Environ. Sci. Technol. 1996, 30, 3551-3557
Disappearance of Aerially Applied Fenitrothion in Rice Crop Waters† ANNA OUBIN ˜ A, IMMA FERRER, JORDI GASCO Ä N , A N D D A M I AÅ B A R C E L O Ä * Department of Environmental Chemistry, CID-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain
The disappearance of fenitrothion under real environmental conditions was studied. Fenitrothion was applied in the rice crop field of the Ebre Delta (Tarragona, Spain) during July 1995 by helicopter spraying at a rate of 148 mL/ha Tionfos 50 LE (50% of pure fenitrothion). For monitoring fenitrothion residues in water, two different analytical techniques were used: enzyme-linked immunosorbent assay (ELISA) and automated on-line solid-phase extraction (Prospekt) followed by liquid chromatography/diode array detection (LC/DAD). The unequivocal identification of fenitrothion, fenitrooxon, 3-methyl-4-nitrophenol, and s-methyl isomer of fenitrothion was achieved by liquid chromatography/mass spectrometry (LC/MS) using an atmospheric pressure chemical ionization (APCI) interface in the negative ionization (NI) mode. The residue levels of fenitrothion in rice crop field waters varied from 119-178 µg/L down to 3.8-1.5 µg/L after 48 h of helicopter application. The half-lifetimes t1/2 were calculated by both analytical techniques being 19.3 for ELISA and 11 h for LC/DAD after treatment, with a disappearance rate of 0.036 and 0.063, respectively.
Introduction The fate of organophosphorus pesticides (OP) in the aqueous environment has led to numerous investigations in the recent years. In this regard, it was reported that degradation is influenced by hydrolysis, particularly at pH > 7 (1, 2), by photolysis (3-5) and by microbial degradation (6, 7). Degradation studies were carried out under laboratory conditions using various types of water although in many cases the concentrations used were so high (mg/L) that they did not reflect environmental situations. Few papers (2, 5, 8) used concentrations of micrograms per liter, similar to those currently found in environmental levels. Degradation studies of fenitrothion (2, 4, 9-11), fenthion (12, 13), chlorpyrifos (1, 14), and temephos (5, 15, 16) were performed estimating, in some cases, the half-lives of these pesticides either under laboratory or natural environmental * Corresponding author telephone: 34 3 400 61 18; fax: 34 3 204 59 04; e-mail:
[email protected]. † This work is dedicated to our colleague and friend, Carmen Molina, who left us too early (March 24, 1996).
S0013-936X(96)00185-X CCC: $12.00
1996 American Chemical Society
conditions. Under laboratory conditions, many different experiments were performed: bottles capped to avoid volatilization of pesticides (2, 5) or waters at various pH (from 6 to 8) and at various water temperatures (11-30 °C). Many of the reported results cannot be comparable, and extrapolation to field conditions is difficult. Consequently, there is certainly a lack of field studies. We reported a field study of fenitrothion following manual spraying (4). Papers dealing with aerial spraying of pesticides were also reported for fenitrothion, which was applied in New Brunswick, Canada (11), for temephos (15, 19); fenthion (13); molinate (17); and chlorpyrifos (18). Aerial applications of organophosphorous pesticides as temephos (15, 19) and fenthion (13) were generally carried out at application rates varying from 30-250 mL/ha. In this paper, we will study the disappearance of fenitrothion [O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate] a systemic contact insecticide that is used for the control of several insect pests and overwintering larvae after aerial application. Fenitrothion exhibits a vapor pressure of 5 × 10-5 mmHg at 25 °C, much higher than other pesticides such as atrazine and cyanazine, which exhibit values of 3 × 10-7 and 2 × 10-9 mmHg, respectively (20). The Henry’s law constant is 0.0036 Pa m-3 mol-1, showing a tendency to volatilize from water surface as reported in previous studies in Canadian lakes (11) and in the Ebre Delta waters (4). Photochemical processes were reported to be very important for fenitrothion degradation with a half-life decrease from above 32 to 1.1-2 days when river water samples were kept in the dark or exposed to sunlight (21, 22). Generally, the analytical techniques used to monitor the pesticide degradation in the aquatic environment involve either gas or liquid chromatography coupled to different detectors. Immunoassays, although implemented for several years for monitoring pesticides in the aquatic environment such as atrazine (23, 24) and chlorpyrifos (18, 25, 26), were scarcely applied to degradation studies. Recently (18), a chlorpyrifos immunoassay kit was used to investigate the dissipation of this product in rice flooded water after aerial application. The test was able to monitor the presence of chlorpyrifos during 14 days at low ppb level. One of the problems that we deal with in the present investigation is that a sensitive ELISA immunoassay kit for fenitrothion does not exist. The only one commercially available offered a very poor sensitivity (0.08 mg/L), and it is used for crops (27). To solve this problem, we decided to use the EnviroGard Parathion Plate Kit for detecting fenitrothion, since enough sensitivity was detected. This is not surprising since the chemical structure of fenitrothion and parathion-ethyl exhibit many similarities. This paper reports the results obtained after analyzing water samples collected from a rice crop field after aerial spraying of fenitrothion. The main objectives of this research were to evaluate the dissipation and persistence of fenitrothion in natural waters (rice crop field waters) by (i) measuring the amount of fenitrothion reaching the water after helicopter spraying by two independent analytical techniques: ELISA for parathion, which presents a LLD (lower limit of detection) of 0.17 µg/L for fenitrothion, and on-line-SPE (Prospekt) LC/DAD; (ii) to monitor possible
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transformation products (TPs) formed during the sampling period by LC/MS using an atmospheric pressure chemical ionization (APCI) interface in the negative ionization (NI) mode; and (iii) to investigate the behavior of fenitrothion after aerial spraying. The results reported in this paper follow previous research from our group in the degradation studies of organophosphorous pesticides (4, 5, 19), and they are also particularly useful for compliance with the Common Measures for the Control of Pollution adopted by the Contracting Parties to the Convention for the Protection of the Mediterranean Sea against Pollution. One of these measures indicates the necessity to carry out an immediate action for monitoring the presence of organophosphorous pesticides in “hot spot” areas and, if concentration levels so warrant, to take the necessary measures for the reduction of pollution (28). Certainly the Ebre Delta area (Tarragona, Spain) is a large estuarine area that is a “hot spot” due to the rice crop cultivation (18 000 ha). In order to eliminate rice stem bores such as Chilos suppresalis from this area, the commercial product Tionfos 50 LE was used. The product, which contains 50% fenitrothion, is applied in large amounts during the summer by aerial spraying (4050 t/yr).
Materials and Methods Chemicals. Methanol, acetonitrile, HPLC-grade water, and hydrochloric acid were obtained from Merck (Darmstadt, Germany). Fenitrothion was acquired from Promochem (Wesel, Germany). Acetic acid was purchased from Panreac (Barcelona, Spain). Empore 3M C18 extraction disks were acquired from J. T. Baker (Deventer, The Netherlands). The EnviroGard Parathion Plate Kit was obtained from Millipore Corp. (Bedford, MA). Pesticide Application. Tionfos 50 LE, Agride´s Macia` (Reus, Tarragona, Spain), which contained 50% fenitrothion, was applied by helicopter spraying over the rice crops fields in the area of the Ebre Delta (Tarragona, Spain) at a concentration of 4% of active pure mater mixing 40 L of Tionfos with 500 L of water, obtaining 3.7% diluted fenitrothion. In this way, 2 L/ha formulated diluted product was applied by helicopter, which corresponds to 74 mL of pure fenitrothion or 148 mL of Tionfos 50 LE. This is a common aerial application rate of organophosphorus pesticides, as previously reported for Abate 50E (15, 16) or fenthion (13) and fenitrothion (11). It is quite difficult to estimate which is the amount of fenitrothion reaching the surface of the water after aerial application. When the pesticide is applied in a lake, as in Canada (11), the samples were usually taken from the surface of the lake and 1 m above the bottom (the depth of the lake varied between 11 and 15 m) (11). The irrigation ditch that was monitored in this paper is close to the rice crop fields, and fenitrothion was already monitored in the same irrigation ditches during the winter following manual spraying (4). When the sampling is carried out in a rice crop field or stagnant pond (15, 16), the water column is not so deep and usually varies from 10-15 cm up to 30-35 cm, at the maximum. In this respect, the irrigation ditch was treated in a similar way as a rice crop field, although the water column is somewhat deeper (50 cm) and the amount of active ingredient reaching the water surface was estimated similarly as reported before (15, 16). Calculated residue concentrations of fenitrothion for this applied
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concentration were 125-100 µg/L for water that is in the first 5-10 cm deep to 40-30 µg/L for waters that are 30 cm deep. The Ebre Delta area, which contains rice as a main crop (18 000 ha), suffers during 2 weeks different types of aerial applications (helicopter and airplane) of fenitrothion and distinct organophosphorus pesticides from early morning till afternoon. The samples collected and reported in these experiments were performed on July 26-July 28, 1995. Sampling. The sampling was carried out in the same irrigation ditch that provided freshwater from another rice field. The collection of water samples consisted of 3-5 grab samples from the irrigation ditch, and they were polled together to a final volume of 1 L. The ambar bottles used to transport the collected water were previously cleaned up 2-3 times with the same water of the irrigation ditch. Water samples were collected at the upper layer of the surface, between 5 and 20 cm without any field spillage. Water sample pH varied between 7.8 and 8.2, water conductivity varied between 1220 and 1345 µS, and the water temperature varied between 25 (early morning) and 30 °C during the day. Although paddy water pH follows a diurnal pattern in rice fields due to photosynthesis of algae, it can range from pH 7 (early morning) to pH 9 (late afternoon (29) or from pH 7.8 to pH 8.8 in the rice crop field waters of the Ebre Delta (19). We collected the water from an irrigation ditch nearby the rice crop fields, but it is not a crop field. So, the pH values exhibited low fluctuation, from pH 8.0 to pH 8.2, which is common in the estuarine areas of the Ebre Delta. Samples were collected 0, 1, 2, 3, 4, 5, 8, 10, 24, 30, and 48 h after spraying, plus a blank was collected just before the treatment started. Also a sample of the formulation applied was collected in order to know whether the concentration found in the ditch water followed expectations or problems already existed when the mix of fenitrothion in the tanks took place, which is a common mistake. Sample Preparation. Immediately after sample collection, the samples were acidified with HCl to pH 4 and transported on ice to a field laboratory located 10 min from the sampling area and were extracted into C18 Empore disks, following the protocol described. Water samples were first filtered through fiberglass filters (Millipore Corp. Bedford, MA) of 0.45 µm in order to remove suspended particles as described (1). Aliquots of these samples were acidified to pH 4.0 in order to avoid the degradation and were analyzed directly by ELISA. The SPE off-line method used a Millipore 47-mm filtration apparatus. The membrane extraction disks were manufactured by 3M (St. Paul, MN) under the trademark Empore and are distributed by J. T. Baker and Analytichem International. The disks used in these experiments were 47 mm in diameter and 0.5 mm thick. Each disk contains about 500 mg of C18 bonded silica material. The disk, placed in a conventional Millipore apparatus, was washed with 2 × 10 mL of methanol and 15 mL of water under vacuum avoiding the solid phase to become dry, and 500 mL of water was extracted with the vacuum adjusted to yield a 30-min extraction time. The disks containing the pesticides were used for the transportation and storage at -20 °C. In this respect, it has been shown that C18 material can be used for storage of several organophosphorous pesticides, among them fenitrothion, during a period up to 8 months at -20 °C (16). Two months later, the disks were thawed, and the pesticides trapped in the disk were eluted with 2 × 10 mL
of methanol. The samples were re-dissolved in up to 500 mL of MilliQ water in order to reach the same fenitrothion concentration in water as the original samples for fenitrothion determination by Prospekt- LC/DAD. The aim of this work was the comparison of ELISA procedures and on-line solid-phase extraction, so that, for carrying out the later method, the re-dissolution of the extract sample was necessary. The two last samples of estuarine water taken at 30 and 48 h, respectively, were eluted from C18 with 2 × 10 mL of methanol, rotaevapored until 0.5 mL, and evaporated carefully until dryness with a gentle stream of nitrogen. Afterwards, methanol was added up to a volume of 500 µL, and 20 µL was injected into the mass spectrometer. Sample Analysis. (A) ELISA Procedures. Immunoassay experiments were carried out in a 96-well microplate washer SLT 96PW (SLT, Salzburg, Austria), and the absorbances were read at 450 nm in a microtiter-plate ELISA reader Multiskan Plus (Labsystems, Helsinki, Finland). Data acquisition and calculations were performed using the commercial software package Genesis (Labsystems). A four-parameter logistic equation was used for the calibration curves. The plates coated with antibody were filled with 100 µL (well of standard)-1 sample-1 and 100 µL/well of Parathion-enzyme conjugate. The wells were covered to prevent evaporation and incubated at room temperature for 60 min. The plates were washed five times with distilled water and then, 100 µL (well of substrate)-1chromogen-1 was added. After incubation for 30 min at room temperature, the reaction was stopped with a stopping solution (100 µL/well) and mixed thoroughly. This turned the solution yellow, and the optical density at 450 nm was determined. Standards were prepared by dilution from a 100 mg/L solution of fenitrothion in methanol stored at -20 °C. A standard series were prepared by making several dilutions of the stock solutions to yield the following fenitrothion concentrations: 0, 0.1, 1, 10, 100, 1000, and 10000 µg/L. The standard series were made up in distilled water. All standards were analyzed in triplicate. (B) Prospekt LC/DAD. Preconcentration of the water samples prior to LC analyses was performed by an on-line SPE system as reported in a previous work from our group (5). The LC analyses were performed with a Waters 600MS solvent delivery unit provided with a 20-µL injection loop and were combined with a Waters 996 photodiode array detector (Waters, Millipore, MA). A 25 cm × 4.6 mm i.d. analytical column packed with 5 µm of C8 (J. T. Baker, Deventer, The Netherlands) was used. The 10-mL water sample was preconcentrated at a flow rate of 2 mL/min and introduced into the chromatographic system. C18 cartridges were conditioned with 10 mL of acetonitrile and 10 mL of HPLC-grade water. A gradient elution was employed starting from a mobile phase containing 30% A (acetonitrile) and 70% B (water) to one containing 60% A-40% B in 8 min; isocratic until 20 min and from these conditions to 100% A in 10 min at a flow rate of 1 mL/min. Quantitation was done using the external standard calibration method with the detector set at 270 nm. Calibration graphs were constructed for fenitrothion by analyzing spiked aqueous samples prepared with MilliQ water between the range of 0.1 and 160 µg/L. On the other hand, a degradation study under laboratory conditions was carried out spiking Ebre Delta water (without acidity) with a known amount of fenitrothion formulate. In this case, PLRP-S cartridges were used in order to retain 3-methyl-4-nitrophenol in polymeric material, as C18 sorbent is not a suitable
TABLE 1
Typical Fragment Ions of Fenitrothion and Their Degradation Products in LC/APCI-MS in NI Mode of Operationa compound
Mn
fenitrothion
277
s-methyl isomer of fenitrothion fenitrooxon
277 261
3-methyl-4-nitrophenol
153
m/z and tentative ions
RA
[OC6H3NO2CH3][SC6H3NO2CH3][M - CH3][OC6H3NO2CH3][M - CH3][OC6H3NO2CH3][M - CH3][M - H]-
45 100 45 100 45 100 5 100 5
152 168 262 152 262 152 246 152 136 [M - OH]-
a Experimental conditions: Cone set at 20 V and corona at 3.5 kV. Carrier stream: acetonitrile-water (50:50) containing 1% of acetic acid at a flow rate of 1 mL/min. Mn, nominal mass. RA, relative abundances.
one for such compounds as reported (4). This compound was not retained at all in C18 cartridges, but in PLRP-S a recovery of 77% was obtained. (C) LC/APCI-MS. Liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry with negative mode of operation was used for the determination of the degradation products of fenitrothion. The eluent was delivered by a gradient system from Waters 616 pumps coupled to a Waters Model 600S controller (Waters, Milford, MA). A VG Platform from Fisons Instruments (Manchester, U.K.) equipped with an APCI interface was used. Full details about this interface is published elsewhere (30). A total of 20 µL of the two last samples collected was injected in the mass spectrometer, and the chromatograms were recorded under SCAN and SIM conditions in negative ion mode of operation. For SCAN conditions, the m/z ranged from 142 to 300 and for SIM conditions four ions corresponding to the typical fragments of fenitrothion and its TPs were selectioned: 152, 168, 246, 262 (see Table 1). The same analytical column than that was used for online analysis was used for LC/APCI-MS analysis. The elution was carried out with acetonitrile and HPLC water acidified with 0.5% of acetic acid each and with the same gradient as mentioned before at 1 mL/min. Also, the sample corresponding to the formulated product applied was injected into LC/APCI-MS in order to confirm the presence of the s-methyl isomer of fenitrothion at this high level of concentration.
Results and Discussion General Remarks. The difference in persistence and also toxicity between parathion and fenitrothion led fenitrothion to be widely applied for rice control while parathion is not used for this purpose. Today in developing countries the situation is not the same, and parathion is generally applied for tomatoes and other agricultural applications by manual spraying as for example in Brazil (31). The difference between parathion-methyl and fenitrothion is a methyl group in the ortho position. The presence of this methyl group gives fenitrothion more thermal instability and lower half-life under controlled environmental conditions. Fenitrothion also has a quantum yield 20 times higher than parathion (0.01 versus 0.0005), which means that fenitrothion will exhibit more photochemical degradation than parathion (32). Fenitrothion volatilization is much higher than parathion volatilization (being at least 1 order of magnitude) (11).
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TABLE 2
Specificity of Parathion-ethyl Envirogard ELISA in Distilled and Estuarine Water (n ) 6) distilled water compounds parathion-ethyl fenitrothion fenitrooxon 4-nitrophenole
LDDa (8)d
0.011 0.17 (3) >10000 200
estuarine water
IC50b
%CRc
LDD
IC50
%CR
0.45 (5) 24.6 (7) >10000 3000
100 1.83 10000
0.54 (4) 25.3 (5) >10000
100 2.08
