Environ. Sci. Technol. 2005, 39, 3896-3903
ELISA as an Affordable Methodology for Monitoring Groundwater Contamination by Pesticides in Low-Income Countries B E A T R I Z M . B R E N A , †,‡ LOURDES ARELLANO,† CATERINA RUFO,§ MICHAEL S. LAST,| JORGE MONTAN ˜ O,+ EDUARDO EGAN ˜ A CERNI,@ GUALBERTO GONZALEZ-SAPIENZA,§ AND J E R O L D A . L A S T * ,⊥ Ca´tedra de Bioquı´mica Facultad de Quı´mica, Ca´tedra de Inmunologı´a Facultad de Quı´mica, and Departamento de Geologı´a, Facultad de Ciencias, University de la Repu ´ blica, Montevideo, Uruguay, Laboratorio de Higiene Ambiental and Laboratorio de Bromatologı´a, Intendencia Municipal de Montevideo, Montevideo, Uruguay, and Departments of Statistics and Pulmonary and Critical Care Medicine, University of California, Davis, California, 95616
The traditional instrumental technology for pesticide residue analysis is too expensive and labor-intense to meet the regional needs concerning environmental monitoring. ELISA methodology was used for a pilot scale study of groundwater quality in an agricultural region a few kilometers southwest of Montevideo, the capital city of Uruguay. The study spanned 2 years and examined concentrations (detection limits are given in [ppb]) of two triazine herbicides (simazine [0.3] and atrazine [0.4]) and the carbamate insecticide carbaryl [10] and its major metabolite 1-naphthol [17]. In general, pesticide concentrations were below detection limits in the samples tested and in all cases were well below the maximum contaminant levels set by the U.S. EPA. 1-Naphthol was detected frequently by ELISA, but the assay may have tended to systematically overestimate this analyte. To our knowledge, this is the first study of its type in Uruguay and perhaps the first systematic approach to monitoring for organic pesticides in groundwater water sources in the temperate region of South America.
Introduction There is a growing awareness in the countries in the southern region of South America (the Mercosur)sArgentina, Brazil, Chile, and Uruguaysthat there are potential serious envi* Corresponding author phone: (530) 752-6230; fax: (530) 7528632; e-mail:
[email protected]. † Ca ´ tedra de Bioquı´mica Facultad de Quı´mica, University de la Repu ´ blica. ‡ Laboratorio de Higiene Ambiental, Intendencia Municipal de Montevideo. § Ca´tedra de Inmunologı´a Facultad de Quı´mica, University de la Repu ´ blica. | Department of Statistics, University of California. + Departamento de Geologı´a, Facultad de Ciencias, University de la Repu ´ blica. @ Laboratorio de Bromatologı ´a, Intendencia Municipal de Montevideo. ⊥ Pulmonary and Critical Care Medicine, University of California. 3896
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ronmental consequences to the large-scale use of pesticides in these major agricultural countries (1). They share a temperate climate and a crop selection similar to those of the temperate zones of the United States and Western Europe. Livestock and agricultural production constitute the basis of the Uruguayan economy. In Uruguay, much of the agriculture is still being done on small family farms, especially in the zones in and around the densely populated capital city, Montevideo (where more than half of the total of 3.2 million people in Uruguay live). The use of agricultural chemicals on these small family farms, which grow vegetables and fruits for sale to consumers in the urban area, is essentially unregulated, although there are processes in place to educate farmers in the proper use of pesticides and herbicides. The lack of control at the source could theoretically allow the dispersion of pesticides in the environment, where these chemicals could end up contaminating essential water resources. The situation remains uncontrolled due to the lack of public (and policymaker) perception that there might be a problem, which in turn is caused by the lack of hard data on whether pesticides are present in drinking water sources such as groundwater in the region, and the relatively low total use of these chemicals as compared with larger countries. Therefore, it is difficult to predict which, if any, of the pesticides in current use in Uruguay may present a health hazard to the general population (2). A major Latin American Symposium on Environmental Chemistry sponsored by the International Union of Pure and Applied Chemistry was convened in Montevideo in 1998 to address the regional needs for environmental protection (3). The symposium participants concluded that there was an urgent need for reliable data on the actual occurrence of chemical contamination in the environment. Such fundamental data are needed to trace sources of environmental impact, understand the consequences of contamination on the ecosystem, and constructively approach the causes and prevention of pollution. To accomplish these goals, the participants recommended initiating specific actions, including a large-scale monitoring program to survey the environment. They identified the monitoring of water quality as a most urgent priority. Water quality is particularly important since most pollutants are emitted directly into water sources or finally end up in water as a consequence of atmospheric precipitation or leaching from soil. High quality data and an interdisciplinary approach are essential for environmental studies (4). Therefore, it is necessary to support and reinforce existing training programs in the field before the more ambitious goal of developing a regional monitoring system can realistically be achieved. Such a coordinated international interdisciplinary program has not yet materialized and is unlikely to be a high priority under the current severe economic hardships that most of the countries in the region are experiencing. The traditional instrumental technology for pesticide residue analysis is too expensive and too labor-intense to meet the region’s needs (5-7). Major data gaps on pesticides in the environment exist because of the lack of ability to analyze large sample loads rapidly and cost-effectively. Without better, faster, and cheaper analytical methodology, Uruguay and the other Mercosur countries cannot afford to gather the requisite data to control the quality of their rivers and groundwater sources and to collect the data to make possible the decision required to create an effective regulatory environment for the prevention of drinking water contamination. To approach this question in a cost-effective and scientifically valid manner, we have initiated a project to 10.1021/es048620d CCC: $30.25
2005 American Chemical Society Published on Web 04/21/2005
build in the University of the Republic the capability of developing immunoassays to monitor environmental contamination according to the needs of Uruguay. This is of paramount importance since immunoassays (ELISAs), which are versatile, fast, adaptable to laboratory or field situations, sensitive, and specific, are also cost-effective (8). ELISA results can be confirmed and validated by the selective use of instrumental analysis to independently evaluate key findings. The relatively low cost per assay by ELISA is especially relevant for developing economies, such as Uruguay and the other countries of the Mercosur, where the high cost of classical instrumental analysis and the accompanying requirement for highly trained technicians to perform assays and to maintain the equipment are strong limitations to the widespread use of these techniques. To approach the issue of whether groundwater sources are being affected by agricultural chemicals in Uruguay (2), we started a program in cooperation with the local government in the province (the Intendencia of Montevideo). We developed the basis for a new network of institutions with interests in water quality to begin to monitor by ELISA drinking water wells in the rural areas in the west of the city to examine the potential for exposure of people to pesticide residues via drinking water. The rural area of Montevideo is characterized by a high use of its water resources due to its intensive agriculture. The production in this area of only 16,000 ha accounts for more than half of the total amount of green vegetables consumed by the population of the country. All of the farming in this region is done on small family farms or orchards. Crops are generally irrigated, but in addition, fairly significant rainfall amounts are accumulated during the summer months (December, January, and February). Chemicals are applied as deemed necessary. Because of the extensive rainfall that washes away pesticides, conditions are conducive to their overuse by comparison with other countries in which pesticide usage on crops is more strictly regulated. Previous studies have focused on the physicochemical and microbiological characterization of underground water, but there is almost no information concerning the presence of pesticides. There are concerns that there might be an excessive use of fertilizers and agrochemicals in this area that could negatively affect the sustainability of the natural resources and the health of the people. Therefore, a survey of the presence of pesticides in underground water in this region is a first step toward deciding whether corrective measures are needed. The main goal of this work has been to perform a preliminary analysis of whether there is measurable contamination of the water resources with agrochemicals in the study area. Water from the Punta Espinillo aquifer shows high levels of nitrates, suggesting that contamination from agricultural activities may have taken place. A complete and rigorous pesticide survey was impossible to perform using classical instrumental analysis due to limited resources, so ELISA methodology was chosen to provide a usable tool for screening the actual situation. The study took place over a period of 2 years. The first activity was to transfer the ELISA assays for atrazine, simazine, carbaryl, and 1-naphthol from the University of California to the University of the Republic, Uruguay. This paper reports some of our results from the first and second years of field studies by this program. To our knowledge, it is the first study of its type in Uruguay and perhaps the first systematic approach to monitoring organic pesticides in groundwater drinking water sources in the region, although there have been at least two previous reports of pesticide analysis in groundwater from Argentina (9, 10).
Materials and Methods Study Area. Uruguay is a small country (about 176 000 km2, slightly smaller than the size of the state of Washington or
FIGURE 1. Map of South America showing Uruguay and the study area at the southwest part of Montevideo. Location of wells sampled for this study. the United Kingdom) in the southern part of South America, bordering on the Atlantic Ocean, Brazil, and Argentina (Figure 1). It has a warm temperate climate and a moderate annual rainfall of about 1000 mm (40 in.), which is well-distributed throughout the year. The autumn months tend to be slightly wetter than the other seasons. The study area at Punta Espinillo is located at the southwest of the Department of Montevideo, only a few kilometers from the capital city of Uruguay, at the left side of the mouth of the Santa Lucia River where it empties into the Rı´o de la Plata (Figure 1). The area is divided into small, family owned, production units (farms) of about 10 ha, mostly used for cultivation of green vegetables with watering. The water is obtained from a fissured aquifer, and it is used for drinking and irrigation purposes. Despite the high storage capacity of the aquifer, the intensive use of the water has led to a decrease in its flow. This overexploitation of the aquifer, combined with the extensive use of agrochemicals in the region, has created additional problems of water quality due to the high content of nitrates and high salinity (see Table 1). Water Sampling. Water samples were collected from 23 groundwater wells of the Aquifer Punta Espinillo. Figure 1 shows the location of the sampling spots. After water was pumped from a well for at least 15 min, samples were taken in clean glass bottles, transported refrigerated to the laboratory, and stored at 4 ˚C. Three liter samples were taken from each well and split into two 1 L bottles for ELISA and instrumental analysis. At least one field replicate sample was taken for every 12 samples, and the results agreed within one standard deviation. For each season reported, all the samples were taken within 4 weeks, so they are independent and identically distributed samples. Samples were extracted with solvents within 24 h for instrumental analysis. ELISA analysis was performed directly on the refrigerated water samples within a maximum period of 4 days after collection. Pesticide Analyses. ELISAs Used. Four different competitive format immunoassays, specific to the pesticides carbaryl, its major metabolite 1-naphthol, atrazine, and simazine, were used to analyze all water samples. ELISA protocols were developed in Dr. B. Hammock’s laboratory at UC Davis and performed with some modifications as follows. All samples were analyzed in triplicate and in three serial dilutions (1:2 to 1:8). The absorbance was read at 450 nm in a microtiter plate reader (Multiskan MS, Labsystems). Inhibition curve values were fitted to a four-parameter logistic equation using Microcal Origin version 6.0 (Microcal Software, Inc.) software package. Appropriate quality control/quality assurance procedures were used. Data are reported as the average of three replicate analyses performed on different days. Each ELISA analysis VOL. 39, NO. 11, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Physicochemical Analysis of Groundwater well ID
pH
conductivity (µS/cm)
hardnessa (mg/L)
HCO3-
Cl-
SO4-2
NO3-
Ca2+
Mg2+
Na+
K+
20 27 30 32 33 36 42 46 47 49 53 54 57 58 59 61 62 65 66 69 72 74
7.4 7.3 7.2 7.2 7.2 7.5 7.3 7.6 7.6 7.6 7.6 7.5 7.4 7.4 7.5 7.4 7.4 7.3 7.4 7.2 7.5 7.5
1742 2262 1698 1547 1410 1260 1898 1802 1582 1370 1880 1736 1295 779 1512 1447 1466 1726 1314 2137 1536 1583
201 282 226 180 109 147 319 137 135 126 213 199 188 199 188 183 169 223 162 349 103 167
611 529 599 641 585 543 640 621 561 552 636 550 558 594 576 587 558 511 565 479 637 546
109 164 102 67 54 42 169 132 133 62 151 171 49 135 77 83 84 187 58 327 75 135
85 165 65 55 45 30 95 60 60 70 90 50 40 70 50 40 50 55 50 80 65 50
111 310 106 40 53 38 98 84 53 40 111 84 38 133 75 40 75 84 49 44 53 71
43 62 54 43 26 34 72 31 30 29 49 44 39 44 43 39 36 48 37 76 26 37
23 31 22 33 11 15 34 15 15 13 22 22 22 22 20 20 19 25 17 39 11 18
384 436 304 295 296 248 394 431 382 284 453 398 248 388 290 340 336 363 303 409 405 367
4 6 8 5 4 4 5 5 5 2 4 4 2 2 3 2 3 4 3 5 3 3
a
Hardness expressed as mg/L CaCO3.
included three well replicates in three serial dilutions, and every sample was also analyzed spiked at concentration values near their respective ELISA measuring range. Recoveries of the spiked samples were between 70 and 130%. Ten percent of the samples to be analyzed by instrumental analysis were also spiked and then split for intercalibration studies between ELISA, GC/MS, and HPLC. The recoveries by instrumental analysis were between 72 and 119%. For every 12 samples, a blank of Milli-Q water contained in sampling bottles was analyzed; the results of these control determinations were a uniform nondetection of the analyte tested both for ELISA and instrumental analysis. Carbaryl. The competition assay for carbaryl was a modification from the ELISA previously described by Marco et al. (11). Briefly, 96 well microtiter plates were coated with 100 µL/well of a 1:800 dilution of N-(2-naphthoyl)-6aminohexanoic acid conjugated to OVA in a 0.1 M carbonatebicarbonate buffer (pH 9.6) overnight at 4 °C. The plates were washed with sodium phosphate buffer (1 × PBS) containing 0.05% Tween 20 (PBST) 3 times. Then, 50 µL/well of different carbaryl standards, prepared in distilled water, or 50 µL of the sample altogether with 50 µL/well of a 1:2000 dilution of antibody Ab 2114 in 2 × PBST, was applied. After a 1 h incubation period, wells were washed 3 times with 1 × PBST, and then anti-rabbit IgG conjugated to peroxidase, diluted 1:4000 with PBST, was added to each well. After a 1 h incubation period, the plates were washed, and 100 µL of the peroxidase substrate (0.4 mL of a 6 mg/mL DMSO solution of 3,3′,5,5′-tetramethylbenzidine and 0.1 mL of 1% H2O2 in water in a total of 25 mL of 0.1 M citrate acetate buffer pH 5.5) was dispensed into each well. The enzyme reaction was stopped after 30-40 min by the addition of 50 µL of 2 N H2SO4. 1-Naphthol. The competition assay for 1-naphthol was performed as previously described by Kra¨mer et al. (12). Briefly, 96 well microtiter plates were coated with 100 µL of a 2 µg/mL solution [(5-hydroxy-2-naphthylenyl)oxy] acetic acid conjugated to BSA in a 0.1 M carbonate-bicarbonate buffer (pH 9.6) overnight at 4 °C. The plates were washed with PBST 3 times, and then 50 µL/well of different 1-naphthol standards in PBST or 50 µL of sample altogether with 50 µL/well of a 1:3200 dilution of antibody Ab 3907 in PBST was applied. After a 1 h incubation period, the wells were washed 6 times with 1 × PBST, and then anti-rabbit IgG conjugated 3898
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to peroxidase, diluted 1:1000 with PBST, was added to each well. After an hour incubation period at room temperature, the plates were washed and developed as described for the carbaryl assay. Atrazine. The competition assay for atrazine was performed as previously described (13). Briefly, 96 well microtiter plates were coated with 150 µL of a 1:10000 dilution of hapten 2-carboxypentylamino-4-chloro-6-isopropylamino-1,3,5-triazine conjugated to OVA (13) in 1 × PBS buffer (pH 7.5) overnight at 4 °C. The plates were washed with PBST 3 times, and then 50 µL/well of different atrazine standard concentrations prepared in distilled water or 50 µL of the sample together with 100 µL/well of a 1:150 dilution of AM7B2.1 (cell culture supernatant) (14) in 3 × PBST was applied. After a 1 h incubation period, the wells were washed 6 times with 1 × PBST, and then anti-mouse IgG conjugated to peroxidase, diluted 1:4000 with PBST, was added to each well. After an hour incubation period at room temperature, the plates were washed and developed as previously described for the carbaryl assay. Simazine. The competition assay for simazine was performed as previously described (15). Briefly, 96 well microtiter plates were coated with 150 µL of 1/:16 000 dilution of hapten 2-carboxyethylamino-4-chloro-6-ethylmino-1,3,5-triazine conjugated to OVA in 1 × PBS buffer (pH 7.5) overnight at 4 °C. The plates were washed with PBST 3 times, and then 50 µL/well of different simazine standard concentrations prepared in PBST or 50 µL of the sample altogether with 100 µL/well of a 1:128 000 dilution of Ab 2282 (8) in PBST was applied. After a 1 h incubation period, the wells were washed 6 times with 1 × PBST, and then anti-rabbit IgG conjugated to peroxidase, diluted 1:2500 with PBST, was added to each well. After an hour incubation period at room temperature, the plates were washed and developed as previously described for the carbaryl assay (16). Instrumental Analysis. Captan, folpet, endosulfan, dodecachlor, cypermethtrine, ethyl and methyl-chlorpyrifos, diazinon, dimethoate, ethyon, iprodione, λ-cyalothrine, malathion, methyl-parathion, carbaryl, carbofuran, 1-naphthol, simazine, and atrazine were analyzed according to the EPA method 600/8-800-038 section 10A (Manual of Analytical Methods for the Analysis of pesticides in Human and Environmental Samples). Comparative analysis of 1- naphthol by ELISA and by GC/MS was done with a Hewlet Packard
TABLE 2. Characteristics of ELISAs Used in This Study assay
antiserum
coating antigen
IC50a (ug/L)
SD
DLb (ug/L)
SD
atrazine
AM 7B2.1
0.83
0.0 8
0.26
0.0 4
simazine naphthol carbaryl
Ab 2282 Ab 3907 Ab 2114
2-carboxypenthylamine-4-chloro-6-isopropylamine1,3,5-triazine-OVA 2-carboxyethylamine-4-chloro-6-ethylamine-1,3,5-triazine-OVA 5-hydroxy-2-(naphthylenyl)oxy-acetic acid-BSA N-(2-naphthoyl)-6-aminohexanoic acid-OVA
2.03 63 18
0.0 5 28 8
0.4 17 10
0.1 8 6
a Reported values are the average of the parameters from the four-parameter logistic equations used to fit the standard curves. SD: standard deviation of the experimentally determined detection limit from five or more independent ELISA plates. b DL: detection limit is defined as the concentration giving 80% of the maximum absorbance.
TABLE 3. Pesticide Analysis by ELISA (Spring 2002 to Summer 2003)a spring 2002 1-naphthol well ID
concn (ug/L)
20 27 30 32 33 36 42 46 49 53 54 57 58 59 61 66 69 72 74 DL (ug/L)
74 66 74 72 82 89 112 105 100 120 17
summer 2002 to 2003 atrazine
carbaryl
concn (ug/L)
concn (ug/L)
concn (ug/L)
9 10 7 7 15 14
ND ND ND ND ND ND
10 10
ND ND ND ND 0.3
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 10
ND ND 70 128 ND ND 27 69 ND 63 110 132 ND 33 201 ND 59 ND 17
SD
7 18
0.3
1-naphthol
atrazine SD
19 12 10 12 19 34 43 12 76 12
concn (ug/L) ND ND ND ND ND 0.92 ND ND ND ND ND 0.3
simazine SD
0.01
concn (ug/L)
SD
ND ND ND ND ND ND ND ND ND ND ND ND 0.9 ND ND ND ND ND 0.4
0.1
ND: nondetected; DL: detection limit; -: not analyzed. The standard deviations reported were derived from the three replicate ELISA analyses performed for each sample in different plates (see Materials and Methods). a
model 6890 gas chromatograph equipped with an HP Selective Mass Detector 5973, after extraction according to EPA Method 8270 (16). The column was a DB-1701, 30 m × 0.25 mm × 0.15 µm film. Helium was the carrier gas at a constant flow (0.9 mL/min). The temperature gradient was from 70 °C (2 min) to 130 °C (5 min) at 25 °C/min and from 130 to 180 °C at 2 °C/min using the SCAN mode for identification and SIM for quantification. Statistical Treatment of Data. We pursued a nonparametric method for testing relationships between measured quantities to make our tests less sensitive to small errors in the measured values. This was especially important for the ELISA assays since some of the measurements were near the detection limit. Additionally, nonparametric analysis does not require the assumption that the data follow any known distribution to allow for hypothesis testing, and the statistical tests used have increased power on some distributions. The test procedure chosen actually examined how strongly monotonic (strictly, increasing, or decreasing) the relationship between the measured quantities is, as opposed to the usual correlation coefficient (or regression analysis), which detects the strength of the linear relationship between the quantities being measured. For each quantity measured, the observations were ranked, with 1 corresponding to the largest observed value, 2 the second largest, and so forth. The correlation between the ranks of two quantities known as Kendall’s rank correlation was used, Kendall’s tau statistic was calculated, and the significance of the correlation was determined (17).
P values were obtained by simulation. For each pair of n measurements, 10,000 pairs of n independent random observations were drawn from a uniform (0, 1) distribution. The correlation of the ranks of each pair of n random observations was computed. This procedure gave an approximate distribution for the correlation of ranks of n pairs of independent random variables. The proportion where the absolute value of the correlation is greater than the correlation of the ranks of the data gave a two-tailed p value. One-tailed p values were obtained, as appropriate, by examining the proportion that was either greater than or less than the observed correlation of the ranks of the data. Any well with any missing data was ignored in any comparison that would have used the missing data. In a few cases, outliers were removed for the analysis; these exceptions are noted in the text when they occurred. Cluster analysis was used to examine correlations between pesticide occurrence in wells and the hydrogeological data. The k means function (which fits a model to the data assuming k groups have the same variance but different means) in the mva (from multivariate analysis) package of R, a common open source statistical computing package (17), was used to perform this analysis. Groups of individual wells were divided into clusters by finding the k mean that would minimize the sum of squared differences between the observation and the nearest mean and then assigning each observation to a cluster corresponding to the closest of the K mean. For the hydrogeological data, where measurements were available for 14 different VOL. 39, NO. 11, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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variables, each variable was rescaled to have a mean of zero, and a standard deviation of 1, so that each category would contribute equal weight to the clustering. Given the limitations of our data, the analysis was designed to be exploratory and to suggest new hypotheses, not to be definitive. Therefore, we chose to take an approach that involved more descriptive and less inferential methodology. While we may have been able to better approximate reality by appropriately weighting the data, this approach better allowed us to examine relationships independently of expectations.
Results Physicochemical Parameters of Water Samples. Samples of water from each well studied were analyzed for their pH, conductivity, and hardness. Analyses were also performed for the content of various anions and cations: HCO3-, Cl-, SO42-, NO3-, Ca2+, Mg2+, Na+, and K+. The results of these analyses are presented in Table 1. Most of the well waters tested can be characterized as hard (100-200 mg/L CaCO3) and only six of them as very hard (more than 200 mg/L CaCO3). All the samples analyzed except for the one from well 69 can be classified as calcium bicarbonated according to Piper Diagrams. The water from well 69 corresponds to high-chloride water, which can present restrictions for use in agriculture. Pesticide Analyses: ELISAs as Sensitive Screening Methods. We first characterized the ELISAs that we would use in this study. Except for the case of carbaryl, in which we used a different coating hapten that showed a higher limit of detection, the assay characteristics of the ELISAs performed in Montevideo are comparable to the original assays as reported earlier by the authors of these tests (Table 2). The possible influence of the matrix effect was studied, and in agreement with previous results (16), we found no significant differences in terms of slope and IC50 when the immunoassays were run in surface water, phosphate-buffered saline-0.05% Tween 20 (PBST), and well water. Since this result indicates that there are no matrix effects, the assays were performed directly upon environmental water samples without any additional cleanup steps. Survey activities began with a preliminary screening using only ELISA methods; these assay data were later expanded by standard instrumental analysis by GC and HPLC. A preliminary screening by ELISA was performed on samples from 28 wells during spring and summer of the first year (Table 3). In these samples, simazine and atrazine were each detected in only a single sample. In both cases, the concentration was less than 1 µg/L, well below the maximum contaminant level accepted for both compounds of 4 and 3 µg/L for simazine and atrazine, respectively. However, there was frequent detection of 1-naphthol, which was found in 71% of the samples, with an average value of 89 µg/L. Carbaryl was not detected (with a detection level of 10 µg/L for the asssay). This value is far (about 70-fold) below the action level for drinking water (700 µg/L). Nine samples obtained from surface streams in the summer season were also analyzed by ELISA. In these, atrazine was found in only one sample (1.24 ( 0.03 µg/L), and 1-naphthol was detected in four samples at levels of about 90 µg/L. These findings raised the further question of whether the compounds could persist in well water samples from season to season. Thus, we examined carbaryl and 1-naphthol levels in the subsequent autumn and on into the following summer. We performed parallel instrumental analysis of replicate samples and also analyzed other possible contaminant pesticides. A total of 15 well water samples taken in autumn was analyzed by both ELISA and HPLC. Instrumental analysis did not detect any compound, and 1-naphthol was the only compound detected in a single sample (sample 74) by ELISA; it was found at a concentration of 53 ( 15 µg/L. Since it was 3900
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TABLE 4. Overall Results by ELISA % detection atrazine
simazine
carbaryl
1naphthol
2.7 0.3 0.9 4 23
0 10
63 17 201
Well Water spring and summer no. of samples analyzed 37 DL (ug/L) max value determined MCLa (ug/L) max detection (% of MCL) action levelb (AL) (ug/L) max detection (% of AL) autumn no. of samples analyzed 15 max value determined max detection (% of MCL) max detection (% of AL)
2.7 0.4 1.8 3 60
6.7 0 0
0 700 0 0 0
6.7 53
0
Surface Water summer no. of samples analyzed 16 DL (ug/L) max value determined MCL (ug/L) max detection (% of MCL) action level (AL) (ug/l) max detection (% of AL)
0 0.4 3
6.3 0.3 1.2 4 30
0 10
31 17 174
700 0
a Maximum contaminant level (MCL) for drinking water: the highest level of a contaminant that is allowed in drinking water. MCLs are enforceable standards (EPA 816-F-02-013, July 2002). b Action levels are health-based advisory levels for chemicals in drinking water that lack MCLs. They are used by the California Department of Health Services (DHS) to provide guidance to drinking water systems.
not detected in the same sample by HPLC (fluorescence detector, LD 10 µg/L), the question arose as to whether this was a false positive result by ELISA. To clarify the situation, three new samples were taken from the same wells and analyzed by ELISA, HPLC, and the more sensitive GC/MS method after solvent extraction. The samples contained 1-naphthol as determined by GC/MS, at levels from 10 to 30% of the values detected by ELISA. This could in part be due to the fact that we had high standard deviations for the results (20-30 µg/L), which were close to the ELISA detection limit. However, it is clear that the ELISA test overestimated the concentration of 1-naphthol, a problem that has been reported earlier (16). It should be noted that these inaccuracies are occurring at concentrations several orders of magnitude lower than might have any potential adverse health effects and that even with such an overestimation in the ELISA assay, there would be no public health implications of such an erroneous value (which was found in only one of 15 wells assayed in the original round of assays performed). In the following summer, ELISA and HPLC for carbaryl and 1-naphthol were performed in parallel in nine wells and in seven surface waters from small streams in the study area. In these analyses, 1-naphthol was detected only by ELISA in four well samples, at a level of about 50 µg/L (with a maximum concentration of 70 µg/L). Consistent with the previous results, it was not detected by HPLC (with a detection limit of 10 µg/L), which further shows that the ELISA can overestimate the concentration of 1-naphthol in a subfraction of samples assayed. The overall ELISA results are presented in Table 4, reported as a percentage of maximum contaminant level or action level, which shows that 1-naphthol is the most frequently found compound particularly in spring and summer and that there were no samples above or close to those limits. GC and HPLC analyses were performed on the 15 well samples taken in the autumn. None of the compounds
TABLE 5. Detection Limits for Instrumental Analytical Methods Used and Maximum Contaminant Level (MCL) of the Corresponding Compounds pesticide
DL (µg/L)
MCL (µg/L)
atrazine carbaryl carbofuran cypermethrine ethyl chlorpirifos methyl clorpirifos diazinon dimethoate ethion iprodione lambda cyalothrine malathion methyl parathion 1-naphthol captan folpet endosulfan dodecachlor
5.0 0.1 1 10 1.0 0.5 2.0 2.0 2.0 5.0 2.0 1.0 0.5 3 0.5 0.5 0.5 1.0
3 700 40 70 20 20 0.1 10 4 300 not available 200 2 not available 1.5 not available not available not available
TABLE 6. Clustering of Wells for Statistical Analysis as Described in Texta R-naphthol clusters
high (128)
medium (50)
57, 58, 66
20, 30, 32, 33, 36, 46, 54, 72
hydrogeological clusters
27, 42, 53, 59, 61, 69, 74
group 1
group 2
group 3
27, 42, 69
20, 30, 32, 46, 47,53, 54, 58, 65, 74
33, 36, 49, 57, 59, 61, 62, 66, 72
correlated clusters (based on measured concentrations of specific ions) a
low (nondetected)
group 1
group 2
group 3
20, 30, 46, 47, 53, 54, 58, 65, 72, 74
32, 33, 36, 49, 57, 59, 61, 62, 66
27, 42, 69
See text for details of how each of these clusters is defined.
studied, which include herbicides, fungicides, and insecticides, were detected in the water samples at the levels shown in Table 5. It should be pointed out that the detection limits for atrazine and diazinon were above the MCL for these compounds with the instrumental analysis methods used. Our findings are probably a reflection of relatively low usage of these agents and relatively high rainfall during the summer months. However, several of these agents are persistent, especially under the conditions in groundwater. For this reason, simazine and atrazine are the most frequently observed contaminants of well water in the United States. Thus, we are either seeing a dilution effect to below detection limits for these assays, a relatively shallow aquifer that recharges very rapidly, relatively low quantities of these specific chemicals being applied to crops, efficient breakdown of these chemicals by soil bacteria and/or solar radiation, or some combination of these factors 1-Naphthol is the main degradation product of carbaryl, so that a simple precusor-product relationship between them would imply that the concentration of both compounds in the water samples should show a correlation if a breakdown of carbaryl is occurring in situ. However, we only detected 1-naphthol and no carbaryl in the water samples. This result suggests that the breakdown of carbaryl is occurring elsewhere and that 1-naphthol enters the aquifer. Thus, the
concentration in a given sample would depend on physical forces such as rate of diffusion and water flux into and out of the aquifer. It should be emphasized that the ELISA technique is very sensitive for most chemicals, more sensitive in some cases than the instrumental methods (for atrazine, the detection limit by instrumental analysis of 5 µg/L is higher than the MCL of 3 µg/L, but by ELISA the DL is 0.3 µg/L), and that no false negatives were found by ELISA when compared to the instrumental methods. Thus, the ELISA method is very suitable for screening a large number of water samples to ask whether there is contamination present. For this purpose, it is preferable to use a sensitive method that may overestimate contamination than the opposite situation of using a method that may produce false negative results. While apparent positive results were found for simazine, and atrazine in a few of the samples tested, in no case were any of the pesticides detected at concentrations above, or even close to, the values that would be in excess of health-based standards in either Uruguay or the United States. Correlations between Hydrogeochemical Parameters and 1-Naphthol Concentration Suggest Two Nonequilibrium Zones in the Aquifer. Lacking detailed hydrology, we chose to investigate whether there were any noticeable clusters of hydrogeological data and to determine whether there was any relationship between these results and apparent clusters of wells ranked by their levels of 1-naphthol during the summer months (when 1-naphthol levels were highest). Assignment of specific wells to clusters was based upon an arbitrary choice of levels of contaminant in a given cluster, as we will describe in greater detail next. We divided both the 1-naphthol and the hydrogeological data into three clusters (Table 6), based upon the available data, and restricted the total number of clusters by requiring more than one observation at the level chosen (i.e., none of the clusters was based upon single observations). In the case of 1-naphthol, three clusters were found to contain three, eight, and seven wells, with mean values of 128, 50, and nondetected for 1-naphthol, respectively. For the hydrogeological data, we found clusters of three, ten, and nine wells. The total number of clusters differed from that for 1-naphthol since we did not have any missing hydrogeological data. Interestingly, the members of the cluster of size three for the hydrogeological data were all within the cluster of seven wells with nondetection from the 1-naphthol data (underlined wells in the 1-naphthol cluster), suggesting that nondetection of 1-naphthol was determined, at least in part, by physical characteristics of the aquifer. We next divided the hydrogeological data into two different categories, a group of measured ions that we found to be significantly correlated with each other and another group that was not significantly correlated with each other. This clustering based on the correlated ions yielded groups sizes of ten, nine, and three. Clustering based on the remaining categories yielded group sizes of ten, eight, and four. We then ordered the observations based on how close they were to the center of the smallest cluster for each scheme. This created very different orderings (correlation ) 0.285, p ) 0.196; two-sided, Monte Carlo simulation). When clustering on the highly correlated hydrogeological data, we found that the cluster of size three was a subset of the cluster centered on nondetection for the 1-naphthol (underlined values in the 1-naphthol cluster, same wells as the corresponding cluster for the hydrogeologic data) and that when those wells for which we had hydrogeological data and no 1-naphthol results were ignored, six of the wells (of a total of 11 wells) with detectable levels of 1-naphthol were in the group 2 cluster of the correlated hydrogeological data, and five of these wells were in the cluster of the medium 1-naphthol levels (mean ) 50.1), as indicated by bold font in the VOL. 39, NO. 11, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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1-naphthol cluster. This correlation is highly significant (p ) 0.009). In the case of the wells with uncorrelated hydrogeological data, the cluster of size 4 was contained entirely by the 1-naphthol cluster centered on 17.1 (nondetectable level, p ) 0.01), as indicated by italic font in the 1-naphthol cluster (wells 27, 42, and 69, the same wells as clustered on hydrogeological data as shown by underlining, and also well number 53). Thus, it appears that the hydrogeological data do correlate with the 1-naphthol content of the wells. Insofar as the hydrogeological data (separated into two clusters, one of which has a high correlation between the measured values for mineral content and related variables and the other does not) is a surrogate for two aquifers that are not in equilibrium with one another, it would appear that the 1-naphthol content of the wells is largely determined by migration of 1-naphthol from the surface to the specific aquifer from which the well draws its water supply. Our data also suggest that a different explanation is required to explain the small cluster of wells (57, 58, 66) that contain a mean concentration of 128 µg/L 1-naphthol. These three wells had no discernible pattern in the hydrogeological clustering, nor were they adjacent spatially. Well 57 is of special note, as it is adjacent to well 59, which had a low level of 1-naphthol. In addition, the three wells in this cluster had extremely high levels of 1-naphthol in comparison to the other wells (a mean value of 3 and 7.5 times the mean values of the other two clusters). Further investigation of the underlying hydrogeology and serial sampling of these wells will be necessary to understand why they contain such different amounts of 1-naphthol as compared with the other wells examined in this study.
Discussion ELISAs were performed to determine levels of the triazine herbicides simazine and atrazine, the pesticide carbaryl (a major chemical in use in Uruguay; over 11 400 kg of this insecticide is imported annually), and 1-naphthol, the major environmental breakdown product from carbaryl in soil or water. On the basis of importation data for Uruguay (none of the agricultural chemicals we studied are manufactured in Uruguay), 191,529 kg (active ingredient basis) of atrazine (2003 data) and 37,408 kg of simazine (2002 data) were used in Uruguay. While we can‘t attribute exact usage amounts to the study area, a reasonable estimate would be about half of all agricultural chemicals used on vegetables in the country occurs in the area around Montevideo. Traditional instrumental analyses were performed for captan, folpet, endosulfan, and dodechachlor, all chemicals used in Uruguayan agriculture (the first two are fungicides, the latter two insecticides) as well as for atrazine, carbaryl, carbofuran, 1-naphthol, cypermethrine, ethyl and methyl chlorpyrifos, diazinon, dimethoate, ethion, iprodione, lambda cyalothrine, malathion, and methyl-parathion. None of the fungicides, herbicides, or pesticides studied was found at levels exceeding their water quality standards in this study. However, the amounts of 1-naphthol, for which there is no water quality standard, found in some of the wells during the spring and summer months are quite interesting. 1-Naphthol is itself a mildly toxic compound, with known adverse effects in mice at oral doses of 200 mg/kg (18). 1-Naphthol is more toxic than carbaryl to many species (e.g., cyanobacteria (19)). In this context, it should be noted that samples of water from some of these wells were tested for acute toxicity to daphnia using EPA standard protocols, 1 year before the samples reported in this paper were taken. No acute toxicity was found, consistent with the very low levels of pesticides and fungicides found by chemical and immunoanalysis in the current study. Carbaryl is broken down in the environment by various pathways, of which the most important in water are chemical 3902
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breakdown by hydrolysis, with a half-life at 25 °C of about 10-17 days at pH 7 and about 3 h at pH 9, and aqueous photolysis, so that hydrolysis is much faster in summer than other seasons. Xu (20) has calculated the half-life of carbaryl for aqueous photolysis at 40° north to be 64 h in spring, 52 h in summer, and 102 h in fall. The major product of both hydrolysis and photolysis is 1-naphthol. Carbaryl is also efficiently metabolized to 1-naphthol, with a half-life of about 4 days (21), by bacteria and other soil microorganisms. Our findings of relatively high levels of 1-naphthol in several of the wells tested, while carbaryl was not detected, suggest that extensive breakdown of carbaryl had occurred prior to sampling. There was no significant correlation between the amounts of 1-naphthol detected in a well and the depth of the well, and in the surface waters only naphthol was detected frequently. Thus, it appears that the breakdown of carbaryl occurred prior to its entering the groundwater aquifer, probably catalyzed by enzymes from microorganisms in the soil. Alternatively, 1-naphthol could also arise from photodecomposition of carbaryl on plant or soil surfaces after washoff (22). The relatively frequent detection of 1-naphthol might also suggest that it is more persistent that the other chemicals studied under the conditions in this region, and it may serve as an indicator that short-lived agrochemicals also reach this aquifer. We are assuming in these arguments that all of the 1-naphthol we detected by ELISA arose from the breakdown of carbaryl; lacking rigorous mass balance studies, which were not performed, we recognize the possibility of the apparent amounts of 1-naphthol detected arising from other sources, including a cross-reaction with other species or other analytical artifacts. The groundwater is stored and circulates through fractures developed in the crystalline rocks of the basement, which is constituted mainly of methamorphic rocks of low to medium degree. The fractures have a high capacity of storage for water, and in some cases, they contain sandy materials that allow them to improve their hydrogeological characteristics. Most of the rocks in the basement are covered by very fine materials of low permeability, so the main recharge of the aquifers is done through infiltration from the streams and rivers. There were significant correlations between water content of Ca2+ and Mg2+; conductivity; hardness; pH; and water content of SO42-, NO3-, K+, Na+, and Cl- in the wells sampled. Presumably, these parameters all measured dissolved solids in the aquifer. Interestingly, there was no correlation between conductivity and HCO3-, suggesting no relationship of bicarbonate content to total dissolved solids of the water. The area of Montevideo studied is close to the Rio Plata, which often contains brackish or saltwater depending on the season. The Na+ and Cl- content of samples was highly correlated (p < 0.01). Thus, we initially assumed that Na+ and Cl- in the well water arose from incursion of the river water into the aquifer. However, there was a significant correlation (p < 0.01) between nitrate content and either Na+ or Cl- content in the samples, suggesting that Na+ and Cl- could also arise from agricultural activities. The Ca2+ and Mg2+ content were highly correlated with each other (water hardness); neither the Ca2+ nor the Mg2+ content was correlated with the Na+ or K+ concentration. We assumed that the nitrate content of the well water was a marker for agricultural activities (fertilizer application). However, there was no significant correlation observed between the NO3content of water samples and the simazine, atrazine, or naphthol content. The current findings are reassuring from the perspective of public health. The field data indicate a baseline level of low contamination of groundwater in this farming area using current practices. Future analyses can be compared and correlated with any changes in the practices or effects of longer periods of use. In future studies, we will also perform
additional assays for other pesticides in wide use in Uruguay that were not examined in this study, as reagents become available for expanded ELISA analyses. We will also try to begin to develop a more regional interest in the use of ELISA for environmental analysis by offering short courses, training in the methodology, and ELISA kits to investigators elsewhere in the Mercosur region with interests in collaboration.
Acknowledgments This work was supported by Grant D43TW005718 from Fogarty International Center of U.S., NIH; by an NIEHS Superfund Basic Research Grant, P42-ES04699; and by the Fulbright Commission, Grant ASJ 4-1362 from the Bureau of Educational Affairs of the U.S. Department of State. We acknowledge the instrumental analysis and technical assistance from the Laboratory of DGSSA, Ministerio de Ganaderı´a y Agricultura: Ana Laura Chouhy, Susana Franchi, and Manuel Padro´n. We also thank Cristina Cacho, Laboratory of Environmental Hygiene, for technical assistance and Bruce Hammock and Shirley Gee for the gift of reagents used for ELISA and many helpful discussions.
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Received for review September 3, 2004. Revised manuscript received March 10, 2005. Accepted March 10, 2005. ES048620D
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