysis for improved identification of infants exposed to cocaine in utero. J Pediatr 125:435-440 (1994).

(30.) Callahan CM, Grant TM, Phipps P, Clark G, Novack AH, Streissguth AP, Raisys VA. Measurement of gestational cocaine exposure: sensitivity of infants' hair, meconium, and urine. J Pediatr 120:763-768 (1992).

(31.) Clark GD, Rosenzweig IB, Raisys VA, Callahan CM, Grant TM, Steissguth AP. The analysis of cocaine and benzoylecgonine in meconium. J Anal Toxicol 16:261-283 (1992).

(32.) Maynard EC, Amoruso LP, Oh W. Meconium for drug testing. Am J Dis Child 145:650-652 (1991).

(33.) Dempsey D, Moore C, Deitermann D, Lewis D, Feeley B, Neidbala RS. The detection of cotinine in hydrolyzed meconium samples. Forensic Sci Int 102:167-171 (1999).

(34.) Bearer CF, Lee S, Salvator AE, Minnes S, Swick A, Yamashita T, Singer LT. Ethyl linoleate in meconium: a biomarker for prenatal ethanol exposure. Alcohol Clin Exp Res 23:487-493 (1999).

(35.) Ostrea EM, Romero A, Yee H. Adaptation of the meconium drug test for mass screening. J Pediatr 122:152-154 (1993).

(36.) Mahone PR, Scot K, Sleggs G, D'Antoni T, Woods JR. Cocaine and metabolites in amniotic fluid may prolong fetal drug exposure. Am J Obstet Gynecol 171:465-469 (1994).

(37.) Nuesslein TG, Beckers D, Rieger CH. Cotinine in meconium indicates risk for early respiratory tract infections. Hum Exp Toxicol 18:283-290 (1999).

(38.) Shafik MT, Bradway D, Enos HF. A cleanup procedure for the determination of low levels of alkyl phosphates, thiophosphates, and dithiophosphates in rat and human urine. J Agric Food Chem 19:885-889 (1971).

(39.) Aprea C, Sciarra G, Orsi D, Boccalon P, Sartorelli P, Sartorelli E. Urinary excretion of alkylphosphates in the general population (Italy). Sci Total Environ 177:37-41 (1996).

(40.) Davies JE, Peterson JC. Surveillance of occupational, accidental, and incidental exposure to organophosphate pesticides using urine alkyl phosphate and phenolic metabolite measurements. Ann NY Acad Sci 837:257-268 (1997).

(41.) Aprea C, Sciarra G, Lunghini L. Analytical method for the determination of urinary alylphosphates in subjects occupationally exposed to organophosphorus pesticides and in the general population. J Anal Toxicol 20:559-563 (1996).

(42.) Reid SJ, Watts RR. A method for the determination of dialkyl phosphate residues in urine. J Anal Toxicol 5:126-132 (1981).

(43.) Loewenherz C, Fenske RA, Simcox NJ, Bellamy G, Kalman D. Biological monitoring of organophosphorus pesticide exposure among children of agricultural workers in central Washington State. Environ Health Perspect 105:1344-1353 (1997).

(44.) Thier A, Enck J, Klossner C. Plagued by Pesticides: An Analysis of New York State and New York City's 1997 Pesticide Use and Sales Data. New York Public Interest Research Group, 1998.

(45.) Centers for Disease Control. Unpublished data.

(46.) Barr DB, Barr JR, Driskell WJ, Hill RH, Ashley DL, Needham LL. Strategies for biological monitoring of exposure to contemporary-use pesticides. Toxicol Ind Health 15 (1-2):168-179 (1999).

(47.) U.S. EPA. Pesticide summaries and quantitative usage analyses. Washington, DC:U.S. Environmental Protection Agency, Office of Pesticide Programs. Available: http://www.epa.gov/pesticides/op [cited 10 October 2000].

Robin M. Whyatt(1) and Dana B. Barr(2)

(1) Columbia Center for Children's Environmental Health, Division of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, New York, USA; (2) Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia, USA

Address correspondence to R.M. Whyatt, Division of Environmental Health Sciences, Mailman School of Public Health, Columbia University, 60 Haven Avenue, B-1, New York, NY 10032 USA. Telephone: [phone]. Fax: [phone]. E-mail: [e-mail]

We thank R. Bravo and D. Whitehead of the CDC for technical assistance and P. Holahan and A. Viade of New York Presbyterian Hospital for assistance in the meconium collection.

This work was supported by the National Institute of Environmental Health Sciences (NIEHS) P50 ES09600; NIEHS P30 ES09089; U.S. Environmental Protection Agency R827027; the Gladys and Roland Harriman Foundation; the W. Alton Jones Foundation; and the New York Community Trust.

Received 10 August 2000; accepted 13 October 2000.