Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI. ELECTRONIC JOURNAL OF POLISH AGRICULTURAL UNIVERSITIES 2007 Volume 10 Issue 4 Topic: Biology Copyright Wydawnictwo Akademii Rolniczej we Wroclawiu, ISSN 1505-0297 KrzyŜanowski R., Leszczyński B. 2007. DETERMINATION OF (4-CHLORO-2-METHYLPHENOXY)ACETIC ACID RESIDUES IN SOIL UNDER CEREAL CULTIVATION OF WINTER WHEAT, EJPAU 10(4), #40. Available Online: http://www.ejpau.media.pl/volume10/issue4/art-40.html DETERMINATION OF (4-CHLORO-2-METHYLPHENOXY)ACETIC ACID RESIDUES IN SOIL UNDER CEREAL CULTIVATION OF WINTER WHEAT Robert KrzyŜanowski, Bogumił Leszczyński Department of Biochemistry and Molecular Biology, University of Podlasie, Siedlce, Poland ABSTRACT An analytic method for determination of (4-chloro-2-methylphenoxy)acetic acid (MCPA) residues in soil under cereal cultivation was elaborated. Estrification of the herbicide with methanol has been performed and followed by solid phase microextraction (SPME). Obtained results showed traces of MCPA within the studied soil samples up to 8 weeks, after the herbicide treatment. The herbicide moved within the soil profile to 20 cm of depht. Possible application of the SPME/GC-MS method for monitoring of the MCPA residues within soil under winter wheat cultivation is discussed. Key words: chlorophenoxy herbicides, winter wheat, MCPA, SPME, GC-MS. INTRODUCTION Soils are open systems and thus accumulate a numerous anthropogenic compounds. On the other hand, more than 100.000 organic chemicals are used in industry, agriculture and households worldwide [1] and the knowledge about their influence on environment is incomplete or unknown [2]. Chlorophenoxy acids e.g. MCPA, 2,4-D are used widely in agriculture as selective herbicides (Table 1), and have a harmful effect to the environment [3]. Generally the MCPA (4-chloro-2-methylphenoxyacetic acid, CAS No. 94-74-6) is usually applied as a 2-ethylhexylester or dimethylamine salt form, mainly on cereal crops, such as wheat and barley, at the post-emergence growth phase. Sowinski et al. [4] showed that Polish farmers protect winter wheat in 65% and spring cereals in about 90% with commercial formulations contained MCPA. On the other hand, KrzyŜanowski and Leszczyński [5] noted that MCPA applied in form of commercial preparation such as Chwastox well controlled majority of cereal weeds. Although these phenoxyherbicides have been used since late 1950s, not much attention has been paid to their pollution in the field conditions [6,7]. Table 1. Comparison of some properties of the chlorophenoxy herbicides HERBICIDE PROPERTIES MCPA CHLOROPHENOXY HERBICIDES 2,4-D Chemical name CAS (4-chloro-2-methylphenoxy)acetic acid (2,4-dichlorophenoxy)acetic acid
CAS number 94-74-6 94-75-7 Formula Molecular weight 200.62 221.04 Water solubility 825 mg/l at 25 C (acid) 900 mg/l at 25 C (acid) Solubility in other solvents ether, ethanol, toluene, xylene, methanol Ethanol, diethyl ether, toluene, xylene Melting point 118-119 C 140.5 C Vapor pressure 0.2 mpa at 20 C 0.02 mpa at 25 C (acid) Liquid-liquid extraction (LLE) and solid phase extraction (SPE) have been traditionally used for the herbicide residue extractions from environmental samples [8]. However, LLE method requires a large solvent volume and long preparation time, instead SPE is less time-consuming than LLE, but still requires toxic organic solvents for the elution step [9,10]. Thus, a new extraction technique, solid phase microextraction (SPME) has been introduced by Pawliszyn et al. [11,16]. Krutz et al. [9] showed that application of such technique is possible for determination of volatile compounds and special type of DI-SPME combined with GC-MS for analysis the herbicide residues. The chlorophenoxy herbicide residues were found in trace amounts within the plant tissues and surface waters [17], but little is known about their presence in soil under cereal cultivation. In the present paper we report on possible application of the SPME/GC-MS technique for determination of the (4-chloro-2-methylphenoxy) acetic acid (MCPA) residues within the soil under winter wheat cultivation. MATERIAL AND METHODS Reagents Methanol, ethyl ether (Sigma), commercial preparation of Chwastox Extra 300 SL, containing 30% of MCPA as sodium salt and 70% of nonspecified ingredient was purchased from Organika Sarzyna Nowa Sarzyna, Poland. All chemicals were stored in the darkness at 4 C. Apparatus GC-MS analysis was performed on a Shimadzu series GC 17A gas chromatograph equipped with split/splitless injector and interfaced to QP-5050 Shimadzu mass spectrometer. The carrier gas was helium. The GC was equipped with a capillary column BPX-5 (30 m x 0.25 mm I.D. with 0.25 µm film thickness) (Phenomenex, UK) connected to the split/splitless injector. Separation and detection conditions The optimised oven temperature program was at 80 C (5 min), then from 80 C to 280 C (at 20 C/min) and final temperature was held for 5 min. A column head pressure of 56.7 kpa and an injector temperature of 220 C were applied. Helium was used as the carrier gas at 9.8 ml/min. Mass spectrophotometer was operated in the electron impact ionization (EI), at 70 ev. Mass spectra were acquired in the mass range from m/z 50 to 450. Detection of MCPA was also accomplished in selected ion monitoring (SIM) mode, using the following fragment ions m/z 141, 155, 214 (methyl-4-chloro-2-phenoxyacetate). This instrumentation was controlled by software CLASS 5000 with NIST Mass Spectra Database 107 and 21 (NIST, Gaithersburg, MD, USA). Solid phase microextroextration 75 µm CAR/PDMS fiber was conditioned before initial application into injection port of the gas chromatograph by heating at 280 C for 1 hour. Then the fiber was exposed to the stirred sample with addition of 0.5% NaCl for an optimal adsorption time of 20 min at 50 C. When the adsorption was completed the fiber was removed from the sample and introduced into the GC injector where the thermal desorption of the analytes at 220 C for 6 min was carried out. Field experiments The experimental field of winter wheat Roma cv. located near Siedlce (central-eastern Poland) was sprayed out with 300 g/l MCPA (3 l/ha commercial preparation of the Chwastox Extra 300 SL). The spraying was carried out at growth stage 43 of the winter wheat according to the BBCH codes [18]. Then 20 g of soil samples from surface layer about 0.5 cm of depth and deeper levels (every 5 cm down into 30 cm of the depth) were collected after 1, 2, 3, 5, 19, 33, 61, 90
days. Prepartion of the soil sample 5 g of the soil was shaken mechanically for 10 min with 100 ml ethyl ether-water (90:10 v/v). Then the mixture was filtered under a vacuum through a Whatman No 1 filter paper on Buchner funnel and the extracts were evaporated to dryness at 40 C on a rotary evaporator. The residue was redissolved in 1 ml of methanol and then the solid phase microextraction was performed. All the performed assays were done in three independent replications. RESULTS AND DISCUSSION The carried out analyses showed presence of the (4-chloro-2-methylphenoxy)acetic acid within the studied soil. After the GC-MS separation of the soil samples a single peak of the methyl-4-chloro-2-phenoxyacetate was identified after comparison of its MS spectrum with NIST Mass Spectra Database 107 (Fig. 1). Fig. 1. Comparison MS spectrum of the MCPA extracted from the studied soil with MS spectrum of MCPA from NIST Mass Spectra Database 107
Performed analyses showed different level of the accumulated MCPA within profile of the brown soil under the winter wheat cultivation. The MCPA residues were present in the surface layer of the studied soil over two months. During the first week after treatment level of the herbicide residues gradually increased and moved within the studied soil. One of the possible reason of the herbicide movement was raining conditions during the conducted experiment. Concentration of the herbicide was still detectable until the end of the first month after the field treatment of the observation and during the second month completely declined (Fig. 2). When dipper layers of the soil were studied, the highest accumulation of the pesticide was found during the first five days and reached up to 0.36 ľg/l. After the first week of
the treatment amount of the herbicide declined and it was happend much faster within the deeper soil layers (Fig. 2). Fig. 2. Movement of the MCPA within soil profile under the winter wheat cultivation (mean values; ± SD). The obtained results showed that DI-SPME/GC-MS is an accurate and sensitive analytical method for analysis of the MCPA residues in soil under the cereal cultivation. It is the first evidence that such technique might be useful in determination of the chlorophenoxy herbicide residues in soil. Hitherto the SPME was mostly used in extraction from soil, such compounds as organophosphorous pesticides [19,21], organochloride pesticides [22], triazines [23], nitrogen herbicides [24] and other group pesticides [25]. On the other hand, SPME extraction was suggested to be used in degradation of diesel fuel [26], determination of volatile and semivolatile pollutants [27] and photolytic degradation study of soil matrices [28]. Since, the applied MCPA was moved within soil under the wheat cultivation it is important to stress farmers, plant protection and environmental officers that application of the chlorophenoxy herbicides has to be carefully control. It is especially important in cases when the treated commercial fields are located in neighborhood of drinking water reservoirs. CONCLUSIONS The proposed technique of the SPME/GC-MS might be used in routine monitoring and determination of the chlorophenoxy herbicide residues within soils under the winter wheat cultivation.
ACKNOWLEDGEMENTS This work was supported by the Ministry of Science and Higher Education in the framework of a grant no 2P06S 058 30. REFERENCES 1. Koch R., Umweltchemikalien, VCH Verlagsgesellschaft, Weinheim 1991. 2. Welp G., Brummer G.W., Ecotox. Environ. Safety 1999, 43, 83-90. 3. Santos-Delgado M.J., Crespo-Corral E., Polo-Diez L.M., Talanta 2000, 53, 367-377. 4. Sowiński J., Kozak M., Paszkiewicz-Jasińska A., Ochr. Rosl. 2000, 12, 41-43. (In Polish). 5. Krzyzanowski R., Leszczynski B., in: Chemistry for Agriculture 7, Tech. Univ. Press, Wrocław 2006. 6. Van Ravenzwaay B., Pigott G., Leibold E., Food Chem. Tox. 2004, 42, 115-125. 7. Grabinska-Sota E., Wisniowska W., Kalka J., Crop Prot. 2003, 22, 355-360. 8. Tadeo J.L., Sanchez-Brunete C., Garcia-Valcarcel A.I., Martinez L., Perez R.A., J. Chromatogr. A. 1996, 754, 347. 9. Krutz L.J., Senseman S.A., Sciumbato A.S., J. Chromatogr. A. 2003, 999, 103-121. 10. Aguilar C., Penalver S., Pocurull E., Borrull F., Marce R.M., J. Chromatogr. A. 1998, 795, 105-115. 11. Arthur C.L., Pawliszyn J., Anal. Chem. 1990, 62, 2145. 12. Arthur C.L., Potter D.W., Butchholtz K.D., Motlagh S., Pawliszyn J., LC-GC 1992, 10, 656. 13. Arthur C.L., Pratt K., Motlagh S., Pawliszyn J., J. High Resolut. Chromatogr. 1992, 15, 741. 14. Zhang Z., Pawliszyn J., Anal. Chem. 1992, 64, 1960. 15. Eisert R., Levsen K., J. Chromatogr. A. 1996, 733, 143. 16. Lord H., Pawliszyn J., J. Chromatogr. A. 2000, 885, 153-193. 17. Krzyzanowski, R., Leszczynski, B., Herba Polonica 2004, 50, 95-100. 18. Hansen M.S., Bromand B., Schulz H., Faelles decimalscala for vaekstudviklingen af planter (BBCH skala). Statens Planteavlsforsøg, Grøn viden, 146-1995. 19. Magdic S., Boyd-Boland A., Jinno K., Pawliszyn J.B., J. Chromatogr. A. 1996, 736, 219-228. 20. Ng W.F., Teo M.J.K., Lakso H-,A., Fres. J. Anal. Chem. 1999, 363, 673-679. 21. Bouaid A., Ramos L., Gonzales M.J., Fernández P., Cámara C., J. Chromatogr. A. 2001, 939, 13-21. 22. Moreno D.V., Ferrera Z.S., Rodriguez J.J.S., Anal. Chim. Acta 2006, 571, 51-57. 23. Hu X., Hu Y., Li G., J. Chromatogr. A. 2007, 1147, 1-9. 24. Boyd-Boland A., Pawliszyn J.B., J. Chromatogr. A. 1995, 704, 163-172. 25. Möder M., Popp P., Eisert R., Pawliszyn J.B., Fres. J. Anal. Chem. 1999, 363, 680-685. 26. Eriksson M., Swartling A., Dalhamar G., Fäldt J., Borg Karlson A.-K., App. Microb. Tech. 1998, 50, 129-134. 27. Llompart M., Li K., Fingas M., Talanta 1999, 48, 451-459.
28. Concalves C., Dimou A., Sakkas V., Alpendurada M.F., Albanis T.A., Chemosphere 2006, 64, 1375-1382. Accepted for print: 5.12.2007 Robert KrzyŜanowski Department of Biochemistry and Molecular Biology, University of Podlasie, Siedlce, Poland Prusa 12, 08-110 Siedlce, Poland Phone/fax: +48 25 6431367 email: krzyzanowski_r@yahoo.pl Bogumił Leszczyński Department of Biochemistry and Molecular Biology, University of Podlasie, Siedlce, Poland B. Prusa 12, 08-110 Siedlce, Poland Phone/fax +48 25 644-59-59 email: leszczb@ap.siedlce.pl Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article. Main - Issues - How to Submit - From the Publisher - Search - Subscription