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ELISA and HPLC methods for atrazine and simazine determination in


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Ecotoxicology and Environmental Safety 70 (2008) 341–348 www.elsevier.com/locate/ecoenv

ELISA and HPLC methods for atrazine and simazine determination in trophic chains samples$
Irena Baranowskaa,?, Hanna Barchanskaa, Ramadan A. Abukneshab, Robert G. Priceb, Agata Stalmacha
b a Department of Analytical and General Chemistry, The Silesian University of Technology, M. Strzody Street 7, 44-100 Gliwice, Poland School of Health and Life Science, Analytical Research Group, Pharmaceutical Science Division, King’s College London, University of London, UK

Received 18 October 2006; received in revised form 20 June 2007; accepted 30 June 2007 Available online 4 October 2007

Abstract The aim of the research was to determine optimal conditions for atrazine determination in trophic chain samples by means of an antigen-coated tube enzyme-linked immunosorbent assay (ELISA). The ELISA method was used for analysis of a selection of samples and the results and method requirement compared with HPLC. The 2 h competitive ELISA showed a minimum detection limit of 0.05 ng mL?1 and a dynamic range 0.1–2 ng mL?1. Investigation of atrazine concentration in a selection of trophic chain samples indicated that the content of atrazine (mg kg?1) in soil samples was 3.2–85.4, vegetable roots 32.9–148.9, green parts of plants 67.7–136.4, cereals 42.4–91.5 and samples of animal origin 1.3–8.4. The correlation between results obtained by HPLC and ELISA methods was 0.97. In addition, simazine content was determined by the HPLC method in which the detection limits were 0.2 mg g?1 for atrazine and 0.3 mg g?1 for simazine. The content (mg kg?1) of simazine in soil samples was 13.5–15.5, in vegetables roots 29.5–93.7, in green parts of plants 34.6–72.6 and in cereals 158–189. The study demonstrates the utility and convenience of the simple, practical and cost-effective ELISA method in a non-immunoassay laboratory for the analysis of food and environmental samples. The method is ideal for the rapid screening of large numbers of samples in laboratories where access to HPLC facilities is limited or lacking. In addition the investigation demonstrates the presence of signi?cant levels of atrazine and simazine in trophic chain samples collected from different areas of the region. As expected, the highest concentration of both herbicides was found in plants. r 2007 Elsevier Inc. All rights reserved.
Keywords: Atrazine; Simazine; Trophic chains; ELISA; HPLC

1. Introduction Pesticides in the natural environment can be toxic to useful insects, ?sh and other water organisms, birds and mammals including man (Kiziewicz and Czeczuga, 2002). Herbicides from soil are taken up by plants (primary producers) and transferred to higher trophic levels by means of herbivores (primary consumers), then carnivores
$ Any studies involving humans or experimental animals were conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. ?Corresponding author. Fax: +48 237 12 05. E-mail address: irena.baranowska@polsl.pl (I. Baranowska).

(secondary consumers) and tertiary consumers. Pesticides, which are stable in the environment, undergo bioconcentration in compartments of trophic chains. Transfer of herbicides to various trophic chain compartments depends on the type of herbicides, pH, temperature, humidity and organic content in soil, the species of animals and stage of growth (Notten et al., 2005). The bioconcentration of pesticides is therefore considered to be hazardous for organisms on the top of trophic pyramid (Kiziewicz and Czeczuga, 2002). One of the most frequently used groups of herbicides is the triazines. Most of the triazine herbicides are derived from s-triazine a six membered heterocycle with symmetrically located nitrogen atoms which are substituted in

0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2007.06.012

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positions 2,4,6. Stereochemical stability of s-triazines is high and they are therefore stable in the environment. s-Triazines are weakly basic, poorly soluble in water but ? ? are stable in the solid phase and in solution (Pacakova et al., 1996). Commercially, atrazine (2-chloro-4-ethylamine-6-isopropylamine-s-triazine) is available as Gesaprim, Aatrex or Fenantrol, whereas simazine (6-chloro-N2, N4-diethyl1,3,5-triazine-2,4-diamine) is available as Amizine, Gesatop 50 and also Simazin. Triazines are applied at rates 1.12–2.24 kg/ha, which results in soil concentration range of 3–6 mg/kg (Funari et al., 1998; Lesan and Bhanadari, 2003). Atrazine and simazine are soluble in water (33 and 6.2 mg/L for atrazine and simazine, respectively) (Garcinuno et al., 2003). They have relatively low soil adsorption ? coef?cient, Koc 100 for atrazine and 130 for simazine in soil. As a consequence of their high solubility and low adsorption make atrazine and simazine the most commonly detected pesticides in surface and ground waters (Ying et al., 2005). This in combination with their bioaccumulation in trophic chain results in an increased risk of cancer and endocrine disruption resulting from exposure. It is therefore important to monitor the concentration of triazines in environment (Lee et al., 1999; Hayes et al., 2002). If triazines are to be determined in various trophic chains samples using chromatographic methods a range of different procedures for samples such as water, soil, vegetables, milk and meat need to be developed. These procedures include the extraction, clean-up steps, preconcentration followed by the selection of the analytical method. At present, one of the most popular methods for pesticide determination is immunoassays. These methods are sensitive and speci?c, easy to perform and do not required sophisticated equipment. Enzyme-linked immunosorbent assay (ELISA) may be carried out in microtitre plates or on antibody-coated polystyrene test tubes. The second method is easier to perform in non-immunoassay laboratory, because it requires only a spectrophotometer (Price et al., 2006). High speci?city of the method enables reduction of matrix effects and simpli?cation sample cleanup and preconcentration steps (Brecht and Abuknesha, ? ? 1995; Pacakova et al., 1996; Ahmed, 2001; Bruun et al., 2001). Triazines have been determined in fruit extracts (Delaunay et al., 2000), in water (Gascon et al., 1997; Mallat et al., 2001; Abuknesha and Grif?th, 2004), juice and milk (Franek et al., 1995), soil (Xiong et al., 1998; Price et al., 2006). The principal aim of this research was to demonstrate the utility of a coated-tube ELISA for measuring (screening) of atrazine in typical several trophic chain samples and compare results with data obtained by a standard HPLC procedure. The coated-tube ELISA selected for the study is simple to carry out by non-immunoassay experts in a basic laboratory setting and does not require dedicated

instrumentation and may be applied to large numbers of samples. In addition the assay is highly sensitive and suf?ciently speci?c of a screening the triazines.
2. Materials and methods 2.1. Apparatus
Chromatographic determination of atrazine and simazine were carried out on Merck Hitachi high performance liquid chromatograph system, equipped with L-6200 A pump and DAD-L-4500 A detector. The LiChroCART 125–3 mm PurospherSTAR RP-18 endcapped (5 mm) column and pre–column LiChroCARTs C18 (4 ? 4 mm, 5 mm); Merck, Darmstadt, Germany; were used. SPE-12G (SPE) and PhSO3H (500 mg) and C18 (500 mg) SPE columns (J. T. Baker, Inc., Philipsburg, USA) were used for solid phase extraction. Polystyrene ELISA star-bottomed highbind tubes were obtained from Greiner bio-one, GmbH (Gloucestershire, UK) and the tubes were shaken using an Ika-Werke Gmbh & Co.Kg (Germany). Spectrophotometric measurements were made with diode array spectrophotometer Hewlett-Packard HP P 8452 A (RFN) AND PLATE READER Anthos 2001, Salzburg, Austria.

2.2. Reagents
Atrazine and simazine (more than 99%) were bought from Riedel-de Haen (Seelze, Germany). Acetic acid, acetonitrile, acetone, ethyl acetate, methanol, n-hexane, hydrogen peroxide (30%), potassium dihydrogen orthophosphate, sodium acetate, sodium azide, sodium bicarbonate, sodium chloride, sodium tetraborate (analytical grade) were obtained from POCH S.A. (Gliwice, Poland). Acetonitrile, methanol, water for HPLC analysis were purchased from Merck, (Darmstadt, Germany). Polyclonal antiserum to atrazine was King’s College London (Price et al., 2006). Porcine gelatin powder, Tween 20, goat anti-sheep horseradish peroxidase conjugate, 2,20 -azino-bis (3-ethylbenzothiazolone-6-sulphonic acid) di-ammonium salt (ABTS) and thimerosal were purchased from Sigma Chemical Co. (Poole, UK).

2.3. Samples
Six soils and crops were sampled in three locations in agriculture regions to obtain pictures of the trophic chains. The distribution of triazines in different parts of vegetables were considered. Furthermore, one relationship between concentration of triazines in lake water and ?sh (roach, Rutilus rutilus) as well as in different kinds of animals’ tissues was also investigated. The following trophic relations were investigated: No. 1: Soil–carrot root–carrot leaves cultivation has been treated with triazines herbicides every year. No. 2: Soil–carrot root, red bet–carrot root leaves cultivation has been treated with triazines herbicides every year. No. 3: Soil–parsley root, red beet–parsley leaves cultivation has been treated with triazines herbicides every year. No. 4: Soil triticale (wheat/rye hybride)–maize cultivation has not been treated with triazines herbicide within past 2 years. No. 5: Soil–wheat–egg cultivation has been treated with triazines herbicides every year. No. 6: Soil–grass–goat’s milk cultivation has not been treated with triazines herbicide within past 2 years. No. 7: Water–?sh. No. 8: Soil–wheat–fat, meat cultivation has not been treated with triazines herbicide within past 2 years. The soils were chosen to re?ect a wide range of atrazine and simazine treatment history. Samples were collected in early autumn, during the time of harvest (September 2005).

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2.4. Methods
2.4.1. HPLC analysis The conditions of chromatographic analysis have been described elsewhere (Baranowska et al., 2006). The separation of triazines was achieved using by means of stainless-steel column LiChroCART 125–3 Purospher STAR RP-18 endcapped. The mobile phase consisted of water and methanol (50:50). Quantitative measurements were carried out by 224 nm.

2.4.2. ELISA The antigen-coated tube ELISA procedure used in the study was according to the method described previously (Price et al., 2006). The coating antigen reagent was diluted to 1/8000 in 50 mM sodium tetraborate buffer (pH 9.0) immediately before use. The coating (1 mL) was carried out by leaving the tubes to stand for 24 h at 4 1C, and after washing the tubes three times with washing buffer (1 mL of 50 mM sodium bicarbonate solution containing 0.1 M NaCl), the tubes were blocked for 45 min at room temperature with 1 mL of blocking buffer (20 mM sodium phosphate buffer, pH 7.4, containing 0.45% porcine gelatine and 0.1% thimerosal). After washing the tubes, 0.2 mL of the analyte (standard or sample solution in blocking buffer) and 0.8 mL of antibody solution (diluted 1:15,000 in phosphate buffer with gelatine) were added to tubes and the contents were mixed. After incubation for 25 min at room temperature, the tubes were washed, the second antibody–HRP solution (1 mL of 1/2000) was added and after incubation for 2 h, at 37 1C and washing, the color was developed by adding 1 mL of the substrate solution (10 mg of ABTS in 20 mL 50 mM sodium acetate buffer, pH 4.1, and 8 mL of 30% H2O2) was added to test tubes and left to stand in the dark for 30 min. Absorbance at 405 nm was read after stopping the reaction with 100 mL of 0.25% NaN3 (stop solution).

2.5. Processing of samples
2.5.1. Preparation of samples for ELISA analysis Representative samples of extracts from soil, vegetation, material of animal origin were diluted to 1/30 and 1/60 in blocking buffer before the assay procedure. 2.5.1.1. Soil samples. Dry and sieved soil (100 g) was extracted with 100 mL of chloroform (Baranowska et al., 2005). The extracts were evaporated to dryness and the residues dissolved in methanol (3 mL). 2.5.1.2. Vegetables samples. Carrots, parsley (green parts and roots) and red beets were analyzed. Crushed vegetable roots were weighed (15 g) and dry leaves (5 g) and mixed with acetone (50 mL, vegetable roots) and chloroform (30 mL leaves). After 24 h, samples were shaken for 2 h then ?ltered. The extracts were evaporated under a stream of nitrogen and the residues were dissolved in 3 mL of methanol. The samples were diluted and analyzed by the ELISA procedure. 2.5.1.3. Animal samples. Eggs (yolk and white, 50 g) were extracted with acetone (50 mL) and after centrifugation the supernatants were evaporated to dryness (Baranowska et al., 2005). The residues were dissolved in 3 mL of methanol. Milk (100 mL) was extracted with 100 mL of hexane–acetone (2:1, v/v). Subsequently, the layers were separated and the hexane layer was evaporated to dryness, the residue was dissolved in 3 mL of methanol (Baranowska et al., 2005). Pig and duck muscle (50 g) were extracted with acetone (50 mL) and fats were removed with hexane extraction. Fish’s muscles (50 g) were extracted with methanol (50 mL). The ?ltered extracts were evaporated to dryness and the residues were dissolved in 3 mL of methanol. All the extracted samples were assayed by ELISA and the results compared with those obtained by HPLC.

2.5.2. Preparation of samples for HPLC analysis In order to extract triazines form soil, vegetable samples and animal material different types of extraction procedures and solvents were tested. The recovery of triazines was assessed by adding standards to the samples and after the extraction procedure the recovery was calculated. Samples were extracted with chloroform, acetone, acetone–water (10:1 v/v), methanol, methanol–water (10:1 v/v), hydrochloric acid–water (pH1.5), acetate buffer pH 3.2 and pH 5.0. A comparison was made of shaking in a ?ask, ultrasound-assisted extraction, microwave-assisted extraction and Soxhlet extraction. The time of extraction was optimised and SPE clean up procedure was also used. Procedure of soil samples preparation was described elsewhere (Baranowska et al., 2005). However, in some samples the previously used SPE procedure was insuf?cient. In this case, samples after SPE procedure on PHSO3H sorbent were further cleaned on C18 sorbent. The triazine fractions were eluted with 3 mL of ethyl acetate and after evaporation to dryness, the residue was dissolved in 0.5 mL of methanol. Cereals, goat’s milk, grass and green parts of vegetables were extracted according to published procedure (Baranowska et al., 2005, 2006). However, in the case of maize and wheat samples, repetition of SPE procedure was necessary. Details of soil samples preparation was described elsewhere (Baranowska et al., 2005). Shake ?ask extraction with chloroform provided the cleanest extracts and the highest recoveries (97% atrazine, 91% simazine) in a relatively short extraction time. However in some samples the SPE procedure was insuf?cient. In this case the sample after SPE carried out on PHSO3H sorbent was cleaned with C18 sorbent. The triazines were eluted with 3 mL of ethyl acetate and the solvent was evaporated to dryness and the residue dissolved in 0.5 mL of methanol. The optimal conditions of atrazine and simazine extraction from plants included extraction by shaking in chloroform. The extracts were cleaned by PHSO3H columns and the recoveries were 87% and 93%. Cereals, goat’s milk, grass and green vegetables were extracted according to Baranowska et al. (2005, 2006). Repetition of the SPE procedure was necessary for maize and wheat samples. The conditions used to extract herbicides from animal tissue were the same as for ELISA (described above). However the extracts needed to be cleaned further by SPE. After extraction with appropriate solvents, the mixtures were ?ltered and the extracts were cleaned by solid phase extraction on C18 columns. Triazines were eluted with ethyl acetate and the extracts evaporated to dryness and the residues dissolved in methanol (0.5 mL) Water samples (250 mL) were aspirated through C18 SPE columns. Elution was carried out with ethyl acetated (3 mL) and the solvent evaporated to dryness, and the residue dissolved in methanol (0.5 mL)

3. Results The ELISA method dynamic range was 0.1–2 ng mL?1 and the minimum detection limit was estimated to be in the region of 0.05 ng mL?1 (Fig. 1). Assay signal (absorbance values) in duplicate reference points was conveniently high ranging from 1.8 to 0.2 absorbance units and the precision was within the expected variation range of fully optimized ELISA methods. The critical reagents required for the assay (primary antiserum, coating conjugate, the enzymelabelled second antibody and enzyme substrate) are relatively simple to prepare or commercially available and stable for long periods. The coated tubes may be stored ready-to-use for several weeks. The appropriate dilution levels of samples extracts were found to be 1/30 and 1/60. The limit of detection for HPLC method was 0.20 and 0.3 mg mL?1 for atrazine and simazine, respectively.

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90 80 70 60 50 40 30 20 10 0 0 10 20 30

y = 1,0193x - 1,5745 R2 = 0,9719

ELISA [ug/kg]

40

50

60

70

80

90

HPLC [ug/kg]
Fig. 1. Regression plot of atrazine ELISA data compared with HPLC results obtained with the same soil samples.

Table 1 Concentration of atrazine in vegetable, water and soil extracts determined by ELISA Symbol V1 V1 V4 V4 V4 V4 V5 V5 V5 V5 V7 V7 V10 V10 W1 W1 S1 S1 Sample Red beet Red beet Carrot root Carrot root Carrot leaves Carrot leaves Parsley roots Parsley roots Parsley leaves Parsley leaves Maize Maize Grass Grass Water Water Soil Soil Mass (g) 15.0 15.0 15.0 15.0 5.0 5.0 15.0 15.0 5.0 5.0 10.0 10.0 5.0 5.0 250 250 100 100 Solvent Acetone Acetone Acetone Acetone Chloroform Chloroform Acetone Acetone Chloroform Chloroform Acetone Acetone Chloroform Chloroform – – Chloroform Chloroform Dilution 1/30 1/60 1/30 1/60 1/30 1/60 1/30 1/60 1/30 1/60 1/30 1/60 1/30 1/60 1/30 1/60 1/30 1/60 Atrazine (mg/kg) (ELISA) 36.2 76.4 148.9 105.2 136.4 285.0 41.7 56.8 104.3 142.6 150.2 138.5 682.3 447.8 9.6 4.8 36.3 22.4 SD 1.2 9.6 13.2 8.8 27.9 18.7 1.1 6 4.5 5.3 7.5 3.5 35.2 32.5 0.2 0.6 4.4 1 Atrazine (mg/kg) (HPLC) 87 169.6 250 38.8 110 167 367 5.9 40.5 SD 7.5 10.2 20.5 2.2 2.7 21.5 41 0.1 3.4

Table 2 Concentration of atrazine in animal samples determined by ELISA Symbol A1 A1 A2 A2 A3 A3 A4 A4 A5 A5 Sample Goat’s milk Goat’s milk Pig’s meat Pig’s meat Pig’s fat Pig’s fat Duck’s fat Duck’s fat Fish Fish Mass (g) 100.0 100.0 40.0 40.0 50.0 50.0 50.0 50.0 50.0 50.0 Solvent Hexane–cetone (2:1) Hexane–cetone (2:1) Acetone Acetone Hexane Hexane Hexane Hexane Methanol Methanol Dilution 1/30 1/60 1/30 1/60 1/30 1/60 1/30 1/60 1/30 1/60 Atrazine (mg/kg) (ELISA) 5.0 4.4 4.5 1.3 7.7 4.4 4.6 2.3 9.7 8.4 SD 0.1 0.3 0.9 0.1 0.3 0.7 0.6 0.1 0.2 0.5 Atrazine (mg/kg) (HPLC) 5.2 n.d. 3.4 1.74 7.8 SD 0.4 – 0.1 0.1 0.4

All samples were prepared and measured in triplicate and expressed as the mean and standard deviation (Tables 1 and 2). The recoveries of atrazine and simazine from soil (97%, 94%), grass (87%, 93%), carrot roots (94%, 96%) and leaves (87%, 93%), cereals (92%, 93%) and from milk

samples (79%, 76%) are described elsewhere (Baranowska et al., 2006). The recovery of atrazine and simazine from animal fat was in range 52–54% and from eggs 76% and 88% in this study. Comparison of results obtained by the two methods showed a good degree of correlation. The best correlation

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480 430 ELISA [ug/kg] 380 330 280 230 180 130 150 200

y = 1.2125x - 46.822 R2 = 0.9889

250

300 HPLC [ug/kg]

350

400

450

Fig. 2. Regression plot of atrazine ELISA data compared with HPLC results for the same vegetable root samples. Table 3 Distribution of atrazine and simazine along trophic chains Number Trophic chain Atrazine (mg/kg)7SD ELISA 1 Soil Carrot root Carrot leaves Soil Carrot root Carrot leaves Red beet Soil Parsley root Parsley leaves Red beet Soil Triticale Maize Soil Maize Egg Soilc Grass Goat’s milk Lake water Fish (roach) Pig’s fat Pig’ s meat Duck’s fat Duck’s meat
a b

Simazine (HPLC) (mg/kg)7SD HPLC 40.573.4 169.6710.1 250720.5 52.871.4 33.9725 55.279.6 27.372.0 50.672.3 38.872.2 110.072.7 187717.5 79.670.5 108.573.4 167721.3 19.670.5 59.572.4 4873.5 3.370.7 367741.0 5.270.4 5.970.1 7.870.4 3.470.1 n.d. 1.770.1 n.d. 13.171.0 58.172.5 40.771.4 18.570.8 29.572.5 34.672.1 32.972.5 28.574.6 83.575.7 72.572.4 93.774.7 115.577.7 158710.7 189712.7 n.db n.d n.d n.d n.d n.d 3.970.2 5.970.2 2.070.1 n.d. n.d. n.d.

2a

3a

4a

5

6

7 8

36.374.4 148.9713.2 136.4727.9 48.671.1 36.171.2 67.7715.2 32.971.0 43.270.1 41.771.1 104.374.5 36.271.2 85.771.5 91.576.3 150.277.5 22.873.0 42.474.6 31.872.0 3.270.1 447.8732.5 4.470.3 4.870.6 8.470.5 4.470.7 1.270.1 2.370.1 n.d

Both plants grew on the same ?led, cultivated by the same farmer. n.d—Not detected. c Results published in elsewhere (Price et al., 2006).

(0.97) was found when soil, cereals and vegetables roots samples were diluted to 1/30 (Fig. 1). However, for vegetables leaves and samples of animal origin better correlation between the two methods was achieved when samples were diluted to 1/60 (Fig. 2). This is probably due to the greater complexity of matrices of the green leaves and animal samples. The investigation showed that atrazine concentration (mg kg?1) varied in the trophic chain in the range from 3.2 to 85.4 in soil, in vegetable roots 32.9–148.9, in the green

parts of plants 67.7–136.4, in cereals: 42.4 91.5 and in samples of animal origin from 1.3 to 8.4 (Tables 1 and 2). The HPLC method enabled simazine to be determines in addition to atrazine and therefore provided additional data on the level of a second triazine in the samples investigated (Table 3). As can be seen in the table, all samples that contained atrazine also showed signi?cant levels of simazine. It is interesting to note that the speci?city of the ELISA for atrazine is evident in Table 3. The amounts of atrazine by

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ELISA and HPLC are very close and more importantly, the ELISA detectable triazines are not equivalent to the total levels of atrazine and simazine. 4. Discussion The principal ?nding of the study is the effectiveness of a simple and cost-effective ELISA for the analysis of a wide range of trophic chain samples for atrazine content. The study demonstrated that the immunoassay is not only cheaper and easier to carry out, but also the generated data are as reliable as that obtained by the chromatographic method. The competitive tube ELISA was developed in a research laboratory speci?cally for use with samples that are expected to have complex matrices. In the method, samples extracts are diluted to at least 1/30 and only 200 mL volumes are delivered to the assay tubes where further dilution takes place when the antibody solution (0.8 mL) is added. Dilution of sample extract before the actual antibody–analyte binding step and competition with the solid phase hapten is critical in reducing the effects of sample matrix components. Sample dilution can only be acceptable if the ELISA sensitivity is suf?ciently high to meet the dilution effect on target analyte. The present ELISA is suf?ciently sensitive (Fig. 3) to meet the dilution factor and at the same time reduce the deleterious effects of complex matrices. ELISA is in general accepted as a useful screening and measurement method for pesticide residues in feed, feed, water and animal tissue samples (Hammock and Mumma, 1980; Jung et al., 1989; Thurman et al., 1990; Van Emon and Lopez-Avila, 1992; Lucas et al., 1993; Jung et al., 2006). However, the assay format is also an important issue in deciding the utility of the method not only for immunoassay laboratories but also for regional and less specialized facilities with interest in study of the spread of pesticides in local foods and environment. In this respect, the coated tube format used in this study and reported by other workers (Queffelec et al., 2001; Wang et al., 2006) is proposed to be a much more appropriate method than alternative ELISA formats for several reasons. Tubes are
2 1.8 Absorbance at 405nm 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0.01 0.1

easier to handle manually than microtitre plates, reagent volumes are comfortably high and require less experience and allow greater sample dilution with assay reagents and the color may be read in a normal spectrophotometer and no requirement for a plate reader. The advantages of tube ELISA are further strengthened by the fact that assay performance is identical to the ‘‘standard’’ microtitre plate assay (Abuknesha, unreported ?ndings). The particular solid phase competitive ELISA used in the study, the coated-antigen format as opposed to the coated antibody ELISA (Schneider and Hammock, 1992; Harrison et al., 1991, Goodrow et al., 1990; Price et al., 2006) was a deliberate choice. Coating the antigen (in this case a conjugate of an atrazine hapten derivative) allows the use of unprocessed antiserum which is a great deal more stable and requires no puri?cation or labeling since the second antibody–enzyme conjugate (a commercial product) is used for signal development. Assay tubes with coated antigen may be stored for long periods either in liquid or dried form with little change in activity. HPLC is less sensitive and involves more time consuming procedures of sample pre-treatment. In order to remove the matrix, it is necessary to clean up the sample (the SPE procedure causes losses of analytes) while for ELISA method, the samples dilution is suf?cient to eliminate the in?uence of matrix. The HPLC method has the advantage of allowing the determination of many compounds in the same run and the in?uence of other chemically similar compounds is easier to eliminate. The chromatograms of soil extract after ?rst and second SPE procedure are presented in Fig. 4. The chromatograms of duck and pig fat extracts are presented in Figs. 5 and 6. Retention times, addition of standards and UV spectrums (registered by means of DAD detector) enabled identi?cation of atrazine and simazine to be made unequivocally in the chromatograms. In all examined trophic chains, the highest concentration of herbicides appears in plants (especially atrazine concentration in green parts of vegetables is high). In animal tissues, the highest concentration of atrazine is detected in

y = -0.3415 ln(x) + 0.8955

1 Atrazine ng/mL

10

Fig. 3. Calibration curve for atrazine concentration determined by ELISA.

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0.18 0.16 0.14 0.12 Intensity (A.U.) 0.10 0.08 0.06 0.04 0.02 0.00 0 1 2 3 4 5 6 7 8 9 10 1 2 Intensity (A.U.)

0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0 1 2 3 4 5 6 7 8 9 10 1 2

Retension Time (min)

Retension Time (min)

Fig. 4. (a) Chromatogram of soil extract after SPE procedure (PHSO3H sorbent); (b) Chromatogram of soil extract after second SPE procedure (C18 sorbent); 1—simazine (tR—4, 45 min); 2—atrazine (tR—7, 68 min).

Fig. 6. Chromatogram of pig fat extract. Fig. 5. Chromatogram of duck fat extract.

fat. It was also observed that triazines herbicides were detected in agricultural regions that had not been treated with these compounds. This supports the concept that triazines are able to translocate in ecosystem. In addition to the demonstration of the usefulness of the ELISA method for the analysis of a wide range of trophic

chains samples for atrazine, the present study also revealed that the samples tested contained signi?cant levels of the pesticide. Signi?cant levels were found in a wide range of different samples from the trophic chain which suggests possible exposure of consumers including children may be important. Good agricultural practice in the use of

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pesticides calls for the authorized safe use of pesticides under conditions needed for ef?cient and consistent protection of crops but at the same time they should be applied in a manner which leaves the lowest possible level of residues at the point of consumption. This is the expected practice by all European farmers in order to adhere to the acceptable daily intake of pesticides. Protecting the public from pesticide residues in food and feed does not only require regulatory measures and precise guidelines; it also demands the availability of appropriate and practical enabling technologies to carry out suf?cient analysis of samples on frequent basis. The present study demonstrates the ef?cacy of an appropriate technology which allows easy application by a much wider range of agencies and regional laboratories to support the farming community and local agencies achieve improved protection of public health. Funding sources The study was supported by The British–Polish Young Scientists Programme no. WAR/342/27. References
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