3i. Emmi EGEE 0E ALECEELQR E2-CE-EE GEE}- 23E: «BEE‘EEE‘EE. 3E;- {EEEEEEGX‘EEEEE- STE-EELCEA EEEE EEE E3 E} EEE‘ afiEES ‘nsrs foE EEe E egEe WEE EEEEE SEE. ‘ aEEJw JE‘ SEEEJEE 13 4E EEEEEEEEEEEEEEE’ / Em LIBRARY Michigan gram Univcfifly — * ABSTRACT BIODEGRADATION OF ALACHLOR [2-CHLORO-2',6'-DIETHYL-N-(METHOXYMETHYL)ACETANILIDE] IN SOILS BY Sheng-Fu J. Chou Biodegradation by soil microorganisms accounts for the dissipation of Z-chloro-Z',6'-diethy1-N-(methoxymethyl) acetanilide (alachlor) and its possible breakdown product, 2-chloro-Z',6'-diethy1 acetanilide (demethoxymethyl alachlor) in soil. The half-life of alachlor in three soils was 8 to 16 days, while the half-life of demethoxy- methyl alachlor was approximately 2 days. The amount of 14 CO evolved from 14C-ring labeled alachlor in soils 2 averaged only 4.1% of the original label after 50 days of incubation. After this period of incubation an average of 82.1% of the added label could not be extracted with 14 benzenezisopropanol or accounted for as CO However, 2. more than 49% of the added radioactivity after 60 days incubation could be recovered by weak alkali extraction; 17.0% fractioned with humic acid and 31.9% with fulvic acid. Gas chromatographic analysis of the solvent extracts did not show any metabolite peaks during 50 and 60 day incubation periods. Alachlor appears to biodegrade rapidly in soil but Sheng-Fu J. Chou the resulting metabolites appear to be bound to soil organic matter and to degrade slowly. BIODEGRADATION OF ALACHLOR [2-CHLORO-2',6'-DIETHYL-flf(METHOXYMETHYL)ACETANILIDE] IN SOILS BY Sheng-Fu J. Chou A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Sciences 1974 DEDICATION To my parents, brothers, sisters, and wife, whose memory gave me the inspiration to finish this work. To Dr. and Mrs. R. L. Cook and Professor M. H. Wu, whose wisdom enlightened me. ii ACKNOWLEDGEMENT The author wishes to express his sincere gratitude to Dr. J. M. Tiedje for his encouragement, patience and indispensable guidance. Sincere appreciation is also due to his guidance members: Dr. B. G. Ellis, Dr. D. Penner, Dr. A. R. Wolcott, and Dr. J. L. Lockwood. The author also thanks Dr. A. R. Wolcott and Mr. A1 Filonow for use of the gas chromatograph. The financial assistance from the Environmental Pro- tection Agency, Grant no. 8R01EP00801-06, and Regional Research Project NC-96, are acknowledged. iii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 MATERIALS AND METHODS. Soils . . . . . . . . . . . . . . . 3 Incubation systems. 3 Solvent extraction of soils . . . . 4 Analyses. . . . . . . . . . . . . . . . . . . . 5 Alachlor-humic substances interaction . 6 Chemicals 6 RESULTS AND DISCUSSION . 9 REFERENCES . . . . . . . . . . . . . . . . . . . . . . 28 iv LIST OF TABLES Table Page 1 Properties of the soils used in the experiments . . . . . . . . . . . . . . . . . 3 2 Alachlor remaining in three soils as determined by gas chromatography. . . . . . . 10 3 Half-lives of alachlor in several soils . . . ll 4 Percent of original radioactivity as 14CO2 and in benzenezisopropanol extracts from three soils amended with 14C-alachlor . . . . l7 5 Distribution of radioactivity following incubation of 14C-alachlor in Spinks soil . . 21 Figure LIST OF FIGURES Scheme for alkali extraction and subsequent fractionation of soil organic matter com- ponents O C O O O O O O O I O O O O O O O 0 Progressive loss of alachlor in three soils as analyzed by gas chromatography . Alachlor and demethbxymethyl alachlor remaining after incubation in autoclaved and non-autoclaved soil . . . . . . . . 14CO evolution from 14C-ring labeled alac lor in soils . . . . . . . . . . Progressive loss of benzene-isopropanol extractable radioactivity from three soils. vi Page 13 15 19 23 INTRODUCTION It has been estimated that every year the foliage and soils of the United States are doused with approximately one billion pounds of synthetic organic pesticides. In this process more than 800 substances are used in differ- ent registered formulations that now exceed 60,000 in number (Minter et al., 1969). Several synthetic weed killers are constructed around an anilide moiety or one of its chlorinate chemical cousins. These herbicides include a relatively new class, acylanilides, as well as older types, phenylcarbamates and phenylureas, and they comprise a significant proportion of all agricultural chemicals now in use. Alachlor [Z-chloro-Z',6'—diethy1- N-(methoxymethyl) acetanilide], an acylanilide preemergence herbicide, has recently become popular for the control of annual grasses, redroot pigweed (Amaranthus retronexus L.) and yellow nutsedge (Cyperus esculentue L.) in corn (Zea mays L.) and soybeans [Glycine max (L.) Merr.] (Armstrong at al., 1973; Wax at aZ., 1972). Though con- siderable work has been reported on the degradation and fate of other acylanilide herbicides in soils (Bartha, 1971; Bartha and Pramer, 1970; Chisaka and Kearney, 1970; Kaufman and Blake, 1973; Kaufman et aZ., 1971), little information of this type has been published for alachlor. 1 2 Hargrove and Merkle (1971) found that chemical degradation of alachlor in soil, under conditions of low humidity and high temperature, resulted in the formation of Z-chloro- 2',6'-diethy1 acetanilide. However, this intermediate did not accumulate under more natural soil conditions. Beestman and Deming (1972) have reported that microbial degradation was the major route of alachlor degradation in soil with half-lives ranging between 2 to 14 days for several soils. Taylor (1972) found that alachlor degradation in soil was not accompanied by mineralization of the aromatic portion of the herbicide. These findings for alachlor are consistent with the relatively rapid rate of degradation of other acylanilide herbicides (Bartha and Pramer, 1970; Chisaka and Kearney, 1970; Kaufman and Blake, 1973; Kaufman et al., 1971), though in several cases degradation was not complete (Bartha, 1971; Chisaka and Kearney, 1970). This investigation was initiated to determine the rate and extent of alachlor degradation in soil and to character— ize any residues from intermediates which may occur. MATERIALS AND METHODS The degradation of alachlor was studied in Brookston sandy loam, Conover sandy loam, and Spinks sandy loam surface soils obtained from plots on Michigan State Uni- versity experimental farms. The soils had never received applications of alachlor. The soil properties are shown in Table 1. The soils were freshly collected and allowed to dry to approximately 15% moisture before use. Table 1. Properties of the soils used in the experiments Organic Mechanical analysis Soil type pH matter sand silt clay _. (9.) (9.) (as) Brookston sandy loam 7.1 3.38 54.2 33.0 12.8 Conover sandy loam 7.0 2.03 69.0 20.6 10.4 Spinks sandy loam 6.3 1.25 65.0 29.8 5.2 Incubation systems Fifty grams of soil that had passed through a 2 mm sieve were placed in 250 m1 Erlenmeyer flasks. A portion of Brookston soil was autoclaved for three 30 min periods every other day to provide the sterile soil. Sterility was 3 4 confirmed at the end of the incubation by the absence of growth when soil was inoculated onto nutrient agar. One milliliter of a 100 ppmw solution of filter sterilized 14C-ring labeled alachlor (0.01 uCi) was distributed drop— wise on the unautoclaved soil in each flask (equivalent to 4.5 kg alachlor/ha). In the experiments with 2-chloro- 2',6'-diethy1 acetanilide (demethoxymethyl alachlor) and in experiments with autoclaved soil, 2.5 m1 of a 100 ppmw solution of substrate was used. The amended soil in each flask was then moistened by adding 12 m1 of sterilized distilled water, sealed and incubated at 25 C. Replicate flasks were sampled at the indicated time periods for the following analyses. Respired 14CO was trapped in 1 m1 of l N NaOH which 2 was contained in a disposable 2 m1 beaker suspended above the soils. It is assumed that recovery of the liberated 14CO2 in the alkali trap was substantially complete since equivalent levels of NaH14CO3 added to the soils showed 14 95% CO2 trapped within 8 hr. Solvent extraction of soils Alachlor and demethoxymethyl alachlor were extracted from the soil with three 50 m1 portions of benzenezisopropanol (2:1,-v/v). ;For the first extraction, the solvent-soil mixture was allowed to stand in the flask overnight and then was shaken on a rotary shaker at 150 rpm for 30 min. After decanting the solvent the second and third extractions were made by shaking solvent and soil at 150 rpm for 30 min prior S to decanting. Anhydrous NazSO4 was added to the solvent to remove water. The combined extracts were concentrated to 50 or 100 m1 prior to analysis. Analyses All samples were diluted to appropriate volume and analyzed on a Beckman GC-S gas chromatograph equipped with an electron capture detector. A glass column of 1.83 m by 3 mm I.D. and containing 1.5% OV-17 / 1.95% QF-l on 60/80 chromosorb Q was used. The column, inlet, and detector temperatures were 200, 220, and 250 C, respectively. The carrier gas (He) flow was 85 ml/min. The retention time and minimum sensitivity for alachlor were 3.3 min and 0.01. ug, respectively, and for demethoxymethyl alachlor they were 1.9 min and 0.025 pg, respectively. Fifty grams of each soil without added chemicals were also extracted as a con- trol; no interfering peaks were found. The 14 C-label was assayed by liquid scintillation counting using a Packard Tri-Carb Liquid Scintillation Spectrometer, Model 8310. One milliliter of the benzene: isopropanol extract was counted in 15 m1 of a scintillation solution containing 4 g PPO, 0.5 g POPOP/liter of toluene. The 14 CO2 trapped in 1 N NaOH was counted in Bray's solu- tion (1960) containing 4% Cab-O-Sil. All counts were corrected for quenching by external standardization and for machine efficiency. All reported data are the averages of two replicates. 6 Alachlor-humic substances interaction Either 2.5 m1 (experiment III) or 5.0 ml (experiment II) of 100 ppmw 14 C-ring labeled alachlor (0.025 and 0.05 uCi, respectively) were added to 50 g of Spinks soil. Treated samples were incubated and extracted at 0, 10, 14, 20, 40 and 60 days by the benzenezisopropanol extraction procedure previously described. After this solvent extrac- tion, the soils were shaken in 50 ml 0.5 N NaOH for 24 hr. This alkali extract was fractioned according to the scheme shown in Figure 1. Each fraction was assayed for radioactivity. Several fulvic acid fractions were extracted with three 50 m1 portions diethyl ether, the extract dried with anhydrous NaZSO4 and analyzed by the gas chromatographic procedure previously described except that stainless steel columns, 6 ft by 1/8 O.D., containing 5% SE-30 and 4% SE-30 / 6% QF-l on 60/80 chromosorb Q were used. These 14C compounds by thin- ether extracts were also examined for layer chromatography and subsequent radioautography. Each sample was concentrated to 0.5 ml before 50 ul was spotted on a 250 u silica gel H layer. The chromatogram was developed in petroleum ether : chloroform : 95 percent ethanol (7:2:1, v/v/v). Chemicals Analytical grade alachlor and uniformly 14C-ring labeled alachlor were supplied by the Monsanto Chemical .mucocomeoo nouums owcmmuo Hflom mo :ofiuma0wuuwam ucoscomASm use :ofiuumpuxe fiamxam pom oEonom .H ohsmfim pceucou o How woxmmmm m:0fiuumhm« «a III 90:90 fixauofiw an“: venomupxo _Wumwfimfiuomm_ . _w:mHm:Hemsw_ T 1 .:ME ON .w x coon downwanucoo .m-H :8 on woumfiefium .momz cow: eopumpuxm z m.o :fl ©o>Hommfivop .vofiuw Ham mvfiom 0flascv _ouwuflmwuehm % cogpo flagpofle an . k fiefium ufi>H=mV Emqumauonlmm m x oooe eomsmflhonou Anflsnzv .m-H ma on He: 1 .guuz.euflmfieflu< * Mumufimflnuohi Wampmcuomsfl wwwawfiupceu was vomcfiu «topmomop ..:HE ON .wx ooov womsmfiuuaou momz z m.o cu xooam Hoqmmoumomwnononnon . 3 :ofiuumuuxo youmm. . . E _ 8 Co., St. Louis, Mo. The label purity was determined to be 99.7% by thin-layer chromatography. Demethoxymethyl alachlor was prepared by hydrolysis in 5 N HCl according to the method of Hargrove and Merkle (1971). Nanograde benzene and isopropanol were purchased from Burdick and Jackson Laboratories, Muskegon, MI. RESULTS AND DISCUSSION The loss of alachlor in three soils is shown in Table 2 and Figure 2. By the 10th day, more than 50% of added alachlor was gone in the Brookston and Spinks soils, while almost 50% disappeared in Conover soil. By the 50th day, only 1.4% and 1.5% of the original amount was recovered from Brookston and Spinks soils, respectively, and 2.8% from the Conover soil. This indicates a rapid and similar rate of degradation of alachlor in the three soils. The half-life of alachlor in these three soils ranged between 8 and 16 days (Table 3) over three experiments, which is in agreement with the values reported by Beestman and Deming (1972). The slightly slower rate of degradation for experi- ment III may have been due to the more deteriorated state of the microflora in late summer sample and lower incuba- tion temperature. As seen in Figure 3, the degradation of both chemicals in autoclaved soil was minor. Extractable demethoxymethyl alachlor declined only 5.5% after 20 days of incubation in autoclaved soil compared with a loss of 97.8% in the non- autoclaved soil. Similarly, alachlor decreased only 14.4% after 50 days incubation in autoclaved soil compared with 92.8% degradation in non-autoclaved soil. These results show that microorganisms are the major agents of 9 10 . o.H . m.N . ¢.N m N ¢.H m N N.N v H ¢.H om . o.HH . N.NH . N.e N Na v.HH m NH m.oH N v m.¢ om . o.¢N . N.me . m.NH N «N m.vN m He m.am w NH N.mH ON . N.om . m.mm . o.wm m on 5.0m N mm N.mm N Na H.vv OH . N.mm . m.em . N.mm m mm m.wm m cm v.em N em N.vm o E 33 2; 33 E 33 2.33 ao>ocou cepmwoo~m meg» wopo>oooh po~nom~< :owumnsocH >39muwoumfiounu mew An woGNEpouow mm mHNom oops» a“ maflnfimEou hoagumfi< .N oanme 11 .m»«@ ¢.o H v moumofiamou macaw xufiawnmflum> ”moumofiammh mo omwuo>oaou N m mN o.N mNmH .nom cepmxooum N H mmmxmwv endummwmwop mflflmmv uopoeaaoo HNOm open mafiom mopmowfimom ucosfipomxm omfia-wam: :owumnsucH cw .ocou maflom Haho>om :N Hoacumfim mo mo>fla-mam: .m oanme 12 Figure 2. Progressive loss of alachlor in three soils as analyzed by gas chromatography. Brooks ton G O Conover A——A Sp inks D——l:l 13 _ _ _ _ O 0 0 0 8 2 mAHOw 20mm wmm>oumm moqmu<4< w c -. I SO 40 30 20 10 DAYS OF INCUBATION Figure 2 14 Figure 3. Alachlor and demethoxymethyl alachlor remaining after incubation in autoclaved and non— autoclaved soil. O———O Alachlor Z§_______£§ Demethoxymethyl alachlor % SUBSTRATE REMAINING IN SOIL 80 60 40 20 15 I r 1 l \ fi‘ A . AUTOCLAVED C>--—““*D~—————___Q _> h" '1 L o q - NON-AUTOCLAVED _ V . ' A ‘A‘—fl° _L11 1 ‘*%D 0 10 20 30 4O 50 DAYS OF INCUBATION Figure 3 l6 decomposition of both alachlor and demethoxymethyl alachlor in soils. The loss of demethoxymethyl alachlor from Spinks soil (Figure 3) followed the same loss pattern as alachlor except that demethoxymethyl alachlor was degraded at a much faster rate. The half-life was approximately 2 days, a rate of disappearance four times greater than for alachlor. This suggests that it is unlikely that demethoxymethyl alachlor would ever accumulate in soils except in unusual circumstances where biological activities are severely inhibited; this could explain the occurrence of this product in a previous report (Hargrove and Merkle, 1971). The data did not show demethoxymethyl alachlor to be an intermediate since it was never recovered from alachlor amended soil. Nevertheless, the more rapid rate of decompo— sition of this chemical than for alachlor is consistent with its remaining a candidate for an intermediate. In separate studies on fungal metabolism of alachlor, demethoxymethyl alachlor has been shown to be an intermediate (Tiedje and Hagedorn, unpublished data). 14 14 Based on the recovery of the C of alachlor as CO 2, however, the rapid loss of alachlor in three soils was not accompanied by complete respiration of the herbicide (Table 4 and Figure 4). After 50 days of incubation, an average of only 4.1% of the initial radioactivity was trapped in the NaOH. At this rate of metabolism it would take more than 3 years before complete mineralization of .mmpu momz on» :H u NHo.o uozozm HHom eHHHoum may mxmv om Houmoaou :oumxoogm :oHumnsunH mungowHw u nqu wovcoem mHHom manna scum muomnuxo HonamounomH. onencon :H was NoueH we xwm>HuomoHveH HmchHho mo acoohom .e oHnae Figure 4. 14 alachlor in soils. Brookston Conover Spinks 18 C02 evolution from 0—0 A———A 14 C-ring labeled 2 14c EVOLVED AS 14co2 .5 (N N H 19 l I l l L... - i O . D’ it ‘1 > b P- .J (I J J 1 I 0 10 20 30 40 50 DAYS OF INCUBATION Figure 4 20 the herbicide residue would occur. The majority of the radioactivity in the benzenezisopropanol extracts (Table 4 and Figure 5) was accounted for by unmetabolized alachlor since the quantity of alachlor in these extracts as de- termined by gas chromatography (Table 2) was similar to the observed radioactivity. After 50 days, however, the radioactivity of the extract ranged from 12.9 to 14.4% of the original addition in contrast with 1.4 to 2.9% alachlor at this time. Nevertheless, no other products could be detected by gas chromatography in these or in earlier extracts. It is significant that most of the added radioactivity 14 in alachlor could not be recovered as CO or in benzene: 2 isopropanol extracts. This unrecovered radioactivity ranged from 80.8 to 83.5% of the added label after 50 days incuba- tion for experiment I and 63.8% after 60 days incubation for experiment III. An indication of the fate of the unre- 4C from alachlor is shown by the data in Table 5. covered 1 In experiments II and III, even though the rate of degrada- tion was much slower for experiment III (Table 3), the distribution of the 14 C label among the various fractions (Table 5) was similar at a same stage of alachlor degrada- tion in the two experiments. In experiment III 63.8% of the label was not recovered in the benzenezisopropanol extract nor as 14CO2 though 48.9% was recovered by subse- quent alkali extraction. In both experiments the majority of the alkali soluble label fractionated as fulvic acid (approximately 31%) whereas 17% fractioned as 21 .HoceQOHmomHuoconcon :qu :oHHUMHuxo on HOHHQ HmeHm kn wmuomguxo who: moHnEmm HHomHU wecHEHouew Ho: u nzo .xammhmoumaonno mew kn aoHpowHH :H wasom 90HnumHm fleece Ho “coupomn .coHuumpm some :H u woven mo “awoken mm commohmxo ”mopmoHHmoH 03H mo ewmuo>oooh Honuo oumuHmHo Home Henge nqu Nou Hoehuxo :OHumnsucH >9H>Huum nHHz wHow -opm vHum maoescm. wopomhuxm vH HocmmoumomH -ovaH UHESH Scum uHazn :H vHom “oaoncom Heuoe wouomwuxm UHES: mo :oHumemom Houmm oumaquSm :H HmoEuonH HmemeNm venomupxo mostmoH HHom HHom mxchm :H HOngmHm-o mo :OHHmn302H mcHonHom AHH>Huum0vaa mo GOHuanuumHn .m oHan «H 22 Figure 5. Progressive loss of benzene-isopropanol extractable radioactivity from three soils. Brooks ton O——Q Conover ZX——————£§ Sp inks U—U 23 100 AU 8 0 0 6 4 >HH>HHU< H