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Atmospheric transport of atrazine: A simple device for its detection
Environmental Pollution 48 (1987) 77-82
Atmospheric Transport of Atrazine: A Simple Device for its Detection
W. Elling Fachhochschule Weihenstephan, D-8050 Freising 12, Federal Republic of Germany
S. J. Huber, B. Bankstahl & B. Hock Department of Botany, Technical University of Munich, Munich, Federal Republic of Germany (Received 19 February 1987; revised version received 21 April 1987; accepted 24 April 1987)
ABSTRACT Thin-layer chromatography plates were exposed at three different locations in Bavaria for 3-week periods during the growing season of maize. The adsorption of traces of atrazine was detected by thin-layer chromatograph),, G ~ M S , and by an enzyme immunoassay. It was restricted to the sowing season, which coincides with the application of atrazine. The herbicide could not be detected after this time. The simplicity of the adsorption device, combined with a serological assay, render this procedure suitable fi~r detecting airborne organic pollutants.
INTRODUCTION The detection of pesticides is gaining importance, especially under circumstances where wind or water can carry persistent compounds far away from the site where they are applied. Enzyme immunoassays have become an approved technique for quantifying low molecular weight anthropogenic compounds such as herbicides, e.g. paraquat (Niewola et al., 1983, Benner & Niewola, 1986), atrazine (Huber, 1985, Huber & Hock, 77 Environ. Pollut. 0269-7491/87/$03"50 ~ Elsevier Applied Science Publishers Ltd, England, 1987. Printed in Great Britain
78
w. Elling, S. J. Huber, B. Bankstahl, B. Hock
1986), or terbutryn (Huber & Hock, 1985). In general, they are used to detect the contaminants in the soil or in water. We now report a simple technique for detecting pollutants which are translocated by the wind. As a model we used atrazine, a persistent herbicide which is applied in the cultivation of cereals. During the year 1984, an area of almost 4000 km 2 was used in Bavaria to grow maize, corresponding to 19% of the arable land (Statistisches Bundesamt, 1985). Almost the entire cultivation area for maize is treated by the pre-emergence application of striazine-herbicides mainly atrazine. A device was constructed to adsorb airborne atrazine. By means of an enzyme immunoassay we were able to detect traces of atrazine in the air during the period of application. MATERIALS AND METHODS A simple device for adsorbing atrazine was used to collect outdoor samples. A cylindrical hollow body was constructed of perforated plastic (20cm length, 20 cm circumference, out of anti-knabb *~, Fa. Peter van Eyk G m b H & Co. KG, 4057 Briiggen 2) and hung by four wires from a saucer (24 cm diameter) which acted as a roof (Fig. 1). A TLC plate with a fluorescent indicator (KG 60, F254, Merck Co., Darmstadt) was attached to the cylinder, coating material outside, with three strong rubber bands. Water was kept
Fig. 1. Exposuredevice suspended in the crown of a Norway spruce.
Atmospheric transport of atrazine
79
from running onto the silica gel by zigzags at the lower end of the hanging wires. The adsorption devices were exposed for 3 weeks at different locations. Then the TLC plates were removed and immediately placed in polyethylene bags. In the laboratory, the silica gel was removed from the TLC plates and eluted with 30 ml chloroform: The eluate was filtered (Whatman filter No. 1) and then completely dried in a rotary evaporator. The remaining crystalline residue was suspended in ethanol (1 mg/100#l) and subjected to various analyses. Thin-layer chromatography was carried out on TLC plates (KG 60 F254, Fa. Merck, Darmstadt). Samples of 20#1 were applied. The plates were developed with chloroform/acetone (7:3 v/v) and viewed after drying under uv-light (254 nm). G C - M S analyses were run with an LKB 9000S apparatus using helium as the carrier gas. SE 30 (WGA, Griesheim) and OV 101 (WGA, Griesheim) were used as the stationary phase in a ratio of 1 : 1. The column temperature was raised from 150°C to 240°C by 10°Cmin -1. A thermal conductivity detector was used to identify atrazine. Enzyme immunoassays were carried out as described previously (Huber & Hock, 1986). Briefly, polystyrene microtiter plates were coated with antiatrazine antibodies raised in rabbits. The atrazine samples were dissolved in ethanol (1 mg per 500#1) and brought to 10ml with Tris-buffered saline (TBS). The wells were incubated for 2 h with 200 l~l sample. Then the alkaline phosphatase-haptene conjugate was added. After 30min, the unbound components were removed by washing with TBS. The enzyme reaction was started by adding 200/tl substrate solution (0-5 mg 4-nitrophenylphosphate per 1 ml 0.1 g litre-1 carbonate buffer, pH 9"6) and stopped after 1 h by adding 50~1 5 mol litre ~ KOH. The adsorbances were read at 405 nm by the Manureader S (Fa. Poll/ihne, Wennigsen). The atrazine concentrations were calculated on the basis of calibration curves with known atrazine dilutions.
RESULTS The adsorbing devices were exposed for four consecutive periods of three weeks in 1986 (22'4-13'5; 13"5-3'6; 3.6-24.6; 24-6-15.7) at three different locations in Bavaria (FRG). Geographic details are given in Table 1. The first period of exposure coincided with the sowing season of maize. Atrazine is usually applied soon after sowing. The adsorption of atrazine by the exposed devices was verified qualitatively in all of the samples from the first exposure. This was shown by
W. Elling, S. J. Huber, B. Bankstahl, B. Hock
80
TABLE 1 Exposition of the Absorbing Devices
Location
Height above sea-level
Forest of Thalhausen (near 8050 Freising), edge of a forest
500m
Forest of Kranzberg (near 8050 Freising), within closed forest land Waldh~iuser (National Park Bayerischer Wald, near 8352 Grafenau)
500 m
950 m
Vegetation, surroundings
Position
Stand of Norway spruce (c. 65 years old), adjacent to maize and wheat fields Stand of Norway spruce (c. 20 years old), cultivation of maize within a distance of 0.8-> 2 km Open area surrounded by forest, no cultivation of maize within a distance of 5 km
7 m above ground within the crown of a larch 7 m above ground in a Norway spruce
8 m above ground on the mast of a meteorological station
thin-layer chromatography. The largest spot was found in samples placed in a stand of Norway spruce (Forest of Kranzberg). It exhibited the same Ry value of 0.75 and the same dark-violet colour from remitted fluorescence as the standard. Atrazine was further identified by GC analysis. The atrazine peak appeared after a retention time of 5 min at a temperature of 200°C. In the coupled G C - M S system, the herbicide could be identified by its signal at the molecular weight of 214.5. An enzyme immunoassay was used for quantitative determination. Table 2 shows that traces of atrazine were adsorbed by the plates only during the TABLE 2 Quantification of Atrazine by Means of an Enzyme Immunoassay after the Elution from TLC Plates Exposed at Different Locations (~f Table 1)
Position
Forest of Thalhausen edge of a forest Forest of Kranzberg within closed forest land Waldhfiuser (National Park Bayerischer Wald) " ND = not detectable. b Mean + standard error.
Period 22.4-13.5
13.5-3.6
3"6 24"6
24"6 15"7
< 0"5/~g plate- 1
ND"
ND
ND
5 + 2/~gplate ,b
ND
ND
ND
3 + 1 fig plate- ~
ND
ND
ND
Atmospheric transport of atrazine
81
first exposure period. Again, the largest amounts were found in the samples taken from the interior of a forest (Kranzberg) with levels of 5 pg atrazine per plate. During the subsequent exposures, no atrazine could be detected.
DISCUSSION Atrazine has been used as a model compound for detecting pollutants which are transported through the air. For this purpose, a simple adsorption device was constructed which could be exposed in the air. The adsorption of striazines on silica gel surfaces is very strong. Water, e.g. rain, does not remove the herbicides. Quantitative desorption is only possible with nonpolar organic solvents and a few polar ones. Our investigations do not provide any information on how atrazine is transported through the air, e.g. in the form of aerosol particles or by rain. Quite recently, the presence of atrazine and other pesticides has been detected by Glotfelty et al. (1987). In spite of the very low vapour pressure of atrazine (4 × 10 -v mbar at 20°C, Perkow 1983/1985) gaseous transport is possible because of the evaporation of very small droplets. Adsorption on the large surfaces of TLC plates and analysis with the extremely sensitive enzyme immunoassay, with its atrazine detection limit of 0.2 ng per assay, seems to be an ideal combination for detecting pollutants which are found in trace amounts in the air. Standardisation, not only of the shape and structure of the adsorption device, but also of its exposure, has to be considered taking changing air currents into account. Therefore, exposure is recommended on prominent ground to ensure free air access. Furthermore, the adsorption devices should be suspended from freestanding masts, or in the crowns of predominant trees (height 10-12 m) in young stands of Norway spruce. In the latter case, a whorl of branches should be removed approximately 4 m below the tip to allow free air access. Although atrazine was detected only in trace amounts, and only during the restricted period of application, the airborne transport of herbicides is a factor which must be reckoned with. Because of their high effectiveness, they may pose a threat to the ecological balance by combining with other pollutants spread by the same route and intensifying or contributing to tree damage. ACKNOWLEDGEMENTS This work was supported by the Deutsche Forschungsgemeinschaft (grant Ho 383/24-1). We would like to thank Forstamtsrat Hartmut Strunz of the National Park Bayerischer Wald administration at Grafenau for his help in
82
W. Elling, S. J. Huber, B. Bankstahl, B. Hock
setting up the adsorption devices at the location 'Waldh/iuser'. We are grateful for the technical assistance o f Mrs Schindlbeck during the G C - M S analyses.
REFERENCES Benner, J. P. & Niewola, Z. (1986). Paraquat in soil. In: Methods of enzymatic analysis. (3rd ed), Drugs and Pesticides XII, 451-64, Weinheim, FRG, VCH Verlagsgesellschaft mbH. Glotfelty, D. E., Seiber, J. N. & Liijedahl, L. A. (1987). Pesticides in fog. Nature, 325, 602-5. Huber, S. J. (1985). Improved solid-phase enzyme-immunoassay systems in the ppt range for atrazine in fresh water. Chemosphere, 14, 1795-803. Huber, S. J. & Hock, B. (1985). Solid-phase Enzymimmunoassay zum Nachweis von Pflanzenschutzmitteln in Gew~issern. GIT, Frachz. f. d. Lab., 29, 969-77. Huber, S. J. & Hock, B. (1986). Atrazine in water. In Methods of enzymatic analysis. (3rd ed), Drugs and Pesticides XII, 438-5l, Weinheim, FRG, VCH Verlagsgesellschaft mbH. Niewola, Z., Walsh, S. T. & Davies, G. E. (1983). Enzyme linked immunosorbent assay (ELISA) for paraquat. Int. J. ImmunopharmacoL, 5, 211-18. Perkow, W. (1983/1985). Wirksubstanzen der Pflanzenschutz und Schiidlingsbekiimpfungsmittel. 2. Aufl., Berlin, Hamburg, Paul Parey. Statistisches Bundesamt (1985). Statistisches Jahrbuch 1985 fiir die Bundesrepublik Deutschland, Stuttgart, Mainz, W. Kohlhammer.
Atmospheric Transport of Atrazine: A Simple Device for its Detection
W. Elling Fachhochschule Weihenstephan, D-8050 Freising 12, Federal Republic of Germany
S. J. Huber, B. Bankstahl & B. Hock Department of Botany, Technical University of Munich, Munich, Federal Republic of Germany (Received 19 February 1987; revised version received 21 April 1987; accepted 24 April 1987)
ABSTRACT Thin-layer chromatography plates were exposed at three different locations in Bavaria for 3-week periods during the growing season of maize. The adsorption of traces of atrazine was detected by thin-layer chromatograph),, G ~ M S , and by an enzyme immunoassay. It was restricted to the sowing season, which coincides with the application of atrazine. The herbicide could not be detected after this time. The simplicity of the adsorption device, combined with a serological assay, render this procedure suitable fi~r detecting airborne organic pollutants.
INTRODUCTION The detection of pesticides is gaining importance, especially under circumstances where wind or water can carry persistent compounds far away from the site where they are applied. Enzyme immunoassays have become an approved technique for quantifying low molecular weight anthropogenic compounds such as herbicides, e.g. paraquat (Niewola et al., 1983, Benner & Niewola, 1986), atrazine (Huber, 1985, Huber & Hock, 77 Environ. Pollut. 0269-7491/87/$03"50 ~ Elsevier Applied Science Publishers Ltd, England, 1987. Printed in Great Britain
78
w. Elling, S. J. Huber, B. Bankstahl, B. Hock
1986), or terbutryn (Huber & Hock, 1985). In general, they are used to detect the contaminants in the soil or in water. We now report a simple technique for detecting pollutants which are translocated by the wind. As a model we used atrazine, a persistent herbicide which is applied in the cultivation of cereals. During the year 1984, an area of almost 4000 km 2 was used in Bavaria to grow maize, corresponding to 19% of the arable land (Statistisches Bundesamt, 1985). Almost the entire cultivation area for maize is treated by the pre-emergence application of striazine-herbicides mainly atrazine. A device was constructed to adsorb airborne atrazine. By means of an enzyme immunoassay we were able to detect traces of atrazine in the air during the period of application. MATERIALS AND METHODS A simple device for adsorbing atrazine was used to collect outdoor samples. A cylindrical hollow body was constructed of perforated plastic (20cm length, 20 cm circumference, out of anti-knabb *~, Fa. Peter van Eyk G m b H & Co. KG, 4057 Briiggen 2) and hung by four wires from a saucer (24 cm diameter) which acted as a roof (Fig. 1). A TLC plate with a fluorescent indicator (KG 60, F254, Merck Co., Darmstadt) was attached to the cylinder, coating material outside, with three strong rubber bands. Water was kept
Fig. 1. Exposuredevice suspended in the crown of a Norway spruce.
Atmospheric transport of atrazine
79
from running onto the silica gel by zigzags at the lower end of the hanging wires. The adsorption devices were exposed for 3 weeks at different locations. Then the TLC plates were removed and immediately placed in polyethylene bags. In the laboratory, the silica gel was removed from the TLC plates and eluted with 30 ml chloroform: The eluate was filtered (Whatman filter No. 1) and then completely dried in a rotary evaporator. The remaining crystalline residue was suspended in ethanol (1 mg/100#l) and subjected to various analyses. Thin-layer chromatography was carried out on TLC plates (KG 60 F254, Fa. Merck, Darmstadt). Samples of 20#1 were applied. The plates were developed with chloroform/acetone (7:3 v/v) and viewed after drying under uv-light (254 nm). G C - M S analyses were run with an LKB 9000S apparatus using helium as the carrier gas. SE 30 (WGA, Griesheim) and OV 101 (WGA, Griesheim) were used as the stationary phase in a ratio of 1 : 1. The column temperature was raised from 150°C to 240°C by 10°Cmin -1. A thermal conductivity detector was used to identify atrazine. Enzyme immunoassays were carried out as described previously (Huber & Hock, 1986). Briefly, polystyrene microtiter plates were coated with antiatrazine antibodies raised in rabbits. The atrazine samples were dissolved in ethanol (1 mg per 500#1) and brought to 10ml with Tris-buffered saline (TBS). The wells were incubated for 2 h with 200 l~l sample. Then the alkaline phosphatase-haptene conjugate was added. After 30min, the unbound components were removed by washing with TBS. The enzyme reaction was started by adding 200/tl substrate solution (0-5 mg 4-nitrophenylphosphate per 1 ml 0.1 g litre-1 carbonate buffer, pH 9"6) and stopped after 1 h by adding 50~1 5 mol litre ~ KOH. The adsorbances were read at 405 nm by the Manureader S (Fa. Poll/ihne, Wennigsen). The atrazine concentrations were calculated on the basis of calibration curves with known atrazine dilutions.
RESULTS The adsorbing devices were exposed for four consecutive periods of three weeks in 1986 (22'4-13'5; 13"5-3'6; 3.6-24.6; 24-6-15.7) at three different locations in Bavaria (FRG). Geographic details are given in Table 1. The first period of exposure coincided with the sowing season of maize. Atrazine is usually applied soon after sowing. The adsorption of atrazine by the exposed devices was verified qualitatively in all of the samples from the first exposure. This was shown by
W. Elling, S. J. Huber, B. Bankstahl, B. Hock
80
TABLE 1 Exposition of the Absorbing Devices
Location
Height above sea-level
Forest of Thalhausen (near 8050 Freising), edge of a forest
500m
Forest of Kranzberg (near 8050 Freising), within closed forest land Waldh~iuser (National Park Bayerischer Wald, near 8352 Grafenau)
500 m
950 m
Vegetation, surroundings
Position
Stand of Norway spruce (c. 65 years old), adjacent to maize and wheat fields Stand of Norway spruce (c. 20 years old), cultivation of maize within a distance of 0.8-> 2 km Open area surrounded by forest, no cultivation of maize within a distance of 5 km
7 m above ground within the crown of a larch 7 m above ground in a Norway spruce
8 m above ground on the mast of a meteorological station
thin-layer chromatography. The largest spot was found in samples placed in a stand of Norway spruce (Forest of Kranzberg). It exhibited the same Ry value of 0.75 and the same dark-violet colour from remitted fluorescence as the standard. Atrazine was further identified by GC analysis. The atrazine peak appeared after a retention time of 5 min at a temperature of 200°C. In the coupled G C - M S system, the herbicide could be identified by its signal at the molecular weight of 214.5. An enzyme immunoassay was used for quantitative determination. Table 2 shows that traces of atrazine were adsorbed by the plates only during the TABLE 2 Quantification of Atrazine by Means of an Enzyme Immunoassay after the Elution from TLC Plates Exposed at Different Locations (~f Table 1)
Position
Forest of Thalhausen edge of a forest Forest of Kranzberg within closed forest land Waldhfiuser (National Park Bayerischer Wald) " ND = not detectable. b Mean + standard error.
Period 22.4-13.5
13.5-3.6
3"6 24"6
24"6 15"7
< 0"5/~g plate- 1
ND"
ND
ND
5 + 2/~gplate ,b
ND
ND
ND
3 + 1 fig plate- ~
ND
ND
ND
Atmospheric transport of atrazine
81
first exposure period. Again, the largest amounts were found in the samples taken from the interior of a forest (Kranzberg) with levels of 5 pg atrazine per plate. During the subsequent exposures, no atrazine could be detected.
DISCUSSION Atrazine has been used as a model compound for detecting pollutants which are transported through the air. For this purpose, a simple adsorption device was constructed which could be exposed in the air. The adsorption of striazines on silica gel surfaces is very strong. Water, e.g. rain, does not remove the herbicides. Quantitative desorption is only possible with nonpolar organic solvents and a few polar ones. Our investigations do not provide any information on how atrazine is transported through the air, e.g. in the form of aerosol particles or by rain. Quite recently, the presence of atrazine and other pesticides has been detected by Glotfelty et al. (1987). In spite of the very low vapour pressure of atrazine (4 × 10 -v mbar at 20°C, Perkow 1983/1985) gaseous transport is possible because of the evaporation of very small droplets. Adsorption on the large surfaces of TLC plates and analysis with the extremely sensitive enzyme immunoassay, with its atrazine detection limit of 0.2 ng per assay, seems to be an ideal combination for detecting pollutants which are found in trace amounts in the air. Standardisation, not only of the shape and structure of the adsorption device, but also of its exposure, has to be considered taking changing air currents into account. Therefore, exposure is recommended on prominent ground to ensure free air access. Furthermore, the adsorption devices should be suspended from freestanding masts, or in the crowns of predominant trees (height 10-12 m) in young stands of Norway spruce. In the latter case, a whorl of branches should be removed approximately 4 m below the tip to allow free air access. Although atrazine was detected only in trace amounts, and only during the restricted period of application, the airborne transport of herbicides is a factor which must be reckoned with. Because of their high effectiveness, they may pose a threat to the ecological balance by combining with other pollutants spread by the same route and intensifying or contributing to tree damage. ACKNOWLEDGEMENTS This work was supported by the Deutsche Forschungsgemeinschaft (grant Ho 383/24-1). We would like to thank Forstamtsrat Hartmut Strunz of the National Park Bayerischer Wald administration at Grafenau for his help in
82
W. Elling, S. J. Huber, B. Bankstahl, B. Hock
setting up the adsorption devices at the location 'Waldh/iuser'. We are grateful for the technical assistance o f Mrs Schindlbeck during the G C - M S analyses.
REFERENCES Benner, J. P. & Niewola, Z. (1986). Paraquat in soil. In: Methods of enzymatic analysis. (3rd ed), Drugs and Pesticides XII, 451-64, Weinheim, FRG, VCH Verlagsgesellschaft mbH. Glotfelty, D. E., Seiber, J. N. & Liijedahl, L. A. (1987). Pesticides in fog. Nature, 325, 602-5. Huber, S. J. (1985). Improved solid-phase enzyme-immunoassay systems in the ppt range for atrazine in fresh water. Chemosphere, 14, 1795-803. Huber, S. J. & Hock, B. (1985). Solid-phase Enzymimmunoassay zum Nachweis von Pflanzenschutzmitteln in Gew~issern. GIT, Frachz. f. d. Lab., 29, 969-77. Huber, S. J. & Hock, B. (1986). Atrazine in water. In Methods of enzymatic analysis. (3rd ed), Drugs and Pesticides XII, 438-5l, Weinheim, FRG, VCH Verlagsgesellschaft mbH. Niewola, Z., Walsh, S. T. & Davies, G. E. (1983). Enzyme linked immunosorbent assay (ELISA) for paraquat. Int. J. ImmunopharmacoL, 5, 211-18. Perkow, W. (1983/1985). Wirksubstanzen der Pflanzenschutz und Schiidlingsbekiimpfungsmittel. 2. Aufl., Berlin, Hamburg, Paul Parey. Statistisches Bundesamt (1985). Statistisches Jahrbuch 1985 fiir die Bundesrepublik Deutschland, Stuttgart, Mainz, W. Kohlhammer.