A Longitudinal Investigation of Selected Pesticide Metabolites in Urine

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A longitudinal investigation of selected pesticide metabolites in urine

DAVID L. MACINTOSH,a LARRY L. NEEDHAM,b KAREN A. HAMMERSTROMc AND P. BARRY RYANd aDepartment of Environmental Health, University of Georgia, Athens, GA bNational Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA cNational Center for Environmental Assessment, U.S. Environmental Protection Agency, Washington, D.C. dRollins School of Public Health, Emory University, Atlanta, GA

As part of a longitudinal investigation of environmental exposures to selected chemical contaminants, concentrations of the pesticide metabolites 1-naphthol (1NAP), 3,5,6-trichloro-2-pyridinol (TCPY), malathion dicarboxylic acid (MDA), and atrazine mercapturate (AM) were measured in repeated samples obtained from 80 individuals in Maryland during 1995±1996. Up to six urine samples were collected from each individual at intervals of approximately 8 weeks over a 1-year period (i.e., one sample per participant in each of six cycles). 1NAP (median=4.2 g/l and 3.3 g/g creatinine) and TCPY (median=5.3 g/l and 4.6. g/g creatinine) were present in over 80% of the samples, while MDA and AM were detected infrequently (6.6% and <1% of samples, respectively). Geometric mean (GM) concentrations of 1NAP in urine did not vary significantly among sampling cycles. In contrast, GM concentrationsof TCPY were significantly greater in samples collected during the spring and summer of 1996 than in the preceding fall and winter. Repeated measurementsof 1NAP and TCPY from the same individual over time were highly variable. The average range of 1NAP and TCPY concentrations from the same individual were approximately 200% and 50% greater than the respective population mean levels. Geometric mean (GM) TCPY concentrations differed significantly betweenCaucasian( n=42, GM=5.7 g/g creatinine) and African-American (n=11, GM=4.0 g/g) participants and among education levels, but were not significantly different among groups classified by gender, age, or household income. In future research, environmental measurements of the parent compounds and questionnaire data collected concurrently with the biomarker data will be used to characterize the determinants of variability in the urinary pesticide metabolite levels.

Keywords: biological marker, chronic exposure, exposure, longitudinal, NHEXAS, pesticides, time budget.

Introduction sphate, carbamate, and triazine pesticides and their metabolites have biological half-lives onthe order of days Biological markers of exposure to contaminants afford a (EPA, 1992). Thus, urinary biomarkers of exposure to direct means of assessing the extent of human contact with compounds in these pesticide classes are indicative only of chemicals in the environment. Biomarkers of exposure are recent exposure. If the rate of exposure is relatively useful in many aspects of environmental health, including constant, a single urinary biomarker measurement may be analysis of status and trends, epidemiology, and health-risk representative of typical or chronic exposure. The relation- assessment (Sexton et al., 1995). Urine is relatively ship between a single urinary biomarker measurement and convenient to collect and therefore is a potentially useful chronic exposure to such a pesticide is not well understood. medium inwhich to measure biomarkers of exposure. Improved understanding of this relationship is important for Urinary concentrations of selected metals, pesticides, and investigations of health effects potentially related to chronic polycyclic aromatic hydrocarbons (PAH) or their metabo- pesticide exposure. lites have been used to monitor national trends in exposure Inthis paper, we report onthe relationship betweenshort- in many countries, for example the United States (Kutz et term and long-term average levels of biomarkers of al., 1992; Hill et al., 1995a) and Germany (Trepka et al., exposure to several pesticides measured inurinesamples 1997). Biomarkers inurinemay be used to assess exposure collected from 80 individuals in greater Maryland. Analyses in environmental epidemiology studies as well (Schulte, of variability of exposure levels among and within 1992). individuals over time are presented. The implications of Depending on the pharmacokinetics of a compound in our findings with respect to exposure assessment in the human body, a urinary biomarker may represent short- epidemiology and risk assessment are discussed. term or long-term average exposure. Many organopho-

Methods 1. Address all correspondence to: Dr. David L. MacIntosh, Department of Environmental Health, University of Georgia, 206 Environmental Health Eighty individuals randomly selected from five contiguous Building, Athens, GA 30602-2102. counties in Maryland were enrolled in the study in A longitudinal investigation of selected pesticide metabolites in urine MacIntosh et al.

September 1995 (Ryan et al., 1999). Environmental and 1NAP, TCPY, and MDA at 50 g/l and AM at 100 g/l biological samples, including urine, were collected from were prepared at the CDC laboratory and transported frozen participants over a 1-week period during each of six to the field. Frozenfortified samples were thawed inthe sampling cycles at intervals of approximately 8 weeks over field and transferred to a urine sample cup in the residences the following 12 months (i.e., in each of six cycles). of randomly selected participants. Thereafter, quality- Sampling cycles 1 through 6 corresponded to September± assurance samples were treated identically to the primary December 1995, January±March 1996, February±April urine samples in all respects. 1996, April±June 1996, June±July 1996, and July±Septem- Descriptive statistics were generated for each pesticide ber 1996, respectively. Cycle 1 included a 2-week pilot metabolite by cycle, including the number and percentage phase in which samples were collected from nine homes and of samples containing a detectable amount of the analyte, a 4-week period immediately following that was used to and measures of central tendency and dispersion. Samples revise standard operating procedures and train new field containing metabolite concentrations below the LOD staff. were set to 1/2 LOD. Identical data summaries were Urine samples (first morning void, approximately 250 prepared for creatinine-corrected concentrations expressed ml) were collected onthe secondor eighth day of the week in micrograms of metabolite per gram creatinine. These long sampling period in accordance with the respondent's summaries were restricted to urine samples that contained schedule. Urine samples were separated into four fractions creatinine levels greater than 30 mg/dl as samples with for subsequent determination of creatinine, heavy metals lower creatinine concentrations are generally regarded as (As, Cr), PAH, and pesticide metabolite concentrations. too dilute to provide valid results (Lauwerys and Hoet, Split urine samples were frozen and shipped on dry ice to 1993). the National Center for Environmental Health, Centers for For 1NAP and TCPY, mixed generalized linear models Disease Control and Prevention in Atlanta, Georgia. (GLM) were used to test for significant variability of Concentrations of 1-naphthol (1NAP) and 3,5,6-trichloro- mean urinary metabolite concentrations among cycles and 2-pyridinol (TCPY) were determined by GC/MS/MS among individuals. The low number of quantifiable results following Hill et al. (1995b). Atrazine mercapturate (AM) for AM and MDA prevented statistical analyses of these and malathion dicarboxylic acid (MDA) were determined metabolites from being performed. The repeated measure by LC/MS/MS (unpublished). The analytical limit of switch of the SAS procedure PROC MIXED was used to detection(LOD) for each metabolite was 1 g/l. account for potential correlation among metabolite con- 1NAP is a urinary metabolite of both carbaryl and centrations measured in samples collected from the same naphthalene. Carbaryl is a common carbamate insecticide individual over time (Littel et al., 1996). The form of the marketed primarily for use on turf and gardens, while GLM is showninEquation1 where C is the concentra- naphthalene is used in mothballs and is found in cigarette tionof the pesticide metabolite inurine( g/l), Cycle is a smoke and petroleum products (Hill et al., 1995a). TCPY is categorical variable with six levels denoting the sampling the major metabolite inurineof the pesticides chlorpyrifos, cycle, and HIN (household identification number) is a chlorpyrifos-methyl, and triclopyr and has a biological half- categorical variable with a level for each participant. All life of approximately 27 h (Bakke et al., 1976; Nolanet al., statistical models were run on natural log-transformed 1984; Hill et al., 1995a). TCPY is also the principal product metabolite concentrations to account for skewness in the of environmental degradation of chlorpyrifos and may crude data. persist insoil for more than1 year (Racke et al., 1992). Consequently, urinary levels of TCPY may represent exposure to TCPY as well as its parent compounds. MDA C 1 Cycle 2 HIN 1 is a principal metabolite of malathion, an organophosphate pesticide used against insects on fruits and vegetables in agriculture and against household insects (EPA, 1992; Kutz Among respondents that provided a urine sample in at et al., 1992). Malathionhas a half-life onthe order of days in least four cycles, annual average metabolite concentrations many environmental media and in animal models, e.g., the were computed as the meanof the results amongall rat (EPA, 1992). AM is a urinary metabolite of atrazine, a cycles. Generalized linear models were used to examine widely used herbicide inthe UnitedStates (EPA, 1992). associations between annual average metabolite concentra- Field blanks and fortified (i.e., spiked) samples were tions and selected demographic factors including gender, used as quality assurance indicators for sample collection age, race, education, and household income. Four levels of and analysis activities. Field blanks consisted of 18-M age were used: <25, 25±44, 45±64, and greater than 64 water poured from a bulk container to a urine sample cup in years. Educationwas divided intothree categories: at least the residences of randomly selected participants. Following some high school, high school graduate, and college Shealy et al. (1996), fortified urine samples containing graduate and above. Four levels of annual household

Journal of Exposure Analysis and Environmental Epidemiology (1999) 9(5) 495 MacIntosh et al. A longitudinal investigation of selected pesticide metabolites in urine

Table 1. Summary statistics for selected pesticide metabolites inurine( g/l) from multiple samples collected from 80 individuals in Maryland between September 1995 and September 1996.

Analytea N Frequency of Mean GMb MinPercentile Max detection(%) 5% 25% 50% 75% 95% 1NAP 338 85 33.7 4.2 <1 <1 1.6 4.2 9.3 52.0 2500.0 1NAPc 333 85 13.2 3.8 <1 <1 1.6 4.1 8.8 37.0 550.0 TCPY 346 96 6.8 5.1 <1 1.2 3.1 5.3 9.4 17.0 51.0 AM 348 0.3 ±d ±d <1 <1 <1 <1 <1 <1 1.5 MDA 347 6.6 ±d ±d <1 <1 <1 <1 <1 2.0 51.0 a1NAP=1-naphthol; TCPY=3,5,6-trichloro-2-pyridinol; AM=atrazine mercapturate; MDA=malathion dicarboxylic acid. bGeometric mean. cSummary statistics with potential outlier removed. dInsufficient number of results greater than limit of detection.

income were used: <$20,000, $20,000 to $<$50,000, micrograms of metabolite per liter of urine. Of the 350 urine $50,000 to <$75,000, and $75,000 or greater. samples collected and analyzed, 27 (7.7%) had missing data for creatinine or at least one residue. Reasons for missing data included insufficient specimen volume and analytical Results problems such as matrix interference. 1NAP and TCPY were present at detectable levels in the majority of the A total of 376 samples, including 13 field blanks and 13 samples, while AM and MDA were detected infrequently. In fortified samples, were collected over the course of the Table 2, the summary statistics are presented in terms of study. All field blanks contained concentrations of the micrograms of metabolite per gram of creatinine. pesticide metabolites below the LOD, except for three that The arithmetic mean 1NAP concentrations reported in contained 1NAP levels ranging from 2.8 to 53.0 g/l Tables 1 and 2 are strongly influenced by results from one (mean=31.9). Average recovery rates determined from participant. 1NAP concentrations for this participant ranged analysis of the fortified samples were 95% for TCPY (range from 540 to 2500 g/l with a meanof 1396 g/l. This 82±104%), 104% for 1NAP (94±120), 119% for MDA (94± individual had the five highest creatinine-corrected 1NAP 136), and 93% for AM (84±100). Field blank results and concentrations, 860, 1600, 2600, 3000, and 4600 g/g and recovery rates did not vary systematically among cycles or an arithmetic mean concentration approximately ten times field technicians. greater than that for any other participant. Seven other Tables 1 and 2 summarize the results of the analyses of samples, obtained from five participants, had creatinine- 1NAP, TCPY, AM, and MDA concentrations in urine corrected concentrations greater than 100 g/g. On samples obtained from the study participants. These results questionnaires administered in each sampling cycle this are unweighted such that the sample is the unit of participant did not report smoking tobacco, use of carbaryl observation. In Table 1, concentrations are expressed as at work or at home, use of mothballs (some of which use

Table 2. Summary statistics for selected pesticide metabolites inurine( g/g creatinine) from multiple samples collected from 80 individuals in Maryland betweenSeptember 1995 andSeptember 1996.

Analytea N MeanGM b MinPercentile Max 5% 25% 50% 75% 95% 1NAP 323 52.0 3.7 <0.2 <0.5 1.4 3.3 7.2 43.0 4600.0 1NAPc 318 13.0 3.3 <0.2 <0.5 1.4 3.2 7.1 32.0 540.0 TCPY 329 5.8 4.5 <0.2 1.3 3.0 4.6 7.1 16.0 35.0 AM 331 ±d ±d <0.2 <0.2 <0.3 <0.4 <0.6 <1.2 <1.6 MDA 330 ±d ±d <0.2 <0.2 <0.3 <0.4 <0.7 1.5 51.0 a1NAP=1-naphthol; TCPY=3,5,6-trichloro-2-pyridinol; AM=atrazine mercapturate; MDA=malathion dicarboxylic acid. bGeometric mean. cSummary statistics with potential outlier removed. dInsufficient number of results greater than limit of detection.

496 Journal of Exposure Analysis and Environmental Epidemiology (1999) 9(5) A longitudinal investigation of selected pesticide metabolites in urine MacIntosh et al.

Table 3. Frequency of detection (%) of 1-naphthol (1NAP), 3,5,6- trichloro-2-pyridinol (TCPY), atrazine mercapturate (AM), and malathion dicarboxylic acid (MDA) in urine by sampling cycle.

Analyte Cycle 123456 1NAP 84.2 86.4 83.6 80.0 78.0 77.6 TCPY 94.7 83.1 97.0 98.3 98.0 98.3 AM 0.0 1.7 0.0 0.0 0.0 0.0 MDA 3.5 3.4 6.0 13.3 8.0 5.2 naphthalene as the active ingredient) at home, or exposure to environmental tobacco smoke, all potential sources of exposure to compounds degraded to 1NAP in humans. Although potentially a true outlier, we chose to retain this individual in the data set and to explore the influence of their inclusion on subsequent data summaries and analyses. Removal of this participant from the data set yielded a mean 1NAP concentration of 13.2 g/l and a mean creatinine- corrected concentration of 13.0 g/g. Figure 2. Distribution of 3,5,6-trichloro-2-pyridinol concentrations in Results by Cycle urine by sampling cycle. Dotted line indicates arithmetic mean; The number of urine samples collected in cycles 1±6 was whiskers extend to 10th and 90th percentiles; open circles denote values outside of 10th and 90th percentiles. 57, 59, 67, 60, 50, and 58, respectively. The frequency of detectionof the four pesticide metabolites inurineby cycle is presented in Table 3. The detection rate for 1NAP ranged from 78% in cycles 5 and 6 to 86% in cycle 2. TCPY was detected inthe urinefrom at least 95% of the respondents in all cycles except cycle 2 where the detection rate was 83%. The lone sample that contained a detectable concentration of AM was collected in cycle 2, while the detectionrate for MDA peaked at 13% during cycle 5. The distribution of 1NAP and TCPY concentrations by cycle are summarized graphically inFigures 1 and2. Arithmetic mean cycle-specific 1NAP concentrations ranged from 14.9 g/l incycle 1 to 88.8 incycle 3. Incontrast, the median 1NAP concentration ranged from only 2.7 to 4.1 g/l among cycles. Similarly, the interquartile range for 1NAP exhibited a relatively small range among cycles (5.3 to 10.3 g/l) compared to variability of the arithmetic mean. The arithmetic mean TCPY concentration in each cycle was approximately 6 g/l and ranged from 4.2 g/l incycle 2, to 6.8 g/l incycle 4.

Table 4. Number of participants from which a given number of valid 1- naphthol (1NAP) and 3,5,6-trichloro-2-pyridinol (TCPY) measurements were obtained.

Analyte Number of valid measurements 12 34 5 6 Figure 1. Distribution of 1-naphthol concentrations in urine by sampling cycle. Dotted line indicates arithmetic mean; whiskers 1NAP 8 10 8 11 25 17 extend to 10th and 90th percentiles; open circles denote values outside TCPY 9 10 8 8 26 19 of 10th and 90th percentiles.

Journal of Exposure Analysis and Environmental Epidemiology (1999) 9(5) 497 MacIntosh et al. A longitudinal investigation of selected pesticide metabolites in urine

lites over the course of the 1-year study. The number of participants from which a given number (i.e., 1 to 6) of valid 1NAP and TCPY results were obtained is summarized inTable 4. 1NAP andTCPY results from at least four cycles were obtained from 53 of the participants. The average intra-individual range of 1NAP concentrations was 108 g/g, while that for TCPY was 7.0 g/g. Removing the potential outlier from the 1NAP data set reduced the average within-individual range to 38.1 g/g. Among these participants, the arithmetic mean annual average creatinine-corrected 1NAP and TCPY concentrations were 62.2 g/g (14.7 g/g whenomitting the potential outlier) and 5.9 g/g (2.3 to 13.1), respectively. Cumulative distribution functions for average annual creatinine-corrected 1NAP and TCPY concentrations are plotted in Figure 3.

Temporal and Population Variability Figure 3. Cumulative distribution functions for annual average creatinine-corrected 1-naphthol and 3,5,6-trichloro-2-pyridinol con- Geometric mean creatinine-corrected urinary concentrations centrations in urine. of 1NAP did not vary significantly among cycles ( p=0.3897), however, they were significantly different among individuals ( p<0.0001). Omitting the potential Results by Participant outlier from the data set did not change the outcome of At least one urine sample from all 80 participants was the repeated measures GLM with respect to the statistical collected and analyzed for the selected pesticide metabo- significance of sampling cycle or participant effects on

Figure 4. Comparison of geometric mean creatinine-corrected 1-naphthol concentrations by selected demographic factors. Error bars denote one standard error. Sample size for each group is shown in parentheses along x-axis.

498 Journal of Exposure Analysis and Environmental Epidemiology (1999) 9(5) A longitudinal investigation of selected pesticide metabolites in urine MacIntosh et al. creatinine-corrected 1NAP concentrations. For TCPY, mean Discussion concentrations were found to vary significantly ( p<0.0001) among cycles and among individuals. Inthis study, urinesamples were collected from up to 80 individuals at up to six different times or cycles approxi- Demographic Covariates mately equally spaced over 1 year. The concentrations of The geometric means for creatinine-corrected annual 1NAP, TCPY, and MDA measured in these samples, average 1NAP and TCPY concentrations by selected representing exposure to naphthalene or carbaryl; chlorpyr- demographic factors are showninFigures 4 and5. ifos, chlorpyrifos-methyl or triclopyr; and malathion, Geometric mean 1NAP concentrations did not differ respectively, were similar to those found in previous significantly between genders ( p=0.929), betweenraces investigations (Needham et al., 1995). Concentrations of ( p=0.9706), among education groups ( p=0.1426), or AM in urine, representative of exposure to atrazine, to our among groups of annual household income ( p=0.3978). knowledge have not been published elsewhere, thereby Geometric mean 1NAP concentrations varied significantly precluding direct comparison of our results with those from ( p=0.0141) among age groups with levels in the two older other studies. age groups greater thanthose inthe two youngerones. Among urine samples collected from approximately Omissionof data for the potential1NAP outlier caused 1000 adult participants between 1988 and 1994 in the differences among age groups to no longer be significant National Health and Nutrition Examination Survey III ( p=0.1005), but did not substantially affect the conclusions (NHANES III), 1NAP was detected at a rate of 86% and of the other analyses. For TCPY, geometric mean concen- the mean creatinine-corrected concentration was 17 g/g trations were found to differ significantly ( p=0.0260) (Hill et al., 1995a,b). The frequency of 1NAP detection was between races and among the three education groups nearly equal to that in the present study (Table 1), as ( p=0.0127). Differences in TCPY concentrations were was the central tendency, when results from the potential not significantly different between genders ( p=0.8191), outlier inour populationwere omitted, andthe distribution among age groups (0.7524), or among income groups of concentrations. The maximum 1NAP concentration ( p=0.5771). measured inthe NHANES III populationwas 2500 g/l

Figure 5. Geometric mean creatinine-corrected 3,5,6-trichloro-2-pyridinol concentrations by selected demographic factors. Error bars denote one standard error. Sample size for each group is shown in parentheses along x-axis.

Journal of Exposure Analysis and Environmental Epidemiology (1999) 9(5) 499 MacIntosh et al. A longitudinal investigation of selected pesticide metabolites in urine

(1400 g/g creatinine), suggesting that the upper-end reflect applicationof the herbicide triclopyr duringthe extreme values inour data set representtrue, albeit high, growing season. While statistically significant, variability of measures rather than sampling or analysis artifacts. median TCPY concentrations among cycles may not be TCPY was found in 82% of the NHANES III urine substantive in the context of some environmental health samples and 5.8% of the NHANES II urines collected applications. For example, other errors in health risk between1976±1980 (Kutz et al., 1992; Hill et al., 1995a,b). assessments (e.g., the dose±response curve) may be much Inour study, conducted from 1995±1996, TCPY was larger. However, if mean or median TCPY concentrations present at measurable levels in 96% of the samples, for a populationvary systematically by season,thentiming indicating that frequency of exposure to chlorpyrifos, of sample collection should be an explicit component of chlorpyrifos-methyl, triclopyr, or TCPY inthe NHEXAS- longitudinal studies designed to characterize status and Maryland study population was similar to that observed in trends of TCPY levels in urine. the most recent national survey. Mean and median The typical range of intra-individual variability of 1NAP creatinine-corrected TCPY concentrations measured in the and TCPY concentrations in the NHEXAS-Maryland present study (5.8 and 4.6 g/g, respectively) are populationwas greater thanthe correspondingpopulation approximately twofold greater thanthose measured in mean and median concentrations. The average range of NHANES III (3.1 and 2.2 g/g, respectively), however, the 1NAP measurements from a single participant was upper end of the distributions are approximately equal. approximately two times greater thanthe populationmean Less than1% of the urinesamples from the NHANES II level and approximately six times greater than the median population were found to contain detectable concentrations concentration for the population. For TCPY, the average of MDA in urine, compared to 6.6% in the Maryland study range of measurements from a single respondent was population(Kutz et al., 1992). Despite the higher frequency approximately 50% greater thanboth the populationmean of detectioninthe presentstudy, the maximum NHANES II and median concentration. In epidemiology, the objective of concentration of 250 g/l exceeded that observed here by a exposure assessment often is to place an individual into a factor of 5 (Table 1). broad category of exposure relative to other individuals in As noted earlier, biomarkers of exposure can be used to the study (e.g., the upper or lower quintile). Our results document status and trends of exposure within populations indicate that a single measure of urinary 1NAP and TCPY or population subgroups and as inputs to epidemiological levels is not sufficient to characterize accurately the relative investigations of health effects of chemical exposure and magnitude of a person's typical acute or chronic exposure to humanhealth risk assessments.The repeated measures the respective parent compounds. Use of these results to feature of the NHEXAS-Maryland study design affords a determine the minimum number of 1NAP and TCPY results relatively unique ability to investigate temporal variability from anindividual required to achieve a specified degree of of exposure to selected chemicals and the implications with misclassification error will be a component of future respect to potential uses of exposure data. research. We found that log-transformed population mean 1NAP In this paper, we reported on the occurrence of urinary concentrations in urine did not vary significantly among six metabolites indicative of exposure to naphthalene or sampling periods spaced over a year. This result indicates carbaryl; chlorpyrifos, chlorpyrifos-methyl, or triclopyr; that the timing of sample collection may not be an important malathion, and atrazine in repeated samples obtained from a design consideration for studies (e.g., status and trend probability sample drawn from Maryland. 1NAP and TCPY analyses and chronic risk assessments) that intend to utilize were present in over 80% of the samples, while MDA and the population geometric mean concentration of this AM were detected infrequently. Mean and median con- compound. Because the distribution of 1NAP concentra- centrations of 1NAP and TCPY in urine did not vary tions did not vary substantially among sampling cycles, substantially among sampling cycles, while repeated assessments of risk related to acute exposures to naphtha- measurements from the same individual over time were lene/carbaryl may also be insensitive to the time of year in highly variable. In future research, environmental measure- which exposure input data are obtained. ments of the parent compounds and questionnaire data Mean log-transformed, creatinine-corrected TCPY con- collected concurrently with the biomarker data will be used centrations in urine were found to vary significantly among to characterize the determinants of variability in the urinary cycles. Medianlevels differed by nearly a factor of 2 pesticide metabolite levels. betweencycles 2 and6 with anapparentshift toward higher levels in the spring, summer and early autumn. With respect Acknowledgments to chlorpyrifos, this shift may reflect increased rates of residential application for insect control during the warmer This research was supported by U.S. Environmental Protec- months of the year or greater application to agricultural tionAgencyCooperative AgreementNo. CR822038-01 and commodities during the growing season. The shift may also the University of Georgia Agricultural Experiment Station.

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