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Distribution of pesticides and heavy metals in trophic chain
Chemosphere 60 (2005) 1590–1599 www.elsevier.com/locate/chemosphere
Distribution of pesticides and heavy metals in trophic chain Irena Baranowska *, Hanna Barchan´ska, Anna Pyrsz Department of Analytical and General Chemistry, Silesian Technical University, ul. ks. M. Strzody 7, 44-100 Gliwice, Poland Received 23 November 2004; received in revised form 8 February 2005; accepted 14 February 2005 Available online 13 April 2005
Abstract Determination of triazines herbicides (atrazine and simazine) by high performance liquid chromatography (HPLC) in samples of trophic chain were worked out. Determination limits of 0.5 lg g 1 for atrazine, 0.8 lg g 1 for simazine with pesticides recovery of 70–77% in trophic chain samples were obtained. The content of simazine in soils was in range 1.72–57.89 lg g 1, in grass 5–88 lg g 1, in milk 2.32–15.29 lg g 1, in cereals 10.98–387 lg g 1, in eggs 30.14– 59.48 lg g 1, for fruits: 2.45–6.19 lg g 1. The content of atrazine in soils was in range 0.69–19.59 lg g 1, in grass 7.85–23.85 lg g 1, in cereals 1.88–43.08 lg g 1. Cadmium, lead and zinc were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) in the same samples as atrazine and simazine. Determination limits for cadmium 5 · 10 3 lg g 1, for lead 1 · 10 2 lg g 1, and for zinc 0.2 · 10 3 lg g 1, were obtained. The content of cadmium in soil was in range 0.13–5.89 lg g 1, in grass 114–627.72 · 10 3 lg g 1, in milk 8.88–61.88 · 10 3 lg g 1, in cereals 0.20–0.31 lg g 1, in eggs 0.11–0.15 lg g 1, in fruits 0.23–0.59 lg g 1. The content of lead in soils was in range 0.57–151.50 lg g 1, in grass 0.16–136.57 lg g 1, in milk 1.16–3.74 lg g 1, in cereals 1.05–5.47 lg g 1, in eggs 5.79–55.87 lg g 1, in fruits 21.00–87.36 lg g 1. Zinc content in soil was in range 9.15–424.5 lg g 1, in grass 35.20– 55.87 lg g 1, in milk 20.00–34.38 lg g 1, in cereals 14.94–28.78 lg g 1, in eggs 15.67–32.01 lg g 1, in fruits 14.94– 18.88 lg g 1. Described below extraction and mineralization methods for particular trophic chains allowed to determine of atrazine, simazine, cadmium, lead and zinc with good repeatability and precision. Emphasis was focused on liquid–liquid extraction and solid-phase extraction of atrazine and simazine from analysed materials, as well as, on monitoring the content of herbicides and metals in soil and along trophic chain. Higher concentration of pesticides in samples from west region of Poland in comparison to that of east region is likely related to common applying them in Western Europe in relation to East Europe. The content of metals strongly depends on samples origin (industry area, vicinity of motorways). 2005 Elsevier Ltd. All rights reserved. Keywords: Atrazine; Simazine; Heavy metals; HPLC
1. Introduction
*
Corresponding author. Tel.: +4832 237 18 16; fax: +4832 237 12 05. E-mail address: [email protected] (I. Baranowska).
There are many methods for the determination of pesticides and heavy metals in environmental samples. Monitoring of the content of these compounds in soil, plants and food is necessary because of the toxicity of these metals and pesticides (Anklam and Battaglia,
0045-6535/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.02.053
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2001). Moreover, pesticides (for example atrazine, simazine) are able to translocate in whole ecosystem (Biziuk, 2001). There is a risk, that even crops from free pesticides cultivations may be contaminated with these compounds (Uri, 1997). In this situation, it is necessary to monitor concentration of these compounds in soil and their level of accumulation in the food chain. For the determination of pesticides by means of high performance liquid chromatography special samples treatment is needed. Extraction of residues from material depends on the polarity of the herbicides as well as on the type of sample matrix. Acetone, acetonitrile, methanol and ethyl acetate are the most usual organic solvents employed in the extraction of the herbicides residues (Tadeo et al., 2000, review). The extraction may be conducted in microwaves (Xiong et al., 1998; Shen and Lee, 2003) or ultrasonic solvent extraction can be applied (Babic et al., 1998; Gfrerer et al., 2004). Solid phase extraction (SPE) is one of the most popular method of preparing soil and environmental samples. Type of SPE column or disk depends on type of herbicide and matrix (Tadeo et al., 2000, review). Triazines can be separated from soil samples on C18 column using mobile phase of acetonitrile–sodium acetate buffer, pH 7.0 or 1 mM ammonium acetate–acetonitrile (50:50) (Schutz et al., 1994; Hogendoorn and van Zoonen, 2000). For determination triazines and dinitroanilines in soils and crops separation on C18 column with gradient elution was applied (mobile phase acetonitrile:water) (Dean et al., 1996). Determination of atrazine and simazine in corn and fruit is carried out on C18 columns, methanol:water or methanol:acetnonitrile are applied as mobile phases (Tadeo et al., 2000, review). Coupled with off-line concentration and pre-column derivatization liquid chromatographic fluorescence detection (LC-FL) was developed to determine atrazine and simazine in soil, crop and water sample (Gong and Changming, 1998). In the ecosystem there is interrelationship between contents of trace elements in soil, water, plants and air (Voutsa et al., 1996). Soils have been contaminated with heavy metals as a result of industrial activities: mining, automobile battery production, vehicle emission, landfilling of industrial waste (McCrea and Fischer, 1986; Ellen et al., 1990; Atta et al., 1997; Hong et al., 2002). Determination metals in soil and biological samples requires mineralization. After drying and grinding, soil and plants material are usually digested with aqua-regia (Siebe, 1995; Devkota and Schmidt, 2000; Turkdogan et al., 2002; During et al., 2003) or in hot concentrated nitric acid (Brekken and Steinnes, 2004). For plants material dry mineralization is often applied (Angelova et al., 2004), the residue is dissolved in H2SO4, HCl, conc. HNO3 + HClO4 conc. (Turkdogan et al., 2002; Deng et al., 2004). Curdova et al. (2004) conducted wet digestion of plant material under elevated pressure
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and a focused microwave heating in a mixture of 65% HNO3 and 30% H2O2. Leaching the elements from plant material can be carried out by means of ultrasonic (Borkowska-Brunecka et al., 2004). Heavy metals in soils, plants and fruit are usually determined by atomic absorption spectrometry (AAS). ICP-AES or ICP-MS are not as frequently used as AAS to determine metals in soil or biological materials. However, it is a method, that facilitates fast analysis (Frost and Ketchum, 2000; Curdova et al., 2004; Borkowska-Brunecka et al., 2004; Xiao-ping et al., 2004). The present research concern the way of samples preparation, conditions of analysis, monitoring the content of simazine, atrazine and metals (Cd, Pb, Zn) and translocation them along trophic chains. The influence of human activity (industry, agriculture) on contamination the environment was also analysed.
2. Materials and methods 2.1. Apparatus The high-performance liquid chromatography (HPLC) measurements were carried out on a Merck Hitachi L-6200 A instrument. Merck Hitachi L-4500 diode array detector was used. Cadmium, lead and zinc determinations were conducted by means of inductively coupled plasma optical emission spectrometer (ICP-OES), Jobin Yvon, Ultima 2. For mineralization High Performance Microwave Digestion Unit Milestone MLS 1200 MEGA was used. 2.2. Reagents Acetonitrile, ethyl acetate, methanol, n-hexane, acetone, acetic acid, potassium phosphate dibasic, nitric acid (concentrated), hydrogen peroxide (30%), all analytical grade, (POCH S.A.––Poland). Acetonitrile, methanol and water for HPLC analysis (HPLC grade) were obtained from Merck (Darmstadt, Germany). Atrazine and simazine (more than 99.2%) were bought from Riedel–de Haen (Seelze, Germany). Stock standards solution of 1 mg ml 1 were prepared by dissolving 10 mg of respective herbicide in 10 ml of methanol and stored at 4 C. Working solutions were prepared daily, in methanol, by appropriate dilution. For solid phase extraction Bakerbond spe Aromatic Sulfonic Acid (3 ml, 500 mg) columns, Bakerbond spe Octadecyl (C18) (3 ml, 500 mg) columns, Bakerbond spe Poly(styrenedivinylbenzene)copolymer columns (3 ml, 500 mg) were used. Columns were obtained from J.T Baker (Deventer, Netherlands). Stock standards solutions of 1 mg ml 1 of cadmium, lead and zinc were purchased from Tritisol Merck
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(Darmstadt, Germany). Redistilled water from quartz apparatus was used. All glassware, before use, were washed with distilled water, soaked in nitric acid (30%), then rinsed in redistilled water and air-dried. Items were kept in clean place, to avoid contamination.
Table 1 Source of analysed samples Trophic chain
Kind of sample
Origin of sample
1
Soil Grass Milk
Kobio´r, Silesia
2
Soil Grass Milk
Wilkowyje, Silesia
3
Soil Grass Milk
Wartogłowiec, Silesia
4
Soil Grass Milk
Jaszowice, Silesia
5
Soil Grass Milk
_ ´ w, Silesia Zwako
6
Soil Grass Milk
Cielnice, Silesia
7
Soil Grass Milk
Tarno´w, the Malopolska Province
8
Soil Cereal Egg
Wilcza, Silesia
9
Soil Cereal Egg
Wo´jtowa Wies´, Silesia
10
Soil Cereal Egg
Wo´jtowa Wies´, Silesia
11
Soil Cereal Egg
Brzeziny, the Welkopolska Province
12
Soil Strawberry
Pyskowice, Silesia
13
Soil Strawberry
Pyskowice, Silesia
14
Soil Sweet cherries
Chorzo´w, Silesia
15
Soil Sweet cherries
Chorzo´w, Silesia
16
Soil Sweet cherries
Ocieszyn, the Welkopolska Province
2.3. Determination of atrazine and simazine by HPLC method The procedures of preparing soil, grass, cereals, fruits, milk and eggs for herbicide determination by means of HPLC and heavy metals determination by means of ICP were worked out. The stainless steel column (25 cm · 4.6 mm ID) packed with C18 ODS chemical bonded phase, particle size 5 lm was employed to carry out HPLC analysis. Atrazine and simazine separation and determination was performed using isocratic elution at flow rate of 0.8 ml min 1. The mobile phase contained methanol:water (1:1). Absorbance was measured continuously in the 200–400 nm range by diode-array detector. Quantitative measurements of peaks areas by 224 nm were carried out in order to achieve maximum sensitivity. Soil, crops, grass samples were spiked with atrazine and simazine in order to determine the repeatability, precision and recovery. Determination limits for the chromatographic method carried out of 0.5 lg g 1 and 0.8 lg g 1 for atrazine and simazine, respectively. 2.4. Determination of cadmium, lead and zinc by ICP-AES method Determination of cadmium, lead and zinc by means of ICP were performed with following parameters: power 1000 W, normal speed of pump: 20, plasma flow rate 12 l min 1, coating gas flow rate: 0.2 l min 1, nebulisator flow rate: 0.7, nebulisator pressure 3.0 bar. Standards were prepared from 1 mg ml 1 solutions and diluted with water. Wavelengths were 214.438 nm for cadmium, 220.353 nm for lead, 213.856 nm for zinc. Detection limits for cadmium 5 · 10 3 lg g 1, for lead 1 · 10 2 lg g 1, and for zinc 0.2 · 10 3 lg g 1, were obtained. 2.5. Samples Sample materials was selected to obtain a picture of the trophic chains (for example: soil, then grass, that grew on this soil, finally milk from the cow which was fed on this grass), which are listed in Table 1. Samples were collected in industrial areas (Silesia) as well as from eco-regions (the Wielkopolska Province, the Malopolska Province) in Poland (Fig. 1).
2.6. Samples treatment Soil (from filed about one ar) was collected, according to the norm PN-ISO 11466, to plastic bags and
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Fig. 1. Map of Poland.
transport to laboratory as fast as possible, then airdried, pulverized and sieved through 0.102 mm mesh. Grass, cereals from fields (about one ar), strawberries (from different places of plantation, about one ar), sweet cherries, early variety and late variety, (from different places of a orchard) were collected to paper bags. Fruits were air-dried and ground. Milk was collected to clean glass bottles, eggs to plastic boxes. All samples were transported to laboratory as fast as possible.
2.6.1. Samples preparation for HPLC analysis 2.6.1.1. Soil and grass. Hundred grams of sieved soil was placed in 300 ml shake-flask filled with 100 ml of chloroform; 15 g of dry, ground grass was placed in 300 ml shake-flask filled with 100 ml of chloroform. After 24 h the mixtures were shaken over 2 h. Then the mixtures were filtered through filter funnels and the filtrates were evaporated to dryness. Five millilitre of acetonitrile was added into each of the residues (Bakerbond Application
Fig. 2. Chromatogram of milk sample, after spe treatment. (1) simazine (tR = 7.87), (2) atrazine (tR = 11.59).
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Note) and the obtained solutions were then filtrated through soft filters. Solid phase extraction of these samples were performed by using of Bakerbond spe Aromatic Sulfonic Acid columns. The columns were conditioned with 3 ml of acetic acid/HPLC grade water (1:99). Twenty-five millilitre of acetic acid/HPLC grade water (1:99) and 5 ml of soil (grass) filtrate were mixed and aspirated through the column at 5 ml min 1. Next each column was washed with 2 ml of acetic acid/HPLC grade water (1:99), subsequently they was washed with 1 ml of acetonitrile, followed by 3 ml of HPLC grade water, followed by 1 ml of 0.1 M potassium phosphate dibasic. The columns were allowed to air dry under vacuum over about 2 min after each addition. Triazine herbicides were eluted with 2 ml of acetonitrile/0.1 M potassium phosphate dibasic (1:1). 2.6.1.2. Cereals. Fifty millilitre of acetone was poured over 50 g of grounded cereals in 300 ml shake-flask. After 24 h it was shaken over a period of 2 h. Next, it was filtrated through filter funnel and the filtrate evaporated to dryness. Five millilitre of methanol was added to the residue, then SPE was carried out on Bakerbond spe Octadecyl columns. The column was conditioned with 6 ml of methanol, next 6 ml of water. Ten millilitre of water and 5 ml of cereals filtrate were mixed well and aspirated through the column at 5 ml min 1. Next the column was washed with 3 ml of water and allowed to air dry under vacuum for about 5 min. Triazines were eluted with 3 ml of methanol. 2.6.1.3. Cherries. To 100 ml shake-flask 10 g of dry and ground fruit and 20 ml of acetone were added. After 24 h it was shaken for 2 h then filtrated, next SPE was performed. Bakerbond spe Poly(styrenedivinylbenzene)copolymer column was conditioned with 6 ml of ethyl acetate, 6 ml of methanol, finally 8 ml of water. An acetone extract was aspirated through the column at 2 ml min 1. Next column was washed with 3 ml of acetone. Pesticides were eluted with 6 ml of ethyl acetate:acetone (9:1) without vacuum. 2.6.1.4. Strawberries. Fifty grams of strawberries and 20 ml of methanol were placed into shake-flask and shaken for 1 h and then filtrated. The mixture consisting 10 ml of filtrate and 40 ml of water was aspirated through the Bakerbond spe Octadecyl columns, previously conditioned with 5 ml of methanol. Subsequently, the column was washed with 3 ml of water and left to air dry under vacuum for about 10 min. Triazine herbicides were eluted with 3 ml of dichloromethane (Topuz et al., 2005). Next, the solvent was evaporated and the dry residue was dissolved in 1 ml of methanol. 2.6.1.5. Milk. One hundred and fifty millilitre of hexane:acetone (2:1) was poured over 20 ml of milk. The
mixture was shaken for 0.5 h. Next the layers were separated in separating funnel. The lower aqueous layer Table 2 Distribution of simazine and atrazine in trophic chains Trophic chain
Material
Concentration [lg g 1] Simazine
Atrazine
1
Soil Grass Milk
27 ± 1.15 31.93 ± 2.01 15.29 ± 1.07
2
Soil Grass Milk
2.38 ± 0.13 27.15 ± 0.65 n.d.
3
Soil Grass Milk
1.72 ± 0.02 19.03 ± 0.18 2.32 ± 0.02
4
Soil Grass Milk
n.d. n.d. n.d.
n.d. n.d. n.d.
5
Soil Grass Milk
7.71 ± 0.03 17.95 ± 0.07 n.d.
n.d. n.d. n.d.
6
Soil Grass Milk
5.77 ± 0.21 88.32 ± 3.77 2.53 ± 0.56
n.d. n.d. n.d.
7
Soil Grass Milk
3.50 ± 0.03 5.1 ± 0.87 n.d.
3.48 ± 0.05 7.85 ± 0.14 n.d.
8
Soil Maize Egg
45.48 ± 2.10 387.15 ± 20.11 57.49 ± 3.78
n.d. n.d. n.d.
9
Soil Barley Egg
4.45 ± 0.23 10.98 ± 1.05 30.14 ± 2.00
19.59 ± 0.51 43.08 ± 3.50 52.17 ± 4.12
10
Soil Wheat Egg
25.97 ± 0.69 81.59 ± 5.45 50.47 ± 2.13
n.d. 1.88 ± 0.08 n.d.
11
Soil Wheat Egg
57.89 ± 2.47 65.48 ± 3.68 59.48 ± 5.78
n.d. n.d. n.d.
12
Soil Strawberry
2.69 ± 0.18 5.29 ± 0.58
2.74 ± 0.19 n.d.
13
Soil Strawberry
8.23 ± 0.45 6.19 ± 0.13
1.38 ± 0.11 n.d.
14
Soil Sweet cherry
3.90 ± 0.18 2.97 ± 0.07
0.69 ± 0.09 n.d.
15
Soil Sweet cherry
3.52 ± 0.21 3.63 ± 0.12
0.58 ± 0.1 n.d.
16
Soil Sweet cherry
2.69 ± 0.12 2.45 ± 0.19
2.74 ± 0.07 3.04 ± 0.10
n.d.––not detected.
n.d. n.d. n.d. 13.85 ± 0.31 23.85 ± 0.02 1.77 ± 0.05 n.d. n.d. n.d.
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was removed to waste (Burke et al., 2003). The upper layer was added to another portion of milk. The procedure was repeated three times. After this, the upper layer was evaporated and the residue was dissolved in 5 ml acetonitrile. The SPE was performed on Bakerbond spe Aromatic Sulfonic Acid column. The SPE procedure was the same as for grass and soil. 2.6.1.6. Eggs. An egg (yolk and white, without eggshell) was extracted with 50 ml of acetone for 2 h by means of a shaker. Next the mixture was centrifuged, the acetone layer was evaporated to dryness, the residue dissolved in 5 ml of methanol and SPE solid phase extraction on Bakerbond spe Octadecyl columns was performed. The SPE procedure was the same as for cereals. Each time the completeness of extraction was checked. From each sample three samples were prepared simultaneously and all samples were measured three times. An aliquot of 20 ll of the sample was injected for HPLC measurement. 2.6.2. Samples preparation for ICP analysis 2.6.2.1. Soil and milk. 0.5 g of dried, sieved soil and 0.5 g of dried to constant weight milk samples were mineralised in microwaves in HNO3:H2O2 4:1 and 5:3 for soil and milk respectively. The digest was diluted with 20 ml of distilled water, filtrated and transferred to 25 ml volumetric flask. The volume made up with redistilled water to the mark. 2.6.2.2. Grass, cereals, fruits and eggs. Two grams of dry and ground grass, seeds of cereals, dry fruit, 0.5 g of previously dried to constant weight eggs were burnt to ashes in furnace (T = 350 C). Next, 5 ml of the mixture of HNO3:H2O (1:1) was added to the residue. Subsequently, the sample was filtrated and quantitatively transferred to a 25 ml volumetric flask. The volume made up to the mark with redistilled water.
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From each material three samples were prepared simultaneously and all samples were measured three times.
3. Results and discussion From among methods of extraction of triazines described in literature (Tadeo et al., 2000, review), in this research, the best quantitative extraction of atrazine and simazine from soil and grass were obtained using chloroform, whereas from fruit and cereals using acetone. We examined different kinds of SPE column filling. For grass, soil and milk the most suitable were Bakerbond spe Aromatic Sulfonic Acid columns. For cereals and eggs Bakerbond spe Octadecyl (C18) columns were chosen, whereas for fruit (strawberries and sweet cherries) adequate was Bakerbond spe Poly(styrenedivinylbenzene)copolymer columns. The mobile phase methanol:water (1:1) was used previously by authors for determination simazine, propazine, heksasinon, bromacil, metoksuron (Pieszko and Baranowska, 2000). Nevertheless, this mobile phase has not been use before for determination of atrazine and simazine simultaneously. Chromatograms were recorded continuously in the 200–400 nm range, but the determination wavelength at 224 nm was selected as the best because of its optimum sensitivity for determination. The retention time for atrazine and simazine standars was amount 7.76 min and 11.49 min, respectively. However, retention time for soil and biological samples was in range 7.51–8.05 for simazine and 11.50–12.00 min for atrazine and simazine. To create calibration curves standard compounds were diluted with methanol to give concentration 0.5–100 lg g 1. In this range the curves equations were linear.
Fig. 3. Simazine and atrazine in trophic chain samples.
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The regression equations of peak area and injection concentration [lg g 1] were determined with correlation
coefficient of 0.9988 and 0.9998 for atrazine and simazine, respectively. The recovery of simazine from soil,
Table 3 Distribution of cadmium, lead and zinc in trophic chains Trophic chain
Material
Concentration [lg g 1] Cd
1
Pb
Soil Grass Milk
5.89 ± 0.06 (170.16 ± 6.78) · 10 (19.38 ± 9.38) · 10
2
Soil Grass Milk
3
Zn
62.50 ± 0.50 0.16 ± 0.06 1.76 ± 0.43
424.5 ± 15.50 35.20 ± 0.36 20 ± 1.25
1.16 ± 0.04 (292.25 ± 12.23) · 10 3 (15.62 ± 1.38) · 10 3
151.50 ± 5.50 7.69 ± 0.19 1.16 ± 0.64
129.5 ± 6.00 47.67 ± 2.12 27.5 ± 0.62
Soil Grass Milk
1.48 ± 0.09 (263.72 ± 3.75) · 10 (5.88 ± 5.62) · 10
47.46 ± 1.00 136.57 ± 2.50 1.18 ± 0.23
126.9 ± 4.50 45.31 ± 1.25 34.38 ± 1.88
4
Soil Grass Milk
1.48 ± 0.12 (627.24 ± 11.60) · 10 3 (25.00 ± 1.31) · 10 3
52.48 ± 1.00 3.13 ± 0.10 2.65 ± 0.57
128.45 ± 6.00 55.87 ± 2.00 28.75 ± 1.25
5
Soil Grass Milk
0.58 ± 0.02 (473.51 ± 9.25) · 10 (61.88 ± 2.06) · 10
78.50 ± 2.00 8.05 ± 0.33 3.74 ± 0.27
71.5 ± 3.00 55.74 ± 2.87 20.6 ± 31.25
Soil Grass Milk
5.60 ± 0.16 (114.29 ± 4.12) · 10 n.d.
137.50 ± 2.50 3.39 ± 0.12 2.49 ± 0.14
396 ± 24.00 42.22 ± 0.87 30.63 ± 0.62
Soil Grass Milk
4.68 ± 0.13 (152.16 ± 6.14) · 10 n.d.
6
7
3 3
3 3
3 3
3
4
57 ± 1.03 7.86 ± 0.56 1.47 ± 0.11
97.74 ± 4.15 38.48 ± 0.89 25.12 ± 0.75
8
Soil Maize Egg
0.22 ± 0.02 0.27 ± 0.03 0.12 ± 0.01
0.57 ± 0.05 4.56 ± 0.12 5.79 ± 0.52
24.37 ± 2.46 28.78 ± 2.48 32.01 ± 2.87
9
Soil Barley Egg
0.13 ± 0.008 0.20 ± 0.01 0.15 ± 0.004
1.11 ± 0.09 1.56 ± 0.18 7.16 ± 0.20
9.15 ± 1.48 14.94 ± 0.38 19.03 ± 1.43
10
Soil Wheat Egg
0.25 ± 0.009 0.31 ± 0.01 0.14 ± 0.007
n.d. 1.05 ± 0.3 9.6 ± 0.20
10.46 ± 1.46 16.65 ± 0.22 15.67 ± 0.02
11
Soil Wheat Egg
1.47 ± 0.3 5.47 ± 0.57 6.78 ± 0.72
26.48 ± 2.38 25.47 ± 2.91 22.47 ± 3.05
12
Soil Strawberry
0.20 ± 0.01 0.23 ± 0.015
58.12 ± 3.45 61.48 ± 4.05
37.33 ± 1.48 14.94 ± 0.44
13
Soil Strawberry
0.37 ± 0.02 0.24 ± 0.01
49.75 ± 2.78 53.46 ± 3.48
34.6 ± 11.91 18.88 ± 0.14
14
Soil Sweet cherry
0.13 ± 0.006 0.59 ± 0.005
85.08 ± 4.21 87.36 ± 5.01
49.73 ± 0.21 15.86 ± 0.19
15
Soil Sweet cherry
0.15 ± 0.008 0.61 ± 0.005
80.69 ± 4.63 82.69 ± 3.65
45.46 ± 3.45 18.46 ± 2.33
16
Soil Sweet cherry
0.25 ± 0.03 0.31 ± 0.05
42.68 ± 1.87 21 ± 1.58
28.79 ± 2.85 15.47 ± 1.47
n.d.––not detected.
n.d. n.d. 0.11 ± 0.01
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Fig. 4. Heavy metals in trophic chain samples.
grass and cereals were 75.6%, 72.45%, 76.78% respectively, whereas for atrazine the recovery carried out 70.1% for soil, 72.45% for grass and 71.45% for cereals. An example of chromatogram of milk sample was shown in Fig. 2. Results and standard deviations of the determination of simazine and atrazine by HPLC method are listed in Table 2. Concentration of herbicides in milk, fruits and eggs are given in lg g 1 wet weight, whereas in soil, grass and cereals in lg g 1 air-dried weight. Simazine was detected in most of the samples, whereas atrazine was detected only in few samples. This is a result of simazine more commonly being used in pesticide preparations in Poland. It should be emphasized, that content of analysed pesticides are caused by their ability to translocation, only one of the cultivation (trophic chain 8) was treated with simazine preparation within past two years. The highest concentration of simazine was detected in grass (5–88 lg g 1) and cereals (10.98–387 lg g 1) (Fig. 3).1 It is related to tendency of these herbicides to accumulate in plants. Very high content of simazine in maize (387 lg g 1) is caused by using herbicides preparation to combat weeds on this field. The concentration of herbicides determined in milk was the lowest (2.32–15.29 lg g 1), what testifies favourable homeostasis system of cows, although it is contaminated as well. Nevertheless, the concentration of simazine in eggs was higher than in soil, but considerably lower than in henÕs feed––cereals. Higher concentration of pesticides in samples from the Wielkopolska Province, in comparison with the Malopolska Province was noticed. It is probably related to 1
Results obtained from trophic chain number 8 were omitted, because this cultivation was treated with pesticides within past two years.
common applying of pesticides in Western Europe in relation to East Europe. Results and standard deviations of the determination of heavy metals in trophic chain samples by ICP method are listed in Table 3. Concentration of metals in milk, fruits and eggs are given in (lg g 1) wet weight, whereas in soil, grass and cereals in (lg g 1) air-dried weight. Comparing the concentration of heavy metals in the analysed trophic chains (Fig. 4), the lowest level cadmium and lead was detected in milk (for Cd 8.88 · 10 3–61.88 · 10 3 lg g 1, for Pb 1.16–3.74 lg g 1). There was lower concentrations of metals in cereals (Cd 0.20–0.31 lg g 1, Pb 1.05–5.47 lg g 1, Zn 14.94– 28.78 lg g 1) in comparison with soil (Cd 0.13–5.89 lg g 1, Pb 0.57–151.50 lg g 1, Zn 9.15–424.5 lg g 1). In interrelation soil–cereals–eggs, the lowest level of cadmium was determined in eggs (Cd 0.11–0.15 lg g 1). Nevertheless, the content of metals strongly depends on samples origin (vicinity of motorway––trophic chain 6, heavy industry––trophic chain 2). Monitoring of heavy metals distribution in soil, plants as well as in food of animal origin makes it possible to determine the level of contamination of foodstuffs, thereby enable to choose the least contaminated food.
4. Conclusion Conducted research proved that, demonstrated HPLC method is suitable for the determination of atrazine and simazine in all analysed materials for the sake of good separation of peaks, without interference other compounds of matrix. Prepared extraction methods for particular trophic chains allowed to quantitative extraction of herbicides and determination with good repeatability and precision.
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The conditions of mineralisation as well as ICP analysis parameters enable determination cadmium, lead and zinc in different kinds of materials, with good precision, at low determination limits. Acknowledgment We thank Dr. Bo_zena Smolec, Institute of Ferrous Metallurgy in Gliwice (Poland), for conducting ICP analysis. References Angelova, V., Ivanova, R., Delibaltova, V., Ivanov, K., 2004. Bio-accumulation and distribution of heavy metals in fibre crops (flax, cotton and hemp). Indust. Crops Products 19, 197–205. Anklam, E., Battaglia, R., 2001. Food analysis and consumer protection. Trends Food Sci. Technol. 12, 197–202. Atta, M.B., El-Sebaie, L.A., Noaman, M.A., Kassab, H.E., 1997. The effect of cooking on the content of heavy metals in fish (Tilapia nilotica). Food Chem. 58, 1–4. Babic, S., Petrovic, M., Kastelan-Macan, M., 1998. Ultrasonic solvent extraction of pesticides from soil. J. Chromatogr. A 823, 3–9. Biziuk, M., 2001. Pestycydy, Wystepowanie, Oznaczanie I Unieszkodliwianie. Wydawnictwo Naukowo-Techniczne, Warszawa. Borkowska-Brunecka, J., Jankowiak, U., Zyrnicki, W., Wilk, K.A., 2004. Effect of surfactant addition on ultrasonic leaching of trace elements from plant samples in inductively coupled plasma-atomic emission spectrometry. Spectrochim. Acta, Part B 59, 585–590. Brekken, A., Steinnes, E., 2004. Seasonal concentrations of cadmium and zinc in native pasture plants: consequence for grazing animals. Sci. Total Environ. 326, 181–195. Burke, E.R., Holden, A.J., Shaw, I.C., 2003. A method to determine residue levels of persistent organochlorine pesticides in human milk from Indonesian women. Chemosphere 50, 529–535. Curdova, E., Vavruskova, L., Suchanek, M., Baldrin, P., Gabriel, J., 2004. ICP-MS determination of heavy metals in submerged cultures of wood-rotting fungi. Talanta 62, 483– 487. Dean, J.R., Wade, G., Barnabas, I.J., 1996. Determination of triazine herbicides in environmental samples. J. Chromatogr. A 733, 295–335. Deng, H., Ye, Z.H., Wong, M.H., 2004. Accumulation of lead, zinc, copper and cadmium by 12 wetland plant species thriving in metal-contaminated sites in China. Environ. Pollut. 132, 29–40. Devkota, B., Schmidt, G.H., 2000. Accumulation of heavy metals in food plants and grasshoppers from the Taigetos Mountains, Greece. Agric. Ecosyst. Environ. 78, 85– 91. During, R.-A., Hoss, T., Gath, S., 2003. Sorption and bioavailability of heavy metals in long-term differently tilled soils amended with organic wastes. Sci. Total Environ. 313, 227–234.
Ellen, G., Loon, J.W., Tolsma, K., 1990. Heavy metals in vegetables grown in the Netherlands and in domestic and imported fruits. Z. Lebensm. Unters. Forsch. 190, 34– 39. Frost, H.L., Ketchum Jr., L.H., 2000. Trace metal concentration in durum wheat from application of sewage sludge and commercial fertilizer. Adv. Environ. Res. 4, 347–355. Gfrerer, M., Chen, S., Lankmayr, E.P., Quan, X., Yang, F., 2004. Comparison of different extraction techniques for determination of chlorinated pesticides in animal feed. Anal. Bionala. Chem. 378, 1861–1867. Gong, A., Changming, Y., 1998. Analysis of trace atrazine and simazine in environmental samples by liquid chromatography––fluorescence detection with pre-column derivatization reaction. J. Chromatogr. A 827, 57–63. Hogendoorn, E., van Zoonen, P., 2000. Recent and future developments of liquid chromatography in pesticide trace analysis. J. Chromatogr. A 892, 435–453. Hong, K., Tokunaga, S., Kajiuchi, T., 2002. Evaluation of remediation process with plant-derived biosurfactant for recovery of heavy metals from contaminated soils. Chemosphere 49, 379–387. McCrea, R.C., Fischer, J.D., 1986. Heavy metal and organochlorine contaminations in the five major Ontario rivers of the Hudson bay lowland. Water Poll. Res. J. Can. 21, 225– 234. Pieszko, C., Baranowska, I., 2000. Determination of selected herbicides and phenols in water and soils by solid-phase extraction and high performance liquid chromatography. J. Chromatogr. Sci. 38, 211–218. Schutz, S., Hummel, H.E., Duhr, A., Wollnik, H., 1994. Structural elucidation and trace analysis with combined hyphenated chromatography and mass spectrometric methods. Potential of using hybrid sector mass spectrometry– time-of-flight mass spectrometry for pesticide analysis. J. Chromatogr. A 683, 141–148. Shen, G., Lee, H.K., 2003. Determination of triazines in soil by microwave-assisted extraction followed by solid-phase microextraction and gas chromatography–mass spectrometry. J. Chromatogr. A 985, 167–174. Siebe, Ch., 1995. Heavy metal availability to plants in soils irrigated with wastewater from Mexico City. Water. Sci. Tech. 12, 34–39. Tadeo, J.L., Sa´nchez-Brunete, C., Pe´rez, R.A., Ferna´ndez, M.D., 2000. Analysis of herbicide residues in cereals, fruits and vegetables. J. Chromatogr. A 882, 175–191, Review. Topuz, S., Ozhan, G., Alpertunga, B., 2005. Simultaneous determination of various pesticides in fruit juices by HPLCDAD. Food Contr. 16, 87–92. Turkdogan, M.K., Kilicel, F., Kara, K., Tuncer, I., Uygan, I., 2002. Heavy metals in soil, vegetables and fruits in the endemic upper gastrointestinal cancer region of Turkey. Environ. Toxicol. Pharmacol. 13, 175–179. Uri, N.D., 1997. A note on the development and use of pesticides. Sci. Total Environ. 204, 57–74. Voutsa, D., Grimanis, A., Samara, C., 1996. Trace elements in vegetables grown in an industrial area in relation to soil and air particulate matter. Environ. Pollut. 94, 325– 335.
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Xiong, G., Liang, J., Shichun, Z., Zhang, Z., 1998. Microwaveassisted extraction of atrazine from soil followed by rapid detection using commercial ELISA kit. Anal. Chim. Acta 371, 97–103.
Distribution of pesticides and heavy metals in trophic chain Irena Baranowska *, Hanna Barchan´ska, Anna Pyrsz Department of Analytical and General Chemistry, Silesian Technical University, ul. ks. M. Strzody 7, 44-100 Gliwice, Poland Received 23 November 2004; received in revised form 8 February 2005; accepted 14 February 2005 Available online 13 April 2005
Abstract Determination of triazines herbicides (atrazine and simazine) by high performance liquid chromatography (HPLC) in samples of trophic chain were worked out. Determination limits of 0.5 lg g 1 for atrazine, 0.8 lg g 1 for simazine with pesticides recovery of 70–77% in trophic chain samples were obtained. The content of simazine in soils was in range 1.72–57.89 lg g 1, in grass 5–88 lg g 1, in milk 2.32–15.29 lg g 1, in cereals 10.98–387 lg g 1, in eggs 30.14– 59.48 lg g 1, for fruits: 2.45–6.19 lg g 1. The content of atrazine in soils was in range 0.69–19.59 lg g 1, in grass 7.85–23.85 lg g 1, in cereals 1.88–43.08 lg g 1. Cadmium, lead and zinc were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) in the same samples as atrazine and simazine. Determination limits for cadmium 5 · 10 3 lg g 1, for lead 1 · 10 2 lg g 1, and for zinc 0.2 · 10 3 lg g 1, were obtained. The content of cadmium in soil was in range 0.13–5.89 lg g 1, in grass 114–627.72 · 10 3 lg g 1, in milk 8.88–61.88 · 10 3 lg g 1, in cereals 0.20–0.31 lg g 1, in eggs 0.11–0.15 lg g 1, in fruits 0.23–0.59 lg g 1. The content of lead in soils was in range 0.57–151.50 lg g 1, in grass 0.16–136.57 lg g 1, in milk 1.16–3.74 lg g 1, in cereals 1.05–5.47 lg g 1, in eggs 5.79–55.87 lg g 1, in fruits 21.00–87.36 lg g 1. Zinc content in soil was in range 9.15–424.5 lg g 1, in grass 35.20– 55.87 lg g 1, in milk 20.00–34.38 lg g 1, in cereals 14.94–28.78 lg g 1, in eggs 15.67–32.01 lg g 1, in fruits 14.94– 18.88 lg g 1. Described below extraction and mineralization methods for particular trophic chains allowed to determine of atrazine, simazine, cadmium, lead and zinc with good repeatability and precision. Emphasis was focused on liquid–liquid extraction and solid-phase extraction of atrazine and simazine from analysed materials, as well as, on monitoring the content of herbicides and metals in soil and along trophic chain. Higher concentration of pesticides in samples from west region of Poland in comparison to that of east region is likely related to common applying them in Western Europe in relation to East Europe. The content of metals strongly depends on samples origin (industry area, vicinity of motorways). 2005 Elsevier Ltd. All rights reserved. Keywords: Atrazine; Simazine; Heavy metals; HPLC
1. Introduction
*
Corresponding author. Tel.: +4832 237 18 16; fax: +4832 237 12 05. E-mail address: [email protected] (I. Baranowska).
There are many methods for the determination of pesticides and heavy metals in environmental samples. Monitoring of the content of these compounds in soil, plants and food is necessary because of the toxicity of these metals and pesticides (Anklam and Battaglia,
0045-6535/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.02.053
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2001). Moreover, pesticides (for example atrazine, simazine) are able to translocate in whole ecosystem (Biziuk, 2001). There is a risk, that even crops from free pesticides cultivations may be contaminated with these compounds (Uri, 1997). In this situation, it is necessary to monitor concentration of these compounds in soil and their level of accumulation in the food chain. For the determination of pesticides by means of high performance liquid chromatography special samples treatment is needed. Extraction of residues from material depends on the polarity of the herbicides as well as on the type of sample matrix. Acetone, acetonitrile, methanol and ethyl acetate are the most usual organic solvents employed in the extraction of the herbicides residues (Tadeo et al., 2000, review). The extraction may be conducted in microwaves (Xiong et al., 1998; Shen and Lee, 2003) or ultrasonic solvent extraction can be applied (Babic et al., 1998; Gfrerer et al., 2004). Solid phase extraction (SPE) is one of the most popular method of preparing soil and environmental samples. Type of SPE column or disk depends on type of herbicide and matrix (Tadeo et al., 2000, review). Triazines can be separated from soil samples on C18 column using mobile phase of acetonitrile–sodium acetate buffer, pH 7.0 or 1 mM ammonium acetate–acetonitrile (50:50) (Schutz et al., 1994; Hogendoorn and van Zoonen, 2000). For determination triazines and dinitroanilines in soils and crops separation on C18 column with gradient elution was applied (mobile phase acetonitrile:water) (Dean et al., 1996). Determination of atrazine and simazine in corn and fruit is carried out on C18 columns, methanol:water or methanol:acetnonitrile are applied as mobile phases (Tadeo et al., 2000, review). Coupled with off-line concentration and pre-column derivatization liquid chromatographic fluorescence detection (LC-FL) was developed to determine atrazine and simazine in soil, crop and water sample (Gong and Changming, 1998). In the ecosystem there is interrelationship between contents of trace elements in soil, water, plants and air (Voutsa et al., 1996). Soils have been contaminated with heavy metals as a result of industrial activities: mining, automobile battery production, vehicle emission, landfilling of industrial waste (McCrea and Fischer, 1986; Ellen et al., 1990; Atta et al., 1997; Hong et al., 2002). Determination metals in soil and biological samples requires mineralization. After drying and grinding, soil and plants material are usually digested with aqua-regia (Siebe, 1995; Devkota and Schmidt, 2000; Turkdogan et al., 2002; During et al., 2003) or in hot concentrated nitric acid (Brekken and Steinnes, 2004). For plants material dry mineralization is often applied (Angelova et al., 2004), the residue is dissolved in H2SO4, HCl, conc. HNO3 + HClO4 conc. (Turkdogan et al., 2002; Deng et al., 2004). Curdova et al. (2004) conducted wet digestion of plant material under elevated pressure
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and a focused microwave heating in a mixture of 65% HNO3 and 30% H2O2. Leaching the elements from plant material can be carried out by means of ultrasonic (Borkowska-Brunecka et al., 2004). Heavy metals in soils, plants and fruit are usually determined by atomic absorption spectrometry (AAS). ICP-AES or ICP-MS are not as frequently used as AAS to determine metals in soil or biological materials. However, it is a method, that facilitates fast analysis (Frost and Ketchum, 2000; Curdova et al., 2004; Borkowska-Brunecka et al., 2004; Xiao-ping et al., 2004). The present research concern the way of samples preparation, conditions of analysis, monitoring the content of simazine, atrazine and metals (Cd, Pb, Zn) and translocation them along trophic chains. The influence of human activity (industry, agriculture) on contamination the environment was also analysed.
2. Materials and methods 2.1. Apparatus The high-performance liquid chromatography (HPLC) measurements were carried out on a Merck Hitachi L-6200 A instrument. Merck Hitachi L-4500 diode array detector was used. Cadmium, lead and zinc determinations were conducted by means of inductively coupled plasma optical emission spectrometer (ICP-OES), Jobin Yvon, Ultima 2. For mineralization High Performance Microwave Digestion Unit Milestone MLS 1200 MEGA was used. 2.2. Reagents Acetonitrile, ethyl acetate, methanol, n-hexane, acetone, acetic acid, potassium phosphate dibasic, nitric acid (concentrated), hydrogen peroxide (30%), all analytical grade, (POCH S.A.––Poland). Acetonitrile, methanol and water for HPLC analysis (HPLC grade) were obtained from Merck (Darmstadt, Germany). Atrazine and simazine (more than 99.2%) were bought from Riedel–de Haen (Seelze, Germany). Stock standards solution of 1 mg ml 1 were prepared by dissolving 10 mg of respective herbicide in 10 ml of methanol and stored at 4 C. Working solutions were prepared daily, in methanol, by appropriate dilution. For solid phase extraction Bakerbond spe Aromatic Sulfonic Acid (3 ml, 500 mg) columns, Bakerbond spe Octadecyl (C18) (3 ml, 500 mg) columns, Bakerbond spe Poly(styrenedivinylbenzene)copolymer columns (3 ml, 500 mg) were used. Columns were obtained from J.T Baker (Deventer, Netherlands). Stock standards solutions of 1 mg ml 1 of cadmium, lead and zinc were purchased from Tritisol Merck
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(Darmstadt, Germany). Redistilled water from quartz apparatus was used. All glassware, before use, were washed with distilled water, soaked in nitric acid (30%), then rinsed in redistilled water and air-dried. Items were kept in clean place, to avoid contamination.
Table 1 Source of analysed samples Trophic chain
Kind of sample
Origin of sample
1
Soil Grass Milk
Kobio´r, Silesia
2
Soil Grass Milk
Wilkowyje, Silesia
3
Soil Grass Milk
Wartogłowiec, Silesia
4
Soil Grass Milk
Jaszowice, Silesia
5
Soil Grass Milk
_ ´ w, Silesia Zwako
6
Soil Grass Milk
Cielnice, Silesia
7
Soil Grass Milk
Tarno´w, the Malopolska Province
8
Soil Cereal Egg
Wilcza, Silesia
9
Soil Cereal Egg
Wo´jtowa Wies´, Silesia
10
Soil Cereal Egg
Wo´jtowa Wies´, Silesia
11
Soil Cereal Egg
Brzeziny, the Welkopolska Province
12
Soil Strawberry
Pyskowice, Silesia
13
Soil Strawberry
Pyskowice, Silesia
14
Soil Sweet cherries
Chorzo´w, Silesia
15
Soil Sweet cherries
Chorzo´w, Silesia
16
Soil Sweet cherries
Ocieszyn, the Welkopolska Province
2.3. Determination of atrazine and simazine by HPLC method The procedures of preparing soil, grass, cereals, fruits, milk and eggs for herbicide determination by means of HPLC and heavy metals determination by means of ICP were worked out. The stainless steel column (25 cm · 4.6 mm ID) packed with C18 ODS chemical bonded phase, particle size 5 lm was employed to carry out HPLC analysis. Atrazine and simazine separation and determination was performed using isocratic elution at flow rate of 0.8 ml min 1. The mobile phase contained methanol:water (1:1). Absorbance was measured continuously in the 200–400 nm range by diode-array detector. Quantitative measurements of peaks areas by 224 nm were carried out in order to achieve maximum sensitivity. Soil, crops, grass samples were spiked with atrazine and simazine in order to determine the repeatability, precision and recovery. Determination limits for the chromatographic method carried out of 0.5 lg g 1 and 0.8 lg g 1 for atrazine and simazine, respectively. 2.4. Determination of cadmium, lead and zinc by ICP-AES method Determination of cadmium, lead and zinc by means of ICP were performed with following parameters: power 1000 W, normal speed of pump: 20, plasma flow rate 12 l min 1, coating gas flow rate: 0.2 l min 1, nebulisator flow rate: 0.7, nebulisator pressure 3.0 bar. Standards were prepared from 1 mg ml 1 solutions and diluted with water. Wavelengths were 214.438 nm for cadmium, 220.353 nm for lead, 213.856 nm for zinc. Detection limits for cadmium 5 · 10 3 lg g 1, for lead 1 · 10 2 lg g 1, and for zinc 0.2 · 10 3 lg g 1, were obtained. 2.5. Samples Sample materials was selected to obtain a picture of the trophic chains (for example: soil, then grass, that grew on this soil, finally milk from the cow which was fed on this grass), which are listed in Table 1. Samples were collected in industrial areas (Silesia) as well as from eco-regions (the Wielkopolska Province, the Malopolska Province) in Poland (Fig. 1).
2.6. Samples treatment Soil (from filed about one ar) was collected, according to the norm PN-ISO 11466, to plastic bags and
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Fig. 1. Map of Poland.
transport to laboratory as fast as possible, then airdried, pulverized and sieved through 0.102 mm mesh. Grass, cereals from fields (about one ar), strawberries (from different places of plantation, about one ar), sweet cherries, early variety and late variety, (from different places of a orchard) were collected to paper bags. Fruits were air-dried and ground. Milk was collected to clean glass bottles, eggs to plastic boxes. All samples were transported to laboratory as fast as possible.
2.6.1. Samples preparation for HPLC analysis 2.6.1.1. Soil and grass. Hundred grams of sieved soil was placed in 300 ml shake-flask filled with 100 ml of chloroform; 15 g of dry, ground grass was placed in 300 ml shake-flask filled with 100 ml of chloroform. After 24 h the mixtures were shaken over 2 h. Then the mixtures were filtered through filter funnels and the filtrates were evaporated to dryness. Five millilitre of acetonitrile was added into each of the residues (Bakerbond Application
Fig. 2. Chromatogram of milk sample, after spe treatment. (1) simazine (tR = 7.87), (2) atrazine (tR = 11.59).
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Note) and the obtained solutions were then filtrated through soft filters. Solid phase extraction of these samples were performed by using of Bakerbond spe Aromatic Sulfonic Acid columns. The columns were conditioned with 3 ml of acetic acid/HPLC grade water (1:99). Twenty-five millilitre of acetic acid/HPLC grade water (1:99) and 5 ml of soil (grass) filtrate were mixed and aspirated through the column at 5 ml min 1. Next each column was washed with 2 ml of acetic acid/HPLC grade water (1:99), subsequently they was washed with 1 ml of acetonitrile, followed by 3 ml of HPLC grade water, followed by 1 ml of 0.1 M potassium phosphate dibasic. The columns were allowed to air dry under vacuum over about 2 min after each addition. Triazine herbicides were eluted with 2 ml of acetonitrile/0.1 M potassium phosphate dibasic (1:1). 2.6.1.2. Cereals. Fifty millilitre of acetone was poured over 50 g of grounded cereals in 300 ml shake-flask. After 24 h it was shaken over a period of 2 h. Next, it was filtrated through filter funnel and the filtrate evaporated to dryness. Five millilitre of methanol was added to the residue, then SPE was carried out on Bakerbond spe Octadecyl columns. The column was conditioned with 6 ml of methanol, next 6 ml of water. Ten millilitre of water and 5 ml of cereals filtrate were mixed well and aspirated through the column at 5 ml min 1. Next the column was washed with 3 ml of water and allowed to air dry under vacuum for about 5 min. Triazines were eluted with 3 ml of methanol. 2.6.1.3. Cherries. To 100 ml shake-flask 10 g of dry and ground fruit and 20 ml of acetone were added. After 24 h it was shaken for 2 h then filtrated, next SPE was performed. Bakerbond spe Poly(styrenedivinylbenzene)copolymer column was conditioned with 6 ml of ethyl acetate, 6 ml of methanol, finally 8 ml of water. An acetone extract was aspirated through the column at 2 ml min 1. Next column was washed with 3 ml of acetone. Pesticides were eluted with 6 ml of ethyl acetate:acetone (9:1) without vacuum. 2.6.1.4. Strawberries. Fifty grams of strawberries and 20 ml of methanol were placed into shake-flask and shaken for 1 h and then filtrated. The mixture consisting 10 ml of filtrate and 40 ml of water was aspirated through the Bakerbond spe Octadecyl columns, previously conditioned with 5 ml of methanol. Subsequently, the column was washed with 3 ml of water and left to air dry under vacuum for about 10 min. Triazine herbicides were eluted with 3 ml of dichloromethane (Topuz et al., 2005). Next, the solvent was evaporated and the dry residue was dissolved in 1 ml of methanol. 2.6.1.5. Milk. One hundred and fifty millilitre of hexane:acetone (2:1) was poured over 20 ml of milk. The
mixture was shaken for 0.5 h. Next the layers were separated in separating funnel. The lower aqueous layer Table 2 Distribution of simazine and atrazine in trophic chains Trophic chain
Material
Concentration [lg g 1] Simazine
Atrazine
1
Soil Grass Milk
27 ± 1.15 31.93 ± 2.01 15.29 ± 1.07
2
Soil Grass Milk
2.38 ± 0.13 27.15 ± 0.65 n.d.
3
Soil Grass Milk
1.72 ± 0.02 19.03 ± 0.18 2.32 ± 0.02
4
Soil Grass Milk
n.d. n.d. n.d.
n.d. n.d. n.d.
5
Soil Grass Milk
7.71 ± 0.03 17.95 ± 0.07 n.d.
n.d. n.d. n.d.
6
Soil Grass Milk
5.77 ± 0.21 88.32 ± 3.77 2.53 ± 0.56
n.d. n.d. n.d.
7
Soil Grass Milk
3.50 ± 0.03 5.1 ± 0.87 n.d.
3.48 ± 0.05 7.85 ± 0.14 n.d.
8
Soil Maize Egg
45.48 ± 2.10 387.15 ± 20.11 57.49 ± 3.78
n.d. n.d. n.d.
9
Soil Barley Egg
4.45 ± 0.23 10.98 ± 1.05 30.14 ± 2.00
19.59 ± 0.51 43.08 ± 3.50 52.17 ± 4.12
10
Soil Wheat Egg
25.97 ± 0.69 81.59 ± 5.45 50.47 ± 2.13
n.d. 1.88 ± 0.08 n.d.
11
Soil Wheat Egg
57.89 ± 2.47 65.48 ± 3.68 59.48 ± 5.78
n.d. n.d. n.d.
12
Soil Strawberry
2.69 ± 0.18 5.29 ± 0.58
2.74 ± 0.19 n.d.
13
Soil Strawberry
8.23 ± 0.45 6.19 ± 0.13
1.38 ± 0.11 n.d.
14
Soil Sweet cherry
3.90 ± 0.18 2.97 ± 0.07
0.69 ± 0.09 n.d.
15
Soil Sweet cherry
3.52 ± 0.21 3.63 ± 0.12
0.58 ± 0.1 n.d.
16
Soil Sweet cherry
2.69 ± 0.12 2.45 ± 0.19
2.74 ± 0.07 3.04 ± 0.10
n.d.––not detected.
n.d. n.d. n.d. 13.85 ± 0.31 23.85 ± 0.02 1.77 ± 0.05 n.d. n.d. n.d.
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was removed to waste (Burke et al., 2003). The upper layer was added to another portion of milk. The procedure was repeated three times. After this, the upper layer was evaporated and the residue was dissolved in 5 ml acetonitrile. The SPE was performed on Bakerbond spe Aromatic Sulfonic Acid column. The SPE procedure was the same as for grass and soil. 2.6.1.6. Eggs. An egg (yolk and white, without eggshell) was extracted with 50 ml of acetone for 2 h by means of a shaker. Next the mixture was centrifuged, the acetone layer was evaporated to dryness, the residue dissolved in 5 ml of methanol and SPE solid phase extraction on Bakerbond spe Octadecyl columns was performed. The SPE procedure was the same as for cereals. Each time the completeness of extraction was checked. From each sample three samples were prepared simultaneously and all samples were measured three times. An aliquot of 20 ll of the sample was injected for HPLC measurement. 2.6.2. Samples preparation for ICP analysis 2.6.2.1. Soil and milk. 0.5 g of dried, sieved soil and 0.5 g of dried to constant weight milk samples were mineralised in microwaves in HNO3:H2O2 4:1 and 5:3 for soil and milk respectively. The digest was diluted with 20 ml of distilled water, filtrated and transferred to 25 ml volumetric flask. The volume made up with redistilled water to the mark. 2.6.2.2. Grass, cereals, fruits and eggs. Two grams of dry and ground grass, seeds of cereals, dry fruit, 0.5 g of previously dried to constant weight eggs were burnt to ashes in furnace (T = 350 C). Next, 5 ml of the mixture of HNO3:H2O (1:1) was added to the residue. Subsequently, the sample was filtrated and quantitatively transferred to a 25 ml volumetric flask. The volume made up to the mark with redistilled water.
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From each material three samples were prepared simultaneously and all samples were measured three times.
3. Results and discussion From among methods of extraction of triazines described in literature (Tadeo et al., 2000, review), in this research, the best quantitative extraction of atrazine and simazine from soil and grass were obtained using chloroform, whereas from fruit and cereals using acetone. We examined different kinds of SPE column filling. For grass, soil and milk the most suitable were Bakerbond spe Aromatic Sulfonic Acid columns. For cereals and eggs Bakerbond spe Octadecyl (C18) columns were chosen, whereas for fruit (strawberries and sweet cherries) adequate was Bakerbond spe Poly(styrenedivinylbenzene)copolymer columns. The mobile phase methanol:water (1:1) was used previously by authors for determination simazine, propazine, heksasinon, bromacil, metoksuron (Pieszko and Baranowska, 2000). Nevertheless, this mobile phase has not been use before for determination of atrazine and simazine simultaneously. Chromatograms were recorded continuously in the 200–400 nm range, but the determination wavelength at 224 nm was selected as the best because of its optimum sensitivity for determination. The retention time for atrazine and simazine standars was amount 7.76 min and 11.49 min, respectively. However, retention time for soil and biological samples was in range 7.51–8.05 for simazine and 11.50–12.00 min for atrazine and simazine. To create calibration curves standard compounds were diluted with methanol to give concentration 0.5–100 lg g 1. In this range the curves equations were linear.
Fig. 3. Simazine and atrazine in trophic chain samples.
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The regression equations of peak area and injection concentration [lg g 1] were determined with correlation
coefficient of 0.9988 and 0.9998 for atrazine and simazine, respectively. The recovery of simazine from soil,
Table 3 Distribution of cadmium, lead and zinc in trophic chains Trophic chain
Material
Concentration [lg g 1] Cd
1
Pb
Soil Grass Milk
5.89 ± 0.06 (170.16 ± 6.78) · 10 (19.38 ± 9.38) · 10
2
Soil Grass Milk
3
Zn
62.50 ± 0.50 0.16 ± 0.06 1.76 ± 0.43
424.5 ± 15.50 35.20 ± 0.36 20 ± 1.25
1.16 ± 0.04 (292.25 ± 12.23) · 10 3 (15.62 ± 1.38) · 10 3
151.50 ± 5.50 7.69 ± 0.19 1.16 ± 0.64
129.5 ± 6.00 47.67 ± 2.12 27.5 ± 0.62
Soil Grass Milk
1.48 ± 0.09 (263.72 ± 3.75) · 10 (5.88 ± 5.62) · 10
47.46 ± 1.00 136.57 ± 2.50 1.18 ± 0.23
126.9 ± 4.50 45.31 ± 1.25 34.38 ± 1.88
4
Soil Grass Milk
1.48 ± 0.12 (627.24 ± 11.60) · 10 3 (25.00 ± 1.31) · 10 3
52.48 ± 1.00 3.13 ± 0.10 2.65 ± 0.57
128.45 ± 6.00 55.87 ± 2.00 28.75 ± 1.25
5
Soil Grass Milk
0.58 ± 0.02 (473.51 ± 9.25) · 10 (61.88 ± 2.06) · 10
78.50 ± 2.00 8.05 ± 0.33 3.74 ± 0.27
71.5 ± 3.00 55.74 ± 2.87 20.6 ± 31.25
Soil Grass Milk
5.60 ± 0.16 (114.29 ± 4.12) · 10 n.d.
137.50 ± 2.50 3.39 ± 0.12 2.49 ± 0.14
396 ± 24.00 42.22 ± 0.87 30.63 ± 0.62
Soil Grass Milk
4.68 ± 0.13 (152.16 ± 6.14) · 10 n.d.
6
7
3 3
3 3
3 3
3
4
57 ± 1.03 7.86 ± 0.56 1.47 ± 0.11
97.74 ± 4.15 38.48 ± 0.89 25.12 ± 0.75
8
Soil Maize Egg
0.22 ± 0.02 0.27 ± 0.03 0.12 ± 0.01
0.57 ± 0.05 4.56 ± 0.12 5.79 ± 0.52
24.37 ± 2.46 28.78 ± 2.48 32.01 ± 2.87
9
Soil Barley Egg
0.13 ± 0.008 0.20 ± 0.01 0.15 ± 0.004
1.11 ± 0.09 1.56 ± 0.18 7.16 ± 0.20
9.15 ± 1.48 14.94 ± 0.38 19.03 ± 1.43
10
Soil Wheat Egg
0.25 ± 0.009 0.31 ± 0.01 0.14 ± 0.007
n.d. 1.05 ± 0.3 9.6 ± 0.20
10.46 ± 1.46 16.65 ± 0.22 15.67 ± 0.02
11
Soil Wheat Egg
1.47 ± 0.3 5.47 ± 0.57 6.78 ± 0.72
26.48 ± 2.38 25.47 ± 2.91 22.47 ± 3.05
12
Soil Strawberry
0.20 ± 0.01 0.23 ± 0.015
58.12 ± 3.45 61.48 ± 4.05
37.33 ± 1.48 14.94 ± 0.44
13
Soil Strawberry
0.37 ± 0.02 0.24 ± 0.01
49.75 ± 2.78 53.46 ± 3.48
34.6 ± 11.91 18.88 ± 0.14
14
Soil Sweet cherry
0.13 ± 0.006 0.59 ± 0.005
85.08 ± 4.21 87.36 ± 5.01
49.73 ± 0.21 15.86 ± 0.19
15
Soil Sweet cherry
0.15 ± 0.008 0.61 ± 0.005
80.69 ± 4.63 82.69 ± 3.65
45.46 ± 3.45 18.46 ± 2.33
16
Soil Sweet cherry
0.25 ± 0.03 0.31 ± 0.05
42.68 ± 1.87 21 ± 1.58
28.79 ± 2.85 15.47 ± 1.47
n.d.––not detected.
n.d. n.d. 0.11 ± 0.01
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Fig. 4. Heavy metals in trophic chain samples.
grass and cereals were 75.6%, 72.45%, 76.78% respectively, whereas for atrazine the recovery carried out 70.1% for soil, 72.45% for grass and 71.45% for cereals. An example of chromatogram of milk sample was shown in Fig. 2. Results and standard deviations of the determination of simazine and atrazine by HPLC method are listed in Table 2. Concentration of herbicides in milk, fruits and eggs are given in lg g 1 wet weight, whereas in soil, grass and cereals in lg g 1 air-dried weight. Simazine was detected in most of the samples, whereas atrazine was detected only in few samples. This is a result of simazine more commonly being used in pesticide preparations in Poland. It should be emphasized, that content of analysed pesticides are caused by their ability to translocation, only one of the cultivation (trophic chain 8) was treated with simazine preparation within past two years. The highest concentration of simazine was detected in grass (5–88 lg g 1) and cereals (10.98–387 lg g 1) (Fig. 3).1 It is related to tendency of these herbicides to accumulate in plants. Very high content of simazine in maize (387 lg g 1) is caused by using herbicides preparation to combat weeds on this field. The concentration of herbicides determined in milk was the lowest (2.32–15.29 lg g 1), what testifies favourable homeostasis system of cows, although it is contaminated as well. Nevertheless, the concentration of simazine in eggs was higher than in soil, but considerably lower than in henÕs feed––cereals. Higher concentration of pesticides in samples from the Wielkopolska Province, in comparison with the Malopolska Province was noticed. It is probably related to 1
Results obtained from trophic chain number 8 were omitted, because this cultivation was treated with pesticides within past two years.
common applying of pesticides in Western Europe in relation to East Europe. Results and standard deviations of the determination of heavy metals in trophic chain samples by ICP method are listed in Table 3. Concentration of metals in milk, fruits and eggs are given in (lg g 1) wet weight, whereas in soil, grass and cereals in (lg g 1) air-dried weight. Comparing the concentration of heavy metals in the analysed trophic chains (Fig. 4), the lowest level cadmium and lead was detected in milk (for Cd 8.88 · 10 3–61.88 · 10 3 lg g 1, for Pb 1.16–3.74 lg g 1). There was lower concentrations of metals in cereals (Cd 0.20–0.31 lg g 1, Pb 1.05–5.47 lg g 1, Zn 14.94– 28.78 lg g 1) in comparison with soil (Cd 0.13–5.89 lg g 1, Pb 0.57–151.50 lg g 1, Zn 9.15–424.5 lg g 1). In interrelation soil–cereals–eggs, the lowest level of cadmium was determined in eggs (Cd 0.11–0.15 lg g 1). Nevertheless, the content of metals strongly depends on samples origin (vicinity of motorway––trophic chain 6, heavy industry––trophic chain 2). Monitoring of heavy metals distribution in soil, plants as well as in food of animal origin makes it possible to determine the level of contamination of foodstuffs, thereby enable to choose the least contaminated food.
4. Conclusion Conducted research proved that, demonstrated HPLC method is suitable for the determination of atrazine and simazine in all analysed materials for the sake of good separation of peaks, without interference other compounds of matrix. Prepared extraction methods for particular trophic chains allowed to quantitative extraction of herbicides and determination with good repeatability and precision.
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