THESI‘ -‘..._- . M war \ mghfig'gn fitete This is to certify that the thesis entitled Fate of MBOCA (4,4'-Methylene-BIS (2-Chloroaniline)) in Soil and Plants presented by Richard Voorman has been accepted towards fulfillment of the requirements for M.S. degree in Crop‘ & Soil Sciences Major professor Date 11'5'8] 0-7639 MSU LIBRARIES 4—1:- RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. G>1981 RICHARD VOORMAN All Rights Reserved FATE OF MBOCA [4,4'-METHYLENE-BIS (2-CHLOROANILINE)] IN SOIL AND PLANTS BY Richard Voorman 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 1981 ABSTRACT FATE OF MBOCA [4,4'-METHYLENE-BIS (2-CHLOROANILINE)] IN SOIL AND PLANTS BY Richard Voorman 14C-MBOCA [4,4'-methylene-bis (2-chloroaniline)] was rapidly bound to soil with most of the binding occurring within the first 24 h after application. After 24 weeks less than 20% of the radioactivity could be extracted from soil and less than 6% could be extracted as parent MBOCA. MBOCA has a water solubility of 13.9 ppm and the MBOCA-soil adsorption isotherm indicates a strong affinity of MBOCA to soil. The predominate metabolite found in the non-sterile soil was identified as 4,4'-diamino-3,3'-dichlorobenzophenone. 14 Very little CO was released during 24 weeks of the appli- 2 cation, indicating little ring cleavage by soil microbes; however, more CO was released in the non-sterile samples 2 than the sterile samples. 14C-MBOCA was absorbed by cabbage (Brassica oleracea L.), bean (Phaseolus vulgaris L.), and sugar beet (Beta vulgaris L.) leaves, but did not move beyond the absorption point. Radio autographs of bean, sorghum (Sorghum vulgare Pers.), orchard grass (Dactylis glomerata L.), and carrot (Daucus Richard Voorman l4 carrota L.) plants exposed to C-MBOCA via hydroponic culture showed considerable radioactivity associated with 14 the roots with only limited translocation of C into upper plant parts. Bean and cucumber (Cucumis sativa L.) plants 14 grown in 14 C-MBOCA treated soil translocated virtually no C-MBOCA into aerial parts, but again considerable radio- activity was found on roots. Radioactivity could not be rinsed off roots with water or acetone, and a small amount of radioactivity was seen in the xylem-phloem layer of the carrot pulp. To my wife, Mary, for her encouragement and patience. ii ACKNOWLEDGMENTS I am deeply indebted to Dr. Donald Penner for his encour- agement and guidance and his invaluable suggestions in the writing of this manuscript. I would like to thank Drs. F. Matsumura, S. A. Boyd, and M. J. Zabik for serving as members of my guidance committee and for their valuable suggestions on my research. I am also thankful to Dr. R. A. Leavitt for use of the gas chromatograph and Dr. M. J. Zabik for assistance with the mass spectrometer. My thanks also go to Pat Mahoney for her unfailing assistance in the laboratory work. I owe too a special thanks to my laboratory co-workers who put up with all my disorder, petty thievery, and music. The financial assistance of the Michigan Toxic Sub- stances Control Commission is also gratefully acknowledged. iii TABLE OF CONTENTS LIST OF TABLES . O O I O O C O O O O 0 LIST OF FIGURES . . . . . . .. . . . . CHAPTER I. FATE OF MBOCA [4,4'-METHYLENE-BIS (2-CHLOROANILINE)] IN SOIL . . . . . Introduction . . . . . . . Materials and Methods . . . . . . Soil Preparation . . . . . . . 4C- Extraction . . . . . . . . Analytical Procedures . . . . . Water Solubility and MBOCA Movement in Adsorption Isotherm . . . . . . Results and Discussion . .. . . . . Binding of 14C-MBOCA to Soil . . . Metabolism of 14c—MBOCA in Soil. . . Evolution of 14C02 . . . . . . . Conclusion . . . . . . . . . . Literature Cited . . . . . . . . II. PLANT UPTAKE OF MBOCA (4',4'-METHYLENE-BIS (2-CHLOROANILINE)] . . . . . . . Introduction . . . . . . . Materials and Methods . . . . . . Foliar Absorption . Root and Shoot Absorption of 14é- -MBOCA from Aqueous Culture . . . Absorption of 14C-MBOCA from Soil . . Results and Discussion . . . . . . Foliar Absorption . . Root and Shoot Absorption of 14C- -MBOCA Aqueous Culture . . . . . . . iv Page vi vii \IG‘U'Iubw LOH \l 21 35 37 37 4o 40 42 42 43 44 44 44 46 Page 14 Absorption of C-MBOCA from Soil . . . . 62 Conclusions . . . . . . . . . . . . 62 Literature Cited . . . . . . . . . . 68 GENERAL REFERENCES . . . . . . . . . . . . 69 LIST OF TABLES Table Page CHAPTER I 1. Cumulative l4CO2 production after 24 weeks . . 36 CHAPTER II 1. MBOCA absorbed from glass surface by cabbage leaf . . . . . . . . . . . . . . 45 2. Absorption of l4C—MBOCA by plant leaves after 5 days . . . . . . . . . . . . . . 45 3. Radioactivity distribution in bean plant . . . 55 4. Radioactivity distribution in plant parts . . . 55 5. Radioactivity in carrot plant . . . . . . . 61 6. Radioactivity distribution in soil grown plants . 67 vi LIST OF FIGURES Figure CHAPTER I 1. Progressive loss of extractability of 14C in Hoytville and Wabash Park soil . . . . . 2. Progressive loss of extractable MBOCA radio- activity over 1 week . . . . . . . . . 3. Progressive increase of soil bound radioactivity from 14c-MBOCA . . . . . . . . . . . 4. Radioautograph of soil TLC plates. MBOCA (A), Trifluralin (B), and Chloramben (C) are shown. "1" is origin, "2" is solvent front . . . . 5. MBOCA adsorption isotherm on Hoytville soil . . 6. Radioautograph of thin layer chromatogram of extract following 4 week incubation. "A and C" are 4 ppm MBOCA added to sterile and non- sterile Hoytville soil, respectively. "B" is MBOCA standard. “1" is origin, "2" is metabo- lite, and "3" is MBOCA . . . . . . . . 7. Radioautograph of thin layer chromatogram of extract following 4 week incubation. "A" is 40 ppm MBOCA added to Hoytville soil, "B" is 4 ppm MBOCA added to Wabash Park soil, "C" is MBOCA standard. "1" is origin, "2" is metabo- lite ’ "3" is MBOCA o o o o o o o o o 8. Radioautograph of thin layer chromatogram of extract following 72 h MBOCA soil incubation. "A" 1 ppm, "B" 10 ppm, "C" 100 ppm, "D" stan- dards. "l" is origin, "2" is N,N-diacetyl MBOCA, "3" is N-monoacetyl MBOCA, "4" is metabolite, and "5" is MBOCA . . . . . . vii Page 12 15 17 19 23 25 27 Figure 9. Beta camera scans of TLC plates. "A" 40 ppm MBOCA amended Hoytville soil at zero time extraction, "B" 4 ppm MBOCA amended Wabash Park soil at zero time extraction, "C" 40 ppm MBOCA amended Hoytville at 4 week extraction, "D" 4 ppm MBOCA amended Wabash Park soil at 4 week extraction . . . . . . . . . . 10. Mass spectrum of MBOCA metabolite . . . . . 11. Progressive loss of extractable parent compound. CHAPTER II 1. Plant leaves (top) and radioautographs (bottom). Cabbage (A) and sugar beets (B) were exposed to l4C-MBOCA for 5 days . . . . . . . . 2. Sorghum seedlings (top) and radioauiographs (bottom). Roots were exposed to 4C-MBOCA for 8 days. Roots of "A" were water rinsed, roots of "B" were acetone rinsed . . . . . 3. Bean plant (top) and radioautogfiaph (bottom). Plant roots were exposed to C-MBOCA for 8 days . . . . . . . . . . . . . 4. Carrot plant (top) and radioautograph (bottom). Root was exposed to l4C-MBOCA for 8 days. Car- rot is sliced longitudinally for radioauto- graph . . . . . . . . . . . . . . 5. Orchard grass (top) and radioautograph (bottom). Roots were exposed to C-MBOCA for 4 weeks . 6. Carrot plants (top) and radioautograph (bottom). Roots were exposed to l4C-MBOCA for 4 weeks . 7. Bean plants (top) and radioautograph (bottom). Plants were raised in C-MBOCA treated soil . 8. Cucumber plants (tOp) and radioautograph (bot- tom). Plants were raised in l4C-MBOCA treated soil . . . . . . . . . . . viii Page 29 31 34 48 50 52 54 58 60 64 66 CHAPTER I FATE OF MBOCA [4,4'-METHYLENE-BIS (2-CHLOROANILINE)] IN SOIL Introduction MBOCA [4,4'—methylene-bis (2-chloroaniline)] has been widely used as a crosslinking agent in the production of diisocyanate based polymers and epoxy resins. It is used in these materials to manufacture semirigid polyurethane foam and hard urethane rubber. The compound was developed and marketed by the DuPont Company beginning in the mid-19505 and was later manufactured by a small chemical company in Adrian, Michigan in the 19705, with production of up to 500,000 kg of MBOCA annually. MBOCA has been proven a carcinogen in rats, mice, and dogs (Stula et al., 1975; Russfield et al., 1975; Stula et al., 1977) and has been reported to be absorbed through the skin of workers exposed to it (Linch et al., 1971). In 1978 NIOSH recommended that MBOCA be treated as a potential human carcinogen. In response to this report an investigation by State of Michigan agencies found extensive environmental contamination of MBOCA on several hundred hectares of land surrounding the MBOCA plant in Adrian (Agency memoranda, 1979). MBOCA was found in the urine of plant workers and was also found in the urine of young children living in the contaminated area (Agency memoranda, 1979). MBOCA levels as high as several parts-per-million (PPm) were found in gardens and community recreation areas (Agency memoranda, 1979). Since MBOCA was not intended for use in the outdoor environment, no research has been done on its fate in the environment. However, previous studies of pesticides with structures similar to MBOCA may offer insight in hypothesiz- ing the fate of MBOCA in soil. The widely used herbicide trifluralin (1,l,l-trifluoro-Z,6-dinitro-N,N-dipropyl-p- toluidine) is a tertiary dinitro aniline compound. It is rapidly bound to soil, does not leach, and normally does not present problems of persistence in soil (Probst et al., 1966). Research on the chloroacetanilide group of herbicides is especially applicable, as some of its members degrade in soil to chloroanilines and these appear to be very rapidly bound in soil. Bartha (1971) found that after 13 days only 23% of added 4-chloroaniline (4CA) could be extracted from soil. Research by Hsu and Bartha (1974a) found that most 4CA was bound into the organic matter complex in the soil. N-acetylation of anilines by soil microorganisms has been demonstrated (Kaufman, 1973) and raises additional concern over the presence of MBOCA in the environment. It has been proposed that in humans, MBOCA becomes carcinogenic only after metabolic activation by N-hydroxylation or N-acetylation. The objectives of this study were to determine the fate of MBOCA in soil by measuring persistence, adsorption to soil, leaching, and metabolism. Materials and Methods Soil Preparation Soil used in this study was collected from two sites. A Hoytville soil, free of MBOCA, was collected 14 km northeast of Adrian. Physical properties of the soil were: 48.6% sand, 20% silt, 31.4% clay, 7.6% organic matter, and pH 7.4. A second Hoytville soil known to be contaminated with MBOCA was collected in Wabash Park in Adrian about 100 meters from the MBOCA manufacturing plant. This soil will be referred to as Wabash Park soil. Soils were maintained in moist condition (50% field capacity). Soil was amended with 14 14 C-MBOCA (uniformly ring-labeled, sp. act. 10.9) or C-trifluralin (uniformly ring-labeled, sp. act. 2.5) in l-kg batches by spraying an ethanol solution (25 ml) containing 12 uCi of the chemical on the soil as it was blending in a 19-L rotary mixer. The following treat- ments were used: 4 ppm or 40 ppm MBOCA to Hoytville soil, 4 ppm MBOCA to sterile Hoytville soil, 4 ppm MBOCA to Wabash Park soil, and 4 ppm trifluralin to Hoytville soil. For the sterile soil treatment, soil was sterilized by autoclaving for two 1-hour periods 24 hours apart. Following treatment the soils were divided into 25-9 samples and added to 125-m1 erlenmeyer flasks. Sterile samples were briefly autoclaved at 100°C for 5 min to assure sterility. A disposable 2-m1 plastic beaker containing 1 m1 of 0.1 N NaOH was suspended in each flask to trap respired C02. Each flask was considered an experimental unit and units were analyzed in triplicate over a period of 24 weeks. A short term extraction study was also conducted to characterize the first week of incubation. Three 120-g batches of field moist soil (50% FC) were amended with l, 10, 14 and 100 ppm C-MBOCA, respectively, by pipetting 1 ml of the appropriate solution over the soil and mixing. Five uCi of l4C-MBOCA was incorporated with an appropriate amount of MBOCA. Each batch was divided into 5 9 portions and these added to 20 by 150 mm screw cap tubes. Caps were placed loosely on the tubes and the tubes periodically aerated. At the appropriate time samples were extracted and analyzed in duplicate. 14C-Extraction Soils from the long-term study were extracted twice in the flask on a wrist action shaker for 20 min. Fifty milli- liters of ethyl acetate/methanol (60:40) were used each time. The extract was filtered into a 250 m1 round bottom flask and evaporated en yagug until a few ml of water remained. The residual water was transferred to a 16 x 125 mm screw cap tube and the flask rinsed with 2 x 5 m1 ethyl acetate and also transferred to the tube. One milliliter saturated ammonium sulfate solution was added; the tube was shaken, centrifuged, and the aqueous layer discarded. The lO-ml soil extract was light to dark yellow from organic soil constituents. At the appr0priate time samples from the short-term study were extracted with 10 ml of ethyl acetate-methanol (60:40) by shaking for 15 min. Analytical Procedures Radioactivity in the extract and the NaOH solution was assayed by liquid scintillation spectroscopy (LSS) using external standard quench correction (Beckman 8100 LS). The scintillation solution used was either ACS (Amersham) or NEN 963 (New England Nuclear). Following extraction some soil samples were combusted in a biological oxidizer (Harvey Inst. Co.) to account for bound radioactivity. One gram samples were used and the evolved 14CO2 trapped in OCS/Oxisorb II (2:1) (Amersham and New England Nuclear, respectively) and assayed by LSS. An aliquot of the extract was reduced in volume, spotted on a silica gel thin layer chromatography (TLC) plate (Merck 60), and developed in chloroform/ethyl acetate (70:30). The plate was then radioautographed or examined with a beta camera TLC scanner to determine the distribution of radioactivity. Quantitation of MBOCA in the long-term study was accomplished by gas liquid chromatography (GLC) with electron capture detection (Tracor 560). A dipentafluoropropionyl derivative of MBOCA was made and the sample diluted 1:200 for injection. It was not necessary to derivatize the trifluralin samples. A 1.8 m 3% OV-210 column at 230° was used for MBOCA and a 1.8 m 10% carbowax 20 M col- umn at 200°C was used for the trifluralin. Nitrogen carrier gas at 20 ml/min was used for all samples. Mass spectra were obtained on a DuPont 321 mass spectrometer by direct probe at 70 ev. Results were statistically analyzed by analysis of vari- ance with separation of means using Duncan's multiple range test at the 5% level. Water Solubility and MBOCA Movement in Soil Water solubility of MBOCA was determined by making a l4C-MBOCA at 24 i 2°C. The saturated aqueous solution of solution was shaken for 72 h in foil wrapped tubes and then centrifuged at 30,000 x g for 1 h to sediment particulate MBOCA. The supernatant was assayed for radioactivity and the MBOCA concentraton calculated from the specific activity. All treatments were replicated five times. Mobility of MBOCA in soil was estimated by using a simple soil TLC system. The Hoytville soil was sieved to less than 100 microns. A water slurry was made (2 drops ethanol added to reduce bubbles) and then spread on glass plates at 400 micron thickness. Ten microgram amounts of l4C-MBOCA, 14 4C-trifluralin, and C-chloramben (3-amino—2,5-dichloro- benzoic acid) were spotted on the plates. A paper towel was wrapped around the base of the plate and the plate developed for 10 cm with water. The plate was radioautographed and the Rf values determined. Adsorption Isotherm An adsorption isotherm for MBOCA on the Hoytville soil was also developed. A saturated aqueous solution of MBOCA was prepared and diluted to 1,3,6, and 10 ppm in 50 ml water in 125-ml foil wrapped flasks. Ten grams of dry, sieved (<2 mm) Hoytville soil was added to the water and MBOCA allowed to equilibrate for 64 h on a wrist action shaker at 24 i 2°C. The mixture was centrifuged and the equilibrium concentration of MBOCA determined by radioassay. Mixing and equilibrations were done in silane treated glassware. Results and Discussion 14 Binding of C-MBOCA to Soil 14 The extraction efficiency of C-MBOCA applied to soil at zero time was 85-95%, but decreased rapidly over the first few weeks (Figure 1). It is interesting to note the decrease in 14C extractability between the Hoytville and Wabash Park soils receiving 4 ppm MBOCA. The decrease occurred in both at the same rate and to the same level, even though the Wabash Park soil already contained a large amount of MBOCA. In the 14 sterile soil extractability of C decreased more slowly, and in the soil with 40 ppm MBOCA the loss of extractability of 14C was slower as would be expected due to a higher MBOCA Figure 1.--Progressive loss of extractability of 14C in Hoyt- ville and Wabash Park soil. 4 ppm Trifluralina * *c 40 ppm MBOCA [3 [lb 4 ppm MBOCA, sterile fir firb 4 ppm MBOCA 0 On 4 ppm MBOCA, Wabash Q Ca Park soil aLetters following legend indicate statistical separation of lines using Duncans multiple range test (P<0.05). >._._>:U6) on montmorillonite clay and considerably greater than the K of parathion (1.1 x 102) under similar conditions d (Green, 1974). 21 l4C-MBOCA in Soil 14 Metabolism of C extracted from the various l4 Radioautography of the soils showed the presence of a C-metabolite with a rf of 0.42 after 3 days (Figure 6). The metabolite was not formed in sterile soil so is presumably a product of microbial metabolism. It was formed even with 40 ppm MBOCA in the l soil (Figure 7) and was extracted in greater quantities than the parent MBOCA (Figure 9). This metabolite did not co- chromatograph with the mono- or diacetyl derivative of MBOCA :3 (Figure 8). The metabolite reacted with Ehrlich's reagent E to produce a bright yellow color, indicating the presence of primary aromatic amines. The portion of the plate containing the metabolite was scrapped, extracted, and introduced by direct probe into the mass spectrometer. An M+ of 280 was found with isotopic peaks indicating two chlorines; a major ion at m/z 154 with isotopic peaks indicated one chlorine (Figure 10). MBOCA has an M+ of 266 and no peaks below m/z 200. Based on this information the following structure was proposed: Cl C o H H2 c O NH2 The 154 ion results from cleavage at the bridge and formation of the 4-amino-3-chlorobenza1dehyde ion. The 22 Figure 6.—-Radioautograph of thin layer chromatogram of extract following 4 week incubation. "A and C" are 4 ppm MBOCA added to sterile and non-sterile Hoytville soil, respectively. "B" is MBOCA stan- dard. "1" is origin, "2" is metabolite, and "3" is MBOCA. 23 24 Figure 7.--Radioautograph of thin layer chromatogram of extract following 4 week incubation. "A" is 40 ppm MBOCA added to Hoytville soil, "B" is 4 ppm MBOCA added to Wabash Park soil, "C" is MBOCA standard. "1" is origin, "2" is metabolite, "3" is MBOCA. 25 26 Figure 8.--Radioautograph of thin layer chromatogram of following 72 h MBOCA soil incubation. "A“ 1 "B" 10 ppm, "C" 100 ppm, "D" standards. "1" origin, "2" is N,N-diacetyl MBOCA, "3" is N- monoacetyl MBOCA, "4" is metabolite, and "5" MBOCA. extract PPm: is is 27 28 Figure 9.--Beta camera scans of TLC plates. "A" 40 ppm MBOCA amended Hoytville soil at zero time extraction, "B" 4 ppm MBOCA amended Wabash Park soil at zero time extraction, "C" 40 ppm MBOCA amended Hoytville at 4 week extraction, "D" 4 ppm MBOCA amended Wabash Park soil at 4 week extraction. MBOCA ORIGIN ;\ MBOCA ORIGIN E3 METABOLITE ORIGIN MBOCA METABOLITE ORIGIN MBOCA’ D 30 Figure 10.--Mass spectrum of MBOCA metabolite. 3' our cop co 3I 4a.. zux ao— oauopun qo on” up; 32 benzophenone metabolite was confirmed by a positive reaction with 2,4-dinitro phenyl hydrazine (yellow-orange color) indi- cating the presence of a carbonyl. There is precedence for bacterial metabolic reactions producing derivatives of this type, as soil bacteria have been known to produce the benzophenone derivative from DDT (Wedemeyer, 1967). The material at the origin made up a large portion of the radioactivity in most samples. It was not colloidal material and was not soluble in dilute acid or base. It migrated up the TLC plate if developed in a very polar sol- vent such as ethanol. Much of the colored material in the extract (mentioned earlier) was retained at the origin, and perhaps the MBOCA was simply complexed with this material. Since this occurred at zero time as well, MBOCA could have reacted with the material during extraction. This can indeed occur, since a similar chromatogram was obtained when MBOCA was added to the extract of a soil blank. It is impos- sible to state whether the radioactivity at the origin was indeed free MBOCA in the soil, or if it was already com- plexed when extracted. To determine the persistence of MBOCA in soil, the amount of extractable MBOCA was determined over the 24 week period (Figure 11). In all soils the parent MBOCA decreased very rapidly. Even at the zero time analysis only 60-70% of the parent MBOCA could be recovered, and this decreased to 33 Figure ll.-—Progressive loss of extractable parent compound. 4 ppm trifluralin * * 4 ppm MBOCA, sterile soil 40 ppm MBOCA oar} 0E1)? 4 ppm MBOCA MATERIAL ADDED TOTAL °/o 100 90 80 34 // \ g\-\ ———-——"—D I 6\ 9: 9W8 4 I8 l2 I6 20 24 WEEKS OF INCUBATION 35 only a few percent after the 24 week period. This occurred to a limited degree in the sterile soil, as well. Hoytville soils receiving 40 ppm or 4 ppm MBOCA behaved quite similarly; MBOCA extractability decreased at similar rates and resulted in the same percentage of extractable MBOCA. Overall, MBOCA concentrations decreased very rapidly to quite low levels. The levels in the Hoytville soil samples receiving 4 ppm MBOCA decreased to about 10-20 ppb, just above the detection limit of 5 ppb for this procedure. Though it could not be determined with certainty if the metabolite interfered with the quantitative determination of MBOCA, the absence of metabolic conversion could explain the higher levels of MBOCA found in the sterile treatment com- pared to non-sterile 4 ppm MBOCA treatment. Evolution of 14C09 The production of 14CO2 from ring-labeled MBOCA can occur only with cleavage and oxidation of the rings. Radio— 14 activity, presumably from C02, was found in the NaOH sampling vials, but rarely amounted to more than 1% of the total added radioactivity (Table l). Traps in the sterile samples collected virtually no radioactivity. Implicit in this is biological mediation of the MBOCA oxidation. It is not known whether the 14 CO2 was produced from parent MBOCA or from a metabolite; perhaps it may have been oxidized only with the slow microbial oxidation of the recalcitrant humic matrix. 36 14 Table 1.--Cumu1ative CO2 production after 24 weeks. l4CO2 Recovered Treatment Soil (% of Total Applied)a 4 ppm MBOCA Hoytville 0.878 b 4 ppm MBOCA Sterile Hoytville 0.007 a 40 ppm MBOCA Hoytville 0.429 ab 4 ppm MBOCA Wabash Park 0.772 b 4 ppm trifluralin Hoytville 0.444 b aMeans followed by different letters indicate statistical separation. 37 14 Production of CO varied between replications but was 2 consistent, however, within flasks where vials were sampled and replaced as would be expected if CO2 release was due to microbial activity. Variability could have occurred if some samples became anerobic. Degradation of aromatic molecules, especially with halo- gen substituents such as those found in MBOCA, is often dif- ficult for microbes. Conclusion In summary MBOCA levels in soil, under laboratory con- ditions, decreased very rapidly. MBOCA was bound in the soil matrix and was also metabolized by microorganisms in soil. Soil binding was the major fate of MBOCA and occurred probably as both reversible and nonreversible adsorption. MBOCA had a great affinity for soil compared to water, probably as a result of its low water solubility and active amine groups. The nature of the bound MBOCA was unknown but the bound mater- ial probably would have eventually become an indistinguish- able part of the humis matrix. Biological metabolism resulted in the benzophenone derivative and very limited oxidation of some form of 14C- MBOCA to 14C02. Literature Cited Bartha, R. (1971). Fate of herbicide-derived chloroanilines in soil. J. Agr. Food Chem. 19:2, 385-387. 38 Green, R. E. (1974). Pesticide-Clay-Water Interactions. In Guenzi, W. P., Ed. Pesticides in soil and water. Madison, WI: Soil Science Society of America, 3-37. Hsu, T. S. and Bartha, R. (1974a). Interaction of pesticide- derived chloroaniline residues with soil organic matter. Soil Sci. 116, 444-452. Hsu, T. S. and Bartha, R. (1974b). Biodegradation of chloroaniline-humus complexes in soil and in culture solution. Soil Science. 118:3, 213-220. Hsu, T. S. and Bartha, R. (1976). Hydrolyzable and non- hydrolyzable 3,4-dichloroaniline-humus complexes and their respective rates of biodegradation. J. Agr. Food Chem. 24:1, 118-122. Kaufman, D. D., Plimmer, J. R., and Klingebiel, U. I. (1973). Microbial oxidation of 4-chloroaniline. J. Agr. Food Chem. 21:1, 127-132. Linch, A. L., O'Connor, G. B., Barnes, J. R., Killian, A. S., and Neeld, W. E. (1971). Methylene-bis-ortho- chloroaniline (MOCA): Evaluation of hazards and exposure control. Am. Indus. Hyg. Assoc. J. 32, 802-819. NIOSH (1978). Special hazard review with control recommenda- tions for 4,4'-methy1ene-bis (2-chloroaniline). U.S. Dept. Health, Education, and Welfare (NIOSH) Publication No. 78-188. Probst, G. W., Golab, T., Herberg, R. J., Holzer, F. J., Parka, S. J., Van der Schans, C., and Tepe, J. B. (1966). Fate of trifluralin in soils and plants. J. Agr. Food Chem. 15:4, 592-599. Russfield, A. B., Homburger, P., Boger, B., Van Dongen, C. G., Weisburger, E. K., and Weisburger, J. H. (1975). Toxicol. Appl. Pharmacol. 31, 47-54. Stula, E. P., Barnes, J. R., Sherman, H., Reinhardt, C. F., and Zapp, J. R. (1977). Urinary bladder tumors in dogs from 4,4'-methylene-bis (2-chloroaniline) (MOCA). J. Environ. Path. Toxicol. 1, 31-50. Stula, E. F., Sherman, H., Zapp, J. A., and Clayton, J. W. (1975). Experimental neoplasia in rats from oral admin- istration of 3,3'-dichlorobenzidine, 4,4'-methy1ene-bis (2-chloroaniline), and 4,4'-methy1ene-bis (2- methylaniline). Toxico. Appl. Pharmacol. 31, 159-176. 39 Wedemeyer, G. (1967). Biodegradation of dichlorodiphenyl trichloro ethane: Intermediates in dichlorodiphenyl acetic acid metabolism by Aerobacter aerogenes. Appl. Microbiol. 15, 1494-1495. Weed Science Society of America (1979). Herbicide handbook, 4th ed. Champaign, IL: Weed Science Society of America. CHAPTER II PLANT UPTAKE OF MBOCA [4',4'-METHYLENE-BIS (2-CHLOROANILINE)] Introduction It was recently shown that the soil in a section of the city of Adrian in southeast Michigan was contaminated with MBOCA [4,4'-methy1ene-bis (2-chloroaniline)] near a small specialty chemical company which manufactured MBOCA (State of Michigan agency memoranda, 1979). MBOCA has been shown to be a carcinogen in rats (Stula et al., 1975), mice (Russfield et al., 1975), and dogs (Stula et al., 1977) and thus, may pose a health hazard to residents of Adrian. Soil near the manu- facturing plant was contaminated into the parts-per-million (ppm) range, as were some residential areas including several garden areas (State memoranda, 1979). It is not known how the MBOCA was distributed, but is assumed to have been from dust from the manufacturing process and from transport of MBOCA away from the plant. In fully evaluating the hazards associated with this con- taminant, one must determine the fate of MBOCA in soil and whether it can be absorbed by plants and enter the food chain. The first problem was addressed in an earlier study and the second one is addressed here. 40 41 If MBOCA was distributed as dust, it could be absorbed through leaf surfaces and into the plant. MBOCA is a fairly nonpolar compound and could gain entry into the plant by partitioning into the epidermal waxes. Since MBOCA is known to be in the soil, the hazard of root absorption exists, as well as the hazard of contamination of root crops such as carrots (Daucus carrota L.). Numerous studies exist on the uptake of synthetic chemi- cals by plants. Chou et a1. (1978) found no absorption of polybrominated biphenyls (PBB) by corn (Zea mays L.) and soy- beans (Glycine max (L.) Merr.) through either the roots or leaves. Though some radioactivity was found on the roots, most of this could be rinsed off in acetone. Probst et a1. (1967) found that most crops absorbed almost no trifluralin (1,1,1-trif1uoro-2,3-dinitro-N,N-dipropyl-p-toluidine), though carrots absorbed a small amount found mostly in the peel, with small amounts found in the pulp. Still et a1. (1980) found that 3,4-dichloroaniline (DCA), a degradation product of propanil (3,4-dichlor0propionanilide), was absorbed by rice (Oryza sativa L.) plants and could be found in the rice grains, albeit only at 0.4 ppm. The uptake of tetrachlorodibenzo-p—dioxin (TCDD) from nutrient solution, soil, and foliage was studied by Isensee and Jones (1971). Soybean plants contained 0.01 ppm TCDD after 14 days in nutrient culture and less than 0.001 ppm after growing to maturity in soil. TCDD was absorbed by the foliage of oat 42 (Avena sativa L.) and soybean plants, but did not translocate beyond the point of application. Fuhrmann and Lichtenstein (1980) conducted a large study on the absorption by oat plants of six insecticides from two soil types. They con- cluded the most important factors were the water solubility of the chemical and the soil type. The more water soluble compounds exhibited greater absorption, especially from a sandy soil which has fewer adsorption sites. The objectives of this experiment were to determine whether MBOCA was absorbed through plant leaves or roots and to assess its translocation and distribution in plants. Materials and Methods Foliar Absorption Foliar MBOCA absorption was evaluated by placing a 14C- MBOCA-treated glass slide in intimate contact with the leaf surface for several hours or days. Ten micrograms of 14C- MBOCA was spotted on a 5 x 10 mm glass coverslip and applied to the leaf of a cabbage (Brassica oleracea L.) plant for 2 to 120 h. Following the appropriate time period, the radioactivity on the coverslip was assayed by liquid scintil- lation spectroscopy. The treated leaf area was rinsed repeatedly with 2 m1 of water and this was radioassayed. The treated leaf area and a nontreated area were then combusted and radioassayed. Replicate plant leaves were also 1yophi- 1ized and radioautographed to evaluate translocation of 43 radioactivity in the leaf. In another study the glass plate was applied to cabbage, sugar beet (Beta vulgaris L.), and green bean (Phaseolus vulgaris L.) leaves for 120 h and assayed as above. Root and Shoot Absorption of l4C-MBOCA from Aqueous Culture Two types of hydroponic experiments were conducted, one in water-nutrient culture and the other in sand with water and nutrients added. For the first type of hydroponic study green bean, sorghum (Sorghum vulgare Pers.), and carrot plants were grown in vermiculite for 10 to 60 days. The roots were rinsed free of vermiculite and immersed in 150 m1 of Hoaglands nutrient solution with 0.1% Tween 80 and 5 ppm (0.5 uCi) 14C-MBOCA. Three replications were made and the plants were maintained in the light in this solution for 8 days. In the second study green bean, orchard grass (Dactylis glomerata L.), and carrot plants were grown in a sand nutrient culture. After 3 weeks of growth the plants were watered once per week with 5 ppm (3.33 nCi/ml) 14C-MBOCA for the next 4 weeks. At harvest the roots were rinsed in fresh water and selected plant roots were also rinsed in acetone. Plants were then either lyophilized and radioautographed or sec- tioned and oxidized to quantitate radioactivity distribution. 44 14 Absorption of C-MBOCA from Soil Hoytville soil (48.6% sand, 20% silt, 31.4% clay, pH 7.4, 7.6% OM) was sifted through a 4 mm screen and then diluted 50% with coarse sand. 14 C-MBOCA was added to soil at 5 ppm (8.3 uCi/kg) by spraying an ethanol solution of MBOCA on 8.3 kg of soil as it was blended in a l9-liter rotary mixer. The soil was placed in eight 12 x 10 x 5 cm styrofoam flats and seeded with green beans or cucumbers (Cucumis sativa L.). Plants were harvested at germination and for 7 weeks follow- ing. At harvest plants were either radioautographed or oxi- dized to determine radioactivity. The experiments was run as a completely randomized design with two replications of each plant sample. Results and Discussion Foliar Absorption 14 C-MBOCA was absorbed by cabbage leaves within 2 h after application to the leaf surface as dry material (Table 1). There was no significant increased absorption from 2 to 120 hours with less than 30% of the applied radio- activity (or 3 ug) absorbed into the leaf. Only about 10-20% of the absorbed radioactivity could be rinsed off the leaf with water and the remainder was found on the glass. Five days after treatment 41.8% of the 14C was associated with the cabbage leaf (Table 2). Other species absorbed less. The amount absorbed into the leaf may be related to the thickness 45 Table 1.--MBOCA absorbed from glass surface by cabbage leaf. . Distribution of Radioactivitya'b Time After Tre tment Total SH Leaf Glass Rinse Recovered (% of Total) (% of Total) (% of Total) 2 21.7 a 71.9 4.0 97.6 24 27.7 a 69.9 2.6 100.2 48 21.0 a 67.3 2.9 91.2 120 27.4 a 66.4 2.3 96.1 aMeans of three replications. bNo statistical separation of means within columns. . 14 Table 2.--Absorpt10n of C-MBOCA by plant leaves after 5 days. Distribution of Radioactivitya Pl t Total an Leaf Glass Rinse Recovered (% of Total) (% of Total) (% of Total) Cabbage 41.8 60.5 1.8 104.1 Beanb 13.8 90.1 0.5 115.3 Beet 24.2 71.6 1.4 97.3 aMeans of duplicate leaves. b One sample only. Note: Radioactivity was not found beyond application point. 46 of the surface wax, the cabbage being most waxy and the bean leaf least waxy. There was no movement of radioactivity beyond the application point as shown in the radioautographs (Figure 1). This was confirmed by combusting a section of 14 the leaf below the application point and finding no C in the radioassay. Root and Shoot Absorption of 14C-MBOCA from Aqueous Culture Plants grown in a soil-free system should favor absorption of l4C-MBOCA supplied to the roots. It is apparent from the radioautographs of beans, sorghum, and carrots treated in this manner that most of the label is associated with the roots, although a limited amount of the radioactivity was translo- cated into the upper parts of the plants (Figures 2, 3, & 4). Translocation of 14C is obvious in the sorghum plants, but barely discernible in the bean plants. Oxidation and radio- assay of plant parts confirmed that there was very little 14 translocation of C-MBOCA from roots of the bean plants (Table 3). The 14C found in the bean pod was negligible. Radioactivity was found on the carrot root epidermis, but l4C found on the interior of the carrot was insignificant (Table 4). Sorghum roots contained large concentrations of 14C and a significant amount was found in the leaves. . . . 14 Neither a water rinse nor an acetone rinse removed C- MBOCA adsorped to sorghum roots (Figure 2). MBOCA is freely soluble in acetone, yet was not significantly removed from 47 Figure l.--P1ant leaves (top) and radioautographs (bottom). Cabbage (A) and sugar beets (B) were exposed to 14C-MBOCA for 5 days. 'vlllllIJIV'VII nut-nu-u-n 'Il‘."ll.l"1r . ...-..- 49 Figure 2.--Sorghum seedlings (top) and radioautographs (bot- tom). Roots were exposed to l4C-MBOCA for 8 days. Roots of "A" were water rinsed, roots of "B" were acetone rinsed. 50 I'll-IIII I. ".3 I'V‘..Q_,._’ 'Ii'T— m". o o 30...... I. ".0. 51 Figure 3.--Bean plant (top) and radioautograph (bottom). Plant roots were exposed to l4C-MBOCA for 8 days. 52 53 Figure 4.--Carrot plant (top) and radioautograph (bottom). Root was exposed to C-MBOCA for 8 days. Carrot is sliced longitudinally for radioautograph. 54 .I ov’o'I'V' 0“. I .I ll.""‘ I .00 55 Table 3.--Radioactivity distribution in bean planta. 14C Distribution Plant Part (mg/Elanztpart) (dpm/plant partb) (dpm/mg) Bean pod 121 36 0.3 Bean pod 62 0 0.0 3rd trifoliate 212 526 2.5 2nd trifoliate 128 202 1.6 lst trifoliate 210 154 0.7 Cotyledon 228 324 1.4 Lower stem 341 2,292 6.7 Root-stem 415 22,362 53.9 aPlant maintained in 1.1 x 106 dpm (5 ppm) 14C-MBOCA water solution for 8 days. bSubtracted background CPM = 65. Table 4.--Radioactivity distribution in plant partsa. l4C Distribution Fr. wt. Plant Plant Part (mg/plant part) (dpm/plant (d m/m ) part) p 9 Carrot Interior of root 150 35 0.2 Interior of root 200 32 0.2 Root epidermis 120 3,634 30.3 Sorghum Leaf 65 205 3.2 Leaf 110 875 8.0 Leaf 180 y 754 4.2 Roots 55 189,983 3,454.0 aPlants maintained in 1.1 x 106 dpm (5 ppm) MBOCA water solution for 8 days. 56 the roots after three rinses, including 5 min in the final rinse. This was in contrast to the PBB study of Chou (1978) who was able to remove most of the radioactivity with three brief acetone rinses. In the second study plants were maintained in sand to avoid disruption of the root system. The bean plants again 14C to the shoots while the orchard translocated very little grass absorbed a discernible amount of MBOCA similar to the sorghum (Figure 5). These results appear similar to the DCA study on rice by Still et a1. (1980). They found a uniform, detectable level of DCA in the rice plant and grain but were unable to determine the nature of the DCA in the plant. The radioautographs in Figure 6 distinctly show a small amount of radioactivity along the xylem-phloem layer near the center of the pulp of the carrot root. This region was not quantitated by itself, but random sampling of areas inside the pulp showed very low levels of radioactivity (Table 5). Probst et a1. (1967) found that trifluralin was absorbed by carrots with 69% being found on the peel and 31% in the pulp. Ten percent of the trifluralin was found in the xylem-phloem layer near the center of the pulp. In summary, it appears that MBOCA is tightly sorbed to the root surface. Some MBOCA is also able to move with the transpiration stream into and throughout the plant, probably related to the water solubility of MBOCA. MBOCA has water solubility similar to parathion (diethyl-p-nitrophenyl 57 Figure 5.--Orchard grass (top) and radioautograph (bottom). Roots were exposed to l4C-MBOCA for 4 weeks. 58 59 Figure 6.--Carrot plants (top) and radioautograph (bottom). Roots were exposed to l4C-MBOCA for 4 weeks. 60 a“... 0’ 61 Table 5.--Radioactivity in carrot plant. l4C Distribution Fr. wt. Plant Part (mg/plant part) (dpm/plant part) (dpm/mg) Epidermis 132 9,689 73.40 Epidermis 234 15,161 64.79 Epidermis 161 2,005 12.45 Interior 231 136 .59 Interior 135 13 .10 Interior 139 93 .67 Interior 124 46 .37 Interior 237 129 .54 62 monothiophosphate) which was used in the study by Fuhrmann and Lichtenstein (1980). They found a limited but detect- able absorption of parathion by oat plants. 14 Absorption of C-MBOCA from Soil Much radioactivity was associated with the bean and 14C found in the shoots cucumber roots with very little (Figures 7 & 8). No radioactivity was found in the bean pods (Figure 8). The presence of radioactivity in the cucum- ber leaves may be related to contact with the soil surface during watering of the plants. Virtually no radioactivity was found in the plant shoots compared to that found in the roots (Table 6). The bean root contains an extraordinary amount of label (62.4 dpm/mg) com- pared to that added to the soil (18.4 dpm/mg) indicating concentration of the MBOCA around the root. In the sandy soil some of the MBOCA may have migrated with the capillary water flow to the roots and then adsorbed to the surface of the roots. Conclusions MBOCA was absorbed by plants but its movement within the plant was limited. When absorbed by leaves the MBOCA was limited to the absorbtion point and was probably isolated to the leaf cuticle. MBOCA absorbed by plant roots generally remains on the root surface with translocation of a small 14 fraction of the C label into the shoot. MBOCA absorption 63 Figure 7.—-Bean plants (top) and radioautograph (bottom). Plants were raised in C-MBOCA treated soil. 65 Figure 8.--Cucumber plants (top) and radioautograph (bottom). Plants were raised in 14C-MBOCA treated soil. 67 Table 6.--Radioactivity distribution in soil grown plantsa. l4C-Distribution Plant Part (mg/:1anztpart) (dpm/plant part) (dpm/mg) Bean Leaf 135 121 0.9 Bean Apex 160 93 0.6 Bean Lower stem 220 177 0.8 Bean Roots 175 10,927 62.4 Cucumber Leaf 130 114 0.9 Cucumber Leaf 230 109 0.5 aTwo week old plants grown from seed in soil amended to 5 ppm (18,400 dpm/g) 14C-MBOCA. 68 from soil by plants was very limited, probably because of the soil adsorption of MBOCA. Literature Cited Chisaka, H. and Kearney, P. C. (1970). Metabolism of pro- panil in soils. J. Agr. Food Chem. 18:5, 854-861. Chou, S. F., Jacobs, L. W., Penner, D., and Tiedje, J. M. (1978). Absence of plant uptake and translocation of polybrominated biphenyls (PBB's). Environ. Health Perspect. 23, 9-12. Fuhremann, T. W. and Lichtenstein, E. P. (1980). A compara- tive study of the persistence, movement, and metabolism of six carbon-14 insecticides in soils and plants. J. Agr. Food Chem. 28, 446-452. Isensee, A. R. and Jones, G. E. (1971). Absorption of root and foliage applied 2,4-dichlorophenol, 2,7- dichlorodibenzo-p-dioxin, and 2,3,7,8- tetrachlorodibenzo-p-dioxin. J. Agr. Food Chem. 19:6, 1210-1214. Russfield, A. B., Homburger, F., Boger, B., Van Dongen, C. G. Weisburger, E. K., and Weisburger, J. H. (1975). Toxicol. Appl. Pharmacol. 31, 47-54. Still, C. C., Hsu, T. S., and Bartha, R. (1980). Soil bound 3,4-dichloroaniline: Source of contamination in rice grain. Bull. Environ. Contam. Toxicol. 24, 550-554. Stula, E. F., Barnes, J. R., Sherman, H., Reinhardt, C. F., and Zapp, J. R. (1977). Urinary bladder tumors in dogs from 4,4'-methylene-bis (2-chloroaniline) (MOCA). J. Environ. Path. Toxicol. 1, 31-50. Stula, E. F., Sherman, H., Zapp, J. A., and Clayton, J. W. (1975). Experimental neoplasia in rats from oral admin- istration of 3,3'-dichlorobenzidine, 4,4'-methylene- bis (2-chloroani1ine), and 4,4'-methylene-bis (2- methylaniline). Toxicol. Appl. Pharmacol. 31, 159-176. GENERAL REFERENCES GENERAL REFERENCES Bowman, B. T. and Sans, W. W. (1979). The aqueous solubility of twenty-seven insecticides and related compounds. J. Environ. Sci. Health B14(6), 625-634. Chisaka, H. and Kearney, P. C. (1970). Metabolism of pro- ponil in soils. J. Agr. Food Chem. 18(5), 854-861. Fuhremann, Tom W. and Lichtenstein, E. Paul. (1978). Release of soil-bound methyl [14C] parathion residues and their uptake by earthworms and oat plants. J. Agric. Food Chem. 26:3, 605-610. Golab, T., Althus, W. A., and Wooten, H. L. (1979). Fate of [14C] trifluralin in soil. J. Agr. Food Chem. 27:1, 163-179. Liu, S. Y., Minard, R. D., and Bollog, J. M. (1981). Coupling reactions of 2,4-dichloro phenol with various anilines. J. Agric. Food Chem. 29, 253-257. Parris, G. E., Diachenko, G. W., Enitz, R. C., Poppiti, J. A., Lombardo, P., and Rohrer, T. K. (1980). Water borne methylene bis (2-chloroaniline) and 2-chloroaniline contamination around Adrian, Michigan. Bull. Environ. Contam. Toxicol. 24, 497-503. Rhodes, R. C., Belasco, P. J., and Pease, H. L. (1970). Determination of mobility and adsorption of agrichemicals on soils. J. Agr. Food Chem. 18:3, 524-528. Tiedje, J. M. and Mason, B. B. (1974). Biodegradation of nitrilo acetate (NTA) in soils. Soil Sci. Soc. Amer. Proc. 38, 278-283. 69