Atrazine in Rainfall and Surface Water
Research Articles
Research Articles
Determination of Atrazine in Rainfall and Surface Water by Enzyme Immunoassay 1Andrea D a n k w a r d t , 1Susanne Wrist, 2Wolfram Elling, 3E. Michael T h u r m a n , tBertold H o c k 1 Lehrstuhl ffir Botanik, TU Mfinchen (Weihenstephan), D-85350 Freising, Germany 2 Fachbereich Forstwirtschaft, FH Weihenstephan, D-85350 Freising, Germany 3 U.S. Geological Survey, 4821 Quail Crest Place, Lawrence, KS, 66049 USA
Corresponding author: Prof. Dr. Bertold Hock
Abstract Rainwater and surface water from four sites in Germany (Bavaria and Lower Saxony) were analyzed for atrazine by enzyme immunoassay from June 1990 until October 1992. The limit of quantification of the immunoassay was 0.02/~g/L with a middle of the test at 0.2/~g/L. About 60 % of the samples contained measurable amounts of atrazine. Seasonal trends were observed, with the highest concentration in the summer months of up to 4/ag/L for rainwater and up to 15/~g/L for surface waters. The highest concentrations were found in agricultural areas, while in the investigated national parks up to 0.56/~g/L could be detected in rain water. This points to long-range atmospheric transport from agricultural areas to pristine national parks. Samples from forest stands usually showed higher atrazine concentrations than samples from open fields. Deposition rates of 10 - 50/~g/m z 9 yr were observed in the national parks and 10 - 180 ~tg/m2 9 yr at the agricultural sites. Comparison of results obtained by enzyme immunoassay and GC/MS showed a good correlation of r = 0.95.
1
So far, not much is k n o w n about the atmospheric processes and the fate of the pesticides during transport. Generally, photochemical and oxidative inactivation and degradation are possible. The main metabolites of atrazine in soil and aquatic systems are deethylatrazine, deisopropylatrazine, and hydroxyatrazine [13], but little is known about their presence in the atmosphere. The purpose of this study was to determine the presence of atrazine in rainwater of Southern and Central Germany, to monitor seasonal trends, and to infer whether the ban on atrazine in Germany has reduced its concentration in the precipitation. The determination of atrazine was carried out by enzyme immunoassay (EIA), as the samples can be investigated faster and at lower costs than classical analYtical methods like gas chromatography (GC) and high pressure liquid chromatography (HPLC) [14, 15].
Introduction
Atrazine, a s-triazine herbicide, is commonly used on crops of sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum) and corn (Zea mays) as well as on industrial areas. In Europe it is mainly applied on corn. It has been one of the most heavily used herbicides in Germany before its ban in April 1991. The extensive use of atrazine has lead to widespread detection of atrazine and its metabolites in groundwater, rivers and lakes, rainwater and soil [ 1 - 7]. The direct application of herbicides to the soil may lead to the leaching of compounds into groundwater, rivers, and lakes. The influence of herbicides on water quality is important, as 70 % of the German drinking water is obtained from untreated groundwater [8]. Therefore, the EEC drinking water ordinance was set to an upper limit of 0 . 1 / l g / L for a single substance and 0.5/ag/L for the total of all pesticides [9]. Drift during application and evaporation from plant surfaces and soil leads to the presence of pesticides in the atmosphere [10, 11], where they can be transported over some distances [2, 12]. Precipitation clears the atmosphere of the pesticides.
196
2
2.1
Materials and Methods
Sampling sites
Rainwater samples were taken at four sites (-0 Table 1) for the determination of atrazine. Three stations were located in Bavaria, one site in Lower Saxony. The site in Lower Saxony was at the official station of the Umweltbundesamt (Federal Environment Agency), Berlin - Waldhof. The sampler was installed on the roof. W a l d h o f is located in a plain, mostly agricultural area. The station in Freising (Drirnast and Berg) was also set up in an agricultural area, the other two Bavarian stations in national parks (NP). The site Dfirnast is located on a hill at the German Meterological Service station in Weihenstephan (Freising), Berg in a forest near Freising on a hilltop (--) map A in Fig. 1). The station at the Bayerischer Wald national parks was established on the ridge of the B6hmerwald ( ~ map B in Fig. 1) and the station at the Berchtesgarden national park on the slope of the Watzmann (-~ map C in Fig. 1). Rainwater from open fields and forest stands were collected at the stations, except for
ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1 9 6 - 2 0 4 (1994) 9 ecomed publishers, D-86899 Landsberg, Germany
Research Articles
Atrazine in Rainfall and Surface Water
W a l d h o f with sampling only at open fields. The samplers were located in fenced areas where no pesticides had been applied during the last years. Table 1: Sampling sites for the collection of rainwater (NP = national park) Sampling site
Elevation Sample (m) origin
DOrnast (85350 Freising, Bavaria)
470
open field
Berg (85350 Freising, Bavaria)
500
forest stand
78
open field
B&renlochriegel (NP Bayer. Wald, Bavaria)
1 300
open field
B~.renlochriegel (NP Bayer. Wald, Bavaria)
1 300
forest stand
Watzmann (NP Berchtesgaden, Bavaria)
1 500
open field
Watzmann (NP Berchtesgaden, Bavaria)
1 500
forest stand
Waldhof (29394 LQder, Lower Saxony)
Fig. 1: Location of the sampling sites Berg, Freising (A), B/irenlochriegel, NP Bayerischer Wald (B) and Watzmann, NP Berchtesgaden (C). The sections of the maps (1 : 25 000) show an area of 2.5 x 2.5 km
2.2
Sampling
Glass bottles (25 L) with glass funnels containing glass filters were used for the sampling of rainwater from open areas. The surface of the funnel was 680 cm 2. At Dfirnast (Freising) a steel funnel (1 m 2) was used. In the forest stand 16 samplers were set up in a grid of 2 92 m. Each sampler consisted of a glass funnel in a glass measure (1 L, inverse con-
ESPR-Environ. Sci. & Pollut. Res. I (4) 1994
ical form). The sampling surface of all 16 samplers was 4778 cm 2. As the samplers were not closed during dry periods, the total deposition consisting of dry and wet deposition was collected. Evaporation of water from the glass containers was kept to a minimum, because the glass bottles had only a small opening in which the funnel was inserted and the glass measures were covered by the funnels containing glass filters.
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Atrazine in Rainfall and Surface Water
The glass bottles were emptied once a week, and the collected rainwater was transferred to dark glass bottles and mailed from the sampling site directly to the analytical laboratory. The samples were stored at 4 ~ The analyses were carried out within a week of receipt of the samples. The sites Dfirnast and Berg were sampled beginning in June 1990. In 1991 and 1992 the Dfirnast, Berg, and Waldhof sites were sampled from April to November and the national parks from June to October due to inaccessibility because of snow. Rainfall data for the determination of the deposition were obtained either from the local weather stations (Waldhof/Liider, Diirnast/Freising) or from measuring the actual rainfall.
Research Articles
of positive samples should be carried out afterwards by GC, HPLC, and GC/MS, as in the case of our study. 3.1
Enzyme immunoassay
The measuring range of the assay was 0.02 - 2.5/ag/L with a middle of the test (IC 50) at 0.25/ag/L for serum $84 and 0 . 0 2 - 2 lag/L with a middle of the test at 0.15 I~g/L for serum $2 (-0 Fig. 2). Absorption values for zero concentrations were usually above 1. 1.2. absorption
't
1.0 o
0.8-
2.3
Analysis
0.6-
No clean-up of the samples was necessary. Coarse particles were removed by sedimentation and filtration. The pH of the samples was determined and, if necessary, adjusted to pH 7. For the determination of matrix effects one aliquot of the sample was diluted with distilled H/O, another aliquot spiked with atrazine standards. Details of antibody production, enzyme tracer synthesis, and performance of the enzyme immunoassay have been described by WUST and HOCK [16] and DANKWARDTet al. [17]. GC and G C / M S analyses for the validation of the atrazine concentrations obtained by EIA were carried out in the laboratory of Prof. K. B/ICHMANN, T H Darmstadt by Juliane SCHA~ according to a method described by BACHMANN et al. [18] and in the group of Prof. E. M. THURMAN, U.S. Geological Survey, Lawrence, USA as described in earlier reports [19]. In brief, the samples were extracted by solid-phase Cls cartridges from 100 mL to 1 L of water. The cartridges were eluted with an organic solvent and evaporated. Mass spectrometry was carried out in selected ion mode using 3 ions (molecular ion, base peak, and confirming ions). The GC separation was with a 15-m capillary column of methyl silicone with He carrier gas.
3
Results and Discussion
The duration of this study was three years from 1990 to 1992 and included the influence of the ban of atrazine in 1991. Sampling sites were located in agricultural areas as well as at clean air stations in national parks in order to obtain information about atmospheric transport. Open space and forest stands were sampled separately to find out about a possible difference in concentrations and deposition rates of the different type of sites. Determination of atrazine was carried out by enzyme immunoassay. Such tests have been developed for many pesticides [20], and several enzyme immunoassays have been worked out for triazine herbicides [16, 2 1 - 24]. In comparison to classical analytical methods such as gas chromatography and high pressure liquid chromatography, the samples can be investigated faster and at lower costs without any clean-up or concentration steps. A validation
198
0.40.20.00
0.01
0.1
1
10
E
atrazine (l.tg/L) Fig. 2: Absorption curve of serum $2 (E = excess, 1 000 ~g/L)
The suitability of the antibodies for the determination of atrazine in natural water samples was investigated. The antibodies showed a high cross-reactivity for propazine with 116 - 143 % ( ~ Table 2). No interferences are expected since this herbicide is not applied in most European countries. The affinity of the antibodies toward prometryn, deethylatrazine, and ametryn was lower than toward atrazine (2 - 27 %). Prometryn and ametryn play only a minor role as herbicides and were not present in the investigated water samples. The metabolite deethylatrazine can be found in water samples, usually in lower concentrations than its parent compound, atrazine. All other compounds showed crossreactivities below 10 %, most to 1 % or less. Table 2- Cross-reactivities of the sera S 84 and S 21
Substance
Cross-reactivity in % S 84 $2
Atrazine
100
100
Propazine
116
143
Deethylatrazine
13
15
Prornetryn
10
27
2
12
Ametryn
1 only cross-reactivities exceeding 10 % are listed
Natural water samples may vary considerably in their pH and the concentration of humic substances. Thus, the influence of these parameters on the immunoassay was tested. No effect was found for the pH between 4 and 10 and so no pH adjustment was necessary for most samples. However,
ESPR-Environ. Sci. & PoUut. Res. t (4) 1994
Research Articles
Atrazine in Rainfall and Surface Water
values of below pH 4 and above pH 10 yielded atrazine concentrations greatly exceeding the standards. The influence of humic acids on the assay was tested with different concentrations of natural humic acids isolated from the Suwannee River (Horida, USA). Apparently increasing atrazine concentrations were observed after addition of standards containing 100/ag/L of humic acids, concentrations that are rarely found in rainwater and surface water samples. Accuracy and consistency of the enzyme immunoassay are important factors. Therefore, the reproducibility of the assay on the same day, as well as on different days, was investigated (-~ Table 3). Percent coefficients of variation (% CV) ranged from 1.9 to 13.3 %. The assays carried out on the same day showed lower % CV than the day-to-day investigations. Increasing variabilities at different temperatures on different days, slight changes in the assay solutions used, and variations in the personal performance on different days may contribute to this effect. A good reproducibility of the assay was found, however, with % CV mosdy below 12 % for the investigation of natural water samples.
The accuracy of the immunoassay was determined by validation measurements with GC and GC/MS. In 1991, 31 samples were examined (-o Fig. 3), 26 samples in 1992. A good correlation of r = 0.95 was found for both investigations. Slightly higher concentrations were obtained in the EIA, as it could be expected from the cross-reactivities. In addition, the humic substances of the samples may contribute to a slight overestimation, as the samples from the forest stands sometimes had a yellow color. 10 =
O r
._1
Same-day reproducibility Atrazine (pg/L • s)
Waldhof 1 Waldhof 2 Dt3rnast Berg 1 Berg 2 Bayer. Wald 1 Bayer. Wald 2
0.632 0.184 1.336 0.272 0.542 0.283 0.454 Berchtesgaden 0.271 Moosach 0.240
• 0.020 • 0.012 • 0.049 • 0.028 • 0.063 • 0.013 • 0.025 __. 0.026 • 0.026
0.1
0.01 0.01
Day-to-day reproducibility
% CV 3.1 6.7 3.6 10.2 11.7 4.5 5.4 9.6 10.7
Atrazine (ug/L • s) 0.605 0.165 1.206 0.246 0.471 0.281 0.440 0.256 0.208
• • • • • • • • •
0.046 0.020 0.130 0.033 0.009 0.030 0.050 0.020 0.027
O/
o.....................
Table 3: Same-day reproducibility and day-to-day reproducibility of the atrazine immunoassay for natural water samples (based on five different determinations) Sample
ooo
r 0.95
0.1
I
10
I~g/L a t r a z i n e (EIA)
% CV 7.6 12.1 10.8 13.3 1.9 10.6 11.6 7.7 12.9
Fig. 3: Comparison of EIA and GC for the detection of atrazine in water samples from 1991
3.2
Investigation of water samples
A total of 316 rainwater samples and 80 surface water samples were investigated. About 60 % of the investigated samples contained amounts of atrazine higher than the limit of quantification, which is 0.02/~g/L (--' Table 4). The highest concentrations in rainwater (up to 4 pg/L) were
Table 4: Number of positive samples (exceeding 0.02/ag atrazine/L) and highest and median concentration of the positive samples for atrazine at the different sampling sites in the years 1990 - 1992 Sampling site number
1990 max. ~ug/L)
median (pg/L)
number
1991 max. (pg/L)
median /Jg/L)
number
1992 max. (pg/L)
median #g/L)
Dt3rnast open field
15 of 21
3.29
0.16
21 of 32
0.28
0.06
12 of 22
1.49
0.07
Berg forest stand
18 of 18
4.18
0.43
18 of 29
1.14
0.25
19 of 21
3.05
0.20
Waldhof open field
14 of 31
0.49
0.10
9 of 21
0.40
0.06
NP Bayer. Wald open field
10 of 18
0.31
0.06
7 of 15
0.21
0.05
NP Bayer. Wald forest stand
13 of 18
0.41
0.13
11 of 16
0.56
0.08
NP Berchtesgaden open field
4 of 12
0.04
0.03
5 of 16
0.05
0.04
NP Berchtesgaden forest stand
7 of 12
0.23
0.05
4 of 15
0.09
0.06
ESPR-Environ. Sci. & PoUut. Res. 1 (4) 1994
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Atrazine in Rainfall and Surface Water
Research Articles
found at the agricultural sites in Freising (Diirnast and Berg), the lowest concentrations at the Berchtesgaden national park (0.02 - 0.23/~g/L). Intermediate concentrations were observed at the Waldhof site (agricultural) and the Bayerischer Wald national park (0.02 - 0.56/~g/L). Similar concentrations of atrazine in rainwater were reported by other investigators. BRAUN et al. [25] found up to 1.1/lg/L in the Munich area, MOLLER et al. [3] up to 2.2/ag/L at different stations in Bavaria. A seasonal appearance of atrazine was observed. Concentrations started to increase in mid-April to May and the highest concentrations were usually found in June (-~ Fig. 4). In August concentrations declined and in September and the following months no atrazine was detected, except for a second peak at Dfirnast and Berg in November of 1990. The second peak may be due to fall application, or perhaps to the harvest of corn and airborne soil and dust particles containing atrazine. The highest concentrations of atrazine appeared shortly after the application of the herbicide and a few months thereafter. No atrazine was detected in early spring, late fall and during winter. A seasonal appearance of atrazine was also reported in other investigations from Switzerland, Germany and the USA [2, 6, 26]. However, the herbicide was also found in rain during the whole year in a few cases [27, 28]. Our group and several others [2, 5, 6] have found that atrazine was detectable in rain even before its application on corn. The application of atrazine on industrial areas and railroad tracks in early spring might be one reason. On the other hand, it could be due to herbicide resulting from last year's application. As atrazine is mostly bound to the soil, wind erosion and volatilization from soil could be possible. In contrast to late fall and winter, where usually no herbicide was detected, higher temperatures, moist soils and agricultural activities with heavy dust formation may facilitate this effect in spring [2, 5].
A correlation could be found between the amount of rain and the atrazine concentrations in the rain water. Thus, high concentrations often correlated with little rainfall, whereas heavy rain resulted in low atrazine concentrations. The total deposition of atrazine was determined as the product of measured atrazine concentrations and the amount of rainfall. The highest deposition rate for each station was recorded in June, followed by July (-~ Fig. 5). The highest value of 140/ag/m 2 was measured at Diirnast in June 1990, Berg showed 70/~g/m 2. A distinct decline in deposition was observed in the following years due to the ban of atrazine in 1991 leading to deposition rates of 2 0 - 4 5 p g / m 2 in June. The recorded deposition per year was the highest in 1990 ( ~ Fig. 6) with 180/lg/mZ.yr at Diirnast and 150 /~g/m2.yr at Berg. The total deposition during 1990 are presumably higher because measurements were begun only in June of this year. In 1991 and 1992 the deposition was below 100/~g/ma.yr for all sites, with the highest values at Dtirnast and Berg and in the Bayerischer Wald NP (forest stand) with 3 0 - 80/~g/m 2" yr. Similar deposition rates were reported by HERTERICH [29] and OBERWALDERet al. [5]. According to Herterich [29] is the true deposition into a forest stand is usually higher than the recorded deposition rate, as a portion of the herbicides remains adsorbed to leaves and needles of the trees. The high deposition into the forest stand at the Bayerischer Wald NP was unexpected. Deposition rates are similar to the agricultural sites Dfirnast and Berg, although in the national park no pesticides are applied. A transport of atrazine from agricultural areas to the national park via the atmosphere has apparently taken place. The closest corn fields to the park are located at a distance of 20 km to the west. In June 1991 mostly winds from the west were observed, which might have transported herbicides from those corn fields into the national park. In June 1992 the wind did not
atrazine (pg/L) 0.6 0.5 0.4 0.3 0.2 0.1 0.0
[--LJ
J'--LJMay["J--] L--I--]
i~
LJ.J
June
July
August
sampling site I~DOrnast IlWaldhof I~Bayer. Wald ~Berchtesgaden Fig.
200
4: Atrazine concentration in rainwater in 1991 (open field)
ESPR-Environ. Sci. & Pollut. Res, 1 (4) 1994
Research
Articles
A t r a z i n e in R a i n f a l l a n d S u r f a c e W a t e r
atrazine (pg/m 2 * yr) 40
30
20
10
0
April
May
June
July
August
September
sampling site E~]D0rnast ==Berg I~Waldhof ~ B W O F I~BW FS roBe OF ~ B e FS Fig. 5: Deposition of atrazine at the seven sampling sites in 1991 (BW = Bayerischer Wald, Be = Berchtesgaden, OF = open fields, FS = forest stand)
atrazine (pg/m 2 . yr) 200
150
100
50
0
111111 D0rnast
Berg
Waldhof
BW OF
BW FS
Be OF
Be FS
year of sampling E~1990 ==1991 1~1992 Fig. 6: Total deposition of atrazine at the seven sampling sites from 1990 to 1992 (BW = Bayer. Wald, Be = Berchtesgaden, OF = open field, FS = forest stand) come from one certain direction, sources from the Czech Republic may also contribute to the observed herbicide deposition. A transport of atrazine into mountain and forest stands located at some distance to agricultural areas was also reported for the Black Forest [18], the Fichtelgebirge [29], and Swiss mountain lakes [2]. The water samples from forest stands usually showed higher atrazine concentrations and also higher deposition rates as the open field samples (except for D~irnast in 1990). This can be attributed to the filter effect of the trees by adsorbing pollutants (herbicides) on the large surface of their needles and leaves. During the next precipitation event, part of the substances is washed off and can be found in the rain water. Several surface waters were investigated for atrazine in the area of the stations Dfirnast und Berg (Freising). The
ESt'R-Environ. Sci. & PoDut. Res. 1 (4) 1994
Thalhauser Graben was assayed monthly. The highest concentrations were found again in June (4 pg/L in 1990) with little atrazine in April and earlier and in September and the following months. Values have been declining since 1991 (--* Fig. 7). Several ponds and creeks in the area of Freising were investigated twice a year (July and January). No atrazine or only low concentrations were observed in the ones located in forest areas (-* Table 5), while waters near corn fields exhibited high values up to 14/~g/L. Drift-off during application, flushing of soil particles into the water, and washing out of pesticides from the soil leads to the presence of atrazine in the surface waters. The degradation of atrazine in natural waters is relatively slow [30]. The lake in Dfirnast showed 15/~g/L in June 1990. Still 10 lag/L were found one month later. The concentrations declined in winter to 0.05 - 0.08/~g/L. This can
201
Atrazine in Rainfall and Surface Water
Table 5: A t r a z i n e c o n c e n t r a t i o n s ~ g / L ) sampling site
summer
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o f s u r f a c e w a t e r s in t h e a r e a o f F r e i s i n g
1990
summer
1991
winter
1991
summer
1992
winter
Spring D0rnast
< 0.02
< 0.02
< 0.02
< 0.02
< 0.02
Brook D0rnast
< 0.02
< 0.02
< 0.02
< 0.02
< 0.02
0.47
< 0.02
< 0.02
0.11
< 0.02
Fuchsloch Lange Seiche
0.10
< 0.02
< 0.02
0.03
< 0.02
Unt. Rehbuckel
< 0.02
0.07
< 0.02
< 0.02
< 0.02
Ob. Rehbuckel
< 0.02
< 0.02
< 0.02
< 0.02
< 0.02
Tiroler Schlag
0.04
< 0.02
< 0.02
0.05
< 0.02
Eisweiher
0.07
0.07
< 0.02
0.04
< 0.02
Lake DOrnast
14.60
13.49*
4,25
0.08
3.20
Dorfacker
3.29
5.76*
1.69
0.22
0.30
0.08
Moosach
0.09
0.09
0.10
0.06
0.04
3.70* *
1992
0.05
* determined by HPLC; ** determined by G C
atrazine ~gtL) 5
i
4
I! ____
I!11
Im
=l. ,..... I,..._
.[[.
1 2 3 4 5 6 7 8 91011121 2 3 4 5 6 7 8 91011121 2 3 4 5 6 7 8 91011121 2 3 4 5 6 7 8 9
1990
1991
1992
1993
Fig. 7: A t r a z i n e c o n c e n t r a t i o n o f the T h a l h a u s e r G r a b e n ( 1 9 9 0 - 1 9 9 3
be partly attributed to degradation, but uptake by and adsorption to plants and the sediment seems to play an important role in the decrease of atrazine in the water systems [30, 31]. The adsorbed atrazine, however, may be desorbed from the sediments and provide a long-term source for low-level contamination. The concentrations found in the summer months decreased between 1990 and 1992. No atrazine could be detected in a source in the forest of Freising ( ~ Table 5). Yet, another spring in a forest from this area, located at a distance of 5 km from corn fields, exhibited atrazine concentrations of 0 . 2 - 0 . 5 p g / L and deethylatrazine concentrations of 0.4 - 1.1/ag/L over the entire year (Dr. J. MAGUHN, personal communication). No atrazine is applied in this forest, so deposition via the atmosphere may be a possibility. On the other hand, water containing atrazine may be transported through the soil from
202
the neighboring agricultural area due to unfavorable hydrogeological conditions. This again shows how clean areas can be influenced by transportation of herbicide from areas with high herbicide concentrations. The metabolites deethylatrazine and deisopropylatrazine were detected in the samples investigated by G C / M S beside the parent compound. The greatest concentration of 0.2 p g / L for deethylatrazine was found in June in the rain water samples of Berg and Dfirnast and in the brook Thalhauser Graben. Deisopropylatrazine was observed in a few samples up to 0.1 pg/L. Several other pesticides could also be detected. Many of the samples contained terbuthylazine with a maximum concentration of 1.6 p g / L . This s-triazine is frequently used as a substitute for atrazine. Propazine and simazine were found in some cases and metolachlor was observed in many samples with concentra-
E S P R - E n v i r o n . 5ci. & Pollut. Res. 1 (4) 1994
Research Articles
tions of up to 0.84/ag/L. Only samples from the agricultural area contained this herbicide, while in the samples from the national parks none was found. Either metolachlor is not used in the regions around the national park or it is not stable and degraded during the atmospheric transport.
4
Conclusions
The presence of atrazine in rain and surface water was demonstrated in this three-year study. Concentrations of up to 4/ag/L for rainwater and 15/Jg/L for surface water could be detected. Concentrations &dined after the ban of atrazine in 1991, but up to 4/ag/L were still found in rain and surface water in some cases. Transportation of the herbicide via the atmosphere from neighboring countries may contribute to the observed atrazine concentrations, but illegal atrazine application can be assumed as another source of atrazine, especially for surface water. Concentrations of up to 4 p g / L in the Diirnast lake seem very high to be caused by deposition alone, because the deposited atrazine would be diluted by surface water. In this case also all ponds and brooks in one region should show a similar atrazine concentration. As in each summer the surface waters from agricultural areas exhibited higher concentrations than the ponds from the forest, drift-off during application and leaching of herbicide from soil particles flushed into the water seems more probable. The location of the Diirnast lake and the brooks Thalhauser Graben and Dorfacker at a bent near corn fields may add to this effect. No contamination limits exist for rain water. The values of the drinking water ordinance for pesticides of 0.1/Jg/L for a single substance and 0.5 p g / L for the sum of pesticides and their toxic metabolites should be applied if rainwater is collected in cisterns for drinking water. Therefore, from May to July rainwater cannot be recommended as drinking water without special cleaning steps. The influence on organisms of atrazine concentrations of up to 15/Jg/L, as detected in our study, is not fully known. Atrazine as an inhibitor of photosynthesis may influence leaf development. The high concentrations in forest stands, especially in late spring, may effect the development of new leaves and needles. The spruces Picea abies and Piceaglauca cannot detoxify atrazine in contrast to resistant plants like corn and sorghum, as the herbicide is not metabolized by the glutathion-S-transferases of these species [32]. It has been discussed that atrazine and other herbicides may contribute to forest decline [33]. Controversial reports exist for aquatic systems. While atrazine was found as not being toxic for fish and invertebrates in one study [34], other reports disclosed necrotic changes in the liver of rainbow trout after 28 days of exposure to 10 pg atrazine per liter, and 5/lg per liter damaged the kidneys [35, 36]. Inhibition of photosynthesis of some species of phytoplankton was detected at concentrations as low as 1 - 5 pg/L. This was accompanied by the establishment of more resistant species [37]. No effects on aquatic ecosystems were observed below 20 pg/L of atrazine according to a report by HUBER [38]. ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1994
Atrazine in Rainfall and Surface Water
The occurrence of atrazine and other pesticides in rain during spring and summer points to an atmospheric transport of these compounds. Especially the presence of atrazine in rainwater samples from the national parks suggests a medium- or long-range transport of the pesticides. Deposition rates" of 180 p g / m 2 9 yr (1.8 g/ha 9 yr) represent 0.18 % of the amount of atrazine applied in a year per hectare, corresponding to I kg atrazine/hectare. This loading may seem relatively low and according to HERTERICH[29], no effects are expected in general for the vegetation or groundwater quality. Nonetheless, it must be remembered that approximately 1 % of the applied atrazine is washed from soils into surface waters [39]. Thus, 0.2 % represents nearly 20 % of the surface water transport, which is a considerable portion of the total dissipation of the herbicide. Furthermore, it is important to remember that the corn field is the target. Transportation to other areas like forests or water protection zones should be avoided. It is probable that insecticides and fungicides show a similar behavior in the environment as herbicides. Beside their influences on fauna and flora, effects on humans have to be considered, as they come into contact with those pollutants via the air, rain, and dust. Finally, the enzyme immunoassay proved to be a valid tool for the determination of atrazine in natural water samples. The samples could be analyzed in a much shorter time and with less effort as compared to established techniques. No clean-up or pre-concentration steps were necessary. Comparison between samples investigated by EIA and GC/MS showed a good correlation (r -- 0.95), and the methodology is recommended for surveys of water quality.
Acknowledgements We thank the Bundesministerium fiir Umwelt (Federal Environment Ministry) for financial support of this work. We are grateful to Dr. ZmPd., Mr. VOGT, Mr. DONATHand Mr. DATZMANNfrom the NP Berchtesgaden, Mr. JEHL, Mr. KAATZund Mr. ICg-tLHaMMEafrom the NP Bayerischer Wald, Mr. JVIENSCHENDC)RFER,Wl-IWeihenstephan, and the colleagues from the station in Waldhof, who helped with the set up of the stations and the sampling. We appreciate the support of the laboratory of Prof. HUBeR/TU Miinchen-Weihenstephan for HPLCanalyses and of Mrs: Juliane SCHARF from the group of Prof. K. B)~CHMaNN/TH Darmstadt for GC analyses. We thank Dr. BINDERand Dr. HORMANNSD(SRFER/TUMiinchen-Weihenstephan for the immunization of the sheep and the collection of the blood. The joint research between the Lehrstuhl fur Botanik, TU Mfinchen, and the United States Geological Survey is a cooperative study on environmental applications of immunoassays between the two countries.
5
Literature
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Atrazine in Rainfall and Surface Water
[3] MOLLER, C. (1991): PCB und Pflanzenbehandlungsmittel in Niederschliigenund Oberfliichengew~issern.Berichtder Bayerischen Landesanstalt for Wasserforschung, Mtinchen, Schule und Beratung (SUB), Heft 5, IIl-11-III-16 [4] SIEBERS,J.; GOTTSCHILD,D.; NOLTING, H.-G. (1991): Untersuchungen ausgew~ihlterPflanzenschutzmittel und polyaromatischer Kohlenwasserstoffein NiederschliigenSiidost-Niedersachsens - Erste Ergebnisse aus den Jahren 1 9 9 0 / 9 1 . Nachrichtenblatt Deutscher Pflanzenschutzdienst 43, 191- 200 [5] OBERWALDER,C.; GIESSL,H.; IRION,L.; KIRCHHOFF,J.; HURLE, K. (1991): Pflanzenschutzmittel im Niederschlagswasser. Nachrichtenblatt Deutscher Pflanzenschutzdienst 43, 185-191 [6] OBERWALDER,C.; KIRCHHOI~,J.; HURLE,K. (1992): Vorkommen von Pflanzenschutzmittein im Niederschlag Baden-Wfirttembergs. Zeitschrift for Pflanzenkrankheitenund Pflanzenschutz,Sonderheft XIII, 363 - 376 [7] GOH,K. S.; HERNANDEZ,J.; POWEL,S. J.; GREENE,C. D. (1990): Atrazine soil residue analysis by enzyme immunoassay: Solvent effect and extraction efficiency. Bulletin of Environmental Contamination Toxicology 4 5 , 2 0 8 - 214 [8] DIETER, H.H. (1992): German drinking water regulations, pesticides, and axiom of concern. Environmental Management 16, 21-31 [9] AURAND,K.; HASSELBART,K. (1991): Die Trinkwasserverordnung. Erich Schmidt Verlag, Berlin [10] BOEHNCKE,A.; SIEBERS,J.; NOLTING,H.-G. (1990): Investigations of the evaporation of selected pesticides from natural and model surfaces in field and laboratory. Chemosphere 21, 1109-1124 [11] NEURURER,H.; WOMASTEK,R. (1991): Uber das Auftreten von Pflanzenschutzmitteln in der Luft. Die Bodenkultur 42, 5 7 - 70 [12] ELLING,W.; HUBER,S. J.; BANKSTAHL,B.; HOCK,B. (1987): Atmospheric transport of atrazine: A simple device for its detection. Environmental Pollution 48, 7 7 - 82 [13] GRANDET, M.; WEIL, L.; QUENTIN, K.-E. (1988): Gaschromatographische Bestimmung der Triazinherbizide und ihrer Metaboliten im Wasser. Zeitschrift fOr Wasser-AbwasserForschung 21, 21 - 24 [14] BUSHWAY,R.J.; PERKXNS,B.; SAVAGE, S. A.; Lekousi, S. J.; Ferguson, B. S. (1988): Determination of atrazine residues in water and soil by enzyme immnnoassay. Bulletinof Environmental Contramination and Toxicology 40, 547 - 654 [15] THURMAN, E.M.; MEYER, M.; PONES, M.; PERRY, C.A.; SCHWAB,P. A. (1990): Enzyme-linkedimmunosorbent assay compared with gas chromatography/mass spectrometry for the determination of triazine herbicides in water. Analytical Chemistry 62, 2043 - 2048 [16] WOST,S.; HOCK,B. (1992): A sensitive enzyme immunoassay for the detection of atrazine based upon sheep antibodies. Analytical Letters 25, 1 0 2 5 - 1 0 3 7 [17] DANKWARDT,A.; WOST, S.; HOCK. B. (1993): Messung yon Pestiziden in der Atmosphiire und im Niederschlag - Nachweis yon Atrazin im Niederschlag mit Hilfe eines Enzymimmunoassays. Forschungsbericht 104 02 598 des Umweltbundesamtes, Berlin [18] BACHMANN, K.; HILLMANN, R.; PFAFFLIN, D.; SCHARF, J.; WmSIOLLEK, R. (1993): Messung yon Pestiziden in der Atmosph~ire und im Niederschlag. Forschungsbericht 104 02 598 des Umwekbundesamtes, Berlin [19] THURMAN, E.M.; GOOLSBY, D.A.; MEYER, M.T.; MILLS, M. S.; PONES,M. L. (1992): A reconnaissance study of herbicides and their metabolites in surface water of the midwestern United States using immunoassay and gas chromatography/mass spectrometry. Environmental Scienceand Technology 26, 2440 - 2447 [20] HABERER, K.; KRAMER, P. (1988): VerfOgbarkeit immunochemischer Nachweisverfahren fOr Pflanzenschutzmittel im Wasser. Vom Wasser 7 1 , 2 3 1 - 244 [21] SCHLAPPt,J.-M.; FORY, W.; RAMSTEINER,K. (1989): Hydroxyatrazine and atrazine determination in soil and water by enzymelinked immunosorbent assay using specific monoclonal antibodies. Journal of Agricultural and Food Chemistry 37, 1532 - 1538 [22] WITTMANN,C.; HOCK, B. (1990): Evaluation and performance
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[27]
[28]
[29] [30]
[31]
[32]
[33]
[34]
[35] [36]
[37]
[38]
[39]
characteristics of a novel ELISA for the quantitative analysis of atrazine in water, plants and soil. Food & Agricultural Immunology 2, 6 5 - 74 DUNBAR,B.; RIGGLE,B.; NISWENDER,G. (1990): Development of an enzyme immunoassay for the detection of triazine herbicides. Journal of Agricultural and Food Chemistry 3 8 , 4 3 3 - 4 3 7 GIERSCH,T.; HOCK, B. (1990): Production of monoclonal antibodies for the determination of s-triazines with enzyme immunoassays. Food and Agricultural Immunology 2, 85 - 97 BRAUN, F.; SCH~rSSLER,W.; WEHRLE, R. (1988): Kreisl~iufe, Bilanzen und Bewertungen yon Polychlorbiphenylen (PCB), Lindan und Atrazin. In: Gefiihrliche Stoffe in Abwasser und Oberfl/ichenwasser, Bayerische Landesanstalt for Wasserforschung (Hrsg.), Oldenbourg Verlag, Mfinchen R1CHARDS,R. P.; KRAMER,J. W.; BAKER,D. B.; KRIEGER,K. A. (1987): Pesticides in rainwater in the northeastern United States. Nature 327, 129 - 131 GLOTFELTY,D. E. (1985): Pathways of pesticide dispersion in the environment. In: Hilton, J.L. (ed.), Agricultural chemicals of the future (BeltsvilleSymposia in Agricultural Research 8). 425 -435, Rowman & Allanheld Publishers, Totowa, New Jersey Wu, T. L. (1981): Atrazine residues in estuarine water and the aerial deposition of atrazine into Rhode Rive, Maryland. Water, Air, and Soil Pollution 15, 173 - 184 HERTERICH,R. (1991): Atrazin - Atmosphiirischer Eintrag und Immissions-Konzentrationen. Zeitschrift fOr Umweltchemie und Okotoxikologie 3, 196 - 200 BACCI,E.; RENZONI,A.; GAGGI,C.; CALAMARI,D.; FRANCH],A.; VIGHI, M.; SEVERI,A. (1989): Models, field studies, laboratory experiments: An integrated approach to evaluate the environmental fate of arrazine (s-triazine herbicide). Agriculture, Ecosystems and Environment 27, 513- 522 WEHTJE, G. R.; SPALDING, R. F.; BURNSIDE, O. C.; LOWRY, S. R.; LEAVITT,J. C. (1983): Biological significance and fate of atrazine under aquifer conditions. Weed Science 3 1 , 6 1 0 - 618 SSCHRODER,P.; LAMOUREUX,G. L.; RUSNESS,D. G.; RENNEBERG, H. (1990): Glutathione s-transferase activity in spruce needles. Pesticide Biochemistry and Physiology 37, 211- 218 TREVISAN,M.; MONTEPIANI,C.; BARTOLETTI,C.; [OANNILI,E.; DEL RE, A. A. M. (1993): Pesticides in rainfall and air in Italy. Environmental Pollution 80, 31 - 39 MACEK,K. J.; BUXTON,K. S.; SAUTER,S.; GNILKA,S.; DEAN, J. W. (1976): Chronic toxicity of atrazine to selected aquatic invertebrates and fishes. U. S. Environ. Protection Agency,Ecological Res. Series, EPA - 600/3 - 7 6 - 0 4 7 , Contract No. 68 - 01 0092, 6 8 - 0 1 - 1844, Environ. Res. Laboratory, Duluth, MN, pp. 58 NEGELE, R.D. (1989): Studie fiber die Kurz- und Langzeitwirkung von Atrazin auf Regenbogenforellen. Forschungsbericht 116 08 07/02 des Umweltbundesamtes, Berlin VEESER,A. (1990): Licht- und elektronenmikroskopische Untersuchungen zur Toxizit/it yon Atrazin bei Regenbogenforellen(Oncorhynchus mykiss). Inaugural-Dissertation,TieriirztlicheFakult~it, Universitfit M/inchen DE NOYELLES, F.; KETTLE, W. D.; SINN, D. E. (1982): The response of plankton communities in experimental ponds to atrazine, the most heavily used pesticide in the United States. Ecology 63, 1285- 1293 HUBER, W. (1993): Ecotoxicological relevance of atrazine in aquatic systems. Environmental Toxicology & Chemistry 12, 1865 - 1881 LEONARD,R. A. (1988): Environmental Chemistry of Herbicides. Croner, R., Ed. CRC Press, Boca Raton, FL Received: November5, 1993 Accepted: May 2, 1994
ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1994
Research Articles
Research Articles
Determination of Atrazine in Rainfall and Surface Water by Enzyme Immunoassay 1Andrea D a n k w a r d t , 1Susanne Wrist, 2Wolfram Elling, 3E. Michael T h u r m a n , tBertold H o c k 1 Lehrstuhl ffir Botanik, TU Mfinchen (Weihenstephan), D-85350 Freising, Germany 2 Fachbereich Forstwirtschaft, FH Weihenstephan, D-85350 Freising, Germany 3 U.S. Geological Survey, 4821 Quail Crest Place, Lawrence, KS, 66049 USA
Corresponding author: Prof. Dr. Bertold Hock
Abstract Rainwater and surface water from four sites in Germany (Bavaria and Lower Saxony) were analyzed for atrazine by enzyme immunoassay from June 1990 until October 1992. The limit of quantification of the immunoassay was 0.02/~g/L with a middle of the test at 0.2/~g/L. About 60 % of the samples contained measurable amounts of atrazine. Seasonal trends were observed, with the highest concentration in the summer months of up to 4/ag/L for rainwater and up to 15/~g/L for surface waters. The highest concentrations were found in agricultural areas, while in the investigated national parks up to 0.56/~g/L could be detected in rain water. This points to long-range atmospheric transport from agricultural areas to pristine national parks. Samples from forest stands usually showed higher atrazine concentrations than samples from open fields. Deposition rates of 10 - 50/~g/m z 9 yr were observed in the national parks and 10 - 180 ~tg/m2 9 yr at the agricultural sites. Comparison of results obtained by enzyme immunoassay and GC/MS showed a good correlation of r = 0.95.
1
So far, not much is k n o w n about the atmospheric processes and the fate of the pesticides during transport. Generally, photochemical and oxidative inactivation and degradation are possible. The main metabolites of atrazine in soil and aquatic systems are deethylatrazine, deisopropylatrazine, and hydroxyatrazine [13], but little is known about their presence in the atmosphere. The purpose of this study was to determine the presence of atrazine in rainwater of Southern and Central Germany, to monitor seasonal trends, and to infer whether the ban on atrazine in Germany has reduced its concentration in the precipitation. The determination of atrazine was carried out by enzyme immunoassay (EIA), as the samples can be investigated faster and at lower costs than classical analYtical methods like gas chromatography (GC) and high pressure liquid chromatography (HPLC) [14, 15].
Introduction
Atrazine, a s-triazine herbicide, is commonly used on crops of sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum) and corn (Zea mays) as well as on industrial areas. In Europe it is mainly applied on corn. It has been one of the most heavily used herbicides in Germany before its ban in April 1991. The extensive use of atrazine has lead to widespread detection of atrazine and its metabolites in groundwater, rivers and lakes, rainwater and soil [ 1 - 7]. The direct application of herbicides to the soil may lead to the leaching of compounds into groundwater, rivers, and lakes. The influence of herbicides on water quality is important, as 70 % of the German drinking water is obtained from untreated groundwater [8]. Therefore, the EEC drinking water ordinance was set to an upper limit of 0 . 1 / l g / L for a single substance and 0.5/ag/L for the total of all pesticides [9]. Drift during application and evaporation from plant surfaces and soil leads to the presence of pesticides in the atmosphere [10, 11], where they can be transported over some distances [2, 12]. Precipitation clears the atmosphere of the pesticides.
196
2
2.1
Materials and Methods
Sampling sites
Rainwater samples were taken at four sites (-0 Table 1) for the determination of atrazine. Three stations were located in Bavaria, one site in Lower Saxony. The site in Lower Saxony was at the official station of the Umweltbundesamt (Federal Environment Agency), Berlin - Waldhof. The sampler was installed on the roof. W a l d h o f is located in a plain, mostly agricultural area. The station in Freising (Drirnast and Berg) was also set up in an agricultural area, the other two Bavarian stations in national parks (NP). The site Dfirnast is located on a hill at the German Meterological Service station in Weihenstephan (Freising), Berg in a forest near Freising on a hilltop (--) map A in Fig. 1). The station at the Bayerischer Wald national parks was established on the ridge of the B6hmerwald ( ~ map B in Fig. 1) and the station at the Berchtesgarden national park on the slope of the Watzmann (-~ map C in Fig. 1). Rainwater from open fields and forest stands were collected at the stations, except for
ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1 9 6 - 2 0 4 (1994) 9 ecomed publishers, D-86899 Landsberg, Germany
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Atrazine in Rainfall and Surface Water
W a l d h o f with sampling only at open fields. The samplers were located in fenced areas where no pesticides had been applied during the last years. Table 1: Sampling sites for the collection of rainwater (NP = national park) Sampling site
Elevation Sample (m) origin
DOrnast (85350 Freising, Bavaria)
470
open field
Berg (85350 Freising, Bavaria)
500
forest stand
78
open field
B&renlochriegel (NP Bayer. Wald, Bavaria)
1 300
open field
B~.renlochriegel (NP Bayer. Wald, Bavaria)
1 300
forest stand
Watzmann (NP Berchtesgaden, Bavaria)
1 500
open field
Watzmann (NP Berchtesgaden, Bavaria)
1 500
forest stand
Waldhof (29394 LQder, Lower Saxony)
Fig. 1: Location of the sampling sites Berg, Freising (A), B/irenlochriegel, NP Bayerischer Wald (B) and Watzmann, NP Berchtesgaden (C). The sections of the maps (1 : 25 000) show an area of 2.5 x 2.5 km
2.2
Sampling
Glass bottles (25 L) with glass funnels containing glass filters were used for the sampling of rainwater from open areas. The surface of the funnel was 680 cm 2. At Dfirnast (Freising) a steel funnel (1 m 2) was used. In the forest stand 16 samplers were set up in a grid of 2 92 m. Each sampler consisted of a glass funnel in a glass measure (1 L, inverse con-
ESPR-Environ. Sci. & Pollut. Res. I (4) 1994
ical form). The sampling surface of all 16 samplers was 4778 cm 2. As the samplers were not closed during dry periods, the total deposition consisting of dry and wet deposition was collected. Evaporation of water from the glass containers was kept to a minimum, because the glass bottles had only a small opening in which the funnel was inserted and the glass measures were covered by the funnels containing glass filters.
197
Atrazine in Rainfall and Surface Water
The glass bottles were emptied once a week, and the collected rainwater was transferred to dark glass bottles and mailed from the sampling site directly to the analytical laboratory. The samples were stored at 4 ~ The analyses were carried out within a week of receipt of the samples. The sites Dfirnast and Berg were sampled beginning in June 1990. In 1991 and 1992 the Dfirnast, Berg, and Waldhof sites were sampled from April to November and the national parks from June to October due to inaccessibility because of snow. Rainfall data for the determination of the deposition were obtained either from the local weather stations (Waldhof/Liider, Diirnast/Freising) or from measuring the actual rainfall.
Research Articles
of positive samples should be carried out afterwards by GC, HPLC, and GC/MS, as in the case of our study. 3.1
Enzyme immunoassay
The measuring range of the assay was 0.02 - 2.5/ag/L with a middle of the test (IC 50) at 0.25/ag/L for serum $84 and 0 . 0 2 - 2 lag/L with a middle of the test at 0.15 I~g/L for serum $2 (-0 Fig. 2). Absorption values for zero concentrations were usually above 1. 1.2. absorption
't
1.0 o
0.8-
2.3
Analysis
0.6-
No clean-up of the samples was necessary. Coarse particles were removed by sedimentation and filtration. The pH of the samples was determined and, if necessary, adjusted to pH 7. For the determination of matrix effects one aliquot of the sample was diluted with distilled H/O, another aliquot spiked with atrazine standards. Details of antibody production, enzyme tracer synthesis, and performance of the enzyme immunoassay have been described by WUST and HOCK [16] and DANKWARDTet al. [17]. GC and G C / M S analyses for the validation of the atrazine concentrations obtained by EIA were carried out in the laboratory of Prof. K. B/ICHMANN, T H Darmstadt by Juliane SCHA~ according to a method described by BACHMANN et al. [18] and in the group of Prof. E. M. THURMAN, U.S. Geological Survey, Lawrence, USA as described in earlier reports [19]. In brief, the samples were extracted by solid-phase Cls cartridges from 100 mL to 1 L of water. The cartridges were eluted with an organic solvent and evaporated. Mass spectrometry was carried out in selected ion mode using 3 ions (molecular ion, base peak, and confirming ions). The GC separation was with a 15-m capillary column of methyl silicone with He carrier gas.
3
Results and Discussion
The duration of this study was three years from 1990 to 1992 and included the influence of the ban of atrazine in 1991. Sampling sites were located in agricultural areas as well as at clean air stations in national parks in order to obtain information about atmospheric transport. Open space and forest stands were sampled separately to find out about a possible difference in concentrations and deposition rates of the different type of sites. Determination of atrazine was carried out by enzyme immunoassay. Such tests have been developed for many pesticides [20], and several enzyme immunoassays have been worked out for triazine herbicides [16, 2 1 - 24]. In comparison to classical analytical methods such as gas chromatography and high pressure liquid chromatography, the samples can be investigated faster and at lower costs without any clean-up or concentration steps. A validation
198
0.40.20.00
0.01
0.1
1
10
E
atrazine (l.tg/L) Fig. 2: Absorption curve of serum $2 (E = excess, 1 000 ~g/L)
The suitability of the antibodies for the determination of atrazine in natural water samples was investigated. The antibodies showed a high cross-reactivity for propazine with 116 - 143 % ( ~ Table 2). No interferences are expected since this herbicide is not applied in most European countries. The affinity of the antibodies toward prometryn, deethylatrazine, and ametryn was lower than toward atrazine (2 - 27 %). Prometryn and ametryn play only a minor role as herbicides and were not present in the investigated water samples. The metabolite deethylatrazine can be found in water samples, usually in lower concentrations than its parent compound, atrazine. All other compounds showed crossreactivities below 10 %, most to 1 % or less. Table 2- Cross-reactivities of the sera S 84 and S 21
Substance
Cross-reactivity in % S 84 $2
Atrazine
100
100
Propazine
116
143
Deethylatrazine
13
15
Prornetryn
10
27
2
12
Ametryn
1 only cross-reactivities exceeding 10 % are listed
Natural water samples may vary considerably in their pH and the concentration of humic substances. Thus, the influence of these parameters on the immunoassay was tested. No effect was found for the pH between 4 and 10 and so no pH adjustment was necessary for most samples. However,
ESPR-Environ. Sci. & PoUut. Res. t (4) 1994
Research Articles
Atrazine in Rainfall and Surface Water
values of below pH 4 and above pH 10 yielded atrazine concentrations greatly exceeding the standards. The influence of humic acids on the assay was tested with different concentrations of natural humic acids isolated from the Suwannee River (Horida, USA). Apparently increasing atrazine concentrations were observed after addition of standards containing 100/ag/L of humic acids, concentrations that are rarely found in rainwater and surface water samples. Accuracy and consistency of the enzyme immunoassay are important factors. Therefore, the reproducibility of the assay on the same day, as well as on different days, was investigated (-~ Table 3). Percent coefficients of variation (% CV) ranged from 1.9 to 13.3 %. The assays carried out on the same day showed lower % CV than the day-to-day investigations. Increasing variabilities at different temperatures on different days, slight changes in the assay solutions used, and variations in the personal performance on different days may contribute to this effect. A good reproducibility of the assay was found, however, with % CV mosdy below 12 % for the investigation of natural water samples.
The accuracy of the immunoassay was determined by validation measurements with GC and GC/MS. In 1991, 31 samples were examined (-o Fig. 3), 26 samples in 1992. A good correlation of r = 0.95 was found for both investigations. Slightly higher concentrations were obtained in the EIA, as it could be expected from the cross-reactivities. In addition, the humic substances of the samples may contribute to a slight overestimation, as the samples from the forest stands sometimes had a yellow color. 10 =
O r
._1
Same-day reproducibility Atrazine (pg/L • s)
Waldhof 1 Waldhof 2 Dt3rnast Berg 1 Berg 2 Bayer. Wald 1 Bayer. Wald 2
0.632 0.184 1.336 0.272 0.542 0.283 0.454 Berchtesgaden 0.271 Moosach 0.240
• 0.020 • 0.012 • 0.049 • 0.028 • 0.063 • 0.013 • 0.025 __. 0.026 • 0.026
0.1
0.01 0.01
Day-to-day reproducibility
% CV 3.1 6.7 3.6 10.2 11.7 4.5 5.4 9.6 10.7
Atrazine (ug/L • s) 0.605 0.165 1.206 0.246 0.471 0.281 0.440 0.256 0.208
• • • • • • • • •
0.046 0.020 0.130 0.033 0.009 0.030 0.050 0.020 0.027
O/
o.....................
Table 3: Same-day reproducibility and day-to-day reproducibility of the atrazine immunoassay for natural water samples (based on five different determinations) Sample
ooo
r 0.95
0.1
I
10
I~g/L a t r a z i n e (EIA)
% CV 7.6 12.1 10.8 13.3 1.9 10.6 11.6 7.7 12.9
Fig. 3: Comparison of EIA and GC for the detection of atrazine in water samples from 1991
3.2
Investigation of water samples
A total of 316 rainwater samples and 80 surface water samples were investigated. About 60 % of the investigated samples contained amounts of atrazine higher than the limit of quantification, which is 0.02/~g/L (--' Table 4). The highest concentrations in rainwater (up to 4 pg/L) were
Table 4: Number of positive samples (exceeding 0.02/ag atrazine/L) and highest and median concentration of the positive samples for atrazine at the different sampling sites in the years 1990 - 1992 Sampling site number
1990 max. ~ug/L)
median (pg/L)
number
1991 max. (pg/L)
median /Jg/L)
number
1992 max. (pg/L)
median #g/L)
Dt3rnast open field
15 of 21
3.29
0.16
21 of 32
0.28
0.06
12 of 22
1.49
0.07
Berg forest stand
18 of 18
4.18
0.43
18 of 29
1.14
0.25
19 of 21
3.05
0.20
Waldhof open field
14 of 31
0.49
0.10
9 of 21
0.40
0.06
NP Bayer. Wald open field
10 of 18
0.31
0.06
7 of 15
0.21
0.05
NP Bayer. Wald forest stand
13 of 18
0.41
0.13
11 of 16
0.56
0.08
NP Berchtesgaden open field
4 of 12
0.04
0.03
5 of 16
0.05
0.04
NP Berchtesgaden forest stand
7 of 12
0.23
0.05
4 of 15
0.09
0.06
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Atrazine in Rainfall and Surface Water
Research Articles
found at the agricultural sites in Freising (Diirnast and Berg), the lowest concentrations at the Berchtesgaden national park (0.02 - 0.23/~g/L). Intermediate concentrations were observed at the Waldhof site (agricultural) and the Bayerischer Wald national park (0.02 - 0.56/~g/L). Similar concentrations of atrazine in rainwater were reported by other investigators. BRAUN et al. [25] found up to 1.1/lg/L in the Munich area, MOLLER et al. [3] up to 2.2/ag/L at different stations in Bavaria. A seasonal appearance of atrazine was observed. Concentrations started to increase in mid-April to May and the highest concentrations were usually found in June (-~ Fig. 4). In August concentrations declined and in September and the following months no atrazine was detected, except for a second peak at Dfirnast and Berg in November of 1990. The second peak may be due to fall application, or perhaps to the harvest of corn and airborne soil and dust particles containing atrazine. The highest concentrations of atrazine appeared shortly after the application of the herbicide and a few months thereafter. No atrazine was detected in early spring, late fall and during winter. A seasonal appearance of atrazine was also reported in other investigations from Switzerland, Germany and the USA [2, 6, 26]. However, the herbicide was also found in rain during the whole year in a few cases [27, 28]. Our group and several others [2, 5, 6] have found that atrazine was detectable in rain even before its application on corn. The application of atrazine on industrial areas and railroad tracks in early spring might be one reason. On the other hand, it could be due to herbicide resulting from last year's application. As atrazine is mostly bound to the soil, wind erosion and volatilization from soil could be possible. In contrast to late fall and winter, where usually no herbicide was detected, higher temperatures, moist soils and agricultural activities with heavy dust formation may facilitate this effect in spring [2, 5].
A correlation could be found between the amount of rain and the atrazine concentrations in the rain water. Thus, high concentrations often correlated with little rainfall, whereas heavy rain resulted in low atrazine concentrations. The total deposition of atrazine was determined as the product of measured atrazine concentrations and the amount of rainfall. The highest deposition rate for each station was recorded in June, followed by July (-~ Fig. 5). The highest value of 140/ag/m 2 was measured at Diirnast in June 1990, Berg showed 70/~g/m 2. A distinct decline in deposition was observed in the following years due to the ban of atrazine in 1991 leading to deposition rates of 2 0 - 4 5 p g / m 2 in June. The recorded deposition per year was the highest in 1990 ( ~ Fig. 6) with 180/lg/mZ.yr at Diirnast and 150 /~g/m2.yr at Berg. The total deposition during 1990 are presumably higher because measurements were begun only in June of this year. In 1991 and 1992 the deposition was below 100/~g/ma.yr for all sites, with the highest values at Dtirnast and Berg and in the Bayerischer Wald NP (forest stand) with 3 0 - 80/~g/m 2" yr. Similar deposition rates were reported by HERTERICH [29] and OBERWALDERet al. [5]. According to Herterich [29] is the true deposition into a forest stand is usually higher than the recorded deposition rate, as a portion of the herbicides remains adsorbed to leaves and needles of the trees. The high deposition into the forest stand at the Bayerischer Wald NP was unexpected. Deposition rates are similar to the agricultural sites Dfirnast and Berg, although in the national park no pesticides are applied. A transport of atrazine from agricultural areas to the national park via the atmosphere has apparently taken place. The closest corn fields to the park are located at a distance of 20 km to the west. In June 1991 mostly winds from the west were observed, which might have transported herbicides from those corn fields into the national park. In June 1992 the wind did not
atrazine (pg/L) 0.6 0.5 0.4 0.3 0.2 0.1 0.0
[--LJ
J'--LJMay["J--] L--I--]
i~
LJ.J
June
July
August
sampling site I~DOrnast IlWaldhof I~Bayer. Wald ~Berchtesgaden Fig.
200
4: Atrazine concentration in rainwater in 1991 (open field)
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Research
Articles
A t r a z i n e in R a i n f a l l a n d S u r f a c e W a t e r
atrazine (pg/m 2 * yr) 40
30
20
10
0
April
May
June
July
August
September
sampling site E~]D0rnast ==Berg I~Waldhof ~ B W O F I~BW FS roBe OF ~ B e FS Fig. 5: Deposition of atrazine at the seven sampling sites in 1991 (BW = Bayerischer Wald, Be = Berchtesgaden, OF = open fields, FS = forest stand)
atrazine (pg/m 2 . yr) 200
150
100
50
0
111111 D0rnast
Berg
Waldhof
BW OF
BW FS
Be OF
Be FS
year of sampling E~1990 ==1991 1~1992 Fig. 6: Total deposition of atrazine at the seven sampling sites from 1990 to 1992 (BW = Bayer. Wald, Be = Berchtesgaden, OF = open field, FS = forest stand) come from one certain direction, sources from the Czech Republic may also contribute to the observed herbicide deposition. A transport of atrazine into mountain and forest stands located at some distance to agricultural areas was also reported for the Black Forest [18], the Fichtelgebirge [29], and Swiss mountain lakes [2]. The water samples from forest stands usually showed higher atrazine concentrations and also higher deposition rates as the open field samples (except for D~irnast in 1990). This can be attributed to the filter effect of the trees by adsorbing pollutants (herbicides) on the large surface of their needles and leaves. During the next precipitation event, part of the substances is washed off and can be found in the rain water. Several surface waters were investigated for atrazine in the area of the stations Dfirnast und Berg (Freising). The
ESt'R-Environ. Sci. & PoDut. Res. 1 (4) 1994
Thalhauser Graben was assayed monthly. The highest concentrations were found again in June (4 pg/L in 1990) with little atrazine in April and earlier and in September and the following months. Values have been declining since 1991 (--* Fig. 7). Several ponds and creeks in the area of Freising were investigated twice a year (July and January). No atrazine or only low concentrations were observed in the ones located in forest areas (-* Table 5), while waters near corn fields exhibited high values up to 14/~g/L. Drift-off during application, flushing of soil particles into the water, and washing out of pesticides from the soil leads to the presence of atrazine in the surface waters. The degradation of atrazine in natural waters is relatively slow [30]. The lake in Dfirnast showed 15/~g/L in June 1990. Still 10 lag/L were found one month later. The concentrations declined in winter to 0.05 - 0.08/~g/L. This can
201
Atrazine in Rainfall and Surface Water
Table 5: A t r a z i n e c o n c e n t r a t i o n s ~ g / L ) sampling site
summer
Research Articles
o f s u r f a c e w a t e r s in t h e a r e a o f F r e i s i n g
1990
summer
1991
winter
1991
summer
1992
winter
Spring D0rnast
< 0.02
< 0.02
< 0.02
< 0.02
< 0.02
Brook D0rnast
< 0.02
< 0.02
< 0.02
< 0.02
< 0.02
0.47
< 0.02
< 0.02
0.11
< 0.02
Fuchsloch Lange Seiche
0.10
< 0.02
< 0.02
0.03
< 0.02
Unt. Rehbuckel
< 0.02
0.07
< 0.02
< 0.02
< 0.02
Ob. Rehbuckel
< 0.02
< 0.02
< 0.02
< 0.02
< 0.02
Tiroler Schlag
0.04
< 0.02
< 0.02
0.05
< 0.02
Eisweiher
0.07
0.07
< 0.02
0.04
< 0.02
Lake DOrnast
14.60
13.49*
4,25
0.08
3.20
Dorfacker
3.29
5.76*
1.69
0.22
0.30
0.08
Moosach
0.09
0.09
0.10
0.06
0.04
3.70* *
1992
0.05
* determined by HPLC; ** determined by G C
atrazine ~gtL) 5
i
4
I! ____
I!11
Im
=l. ,..... I,..._
.[[.
1 2 3 4 5 6 7 8 91011121 2 3 4 5 6 7 8 91011121 2 3 4 5 6 7 8 91011121 2 3 4 5 6 7 8 9
1990
1991
1992
1993
Fig. 7: A t r a z i n e c o n c e n t r a t i o n o f the T h a l h a u s e r G r a b e n ( 1 9 9 0 - 1 9 9 3
be partly attributed to degradation, but uptake by and adsorption to plants and the sediment seems to play an important role in the decrease of atrazine in the water systems [30, 31]. The adsorbed atrazine, however, may be desorbed from the sediments and provide a long-term source for low-level contamination. The concentrations found in the summer months decreased between 1990 and 1992. No atrazine could be detected in a source in the forest of Freising ( ~ Table 5). Yet, another spring in a forest from this area, located at a distance of 5 km from corn fields, exhibited atrazine concentrations of 0 . 2 - 0 . 5 p g / L and deethylatrazine concentrations of 0.4 - 1.1/ag/L over the entire year (Dr. J. MAGUHN, personal communication). No atrazine is applied in this forest, so deposition via the atmosphere may be a possibility. On the other hand, water containing atrazine may be transported through the soil from
202
the neighboring agricultural area due to unfavorable hydrogeological conditions. This again shows how clean areas can be influenced by transportation of herbicide from areas with high herbicide concentrations. The metabolites deethylatrazine and deisopropylatrazine were detected in the samples investigated by G C / M S beside the parent compound. The greatest concentration of 0.2 p g / L for deethylatrazine was found in June in the rain water samples of Berg and Dfirnast and in the brook Thalhauser Graben. Deisopropylatrazine was observed in a few samples up to 0.1 pg/L. Several other pesticides could also be detected. Many of the samples contained terbuthylazine with a maximum concentration of 1.6 p g / L . This s-triazine is frequently used as a substitute for atrazine. Propazine and simazine were found in some cases and metolachlor was observed in many samples with concentra-
E S P R - E n v i r o n . 5ci. & Pollut. Res. 1 (4) 1994
Research Articles
tions of up to 0.84/ag/L. Only samples from the agricultural area contained this herbicide, while in the samples from the national parks none was found. Either metolachlor is not used in the regions around the national park or it is not stable and degraded during the atmospheric transport.
4
Conclusions
The presence of atrazine in rain and surface water was demonstrated in this three-year study. Concentrations of up to 4/ag/L for rainwater and 15/Jg/L for surface water could be detected. Concentrations &dined after the ban of atrazine in 1991, but up to 4/ag/L were still found in rain and surface water in some cases. Transportation of the herbicide via the atmosphere from neighboring countries may contribute to the observed atrazine concentrations, but illegal atrazine application can be assumed as another source of atrazine, especially for surface water. Concentrations of up to 4 p g / L in the Diirnast lake seem very high to be caused by deposition alone, because the deposited atrazine would be diluted by surface water. In this case also all ponds and brooks in one region should show a similar atrazine concentration. As in each summer the surface waters from agricultural areas exhibited higher concentrations than the ponds from the forest, drift-off during application and leaching of herbicide from soil particles flushed into the water seems more probable. The location of the Diirnast lake and the brooks Thalhauser Graben and Dorfacker at a bent near corn fields may add to this effect. No contamination limits exist for rain water. The values of the drinking water ordinance for pesticides of 0.1/Jg/L for a single substance and 0.5 p g / L for the sum of pesticides and their toxic metabolites should be applied if rainwater is collected in cisterns for drinking water. Therefore, from May to July rainwater cannot be recommended as drinking water without special cleaning steps. The influence on organisms of atrazine concentrations of up to 15/Jg/L, as detected in our study, is not fully known. Atrazine as an inhibitor of photosynthesis may influence leaf development. The high concentrations in forest stands, especially in late spring, may effect the development of new leaves and needles. The spruces Picea abies and Piceaglauca cannot detoxify atrazine in contrast to resistant plants like corn and sorghum, as the herbicide is not metabolized by the glutathion-S-transferases of these species [32]. It has been discussed that atrazine and other herbicides may contribute to forest decline [33]. Controversial reports exist for aquatic systems. While atrazine was found as not being toxic for fish and invertebrates in one study [34], other reports disclosed necrotic changes in the liver of rainbow trout after 28 days of exposure to 10 pg atrazine per liter, and 5/lg per liter damaged the kidneys [35, 36]. Inhibition of photosynthesis of some species of phytoplankton was detected at concentrations as low as 1 - 5 pg/L. This was accompanied by the establishment of more resistant species [37]. No effects on aquatic ecosystems were observed below 20 pg/L of atrazine according to a report by HUBER [38]. ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1994
Atrazine in Rainfall and Surface Water
The occurrence of atrazine and other pesticides in rain during spring and summer points to an atmospheric transport of these compounds. Especially the presence of atrazine in rainwater samples from the national parks suggests a medium- or long-range transport of the pesticides. Deposition rates" of 180 p g / m 2 9 yr (1.8 g/ha 9 yr) represent 0.18 % of the amount of atrazine applied in a year per hectare, corresponding to I kg atrazine/hectare. This loading may seem relatively low and according to HERTERICH[29], no effects are expected in general for the vegetation or groundwater quality. Nonetheless, it must be remembered that approximately 1 % of the applied atrazine is washed from soils into surface waters [39]. Thus, 0.2 % represents nearly 20 % of the surface water transport, which is a considerable portion of the total dissipation of the herbicide. Furthermore, it is important to remember that the corn field is the target. Transportation to other areas like forests or water protection zones should be avoided. It is probable that insecticides and fungicides show a similar behavior in the environment as herbicides. Beside their influences on fauna and flora, effects on humans have to be considered, as they come into contact with those pollutants via the air, rain, and dust. Finally, the enzyme immunoassay proved to be a valid tool for the determination of atrazine in natural water samples. The samples could be analyzed in a much shorter time and with less effort as compared to established techniques. No clean-up or pre-concentration steps were necessary. Comparison between samples investigated by EIA and GC/MS showed a good correlation (r -- 0.95), and the methodology is recommended for surveys of water quality.
Acknowledgements We thank the Bundesministerium fiir Umwelt (Federal Environment Ministry) for financial support of this work. We are grateful to Dr. ZmPd., Mr. VOGT, Mr. DONATHand Mr. DATZMANNfrom the NP Berchtesgaden, Mr. JEHL, Mr. KAATZund Mr. ICg-tLHaMMEafrom the NP Bayerischer Wald, Mr. JVIENSCHENDC)RFER,Wl-IWeihenstephan, and the colleagues from the station in Waldhof, who helped with the set up of the stations and the sampling. We appreciate the support of the laboratory of Prof. HUBeR/TU Miinchen-Weihenstephan for HPLCanalyses and of Mrs: Juliane SCHARF from the group of Prof. K. B)~CHMaNN/TH Darmstadt for GC analyses. We thank Dr. BINDERand Dr. HORMANNSD(SRFER/TUMiinchen-Weihenstephan for the immunization of the sheep and the collection of the blood. The joint research between the Lehrstuhl fur Botanik, TU Mfinchen, and the United States Geological Survey is a cooperative study on environmental applications of immunoassays between the two countries.
5
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ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1994
