Gas chromatographic and gas chromatographic mass spectrometric study of the photodegradation processes of nitrogen-containing herbicides PhD theses Ms. Katalin Lányi Supervisors: Dr. Zoltán Dinya Dr. Lajos Papp University of Debrecen Debrecen, 2002
1. ITRDUCTI AD BJECTIVES The chemistry of pesticides has evolved from some simple compounds like the arsenic, lead and fluoride-compounds, and some naturally occurring organic materials, like pyrethrins and rotenone, to the wide range of compounds used in these days covering almost everything from the broad-spectrum effects to the very specific plant protection. In line with the unexpected spread of new compounds, the demand arose for controlling the utilisation and distribution of pesticides. After the pesticide gets into the soil, various physical and physicochemical processes, and chemical and biochemical reactions determine its fate. Photodegradation processes are involved in dissipation of pesticides in water, soils and plants. The relevance of photodegradation processes in the fate of the herbicides applied to the soil is due to their high water solubility and moderate persistence, which indicate that it might be found as an environmental contaminant in agricultural runoff waters, where photolysis processes play an important role. Triazines, ureas and thiolcarbamates are among the most used herbicides worldwide. Since they can be found in many environmental compartments, their fate in ecosystems and the characterisation of their degradation pathways are to be determined. The aim of this study was to assess the characteristics of phototransformation processes of nitrogen-containing herbicides (triazines: atrazine, cyanazine, terbuthylazine, and terbutryn; ureas: diuron, fenuron, chloroxuron, and methabenzthiazuron; and thiolcarbamates: buthylate, cycloate, EPTC, molinate, vernolate) in vivo. I aimed to determine the degradation rate of individual pesticides, to compare the degradation rates of compounds belonging to the same group in order to assess the structural effects, to find connection between the rate of degradation and the chemical structure of chemicals, and to identify the degradation products. Different additives were used to model the possible degradation pathways occurring within natural circumstances under the sunlight. These additives were photosensitisers (H 2 2, Ti 2 ), as well as, materials naturally occurring in the soils (montmorrilonite, humic acid). In order to gain detailed information about the photoreaction taking place under these circumstances, I conducted experiments for studying the charge transfer processes taking place in the presence of benzophenone. 2
2. MATERIALS AD METHDS The pesticide standards were dissolved in dichloro-methane, and illuminated by a highpressure mercury vapour lamp in a 1-cm silica cuvette equipped with Teflon cap. The degrading energy was 125 W, the degradation of triazines was also conducted by a 15-W mercury vapour lamp, in order to gain information on the effect of illuminating energy. By regular intervals samples were taken from the reaction vessel, and the degradation process was followed by consecutive GC measurements in order to determine the amount of degradation products and the original substance. The completely degraded mixtures were analysed by GC-MS in order to determine the chemical structure of the most important degradation products. 3. EW SCIETIFIC RESULTS 3.1 Chemical structure of the main degradation products The most significant processes of photodegradation of triazines are the partial or complete loss of side-chains, or rather the substitution of the heteroatom-containing side-chain to hydroxyl-group (figure 1.). Besides the consecutive processes, loss of the different side chains takes also place parallely, thus, different metabolites will be formed having mixed sidechains, until the cyanuric acid and 2-amino-4,6-dihydroxy-1,3,5-s-triazine formed by loosing all the side-chains. The presence dimer products, which could be detected during the degradation of all triazines proves the radical character of processes occurring during the photodegradation. The degradation rank of triazines was the following: terbutryn cyanazine atrazine terbuthylazine (figure 2.). The average degradation rate of individual compounds can be seen in the table 1. 3
Figure 1 - General photodegradation pathways of triazines H H R 1 R 1 H H 2 H H H H H H R 1 H 2 H H H 2 H H H H H H H Figure 2 - Degradation pattern of triazines at 125 W degrading energy 100,0 90,0 80,0 70,0 terbutryn cyanazine terbuthylazine atrazine % 60,0 50,0 40,0 30,0 20,0 10,0 0,0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 Photodegradation (hours) Increasing the degradation energy (15 W 125 W) has raised the degradation rate by 2-5 on the one hand, on the other hand, the chlorine containing metabolite which appeared also in the completely degraded mixture during the low-energy experiments has completely disappeared from the mixture, thus, the increased degrading energy favours the formation of less dangerous, nature identical metabolites. 4
Table 1 - Connection between the degradation rate of triazines and the degradation energy Compound Average degradation rate (v), if the degradation energy is 15 W 125 W v 125W/v 15W µg ml -1 h -1 rel. * µg ml -1 h -1 rel. * atrazine 20,6 2,73 45,4 1,30 2,20 cyanazine 17,1 2,28 81,5 2,34 4,77 terbuthylazine 7,6 1,00 34,8 1,00 4,58 terbutryn 63,6 8,48 280,0 8,04 4,40 * : compared to the degradation rate of terbuthylazine, which has the slowest degradation Loss and oxidation of the alkyl-chains are the dominant processes also during the degradation of ureas (figure 3.). Besides that, the substitution of halogen atom to a hydroxylgroup, and hydroxylation of the aromatic ring are the secondary processes. The high ratio of dimer products suggests that in the photodegradation of ureas the radical processes dominate. The well known carcinogenity of azo-compounds assigns outstanding importance to this fact. Figure 3 - General photodegradation pathways of ureas H 2 R 1 H R 1 (H)n ' H H 2 H 2 H R 1 H H H H2 H H H R 1 ' H H ' The degradation rate of ureas was the following: methabenzthiazurone chloroxuron fenuron diuron (figure 4.). The average degradation time of individual compounds can be seen in the table 2. 5
Table 2 - Average degradation rates of ureas Compound Average degradation rate (v) µg ml -1 h -1 rel. * diuron 31,0 1,00 fenuron 55,7 1,80 chloroxuron 164,5 5,31 methabenzthiazurone 766,7 24,73 * : compared to the degradation rate of diuron, which has the slowest degradation Figure 4 - Degradation pattern of ureas at 125 W degrading energy 100,0 90,0 80,0 70,0 diuron fenuron chloroxuron methabenzthiazuron 60,0 % 50,0 40,0 30,0 20,0 10,0 0,0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 Photodegradation (hours) In the case of thiolcarbamates, the most frequent processes are the α- and β-oxidation of alkyl-groups connecting to the nitrogen atom. In the most cases, the -formyl and - dealkylated products were identified in the degradation mixture (figure 5.). The thiolderivatives were formed in very small amounts. Vernolate is the only studied thiolcarbamate having thiopropil-group instead of thioetil connected to the carbonyl-c. The -dealkylation and oxidation of the CH 2 -group connected to the nitrogen occur in this case, too. It can be stated based on the experiments, that the tioalkyl-group shows fair stability under the circumstances of photodegradation. Its partly or completely degraded products can not be detected, or represent only very small part of the mixture. 6
Figure 5 - General photodegradation pathways of thiolcarbamates C SH C S R 1 H C S R 1 ' C C S R 1 HC C S R 1 HC ''' C S R 1 '' C CH 2 C S R 1 C S R 1 ' CH The degradation rate of thiolcarbamates was the following: butylate EPTC vernolate molinate cycloate (figure 6.). The average degradation time of individual compounds can be seen in the table 3. Table 3 - Average degradation rates of thiolcarbamates Compound Average degradation rate (v) µg ml -1 h -1 rel. * butylate 625,0 2,50 cycloate 250,0 1,00 EPTC 438,5 1,75 molinate 277,8 1,11 vernolate 312,5 1,25 * : compared to the degradation rate of cycloate, which has the slowest degradation In the case of experiments with additives, the charge transfer complex formed between the herbicide molecule and benzophenone is expected to promote the degradation, since it increases the efficiency of energy transfer. The results of experiments supported this expectation, since the degradation methabenzthiazurone was the fastest in the presence of benzophenone. H 2 2 can have two different effects: it initiates the formation of hydroxylradicals, but it also consumes them. At lower concentrations the earlier process dominates, at higher concentrations the latter. The H 2 2 concentration used in my experiments (70 µl ml -1 ) belongs clearly to the second category. 7
Figure 6 - Degradation pattern of thiolcarbamates at 125 W degrading energy 100,0 90,0 80,0 70,0 butylate cycloate EPTC molinate vernolate % 60,0 50,0 40,0 30,0 20,0 10,0 0,0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fotobontás (óra) n the score of this, it can be stated that since radical processes play important role in the photodegradation of studied herbicides presence of radical-trap compounds especially in higher amounts can significantly increase the residence time of these xenobiotics in the environment. Ti 2, montmorrilonite and humic acid sensitize by adsorbing the herbicides on their surface, and loosening their bonds. The various, composite aromatic compounds of humic acid, as polyphenols, organic acids, etc. similarly to the benzophenone can also form charge transfer complexes with the organic compounds, further sensitizing them for the photodegradation. At the same time, these additives since they form heterogeneous system with the sample can also have inhibiting effect, since the grains can absorb the light, thus decreasing the amount of degrading energy on the examined molecule. The overall effect depends on the compositions of examined compound, montmorrilonite, or humic acid, and since these latter two are multiparameter attributes, it is hard to predict the effect to be experienced. The overall effect of humic acid utilised in my experiments was the moderate inhibition, thus, the adsorption effect exceeded the sensitizing effect. Since the humic acid herbicide ratio occurring in the soils falls to the same magnitude as utilised in this experiments, presumably this kind of effect can take place in the soils, too. Ti 2 and montmorrilonite have 8
slightly increased the rate of degradation, however, in the case of Ti 2 this effect can not be considered significant (figure 7.). Figure 7 Photodegradation of methabenzthiazurone in the presence of various additives 1,200 MBTA [mg ml -1 ] 1,050 0,900 0,750 0,600 0,450 0,300 hydrogen-peroxyde humic acid control charge transfer complex montmorrilonite titanium-dioxyde 0,150 0,000 0 0,3 0,6 0,9 1,2 1,5 1,8 2,1 2,4 2,7 3 3,3 3,6 3,9 4,2 Óra fotobontás The experiments carried out with additives led to an other important consequence: these materials changed not only the time-course of degradation, but there was also a dramatic shift in the ratio of metabolites. In the presence of additives, the emphasis is on the metabolites of greater size instead of the smaller compounds. The experiments carried out without additives suggested a reassuring picture that the studied herbicides have dominantly natural identical metabolites with low ecotoxicology. The ratio of metabolites representing high toxicity, carcinogenity or other kind of risks was infinestimal, or they were not stable enough for remaining in the reaction mixture. However, the experiment with the additives showed clearly, that the natural or artificial additives can shorten the time of degradation, but their presence can lead to the formation and persistence of less degraded, toxic materials more extraneous for the environment. Moreover, under natural circumstances, many other processes can occur in the soil, on the effect of microbiological processes -oxidation can occur on the nitrogen atom of the studied compound. n the one hand, this leads directly to the formation of nitroso-compounds, which 9
have high toxicity and carcinogenity. The nitrite- and nitrate-ions of soil can react with these nitroso-compounds, thus forming -nitroso-amines, which are even more toxic and carcinogen, that the nitroso-compounds. In the European Union the quantity and quality of - nitroso-amines shall be strictly checked in food and raw materials. Resulting from the tight connection between the plant and the soil, the measurement of -nitroso-amines in the soils is inevitable. Since these processes are dependent on the soil type, local level investigations shall be carried out. In the case of these kind of knowingly utilised xenobiotics like the pesticides in vivo experiments are required for analysing the environmental effect (water, soil, and microorganisms). since the in vitro experiments leave open too much question about the real fate of the compounds in the environment. Since these, this research shall be continued in order to gain more detailed information about the transformation, reactions, metabolites, and derivatives of the nitrogen containing herbicides. 10
Publication of Ms. Katalin Lányi (born Katalin Varró): Referenced publications: 1. Z. Dinya, Gy. Litkei, J. Jekő, K. Varró, S. Antus: GC-LC-MS Studies of the Extracts of Buds of Populus igra. In: Flavonoids and Bioflavonoids 1995 [Proceedings of the International Bioflavonoid Symposium. 9 th Hungarian Bioflavonoid Symposium] (Editors: S. Antus, M. Gábor and K. Vetschera) Akadémiai Kiadó, Budapest, 1996 2. Z. Dinya, F. Sztaricskai, E. Horváth, J.B. Schaág, K. Varró: Studies of the components of Desertomycin complex by means of electrospray and MALDI mass spectrometric techniques. Rapid Communications in Mass Spectrometry Vol. 10. 1439-1448 (1996) 3. Katalin Lányi; Enikő Varga: Elaboration and use of a double channel detection ion chromatographic method in determining ions in aqueous samples. Chromatographia Supplement, Vol. 51. (2000). 4. Katalin Lányi, Zoltán Dinya: Gas Chromatographic Method for Studying the Photodegradation Rate of Some itrogen Containing Pesticides. Chromatographia (közlés alatt) Presentations and other publications: 1. Varró Katalin: Szerves és szervetlen mikroszennyezők meghatározása természetes vizekben HPLC-s és ionkromatográfiás módszerrel. MSc theses, 1993. 5. Dragan Drinic, Zsuzsanna Flachner, Zigrida Shperlina, adya Sozonova, Demi Theodori, Katalin Varró: The future of international negotiations on Long Range Transboundary Air Pollution in the light of a Combined Emission Reduction protocol. EPCEM project riport, 1994. 6. Katalin Varró: Literature study on TRE and TIE methods. EPCEM szakmai gyakorlat riportja az AquaSense BV-nek, Amsterdam, the etherlends, 1994. 7. Z. Dinya, Gy. Litkei, J. Jekő, K. Varró, S. Antus: GC-LC-MS Studies of the Extracts of Buds of Populus igra. International Bioflavonoid Symposium (9 th Hungarian Bioflavonoid Symposium), Vienna, Austria, 1995 8. Zoltán Dinya, László Somsák, Erzsébet Sós, Zoltán Györgydeák, Katalin Varró and J-P. Praly: Stereochemical effects in the EI and CI mass spectra of anomerically disubstituted 11
monosaccharide derivatives. 5 th International Conference on Chemical Synthesis of Antibiotics and Related Microbial Products, Debrecen, 1996 9. Z. Dinya, L. Somsák, E. Sós, Z. Györgydeák, P. Benke and K. Varró: Stereochemical effects in the spectra of anomerically disubstituted monosaccharide derivatives. 14 th International Mass Spectrometry Conference, Tampere, Finland, 1997 10. Lányi Katalin, Lányi István: Helyszíni mérések jelentősége és lehetőségei vizes környezetben. Alföldi Tudományos Tájgazdálkodási apok, Mezőtúr, 1997 11. Katalin Lányi, Enikő Varga: Double channel detection ion chromatographic method for determination of ions in aqueous samples. Advances in Chromatography and Electrophoresis (ACE) Symposium, 1998, Szeged 12. Lányi Katalin, Dinya Zoltán: xigéntartalmú heterociklusos vegyületek fragmentációs sémájának tanulmányozása. 42. Magyar Spektrokémiai Vándorgyűlés, 1999, Veszprém 13. Katalin Lányi; Enikő Varga: Elaboration and use of a double channel detection ion chromatographic method in determining ions in aqueous samples. Balaton Symposium 99 n High-Performance Separation Methods, 1999, Siófok 14. Katalin Lányi; Zoltán Dinya: Gas chromatographic - mass spectrometric method for studying the photodegradation processes of some commonly used pesticides. Balaton Symposium 99 n High-Performance Separation Methods, 1999, Siófok 15. Lányi Katalin: A novel approach for studying the mixability of various pesticides in order to promote new plant growing technologies. II. Alföldi Tudományos Tájgazdálkodási apok, 1999, Mezőtúr 16. Lányi Katalin, Dinya Zoltán: itrogén tartalmú herbicidek fotodegradációs folyamatainak vizsgálata GC-MS módszerrel. 44. Magyar Spektrokémiai Vándorgyűlés, 2001, Baja 17. Katalin Lányi, Zoltán Dinya: Gas Chromatographic Method for Studying the Photodegradation Rate of Some itrogen ContainingHerbicides. Balaton Symposium 01 n High-Performance Separation Methods, 2001, Siófok 12