IAEA-TECDOC-554
RADIOTRACER STUDIES OF FUNGICIDE RESIDUES FOON I D PUNTS
PROCEEDING FINAA F SO L RESEARCH CO-ORDINATION MEETING ORGANIZED BY THE JOINT FAO/IAEA DIVISION OF NUCLEAR TECHNIQUES IN FOOD AND AGRICULTURE AND HEL ANKARADN I , 13-17 MARCH 1989
ATECHNICAL DOCUMENT ISSUED BY THE INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1989 RADIOTRACER STUDIES OF FUNGICIDE RESIDUES IN FOOD PLANTS IAEA, VIENNA, 1990 IAEA-TECDOC-554 ISSN 1011-4289
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Growing world population and food demand have dictated the introduction of intensive agricultural practice n increasina sf o involvin e us g e rangth g e of pesticide chemicals considerabla o t d . le Thi s seha increas foon ei d crop production. However, wit e increasinhth agriculturaf o e gus l chemicaln so crops, there is a major concern from a toxicological standpoint. Such use must not result in the retention of appreciable (and potentially toxic) residue foon so d products. Even when pesticid e conformeus currentlo st y adopted standards of good management practice, undesirable side effects may occu d couldan r timest ,a , conceivably endanger public health o ensurT . e safety-in-use, adequate monitoring programmes shoul availablee db .
In 1984, the Joint FAO/IAEA Division, recognizing the need for coordinated research on the fate and terminal residues of agrofungicides in food plants and further recognizing that such internationally coordinated research efforts have not been undertaken, established the coordinated research programme on "Radiotracer studies of fungicide residues in food plants". In initiating this programme, the Joint FAO/IAEA Division recognized that major knowledge gaps exist in the subject area, that, if filled, would greatl developind yai g nation thein i s r effort o safelst d effectivelyan y utilize fungicide chemicals to maximize food production. The programme was targeted specifically to conduct coordinated research that addressed these needs.
Data generated under this programme are important to national food quality programmes and can assist in evaluating the possible impact of residues in food on the consumer. In this context, radiotracer techniques provide a powerful research tool in detecting, quantifying and identifying chemical residues associated with biological matrices. These techniques also constitut e onleth y effective mean detectinn si quantifyind an g g possible chemical binding to food, plants and soil. The results obtained also provide a significant contribution to the International Joint FAO/WHO Pesticide Residue Programme which establishes international recommendationr sfo acceptable daily intak humanr e fo maximu d san m residue limit foon d feedi s an d . EDITORIAL NOTE
In preparing this material for the press, staff of the International Atomic Energy Agency have mounted and paginated the original manuscripts as submitted by the authors and given some attention to the presentation. The views expressed papers,the statementsin the general madethe and style adoptedthe are responsibility namedofthe authors. necessarilyviewsnot The do reflect governmentsthosethe of of the Member States or organizations under whose auspices the manuscripts were produced. thisin The bookuse of particular designations countriesof territoriesor does implynot any judgement by the publisher, the IAEA, as to the legal status of such countries or territories, of their authorities and institutions or of the delimitation of their boundaries. The mention of specific companies or of their products or brand names does not imply any endorsement or recommendation on the part of the IAEA. Authors themselvesare responsible obtainingfor necessarythe permission reproduceto copyright material from other sources. CONTENTS
Radiotracer studie f maneo s ethylenethioured ban tomatn ai osoiln i fruit d s an ...... s 7 . M.R. Musumeci, S. de Barras Ostiz, T. Bonanho, M.C.D. Suva, E. Flores Ruegg Radiotracer studie f maneo s b residue tomatn si o plants ...... 7 1 . O. Pazmino, M. Bolanos, L. Espinosa, M. Moran, J. Molineros, R. Merino Residue f maneo s tomatn bi theid oan r persistence during cookin storagd gan e ...... 5 2 . D. Kolankaya, A. Oegues, Z. Ayas, M.T. Akay Ethylenethiourea (ETU ethylenethiurad )an m monosulfide (ETM bean )i n plants treated with I4C-maneb ...... 31 A.H.E. Harhash, Hegazi,B. S.M.A.D. Zayed Fat f 14eo C-mane soyabean bi n plants ...... 1 4 . A.H.E. Harhash, S.M.A.D. Zayed, B. Hegazi Degradatio f maneno ethylenethioureo bt largn ai e pepper ...... 9 4 . F. Rarnon, Espinosa,J. Batista,A. ArchibaldW. Radiotracer stud f mancozeyo b residue tomatn i s o plants ...... 5 5 . Lianzhong Zhang, Huanfang Wang, HanhongMo Residues of mancozeb and ethylenethiourea in cucumber treated with 14C-mancozeb under field conditions ...... 1 6 . S.M.F. Calumpang, M.J.V. Barredo, N.P. Roxas, E.D. Magallona Residues of mancozeb and ethylenethiourea in beans ...... 73 M.R. Musumeci, M.C.D. Suva, FloresE. Ruegg, RolimP. Mancozeb residue tomatn i s o plants ...... 9 7 . Johari Ramli,bin Juzu H.B. shad,Ar Maliki Ismail,bin Samsuddin Abdulbin Wahab Persistence and metabolism of mancozeb in brinjal (egg plant) ...... 85 Agarwal,C. H. KumarU. Effect of washing, baking and cooking on aged residues of mancozeb in brinjals (egg plants) ...... 93 Agarwal,C. H. KumarU. Radiotracer study of zineb residues in egg plant-soil systems ...... 97 QianJ. An, Zhang,L. F. Wang,G. Mo, H. Persistence of 14C-ethylenethiourea in brinjal plants and soil (Abstract) ...... 103 H.C. Agarwal, Kumar,U. D.K. Singh Studies of 14C-mancozeb fungicide and I4C-ethylenethiourea in silty clay loam soil (Summary) ...... 105 S.M.F. Calumpang, M.J.V. Barredo, N.P. Roxas, E.D. Magallona An attempt to separate ethylenethiourea and ethyleneurea, degradation products of ethylenebis(dithiocarbamates), using HPLC ...... 107 P.N. Moza, K. Husten
Appraisa Co-ordinatee th f o l d Research Programme ...... l Il .
Model protocol determinatioe th r fo s f ethylenebis-dithiocarbamatno e residues ...... 7 12 . Annex I. Treatment of food plants with 14C-EBDC fungicides and analysi f residueo s s ...... 8 12 . Annex II. Treatment of soil with 14C-EBDC fungicides and analysis of residues ...... 133 Annex III. Methods for the determination of EBDCs and degradation products ...... 136 List of Participants ...... 139
Lis f Publicationo t s produced withi Agrochemicale nth Residued an s s Programme since 1982 ...... 1 14 . RADIOTRACER STUDIES OF MANEB AND ETHYLENETHIOUREA IN TOMATO FRUITS AN SOILN DI S
. MUSUMECIM.R BARROE D . S , S OSTIZ . BONANHOT , , M.C.D. SILVA, E. FLORES RUEGG Radioisotope Center, Biological Institute, Sào Paulo, Brazil
Abstract
residueU ET Mane d an bs were studie tomatoen o d s afte fielr4 d sprayings with 14C-maneb. The residue declined with time; 14 days after the last application, pg/0 fruitmanef 3. o g d 0.0 d ban sha 3 pg/ ETUf go rip n O . e fruits, mane s detectedbwa concentratioU ET n I .o n 6 MS/'S t 0. bu >s nwa 14 tomatoes with a single C-maneb spray, the radiocarbon was associated mainly with the fruit skin; 1.2% were detected in pulp and no activity in seeds. Washing fruits decreased ETU content, but cooking increased ETU levels 14 substantially. In fruits treated with one application of C-ETU, the half-life was 5.7 days with ethyleneurea and ethylenediamine as major 14 14 degradation products soin I .l treated with C-mane r bo C-ETU , 14 C-labelled residues disappeared fairly rapidly. 14C-ETU in soil had a half-life of less than 4 weeks. In soil, most of the radioactivity derived 4 1 from 14C-mane b or C-ETU was found in the top 5 cm layer. Methanol non-extractable activity was substantial in soils treated with 14C-maneb or 14 14 C-ETU. When soil samples were incubated with C-ETU under laboratory conditions, mineralization O occurreevolveC d ddan contribute14 d 27-42% of the initial radioactivity after 35 days.
1. INTRODUCTION Maneb (manganese ethylenebisdithiocarbamate EBDe th C f o e on s )i fungicides, used in Brazil for controlling plant diseases in many crops. In recent years, great interes metabolise rises th tha n ni thesf mo e fungicides on plants since the presence of ethylenethiourea (ETU) on crops after application of ethylenebisdithiocarbamate has been observed (1). ETU can cause thyroid carcinom higt a h dose ratn i s s (2). This research was undertaken to investigate whether climatic conditions in Brazil (high temperature humidityd san ) would promot e formatioeth f no higher levels of ETU in tomato fruits. The behaviour of maneb and ETU in soil samples in the field and in the laboratory was also examined.
2. MATERIA METHODD LAN S
2.1 Chemicals 14 C-maneb, labelled in the 1,2-ethylene position, was purchased from Isotope Trading Enterprise (Institute of Isotopes, Budapest, Hungary); specific activity 83.0 vCi/mg. 14 C-ethylenethiourea, labelled in the 1,2-ethylene position (Institute of Isotopes, Budapest, Hungary); specific activity 117.72 yCi/mg. - Reference compounds: ethylenethiourea (ETU), ethyleneurea (EU) and ethylenediamine (EDA). Formulated maneb as 80% active wettable powder.
2 2. Soil To stud behavioue soiln yth i o soiU ,tw mane ET lf ro d type ban s were used: humic gle=5.7H (p y ; organic matter 4.3%; clay 57%d ;an sil% 12 t sand 31% purpld )an e latoso l5.5= soiH ;(p l organi c matter 2.7%; clay 66%; silt 24% and sand 10%).
3 2. Analysi commercian i U maneET f so d lban preparations Maneb concentration was determined by Keppel method, based on CS evolution (3). ETU in the commercial formulation was determined prior to experimentation by adding 50 ml of freshly distilled ethyl acetate to 5 g of the formulation. The suspension was stirred with a magnetic stirrer for 15 min. and the supernatant decanted. The presence of ETU s detectewa d quantifiean d TLCy b d . Fift e suspensioyth \if o l n were applie silica-gea n o d l thin-layer plate (Merc F-2540 k6 developed )an d in chlorofor butano: m methano: l wate: l r (100:5:1:0.5). After drying, the chromatogram was sprayed with a nitroprusside-ferricyanide reagent (4). Quantification of ETU was achieved by spotting on the same plate 50 yl of graded concentrations of ETU in ethanol and comparing the siz d intensitean spote th sf y o (5).
4 2. Preparatio f no C-mane b formulatio14 n Five hundred mg of the formulated maneb were mixed with 12 mg of 14 C-maneb and 10 ml of dry freshly distilled ethyl acetate were added mixturee tth o . After stirrin min0 1 ethye r ,th fo gl acetate layes rwa removed residue ,th e drie d store dissicatoa an d n i d 4°Ct a r .
5 2. Experiment plantn si s
2.5.1 Applicatio d samplingnan fiele Th .d experimen s conductetwa d durine th g autumn 198 summed 6an r 198 tomatn 7o o plants (Lycopersicon esculentun, Mill, CV. Sta. Cruz), grown at the Biological Institute in two rows with 10 plants each. At a post-bloom stage (initial fruit stage), each plant sprayes aqueoun wa a f o d s l wit m suspensio0 h1 n (10 formulateg 0m d 14 maneb/200 yCi C-maneb/200 ml water), (10 yCi/plant). As in local practice, treatment was repeated 4 times with 7-day intervals. Fruits (3 plants per sampling) were taken for analysis at 0, 7, 15, 28 and 35 days after the first spray, immediately before the subsequent sprays. Ripe fruits from 2 plants were also collected at day 70.
2.5.2 Analysis of carbon-14 activity. For determination of total radioactivity, fruits from 3 plants were mixed and chopped and 100 g of sample were blended wate l witm higt 0 ra h5 h min5 speee r On .fo d 14 hundre g sampledm s were analyse duplicatn i d r totaefo l C-activity by dry combustion (Harvey oxidizer) and liquid scintillation counting (Beckman LS 5801).
2.5.3 Carbon-1 analysisU 4ET . Fruits fro plant3 m s were mixed, chopped an d 100 g duplicate samples were extracted as described by Yip and Onley (6). Each sample was blended with 200 ml of ethanol for 2 min. at high speeblendera n i d ; chlorofor wer) Celitmd g an (10e0 ) the(1 e0 ml n added and the mixture was blended for further 2 min. The mixture was filtered with suctio nlayem c througvolum e Celitf 5 th ro 0. d eha ean measured. The filtrate was reduced under vacuum to 5 ml and the solution quantitatively transferrevolumetril m 0 1 a o ct d flas madd kan e to volume with methanol. Aliquots (100 yl) of each extract were counte determino t d totae eth l radioactivity aliquotl m o Tw f .so extracts were subjecte clean-uo dt columa n pi n containinf o g 0 2 g washed aluminium oxide. The column was first washed with 30 ml of petroleum ether, which was discarded and the column eluted with 20 ml of methanol : acetonitrile : benzene (3:15:82). The eluate was concentrate dryneso t d rotoevaporatoa n o s r unde rresidue vacuuth d ean m methanolf o l m 4 .n take i Aliquotp nu s were applieplateC TL o st d (0.2 mm Silica-ge F-2540 l6 ,d MerckC-ETUan ) streaa cm s , )a 3 ETU( k U ,E standardA anED d s wern alongsideeru plate m Th s developec . ewa 0 1 r fo d wit mixturha chloroforf eo butano: m methano: l wate: l r (100:15:1:0.5) and after drying, 1 cm zones were scrapped and counted.
2.5.4 Analysis of maneb. Duplicates of 100 g sampls of tomato were processed according to Keppel (3) to determine maneb residues in the fruits.
2.5.5 Effec cookinRf o t n summeI . r 1987, developed tomato fruits were sprayed 14 once with C-maneb suspension (24 mg/48 pCi/10 ml HO) and sample rips a d e fruit 5 days1 s after application. e fruiParth s twa f to washemin2 r .fo d under runninsampleg 0 waterp 10 washef ta gso d ,an d and non-washed fruits were blende higt a d h speeblendera n i de Th . homogenate was partially dried in a vacuum oven at 60°C overnight, and g aliquotm 0 10 s subjecte combustioy dr o t dHarvea n i n y oxidizer. Fractions of 100 g of the washed and non-washed fruits were boiled in distillel m 0 5 dmin5 1 wate d allower .an fo r coolo t d . Wate addes rwa d tor losse makfo p seu during boiling.
Cooke non-cooked an d d fruits were extracte d ethanodl m an wit 0 ) 20 h(6 l the filtered extract concentrated to 10 ml. Analysis of ETU was made by TLC as previously described. The plates were developed for 10 cm in ethyl acetate : NH OH : water (90:6:6) and ether : MeOH (9:1).
2.6 Studie carbon-1n so U 4ET 14 14 C-ET + C-ETU U ET solutio Ug m 11. 0 (1 7n yCi/1 Hl 0m 2s 0)wa applied with a Pasteur pipette on tomato fruits in a post-bloom stage of plant4 s (summer 1987). Fruits were collecte aftes hr tim0 r2 t ( ea d application), 4, 7, 14 and 30 days, and analysed for total 14C- 14 activity and for C-ETU as previously described.
2. 7 Soil studies
2 2.7.1 Field experiments Institute smalA th . t a l soiem 3 l 3x plo f to 14 14 Biolögico was used for studying C-maneb and C-ETU behaviour in soil. The experiment was conducted from May 1988 to January 1989. Twenty polyethylene tubes (3 cm diameter by 30 cm long) were driven into a humic gley soil. About 0.5 cm of each cylinder was left protruding above the surface for protection against run-off. Ten cylinders were 14 treate aqueoun a f pipettiny o db sl m solutio5 0. g f no C-ET U (10g 0m ETU -»-11.7 yCi/1 ) ontsoie 0ml th ol surface e remaininTh . 0 1 g
10 14 cylinders were treated f eaco C-mane hl m wit 5 bh0. solutiog m 4 (7 n maneb [148 viCi]/10 ml methanol). Cylinders were removed for analysis weeks5 3 e soi d Th .lan fro3 1 m, 4 eac, afte1 h , r0 cylinde divides rwa d into 4 layers: 0-5; 5-10; 10-20; 20-25 cm. The moisture content was determined for each layer. Triplicate 100 mg samples were analysed for 14 total C-residue combustioy dr y sb d liquinan d scintillation counting. layem Soic 5 rl 0- wersample fro) e g emth 5 Soxhle(2 s t methanolf o l m wits 0 extractehr , h15 2 1 extract r fo d s concentrateo t d 10 ml under vacuum and the radiocarbon measured. Aliquots (200 yl) of extracts were analyse TLCy b d . Non-extracted radiocarbo soin ni l (after Soxhlet extraction) was determined by dry combustion and liquid scintillation counting.
2.7.2 Mineralization studie earbon-1f so 4 mane d carbon-1ban 4 soig ETU n l Te . samples (humic glepurpld yan e Latosol soil) were placed into wide necked screw capped jars. Wate3 fiel s adde2/ rwa do t dcapacity a d ,an 14 14 week later 100 yl of C-ETU or C-maneb solution were added. 14 Control soil non-biologicar sfo O C evolutiol n were preparey b d 14 autoclavin days3 120°t a gr .Cfo s trappe wa Evolve n O i dC d ethanolamin d incubatioean lighty da r hels s ,fo 35°Ct nwa a hr d 2 ,1 35 days; traps were collected weekl) (7 analysed yan C LS y db
3. RESULT DISCUSSIOD SAN N
1 U conten3. ET Mane d commerciaf tan o b l preparations In an open package, maneb concentration declined quickly from 83% to 68% over a period of 4 months, and to 60% after 21 months. ETU also decreased from 2 ppm to 0.01 ppm over a period of 21 months.
2 3. Experiment tomatn so o fruits Maneb residues decrease tomatn i d o fruit sovem fropp ra 0 m 3. 30. o t 2 period of 5 weeks (Table I). This declined further to 0.6 ppm after 10 weeks. Partially ripe fruit weeks5 ( s ) containe whicU ET d hf 0.0o m 3pp decline non-detectablo t d e levels afte week0 r1 s (Tabl . ThieI) s decline is in agreement with the results obtained by other authors (6, . 8Washin,9) g fruitmin2 r .sfo unde r running water decreaseU ET e th d content (Table II). However, cooking tomato fruit treated wite hon 14 14 application of C-maneb led to a substantial increase of C-ETU (Table II). In case of unwashed fruits, the increase was 67%, while the washed fruits contained 117% increase residuesU ET d radiocarboe Th . n
11 Table I - Maneb/ETU residues in tomato fruits
Days after Maneba 14C-ETU Cumulative Number of the first Pg/g Vg/g rainfall applica- application (mm) tions
0 30.2 0.60 0.1 1
7b 15.6 0.31 58.0 1
Ub 28.1 2.2 29.8 2
21b 20.7 1.2 8.0 3
28 12.0 0.2 7.4 4
35 3.0 0.03 4.3 4
70 0.6 nd 17.3 4
a - based on CS£ evolution
subsequen- b t sprays
Table II - Effect of cooking on 14C-maneb and 14C-ETU residues in tomato fruit
Fruits collected Before cooking After cooking 2 weeks after one C—maneb a ETU ETU applicatiof no dpm/ga vg/gk dpm/TLC Pg/g dpm/TLC Vg/g l^C-maneb plate plate
Non-washed fruits 16205 23.9 1001 1.48 1678 2.47
Washed fruits 7350 8.2 539 0.60 1189 1.3
determined by dry combustion evolutio2 CS - n method
12 Table III - ETÜ residues in tomato fruits treated with C-ETU 14
Days after Total 14C ETU treatment dpm/g Vg/g %
0 16,66 10.0 100
4 0 15 , 5 1 3,5 35
7 6,059 1.0 10
14 4,360 0.63 6.3
30 3,090 0.22 2.2
content in different parts of washed fruits showed that 26.6 vg/g of 14 C-maneb was present in the fruit skin, and translocation to pulp was very low (0.04 vg/g). Seeds had no detectable radioactivity. In 14 fruits treated with C-ETU, ETU concentration decreased from 10 Vg/g from samples collected at 0 time to 0.22 vg/g in fruits 30 days after treatment (Tabl daye7 5. III)s s half-lifwite wa . Th ha U ET f eo correlation coefficient of 0.84. The major degradation products of ETU detected by TLC were ethylenediamine and ethyleneurea.
3 3. Soil studies 14 14 C-analysis of soil treated with C-maneb showed that almost one third of the fungicide applied disappeared from soil after 4 weeks (Table IV). Distribution among the soil layers showed that over 97% of 14 layerp to e . th compoune n th i C-ET s wa dU levels declined fairly rapidly. Afte week3 r1 s radioactivity dos e correspondeth ef o % 13 o t d applied (Table IV). Afte weeks3 r1 C analysi,TL soif so l extracts 14 (treated with C-maneb) showed a radiocarbon distribution of 20% ethyleneurea; 15% ethylenediamine; 13% ethylenethiourea; 21%
unidentified metabolite: Rf 0.60 in ethyl acetate : NH OH : H_0 (90:6:6); R 0.70 in ether : MeOH (9:1). Soil extracts (treated with 14 C-ETU) after 13 weeks showed no detectable ETU with the major 14 C-activity associated wit unidentifien ha d product. Tabl eshowV s 14 that most of the activity associated with C-maneb was 14 methanol-unextractable while C-ETU showed initial substantial bindin soio gt l which increased with time, probabl formatioo t e ydu f no
13 residueU Tabl ManeET - d V eI ban s (vg/g n soi)i l after applicatiof no 14C-maneb or 14C-ETU
Exposure time (weeks) Soil depth 0 4 13 35 (cm) ManeU ET bManeU ET b Maneb ETU Maneb ETU
0-5 62.0 83.0 39.0 37.0 0 9. 35.14.0 0 1.6
1 12.1. 5-10 0 00. 0 0. 0.9 2.0 0.170.0
0 0. 5 0. 10 - 20 00. 0 0. 0.3 0.0 0.0 0.0
0 0. 3 0. 5 2 20- 0 0. 0 0. 0.1 0.0 0.0 0.0
TOTAL 62.0 83.0 40.9 49.0 36.3 11.0 15.7 1.6
Total rainfall 0 ra:88 m
Table V - Methanol extractable and non-extractable radiocarbon in soils treated with 14C-mane 14d C-ETban U
Treatment Tim exposurf eo e (weeks) 4 13 35
Extractable 12.0 15.0 10.0 4.0
14C-maneb Non-extractable 88.0 85.0 90.0 96.0
Extractable 69.0 11.2 8.8 14 C-ETU Non-extractable 31.0 87.8 91.0 — —
products which also strongly bind to soil. Soil incubated for 35 days 14 14 with C-ETU showeradioactivite th f do tha% t42 y O evolveC s a d from purple Latoso % frol27 m soi d humilan c gley. Only 2.4f %o 14 CO evolved from soils incubated with 14 C-maneb. No 14CO evolution was detected when either 14C-ET U or 14C-maneb were incubate sterilizen i d d soils.
14 REFERENCES
(1) Engst, R., Schnaak, W. Residues of dithiocarbamate fungicides and their metabolite plann so t food. Residue Revie 2 (1974w5 . )45
) (2 Ulland, B.M., Weisburger, J.H., Weisburger, E.K., Rice, J.M., Cypher, R. Thyroid cancer in rats from ethylenethiourea intake. J. Nat. Cancer Inst. 49 (1972) 583-584.
(3) Keppel, E.G. Modification of the carbon disulfide evolution method for dithiocarbamate residues Assoc. J . . Off. Anal. Chem (19692 .5 ) 162-167.
(4) Vonk, J.W., Sijpesteijn, K. Tentative identification of 2-imidazoline atransformatioa s n produc ethylenebisdithiocarbamatf to e fungicides. Pestic. Chem. Physiol (1971.1 ) 163-165.
(5) Onley, J.H., Yip, G. Determination of ethylenethiourea residues in foods, using thin-laye chromatographys ga d ran Assoc. J . . Off. Anal. Chem (1971. .54 ) 165-169.
(6) Yip, G., Onley, J.H., Howard, S.F. Residues of maneb and ethylenethio- ure fieln ao d sprayed lettuc kaled Assocean . J . . Off. Anal. Chem4 .5 (1971) 1373-1375.
) Musumeci(7 , M.R., Rüegg, E.F. Degradacao microbian fungicido ad a metalaxi soloo ln . Fitopatologia Bras .(1984£ ) 583-591.
(8) Newsome, W.H., Shields, J.B., Villeneuve, D.C. Residues of maneb, ethylenethiuram monosulfide, ethylenethiourea and ethylenediamine on beans and tomatoes field treated with maneb. J. Agr. Food Chem. 23 (1975) 756-758.
) (9 Rhodes, R.C. Studies with manganese 14C-ethylenebis(dithiocarbamate) •^C-maneb fungicides -^C-ET plantsn i U , soiwaterd an l Agr . J .. Food Chem. 25 (1977) 528-533.
Next page(s) left blank RADIOTRACER STUDIES OF MANEB RESIDUES IN TOMATO PLANTS
O. PAZMINO, M. BOLANOS, L. ESPINOSA, M. MORAN Ministr f Agriculturyo Livestocd ean k J. MOLINEROS, R. MERINO Atomic Energy Commission Quito, Ecuador
Abstract
Maneb concentration commercian si l preparations showe appareno dn t significant decreases ove periora week9 1 f so d while ethylenethiourea showed slight ovem increasee sampp th r e 0 period42 o st e frostudA 0 th . m26 n o y 14 14 fate of C-maneb and C-ETU in tomato plants under greenhouse conditions showed tharesidueU tET leaven i s s increase maximua o t d6 day 5 f 1.3 mo sm 7pp afte lase th rt applicatio slowld nan month4 b y pp declines 2 afte0. e o rth t d last application. The fruits showed ETU residues of 1.12 ppm after 56 days following the last application and decreased to undetectable levels after 4 months. Cooking of fruits resulted in a decrease of extractable 14 C-activity whil concentratioU eET extrace th n i n t increased 50%o t fro 1 .3 m
1. INTRODUCTION In Ecuador, ethylenebisdithiocarbamates (EBDCs) are applied routinely to solanaceas crops, green vegetables, fruits and recently ornamental plants. No information is currently available on residues of EBDCs or their toxic decomposition product ethylenethiourea (ETU foon i ) d commoditiee th n i d san environment mose th t s toxicologicalli U ET . y important decomposition product of EBDCs, and has been reported to be carcinogenic and mutagenic in mammals (1, 2). Degradation of EBDCs to ETU may be increased in tropical and subtropical regions since EBDCs are unstable in the presence of moisture and at higher temperature (3).
Tomatoes were selected for this study because of their high consumption in Ecuador. This researc conducteds hwa studo T decompositio e ) yth i : a f no commercial preparation of maneb under local storage conditions; ii) To 14 determine residues of maneb and ETU in tomato plants sprayed with C-maneb
17 following local practice conditions; and iii) to evaluate the effect of cooking on the decomposition of maneb to ETU in tomatoes spiked with 14 C-maneb.
2. MATERIA D METHODLAN S
2.1 Pesticides 14 C-ethylene-labelled maneb, specific activity 83.0 yCi/mgs ,wa obtained froe Institutmth Isotopef eo e Hungaria th f o s n Academf o y Sciences, Budapest, Hungary. Ethylenethiourea, analytical standard (Environmental Protection Agency, USA). Mane % W.P80 b. (Celamerck Ecuatoriana, Quito, Ecuador). Adsee 775, Adjuvant (Witco Chemical Corporation, USA).
2 .2 Major equipment Varian Series 2000 liquid Chromatograph . U.Vnm .4 detecto25 t a t rse Column: Altex Ultraphase ODS 5 urn m c 5 2 Revers x d i e m n Phas 6 e4. Mobil Phase: Methanol HPLC grade Liquid scintillation counter (Packard TRICARB 300) Biological oxidizer, Ox-400 (R.J. Harvey Instruments)
2 . 3 Analysis of commercial formulations monitoo T e effecrth storagf e stabilitto th n eo commerciaf yo l formulations, 5 g of maneb 80% W.P. were analysed at 1-month intervals for 5 months from opened and closed packages. ETU was extracted with dry ethyl acetat quantificatiod ean mads nwa HPLCy eb . Manes bwa
determined by the CS? evolution method (4).
4 2. Experimen tomatn to o plants
14 2.4.1 Preparation of C-maneb suspension 14 8.3 mg of C-maneb were mixed with 5.051 g of maneb 80% W.P. and repeatedly washe ethyf o d l lwitm 0 acetateh3 e combineTh . d washings were mad totae o volumeet th ld radioactivit,an s determinedywa . ETU-free material was dried, suspended in water (500 ml) and sprayed on tomato greenhouseplante th n i s e ETU-freTh . e specifimanea d bha c activit 8 dpm/vg37 f o y.
18 2.4.2 Application 14 Usin Camaga g Uni. Spray e ,C-maneth b suspensio s sprayenwa n o d tomato plant time4 ( s weeklt sa y intervals rate th f eo t )a 10 yCi/plant (60.74 mg/plant).
Condition applicatiof so locan i n l practice: - Application rate: 2.37 kg a.i./ha (76 mg/plant) Numbe- applicationsf ro weeklt (a :4 y intervals starting wheplante nth s ar monthe2 s old)
2.4.3 Samplin analysid an g s Sampling time showe sar Tabln ni . eI Thre e tomato plants were collected in aluminium foil at each sampling time, placed in plastic bags and frozen until analysis. Fruit samples were collecteh d 5t froe mth sampling when tomato fruits appeared. Soil and percolation water samples were collected at the end of the experiment.
14 C-activit determines duplicatg ywa m 0 5 n i de sample combustioy sb n and LSG. Determinatio residueU ET f no mads swa e accordin Onleo t g t ye al (5). Following clean-up, extracts were spotteplateC TL d n an so d
Tabl Time- eI samplinf so tomatf go o plants
Sample Sampling time
1st 1 hour after the first application
2nd 8 days after the first application
3rd 8 days after the second application
4th 8 days after the third application
5th 56 days after the fourth application 6th 77 days after the fourth application
7th 86 days after the fourth application
8th 101 days after the fourth application day1 12 s afte fourte rth 9th application
19 developed in chloroform : n-butanol : methanol : water (100:5:1:5) and spots made visible using Crete's reagent. R values were also determined by counting 1 cm zones in LSC, using Scintilene as a cocktail.
The amount of maneb was determined in 5 g samples by CS evolution method (4). After plant extraction, non-extractable residues were quantified in the remaining plant material by combustion and LSC.
14 Soil C-activity was determined in 200 mg of homogenized samples by combustio d countinnan LSCn i g quantifo .T y extractable residuesg 0 ,5 were Soxhlet extracte methanof o cycles0 1 dl m e r wit 0 On lfo .h 10 hundred mg of the extracted soil were combusted and counted to quantify 14 non-extractable residues. C-activitf o measures l m ywa 1 n i d percolation water and maneb was determined in 50 ml of water by Keppel method (4).
2.4.4 Effect of cooking on maneb residues in tomato fruits o determinT effece eth cookinf e stabilito t th paren e n th o g f tyo compound sample,2 tomatf so o fruit were spiked eac manef ho witg bm h2 80%. One sample was cooked for 15 min. in distilled water and the other uses controls wa a d . Both samples wer eS evolutioC analyse e th y b dn method (4). This experimen alss twa o conducted using C-maneb1.4
3. RESULTS AND DISCUSSION commercian i 1 U 3. ManeET d lban preparations The changes in the amounts of maneb and STU during storage are given in Table II. The commercial formulation of maneb 80% had only 60-66% of active ingredient. Over a period of 19 weeks, no apparent significant decreases coul observede db U levelET . s showed slight increases during storage, but there were no substantial differences between closed and open packages.
2 Residue3. tomatn si o plants 14 The amount of C-activity in the tomato plants increased until the last applicatio started an n slowlo t d y declin 12.8o et % afte monthr4 s (Table III). A parallel procedure to determine maneb by Keppel's method ) gav(4 e much lower values (Table IV) expectee b . o Thit s dsi since eth 14 C-data include the parent chemical and 14C-labelled degradation products possibls wa t I plantn deteco .i et U stET afte secone th r d
20 Table II - Effect of storage time on the stability of maneb 80% commercial formulation formatiod san U ET f no
Tim samplinf eo g Open packages Closed packages (days) U ET Maneb Maneb ETU a.i.% ( ) (ppm) (% a.i.) (ppm)
0 66.3 260 66.3 260
36 64.51 310 0 31 71.5
71 60.10 631 63.4 350
4 10 0 35 63.63 0 35 64.5
133 61.27 420 67.8 340
Datdeterminationsmea4 e f ar ano .
Table III - 1/(C-activity in tomato foliage (determined by combustion - LSC)
Cumulative Sample No.* Maneb applied 14C-maneb % Recovery (mg) (mg/plant)
1 60.72 2.86 4.7
2 60.72 5.59 9.2
3 121.4 45.28 37.3
4 182.6 141.20 77.5
5 242.8
6 242.8 81.01 33.4
8 242.8 41.70 17.2
9 242.8 31.11 12.8
From Table I.
21 Table IV - Maneb residues in tomato foliage (determined by CS2 evolution method)
Sample No.* Maneb applied Maneb Recover% y (mg) (mg/plant)
1 60.72 2.14 3.5
2 60.72 1.7 2.8
3 121.4 11.24 9.3
4 182.16 28.91 15.9
5 242.8 13.57 5.6
6 242.8 14.59 6.0
8 242.8 18.88 7.8
9 242.8 3.22 1.3
From Table I.
application (0.012 ppm). ETU residues in leaves increased to 1.37 ppm in sampld slowlan e5 y bega decreaso nt e reachin s lowesit g t levef lo 0.2 ppb in sample 9.
3 3. Residue tomatn i s o fruits 14 Total C-activity decreased to 1.3 ppm in fruits 4 months after the last application (Table V). ETU residues reached a maximum of 1.12 ppm in sampldecreased an e5 undetectablo t d e th f eo quantitied en e th t sa experiment (Table V).
3.4 Residues in soil and percolating water 14 The total C-activity recovered from soil did not change appreciably from the 2nd through the 9th sample (Table VI). The amount of extractabl samplel eal activitn i soie s s ver th w l (0.9wa n lo yi y 5- 1.96%) while "bound" residues increased d 2n slowle th n yi fro3 m4. 14 sample to 31.7% in the last sample. C-activity in percolating water 14 contained only trace amounts of C-activity (0.04% in sample 1 and 0.01% in sample 6).
22 Tabl 14- C-activit eV levelU ET tomatn d i s yan o fruits
U ET ^c-activity equivalent Sample determine combustion-LSy db C determine Onley-Yiy db p extraction Number* and TLC/LSC m pp 14C-roaneyg ETU/fruib t ppm (mg/plant)
8 7. 5 87.0.551 6 1.12
6 0.213 3.2 11.8 0.16
5 73. 15.0.203 4 0.24
8 0.130 2.4 N.D. -
9 0.053 1.3 N.D. -
* From Table I.
Tabl- I eV l^C-activity in soil
Sample l^C-maneb applied l^C-activity recovered % Recovery Number (mg/pot) (mg/pot)
1 60.72 9.86 16.23
2 60.72 12.80 21.08
3 121.4 30.61 25.21
4 182.16 34.9 19.16
5 242.8 47.31 19.48
6 242.8 53.53 22.04
8 242.8 58.09 23.95
9 242.8 58.45 24.07
From Table I.
23 Table VII - Effect of cooking on maneb and ETU residues in tomato fruits
Sample 14C-maneb applied Extractable 14C-activity Extractable 14C-ETU (mg) (mg) (mg)
Uncooked 24.67 10.61 3.33 (66.6 ppm)
Cooked 24.67 5.5 2.75 (55 ppm)
3.5 Effect of cooking on maneb and ETU residues in tomato fruits When maneb-contaminated tomatoes were cooked in water, they lost over theif o % r 90 residues presumabl watero yt determines ,a Keppey b d l 14 14 method (4). Using C-maneb, extractable C-activit reduces ywa d to about 50% after cooking and ETU concentration in fruit declined from m (Tablpp 5 e5 maintaines VII)o 6i t 7 t I . d that substantial activity watee s th los rwa analysedt o t whicno s hwa . HoweverU ,ET concentration in the extractable fraction increased from 31 to 50%.
REFERENCES
(1) Chernoff, N., Kavlock, R.J. Rogers, E.H., Carver, B.D. and Murray, S., J. Toxicol. Environ. Healt h5 (1979 ) 821.
) (2 Khera, K.S. Ethylenethiourea: Teratogenicity stud ratn d i y san rabbits. Teratolog (19737 y_ ) 243.
(3) Blazquez, C.H., J. Agr. Food Chem. 21 (1973) 330.
(4) Keppel, G.E., J. Assoc, Offic. Anal. Chem. 52 (1969) 162.
(5) Onley, J.H., Giuffrida, L., Ives, N.F. Watts, R.R. and Stornerr, R.W., J. Assoc. Offic. Anal. Chem (19770 .6 ) 1105.
24 RESIDUE MANEF SO TOMATN BI THEID OAN R PERSISTENCE DURING COOKING AND STORAGE
D. KOLANKAYA, A, OEGUES, Z. AYAS, M.T. AKAY Facult f Scienceyo , Hacettepe University, Ankara, Turkey
Abstract
Tomato plants, grow greenhousn ni e were sprayed wit aqueoun ha s suspen- 14 sion of ETU-free C-maneb (5 mg/10 yCi/plant) three times at weekly intervals. Thirty five days after the first application, maneb residues were highes leaven i t s (352 ppmlowesd an ) fruitn ti s (0.12 valueÜ ppm)ET .s were fruie th generall td concentratioan w ylo s 0.0nwa note s 1i t ppmdI tha.U tET residues were only those generated in 35 days from ETU-free maneb. Cooking fruits reduced maneb concentration by 27% and increased ETU from 0.010 to 0.014 ppm. Analysis of commercial preparations of maneb over a period of 18 months showed a decline of maneb concentration by 10% and 40% in closed and open packages, respectively.
1. INTRODUCTION Ethylenebisdithiocarbamate fungicides (EBDCs) are commonly used for the control of a variety of fungus diseases of major vegetables, fruits, crops and hoffs. The three common EBDCs, maneb, mancozeb and zineb, have been Turkeyn registerei e us . r Thesfo d e fungicide knowe sar degrado nt o et several compounds including ethylenethiouram monosulfide (ETM), ethylenethio- urea (ETU) and ethylenediamine (EDA) (1, 2). Studies have shown that a major metabolite, ethylenethiourea (ETU) carcinogenis ,i c (3), mutagenid an ) (4 c teratogenic (5).
w studieFe s have been conducte degradatione th n o d , metabolisd man persistenc EBDCf eo s under tropica subtropicad lan l climates. High temperatures and moisture would promote the formation of ETU. Maneb, like other dithiocarbamates especialls ,i y sensitiv airoxygee o et th , n humiditni y and temperature (6). In spinach, up to 26.5% of EBDC was converted to ETU by boiling (7). This research aimed at investigating the effect on residues of cookin fryind gan g maneb-treated tomatoes boilind ,an g maneb-treated potatoes stabilite Th .commerciae th f yo l formulatio alss nwa o studied under local storage conditions.
25 2. MATERIALS AND METHODS
1 Radiolabelle2. d materials 14 14 Maneb-ethylene (10.5C - 9 mCi/mMol ETU-ethylened )an mCi0 /(2 C - 14 mMole) were purchased from Izinta, Budapest, Hungary. ETU-free maneb- C / maneb was prepared according to the FAO/IAEA Protocol (8). The technical compound (Dikota 0 manebn%8 M-2obtaines 2 )wa d fro locama l supplier. These compounds were checked for ETU by TLC at 1-month intervals.
2.2 Application to plants 14 A suspension of 5 mg technical maneb + 10 pCi C-maneb was sprayed with an atomizer onto each plant in a plastic tent. Three applications were mad weeklt a e y intervals plante Th . s were collected befor afted ean r application kepd san -20°t t a C until analysis.
2.3 Analysis s determinewa U ET d radiometricall C plateTL . n yo sal accordint e p Yi o gt (9). Ten grams of each plant were blended in 100 ml methanol for 2 min. and Soxhlet extracte hrs0 2 extrac e vacuun r .i Th fo dl s reduce t m ma twa 5 o t d 14 s 35°Ctake e solutiowa totar Th l .nfo m lfilteres 1 nwa 0. C-countind an d g iPackara n d liquid scintillation counter n aliquoA . eacf to h extract (0.1 ml) was applied to TLC plates (250 \m silica-gel, 60-F-254) alongside with 14 C-ETU, cold ETU, 14C-mane technicad ban developes wa lC manebTL de Th . in butanol-ethanol-water (120:33:57 v/v). One cm zones of the chromatograms were scrappe d addescintillatioa an do t d n vial containin dioxin-basel m 0 1 g d scintillation cocktail. Visualizatio s achievewa U ET d f no usin g nitroprusside
reagent. Mane determines bwa ? analysiCS y b d s (10).
2.4 Cooking experiments 14 n gram Te tomatf o s o were treated with ETU-free mane + b C-maneb prepared according to a standard protocol (8). The following procedures were followed: 14 blenden I ) da sample tomatof so , mane d ban C-ET U were determined. 14 ) b Blended sample tomatf o s maned o d an wer C-ETban . ehr cookeU1 r fo d were measured. c) Samples were methanol-extracted with Soxhlet and extracts tested by thin 14 layer chromatograph r C-ETUyfo . potatoesr Fo , samples were similarly determined befor d afteean r boilind an g frying.
26 concentrationU Tabl ManeET - ed I ban differenn si t part tomatf so o plantd san soil samples, collecte day5 3 d s afte e firsrth t application
Material Vg Maneb/ yCi Maneb 14C-ETU g sample* equivalent (ug/g)
Leaves 352 1.07/g 0.02
Stem 140 0.70/g 0.11
Fruits 0.12 0.04g 2/k 0.01
Roots 0.16 0.038/kg 0.02
Soil 0.40 0.11/g 0.08
metho2 CS d*
TablEffec- I eI cookinf to ^C-manen go b residue tomatn i s o fruit
Total maneb* yg/g _-^C-maneb uCi __^C-ETU (ppm) Before After Before After Before After cooking cooking cooking cooking cooking cooking
0.157 0.113 0.042/kg 0.031/kg 0.010 0.014
* CS2 method
Table III - Changes in maneb concentration of commercial formulations during storage
Condition recover% y of maneb* of Monthf so storage package 0 6 12 18
Open 79.5 56.5 51.8 47.2
Closed 76.5 74.6 72.9 68.9
* Estimatio C$2y nb evolution method
27 5 Effec2. f storagto e A package of technical maneb (Dikotan M-22 %80 maneb) was opened in January 1987 and at 6-month intervals 20 g were analysed by the CS evolution method (10). Maneb in closed packages was similarly analysed.
3. RESULTS AND DISCUSSION
1 Residue3. tomatn i s o Maneb residues in different parts of the plant, estimated by the CS method are shown in Table I. Most of the fungicide was found on the leaves and stem. Maneb and derived products were also determined by estimating the 14 14 plane th tn i totamaterial C l . C-ETU residues were presenw lo n i t amountfruie th td san containe d onl yconcentratioe 0.0Th m (Tablpp 1 . eI) f no ETU in whole plants declined from 0.1 ppm after 7 days to 0.03 ppm 35 days after the first application.
2 Effec3. cookinf to g 14 Tomato fruits treated with C-maneb were cooke totad an d l manes bwa estimated by the CS evolution method4 1 . Total 14C derived from C-maneb 14 and C-ETU were also determined (Table II). It could be seen that cooking reduce d increase an 40%y d b % maneU 27 . ET dy b
3.3 Effect of storage The recovery of maneb from commercial samples of Dikotan M-22 under local storage condition shows i s Tabln ni e III. Durin months8 g1 , manes bwa closen i degrade % open d10 i packagey n% b dpackages 40 d an s .
4 Mane3. b residue potatn si o When potato, treated with commercial maneb at a concentration of 4.36 ppm s cooke,wa waten i dresidue rth e decline 2.4o t d 2 ppm. When residues weeke on residue ,r th wer fo ee ag lefdecline o t d from 1.4 0.6o t 4 8 ppm.
REFERENCES
(1) Hylin, J.W., Thin-Layer Chromatography of Dithiocarbamate Fungicides. Bull. Environ. Contam. Toxicol (1966.I . )76
) Hylin(2 , J.W., Bull. Environ. Contam. Toxicol 0 (1973.1 ) 277.
(3) Graham, S.L., Hansen, W.H., Davis, J.K. and Perry, C.N., Effect of One Year Administration of Ethylenethiourea on the Thyroid of the Rat. J. Agric. Food Chem. 21 (1973) 324-329.
28 ) Seiler(4 , J.P., Ethylenethiourea (ETU) Carcinogeni,a Hutagenid can c Matabolite of Ethylenebisdithiocarbamate. Mutat. Res. 26 (1974) 189-191.
(5) Khera, K.S., Ethylenethiourea: A Review of Teratogenicity and Distribu- tion StudieAssessmenn a d san Reproductiof to n Risk. C.R.C. Critical Reviews in Toxicol. 18 (2) (1987) 129-139.
) Bontoyan(6 , W.R Lookerd .an , J.B., Degradatio commerciaf no l ethylenebis- dithiocarbamate formulations to ethylenethiourea under elevated temperatur humidityd ean Agric. J . . Foo d) (1973Chem(3 1 ).2 338-341.
(7) Watt, R.R., Sturherr, R.W. and Onley, J.H., Effect of Cooking on Ethylenebisdithiocarbamate Degradation to ETU. Bull. Environ. Cont. Toxicol. 12 (1974) 2-4.
(8) Report on Second FAO/IAEA Research Coodination Meeting on Radiotracer Studies of Fungicide Residues in Food Plants, Neuherberg, Germany F.R., 3-7 May 1987.
) Yip (9 Onley, ,G. , J.HHowardd .an , S.F., Residue manef so ethylened ban - thioure field-sprayen ao d lettuc kaled ean Assoc. J . . Official Anal. Chemists 54 (1971) 1878.
(10) Keppel, G.E., Modification of the carbon disulfide evolution method for dithiocarbamate residues ) (1969 (1 AOA. J 2 .)C5 162-166.
Next page(s9 ) 2 left blank ETHYLENETHIOUREA (ETU) AND ETHYLENETHIURAM MONOSULFroE (ETM) IN BEAN PLANTS TREATED WITH 14C-MANEB
A.H.E. HARHASH Chemistry Department, Facult f Scienceyo , Cairo University B. HEGAZI, S.M.A.D. ZAYED National Research Centre Dokki, Cairo, Egypt
Abstract
e fat magnitudd Th ean residuef eo manef so bean i b n plant (Vicia faba) were determined following fungicidal applications under conditions of local practice. C-maneb, diluted with ETÜ-free cold mane finaa o bt l specific activit mCi/2 applies f o ygwa d three time weeklt sa y intervals totae Th . l dose amounte 12.2o dt 3 mg/plant (25.76 pCi). Residues were extracted with chloroform-ethanol, water, EDTA and the unextractable residues were determined by combustion. Transformation products were identified and quantified by TLC.
date Th a indicate appliee th f o dd % thados3 e s fountth < e wa n i d different plant e partth f .so Chloroform-ethano wated lan r extractable residues showed a gradual increase in leaves, stem and roots of the plant. ETM appeared in water extracts of both leaves and stem. ETU was detected in both chloroform-ethanol and water extracts of leaves and stem 25 days after e firsth twerM treatmentET e d majoan U r ET metabolite. seedy dr se th n si which showed a residue concentration of 0.05 ppm of which only 6% was water and chloroform-ethanol extractable. ETU contributed 25% of the extractable products boune Th .d material remained unidentified.
1. INTRODUCTION Ethylenebisdithiocarbamates (EBDCs) formose mth t important clasf so fungicides for controlling diseases of agricultural crops. This class includes maneb, nabam, mancoze zinebd ban . During recent years, much attentio bees n ha variou e n th pai o t ds findings that ethylenethiourea (ETU) may occur in plant samples following the use of dithiocarbamate fungicides.
31 Higher ETU levels are more likely to form in tropical than in temperate regions. ETU has been shown to have caused or in some cases suspected to have caused various pathological effect experimentan i s l animals. These, including goitrogenic, tumorogeni tetratogenid can c effects, have raised questions concerning the possible harmful effects of terminal residues of EBDCs (1). purpose Th thif eo determinso t wors kwa e fat eth f eo C-mane Vicin bi a 14 faba plant, following foliar application of the fungicide under local field conditions.
. 2 MATERIAL METHODD SAN S
2.1 Chemicals 14 C-maneb (manganese ethylen dithiocarbamates ebi obtaines )wa d from Hungarian Academ Sciencef yo act. ,sp .mCi/mmole0 2 dilutes wa t I d. with ETU-free maneb to make a final specific activity of 1 mCi/500 mg. The specific activity was determined by combustion followed by liquid scintillation counting. Non-labelled maneb used for dilution was synthesized accordin Seidleo (2). gt al .t re
Ethylenethiourea (ETU) was prepared by the reaction of ethylenediamine with carbon disulphide according to Alien et al (3); m.p. 203-204°C.
Ethylenethiuram monosulphide (ETM prepares )wa d accordin. Thoro al gt t ne (4). Aqueous 0.5% nabam solution (200 ml) was mixed with 1/100 equivalent of manganese dioxide. The pH of the solution was about 9.5. Oxyge admittes nwa d intreactioe th o n mixture with continuous agitation min5 1 r . fo After filtration from insoluble material extractes wa M ,ET d from the filtrate according to a known procedure (5), m.p. 125-126°C. The structural formulae of maneb and some products are shown in Fig. 1.
2 Fiel2. d experiment Seeds of Vicia faba (Variety Giza 1) were cultivated under normal field conditions on 1 November 1985. Directly before blooming, the radioactive mane applies bwa suspensioa s a de leas th n tni amoun waterf to . Applicatio s achievenwa mean y paina db f so t brus preveno ht t overdosing and possible runoff. The dose was almost equally distributed on the healthy leaves of the plant. Plants received three applications at intervals of 7 days. At different time intervals, plants (2 plants/
32 H r CHo———N——C .
Hn S CH ———N——Cx 2S H II B (D Maneb (Managnese Ethylenebis (Dithiocarbamate))
H CH2———Nx c _ f»__ ! L ——— 3 S ———NCH / H (S) ETU (Ethylenethiourea)
S H II CH£———N.—— C\ S I / CHE———N——C H II S (3) ETM (Ethylenathiuram Monosulphide)
S H II r , ' S — N — *» \
S s —— N —-C' H II 5 (4) ETD (Ethylenethiuram Disulphide)
FIG. 1. Structural formulae of maneb and some of its products.
sample) were carefully uprooted, immediately froze -20°t na C till analysis. The first sample was taken 25 hr. after application and always a sample was taken directly before the succeeding application (Table I).
3 Extractio2. planf no t material Plant material was chopped and blended with ethanol (200 ml for 50 g). Chlorofor mcelite-50d an (10 wer) ) g 0 ml e0 adde0(1 mixind an ds gwa continued for 5 min. The mixture was filtered and the residue was washed twice with EDTA (0.3 mola rdissolvo t solution ) ml e0 unchange;15 d maneb chloroform-ethanoe Th . l mixtur washes ewa d twice with water (200 then 100 ml). The combined water phases were extracted once with chloroform and the chloroform extracts were combined and concentrated.
33 Table I
Doses Applied on Bean Plants and Sampling Times
Sample Sampling Time jaCi Applied Total jaCi Treatment No. After (mg Maneb) Applied First Treatment (mg Maneb)
1st 9.5 9.5 (4.59) (4.59) hrs4 2 . I II 7 days
2nd 8.35 17.85 (3.89) (8.48) III 14 days
3rd 7.91 25.76 IV 25 days (3.75) (12.23) V 75 days
Chloroform, water and EDTA extracts were measured for their radioactivity ina liqui d scintillation counter. Seed f greeo s beany ndr pod sd san 14 were chopped and extracted as previously described and the C-activity in water and chloroform extracts was determined.
4 Analysi2. extractf so s extracte Th s were analyse unchanger fo d metaboliteds it mane d C ban TL y sb on Silic platel ge a s usin followine th g g systems (6): Syste chlorofor: m1 butano: m methano: l wate: l r (100:5:1:0.5) System 2: méthylène chloride Chromatograms were develope sprayed an d d with chromogenic reagents according to Fishbein et al. (7) to locate spots. Chromatograms were then distancem scrape s elutec e Silic wa 1 th l t d a dge a an swit h methanol and counted in an LSC. Authentic substances were used as
references. Tabl f valueshowS I eI s e it manef sth o s d an b transformation products.
3. RESULT D DISCUSSIOSAN N absorbee C amoun beae Th th f distributio ns to y it b dplan d tan n among different plant parts are presented in Table III. The data indicate that only a small percentag appliee th f eo d dose ente plante rth .
34 Table II
Rf-Values of Maneb and Some of its Transformation Products
Rf-Value in 1 Substance System1 System 2
Maneb 0.0 0.0 ETU 0.29 0.00 ETM 0.80 0.25 ETD 0.47 0.10
Table III
Distributio Manef no b (^C-Activity) Among Different Part Beaf so n Plant
Cumulative Total % Sample Applied Leaf Stem Root Recovered Dose/Plant (MB)
II 4.59 109.2 10.92 1.35 2.64
III 8.48 165 15.84 2.016 2.16
IV 12.23 208.06 13.86 1.665 1.83
Applie* d dos 100e= %
Tabl showV eI distributioe sth f no C-activit aqueoun i y organid san c14 extracts of different parts of treated Vicia faba plants. The radioactivity in both extract f leaveso s increased gradually ove e perioe th rth f o d experiment e increasTh . mors ewa e profoun aqueoue th n i ds extracts stemn I . , the radioactivity in organic extracts increased after new applications and decreased subsequently.
35 TablV eI
Distributio 14f no C-Activit Waten i yd Chloroform-Ethano ran l Extracts
Sample _ Wate14n i C r Extracts* Chloroform-Ethanoln i C 14 * No. Leaf Stem Root Leaf Stem Root Pg PS Pg PS Pg Pg
t applic1s . I 6.95 0.25 0.75 0.04 0.32 0.42 II 27.00 2.50 3.2 0.08 0.33 0.68
2nd applic. III 24.35 1.69 4.25 0.08 2.35 0.52
3rd applic. IV 37.00 0.90 0.4 0.10 0.75 0.75 V 133.40 2.30 1.95 4.15 0.09 0.10
14* C derived materiag fro 0 m5 l
Datmeae duplicatef ar ano s
Tabl eV show distributioe sth f no C-activit aqueoun i y organid san 14c extracts of green and dry seeds. The maximum radioactivity was detected 32 days following the first application of the fungicide. The major part of radioactivit y seedsdr n i ,yunextractabln howevera n i s ,wa e form.
14 Table VI shows the percentage of C-products in organic extracts of bean plants. Analysis of extracts of plant tissues showed the presence of ETU and ETM, in addition to an unknown which remained at the base line and a minor
metabolite (Rf = 0.47 in System 1) believed to be ETD. ETU and ETM were also detecte significann i d t amount aqueoun si s extracts (Tabls wa e U VII)ET . detected in leaves and stems of plants sampled 25 and 75 days following the first application of the fungicide (Table VII). Both water and chloroform extracts showe presence th d tracef eo manef so b whic vers i h y slightly soluble in these solvents. Unknown 1 is non-polar and shows a similar R~ to maneb.
Table VIII shows that ETU and ETM are major metabolites in seeds. The amount botf so h products see increaso t m e with timecontributeU ET . s about 14 extractable th f o % 25 e C-activit seedy dr n si y afte days5 r6 .
36 Table V - -^C-Activity in 100 g of Green or Dry Seeds
Sampling Time Water Extract Chloroform-Ethanol Bound Residue Total after Extract 1st application tag Maneb ppm m ygpp Maneb yg Maneb ppm pg Maneb ppm Equivalent Equivalent Equivalent Equivalent
25 daysa 0.33 0.003 0.04 0.001 1.61 0.016 1.90 0.02
32 days3 3.06 0.030 0.62 0.006 2.00 0.20 5.60 0.06
65 daysb 0.23 0.002 0.07 0.001 4.93 0.049 5.17 0.05
a Green pods seedy Dr s D Table VI
Nature of in Chloroform-Ethanol Extracts of Leaves and Stems
% in Sample
I II III IV V Substance Leaf Stem Leaf Stem leaf Stem Leaf Stem Leaf Stem
Maneb 100 —— 100 —— 93. 1 —— 60. 8 36.5
ETU 17.3 4. 3 34.8 7. 2
ETM 1 .6 1. 2 10.9 3. 2 12. 1 3. 9
ETD 5.3 5.5 11 5. 1 16. 6 11.3
UnknowI n —— 100 —— 100 93. 3 87.4 77.6
1 - V: Sampling times (Table I) 14 Total C-residues in chloroform-ethanol = 100%
Table VII
Natur f -^C-Residueeo Waten i s r Extract f Leaveo s d Steman s s
Sampln i % e I II III IV V Substance Leaf Stem Leaf Stem Leaf Stem Leaf Stem Leaf Stem
Maneb 99.— 3 98. — 6 96. — 4 83. — 5 59.3
ETU 4 10,3. 13.5 1 4. 1
ETM 0.1 0.6 0.9 17.9 2.5 24.3 4.2 39.4 17.0 39.0
— 8 10.1. 6 10. — 11 1. — ETD 5 0. — 0.6
Unknown I 99.4 — 82.1 — 75.7 — 57.2 — 46.8
Samplin: V - I g times (Tabl) I e
Total l^C-residues in water = 100%
38 Table VIII
Percentage of Transformation Products in Seeds
Percentage Radioactivity "&
Days After Appliication
25 days 32 days 65 days
Unkown I 49.6 30.02 23.15
ETU 10.9 13.25 20.1
ETM 19.7 22. 5 28.9
ETD 9.75 7.45 5.26
100% = extractable radioactivity
n conclusionI percentage th , e recover f yo C-residues from plant14 s treated with labelled mane vers bwa y low. Onl yappliee 0.81.55d th an f 8o %d radioactivity coul extractee b d day5 6 d sd aftean followin 5 r2 firse gth t treatment, respectively mose Th .t toxicologically significant compound, ETU, detectes wa considerabln i d e amount latet sa r stageexperimene th f so t (20-30% of chloroform-ethanol soluble materials). Under conditions of local agricultural practices, tota lseey dr residued e amounth f o n 0.0so i m t pp 5 which only 6% could be extracted with water and chloroform-ethanol mixture. ETU, however, contributeextractable th f o % d25 e residue. Thi equivalens i s t to only about 1 ppb in the edible seed. This trace amount of ETU is the result of biological and/or non-biological degradation of the initially applied chemica lfree U residueU whicET ET .s hwa s normaly presene th n i t commercial preparations would probabl yadditionan a lea o t d burdenU lET e Th . bound material could not be identified and it is probably the result of interaction between some degradation products and the endogenous constituents of the seed. The parent chemical may also be present.
39 REFERENCES
[1] Graham, S. L. ; Hansen, W. H. ; Davis, K. J. ; Perry, C. ; Effect . f H o sone-yea r administratiof o n ethylenethiourea upon the thyroid of the rat; J. Agric Food chem. 21 (1973) 324. [2] Seidler, H. M. ; Schnaak, W. ; Engst, R. ; Untersuchungen üben metabolismude r s einigek Insektizid d fungizidun e e r ratteide n . Verteilun d abbaun n gvo uC-markierte m 14 Maneb Nahrunge ,Di 4 (19701 . ) 363.
[3] Allen, C. F. H. ; Edens, C. 0. and Van Allan, J. ; Ethylene thiourea, org. syn. 26 (1946) 34. [4] Thorn, G. D. and Ludwig, R. A. ; Preparation of ethylenethiuram monosulphide . applJ , . chem2 1 (1962 , . ) 90 [5] Pluijgers, C. W. ; Vonk, J. W. ; Thorn, G. D. Re-Examinatio e structurth f o f o n ethylenethiurae m monosulphide; Tetrahedron lett. (1971) 1317.
, ] ResiduBlazquez[6 . H e. C , determinatiof o n ethylenethiourea from tomato foliagd an , soi e, l water , J. Agr. Food chem. 21 (1973) 330.
[7] Fishbein, L. and Fawkes, J. , Thin-layer chromatography f metallio c derivative f o ethylenebis s (dithiocarbamic acid d thei)an r degradation products . J chromatog, 9 1 . (1965) 364.
40 FAT "C-MANEF EO SOYABEAN BI N PLANTS*
A.H.E. HARHASH Chemistry Department, Facult f Scienceyo , Cairo University S.M.A.D. ZAYED . HEGAZB , I National Research Centre Dokki, Cairo, Egypt
Abstract
Seed f soyabeaso n were cultivated under normal field condition Junn i s e 14 1988. C-maneb, diluted with ETU-free cold mane finaa o bt l specific activit mCi/g2 f applies o y ,wa d three time weeklt a s y intervals wit totaha l dose of 6.45 mg maneb/plant. Different parts of plant samples, collected at different intervals, were extracted with ethanol-chloroform, ethylene diamine tetracetat waterd ean .
The overall data indicated that no more than 5% of the applied dose was detected on/i plante nth . Residue leaven si d stesan m showe graduaa d l increas botf eo h chlorofor wated man r extracts. Analysi residuef so thesn i s e extracts showewerM ET e d dsignifican an thaU tET t transformation productn i s addition to two major unknown compounds. The concentration of ETU in both extracts from the leaf increased from 0.0105 ppm after 26 days to 0.163 ppm afte days6 7 rn stem I .U increase ,ET d e froth m r 0.11o 0.00t fo m m 07pp pp same period.
Total residues in seeds declined from 0.074 ppm after 40 days to 0.037 ppm afte days6 7 r . Almost hal thesf o f e residue methanol-unextractables swa e Th . major part of extractable residues in seeds was hydrophobic in nature and characterized in the extracted soyabean oil as ETU, ETM and two unknowns. ETU increasel inoi aftedb afteb pp fro day6 pp 7 7 rday0 r4 m2 so t sfollowin e th g first treatment.
* This wor supportes kwa Internationae th parn di y b t l Atomic Energy Agency, Vienna, under research contract 3735.
41 l. INTRODUCTION Maneb is an important member of ethylenebisdithiocarbamate (EBDC) fungicides use n agriculturao d e EBDCf o th y lea le o sma t dus crops e Th . formation of ethylenethiourea (ETU), now known to cause health hazards (1). Research is, therefore, necessary to provide needed data on persistence and terminal residue EBDCf so ETUd an presense .Th t investigation aimet a d 14 determinatiodéterminât: residuef no f o sC-mane n soyabeai b n plants following local practices.
2. MATERIALS AND METHODS
2.1 Chemicals 14 C-manganese ethylenebisdithiocarbamate, specific activit mCi/g2 y , 14 s obtainewa dilutioy b de th C-labelle f no d fungicide with non- labelled maneb. The radiochemical, specific activity 20 mCi/mmole, was obtained from the Hungarian Academy of Science, Budapest, Hungary. Ethylenethiourea and ethylenethiouram monosulphide were prepared according to known procedures (2, 3, 4) and were used as authentic substance comparisor fo s n purposes.
2.2 Application Sound seed f soyabeaso n were cultivated under normal field conditionn si 14 the middle of June. An aqueous suspension of C-maneb was applied to healthy leaves of the plant under conditions simulating those used in practice e leaveTh . s receive o othetw d r applications separatey b d one-week intervals. At different sampling times, two plants were uprooted and kep -20°t ta C till analysis e dosinTh .d samplin an g g patterne sar give Tabln i n . eI
3 Extractio2. planf no t material Different parts of the plant were extracted and analysed separately. Fifty gram planf o s t material were choppe d blendean d d with ethanol min0 1 hundree r .On fo (20) d 0ml millilitr f o chloroforf eo g 0 1 d man celite-500 wer emixture addeth o d blendint dean s continuegwa r dfo further 5 min. After filtration from insoluble material, the residue was washed twice with 50 ml portions of ethanol : chloroform mixture (1:1). e organiTh c extrac washes twa d twice with water (10e 0eachl th m d )an aqueous extracts were washed onc chloroforml em wit0 10 h combinee Th . d organic and aqueous extracts were concentrated separately and tested for their radioactivity. Unchanged mane extractes bwa d froe insolublmth e
42 Table I: Doses and sampling times
Treatment Sample Dose applied Total Applied Sampling time (mg maneb) No. (yCi) (yCi)after first treatment
1st (2.15) 1 4.469 4.469 24 hours
2nd (2.15) II 4.469 8.938 14 days
3rd (2.15) IT1 4.469 13 .407 26 days
IV 4.469 13 .407 41 days
V 4.469 13 .407 62 days
fraction by shaking with EDTA (0.3 molar solution, 150 ml) for 30 min. on a shaking machine and the radioactivity in extracts was determined.
Seed greef so seedy ndr pod sd wersan e chopped, crushe mortaa n i d ran extracte Soxhley b d twit . methanol-waterapparatu% hr h95 4 2 r sfo e Th . radioactivit thin i y s extrac s directltwa y determine y liquib d d scintillation counting. The unextracted radioactivity was determined by combustio definita f no e weighe extracteth f to d grains afte dryinr rai g methanoe Th . l (5) extrac subjectes twa fractionatioe th o t d n procedure shown in Scheme 1. The residue obtained after evaporation of the alcoholic extract was partitioned between chloroform and water. After separatio chlorofore th f no m layer (chlorofor watee th r, layem1) s rwa acidified with 2N HCl and heated for 2 hr. on a water bath to decompose possible glucosides. After cooling watee ,th r laye s re-extracterwa d with chloroform (chloroforradioactivite th d an ) m2 chloroforf yo , m1 chlorofor wated an rm2 laye determineds rwa .
4 Analysi2. extractf so s The extracts were analysed for metabolites of maneb using thin-layer plates of Silica gel G. For development of the plates, 2 solvent systems wer) (6 e used: System (1): chlorofor n-butano: m methano: l wate: l r (100:5:1:0.5) System (2): méthylène chloride
43 0 gram5 s grain from treated soyabean plants
Extract with methanol-water mixture h (95:524 r )fo Alcohol extract 1570168 ; 100% |
Partition between chloroform and water Lipophilic residues chloroform extract 0.009 ;53.35% (1)______
water extract JO.00 41.69Ï; 7 I
Heat with 2N Hcl for 2h at 100 °C , extract with chloroform Lipophilic residues I chloroform extract 0.00057 ; 3.39% i (2)______
water extract hydrophilic residues 0.004 ; 23.8%
Schem . Fractionatioe1 f methanono l extractable 14C-residue seedn si s from treated soyabean plants 76 days followin firse gth t application.
5 Radiometri2. e measurements Radioactivity in solution was measured by direct counting in a liquid scintillation counter (LSC). Tissues and seeds were dried and combusted followed by liquid scintillation counting.
Silic platel ge a s were s scrapeelute e Silicm zoneswa c th l 1 d d( ge da) an with methanol and then counted. Data were corrected for quenching, using an internal standard.
3. RESULT D DISCUSSIOSAN N
e leveTh f maneo l b residue n soyabeai s n plants, previously treated with 14C-maneb, was determined at different time intervals. The overall data indicate that onl a smaly l amoun f applieo t d radioactivity coul e recovereb d d by extractio f plano n t tissues (less than 5%) .A simila r behavious rwa 14 observe fabn i d a bean plants, treated with C-maneb, where only less than 3% of the applied dose penetrated the plant (7).
44 Tabl 14: C-activityII e * extract f soyabeao s n plant treated with
Chlorof orm-ethano* * l 4 1 Water extract extract C-residue Sampl. No e leaf stem leaf stem leaf stem ppm Total EDTA Total EDTA ppm ppm ppm ppm First application I 0.357 0.021 0.018 0.023 1.496 n.d 0. 253 n.d Second application II 0.375 0.027 0.012 0.013 0.802 n.d 0.86 n.d
Third application III 0.23 0.021 0.043 0.022 0.744 0.134 0.056 0.0021
IV 0.188 0.012 0.035 0.005 0.629 n.d n.d n.d
V 0.54 0.062 0.42 0.008 0.452 0.064 0.041 0. 0014
* Amount determine g sample50 n i ds ** Remaining after exhaustive extraction N.d = not detected
Table III: Residues of l^C-maneb in seeds from treated soyabean plants
-res idues Days Total after Methanol-extractable Non-extractable recovered 1st application 14C-activity ppm % % % ppm
40 0.024 32 .43 0.028 37 .8 70 .23 54 0.019 46 .56 0.020 47 .7 94.26 76 0.017 45 .4 0.020 54 .05 99 .45
n generalI e totath , l amoun f residueo t n lea i sf treate o f d soyabean plants showe n increasa d e bot n chlorofori h waten i d ran mextract s (Table II)n stemI e radioactivit. th , watee th n ri y extract showe a slighd t increase and a slight decrease in the level of radioactivity towards the end of the experiment. In leaf and stem, the unextractable residues showed a decrease in the late sampling times. The parent compound constituted only a small percentage (14-18 n lea3.4d %i an fstem)n %i .
45 Table IV: Lipophilic and hydrophilic l^C-residues in seeds from treated soyabean plants
C - Residues
Fraction y da 0 4 y da 6 7 1 5 4y da
ppm ' % m pppp m ' I % % Total 14C-activity of alcoholic extract 0.024 100 9 0.01 0 !0.01610 8 100 i chloroform (1) 0.017 70.8 0.009 147.36 ,' 0.009 53.35 i i ! chloroform (2) 0.0006 2.5 0. 0005 ' 2.63 ! 0.00057 3. 39 i i water 0 .005 20.8 0.006 i 31.5 0.001 7 4 23. 8
. 0 Total 0226 94. 16 0.0155 |81.57 j 0. 0135 80. 77 _
Table V: Distribution of chloroform-ethanol soluble l^C-residues in soyabean leave stemand s s
% of maneb and metabolites in extract i Substance I [I I]CI IV ______V _ L S Y S L S L | S
i 100 Maneb 100 lL~0 96 100 88 100 81.25l 76.52 ' 100 i i ETU _ _ 4. 12 19.96 18.7 ! 16 10.8! .4 2 . 51 2 i i 1 J_FT/ XM 1 1 , iZ1 ? . —— il 1.94 —— i UnknowI n ——— 100 - 100 80.04 —— |71 —— 69 L...... i 14, Total ^ C-residues in chloroform -ethanol extracts = 100% L = Leaf = SteS m
Tabl I showeII s maneb residue seedsn i s A substantia. l amounto t p ,u 14 47%, was methanol extractable. The nature and level of C-residues present e totae seedth i th ne showf lO ar sTabln radioactivit i n. eIV y presentn ,a appreciable amount could be extracted with chloroform (chloroform 1). After acidification followed by extraction with chloroform (chloroform 2), a small amoun radioactivitf to y coul recoverede b d . This material probably resulted from the effect of acid on water soluble radioactive constituents.
46 Table VI: Distribution of water soluble l^C-residues i soyabean leaves
and stems n
* m f %o aneb and metabc)lite n watei s r
Sub. I II III IV V
L Q L S L j S L S L S ! | Maneb 100 100 100 100 86.80 10 3 84.42 100 83. 26 100 i ETU | 4.711.217 2. 513 ! 11.45 10.58 13.64 17.63 ETM I 3.74 3. 1 7.53 i Unknown I — 100 — 95. 35 188.7— 5 73.44 58. 31 Unknown I I ! 15.34 17.66 * Total 14 C-residue n watei s r extracts =100%. L=leaf, S=stem.
Table VII: Nature of l^C-residues in soyabean oil
14 * Percentage C-residue
Product Days After Application 40 days 54 days 76 days
unknown I 58.6 37.87 19. 18
ETU 9. 14 49.44 43.52
ETM 16.71 12.57 16.6
Unknown II 15.59 -• — -- - — 20.7
* Total C-residues in soyabean oil = 100%
Residues in chloroform and in water extracts are presented in Tables V , anrespectivelydVI e datTh a . obtained indicat ETd e ean M ar thaU ET t significant transformation products majoo Tw r . products (unknowd an I n unknown II) were also found. Unknown II was absent in the chloroform extract and should, therefore e organib , c insoluble. Unknow primara e I nseemb o yt s degradation produc e onlth yts compounsincwa t i e d detecte t earlieda r stages in the item. Its percentage decreased with time parallel with the appearance and increase d ETM an n amountdi .U ET f o s
47 Analysi f soyabeao s n oil, gained from seeds TLy b , C showed that i t contains ETETd Uan n Madditio i unknowno tw o t n s (Table VII)s i t I . noteworth o report y t that radioactive residues presene seedth e ar sn i t partiall ye seed partiallbound th an so t d y presen n soyabeai t n oil t latA . e sampling times, ETU constituted a major constituent of residues present in the l (44-497.oi ).
REFERENCES
] Graham[1 , S.L.;Hansen, W.H.; Davis, K.J.; . ;H Perry . C , Effect f one-yeao s r administratio f o nethylenethioure a upoe thyroie th ratn th f . ;o dJ Agri c Food chem.21 (1973) 324.
[2] Alien, C. F. H. ; Edens , C. 0. and Van Allan, J. ; Ethylenethioure , aorg . syn 6 (19462 .. 34 )
] Thorn[3 , G.D. Ludwig d R.A.an , ; Preparatio f o ethylenen - thiuram monosulphide . J Appl, . Chem 2 (19621 .. 90 )
] Pluijgers[4 , C.W.; Vonk, J.W.; Thorn,G.D.; Re-Examination e structurth of f o ethylenethiurae m monosulphide; Tetrahedron Lett., (1971) 1317.
[5 d Turgeon] an Sun; Wimmers; ; . Scintillatio. D ,R. E , . L , n counting of 14 C—Labeled Soluble and insoluble compounds n plani t tissue, Analytical Biochemistry 9 (198816 , ) 424.
; Residu . ] BlazquezH [6 e. C , deteminatiof o n ethylenethiourea from tomato foliage, soil, and water, J. Agr. Food chem. 21 (1973) 330.
] Harhash[7 ; d HegaziZayeE. an . ; A d , B S.M.A.D., , Ethylene- thiourea and ethylenethiuram monosulfide in bean plants 14 treated with C-Maneb (This issue).
48 DEGRADATIO MANEF NO ETHYLENETHIOUREO BT A IN LARGE PEPPER
F. RAMON, J. ESPINOSA, A. BATISTA, W. ARCHIBOLD Centr Investigacionee ad Técnican sco s Nucleates, Universidad de Panama, Panama City, Panama
Abstract
Large peppe s fielrwa d cultivate sprayed an d d three times with 14 C-maneb at weekly intervals. Samples of fruits were collected after each application and samples of leaves and stems were collected 1-2 days and 1-2-3-4 weeks after the last aplication. Appreciable quantities of maneb and maneb-derived residues were found in stems, leaves and fruits. The latter contained only trace quantities of ETU, which are unlikely to present any hazar consumerso t d .
I. INTRODUCTION mosn Panamae I th t f widelo , e maneon ys bi use d fungicidee th r fo s prevention of fungus in tomatoes, lettuce, large pepper, cabbage and other fruits and vegetables. To our knowledge, no studies have been conducted on residues of maneb and its metabolites in large pepper, which is a major produce in Panama.
II. MATERIALS AND METHODS
1 Radiolabelle2. d materials 14 C-maneb (specific activity 83 yCi/mg) and reference 14C-ETU (specific activity 117.72 yCi/mg), both labelled>in 1,2 ethylene carbons, were purchased from the Institute of Isotopes, Budapest, Hungary.
14 2.2 Formulation of C-maneb Commercial maneb % activ,80 e ingredient mads ,wa Procidy eb a Francd ean packe Mely b d o Co., local supplier. 119. werg 3m e thoroughly mixed with 14 7.69 mg C-maneb in aghata mortar with the aid of a pestle, spatula and then with a Vortex mixer. On analysis of commercial maneb it was shown to be 68.14% and not 80% as indicated on the label.
49 3 Applicatio2. d samplinan n g r experimenou r Fo selectee tw health3 1 d y plant f largso e pepper (Capsicum annum), a Cholo variety selected from Aji Monagre, a breed between sweet pepper, Yol Californid an o a Wonder. Plants were opegrown a n i nfield , cultivated at a distance of 60 cm between rows and 40 cm between plants. 14 122.3 mg C-maneb, specific activity 5.02 pCi/mg, was divided into
3 equal portion eacd an sh portio e timf th eo t na mixe 0 ? H d l witm 0 h4 application. Plants were sprayed at weekly intervals for 3 weeks. Each plant received 3 ml of the above suspension (3.08 mg/15.3 yCi) . Samples of fruits were collected 30 min. after each application. Samples of fruits, leaves and stems were collected 1/2 hr., 1-2 days and 1-2-3-4 weeks after the last application. Samples were store freezea n i d -10°t ra C until analysis.
4 Extractio2. d analysinan U ET f so Plants were analyse e combineth y b dd dan procedur ) (1 p Yi Onlef o ed an y Rhode. (2) al e efficienc .t Th e s extractiof o y s checkenwa addiny b d e th g 14 amoun f to C-ET U (93390 dpm) directl S cocktai L equivalenn a o t y d an l t amount to a sample of non-treated large pepper and carried through the extraction procedure. Data showed an extraction efficiency of 91.3%. Aliquots of extracts were quantified by LSC and the cake was re-extracted with 0.4 M EDTA to determine EDTA-extractable activity.
Extracts were thin-layer chrotnatographed in ethanol, chloroform and 14 benzene (1:5:10 v/v). C-compounds were detecte autoradiographyy b d . 14 14 Spots correspondin o t gC-ET d Uan C-ED A (ethylenediamine) were scraped from the TLC plate and quantified by LSC in aquasol-2 cocktail.
14 2.5 Determination of total C-residues 14 Total C-residues were determined by a wet combustion technique. The efficienc methoe th 85.5%e s foun b f datwa l do yo t dAl . a were correcter fo d 14 this efficiency. Bound C-residues left after extraction were not determined.
3. RESULTS AND DISCUSSION 14 Tabl showI e totae sth l amoun f to C-extractabl e residues with 14 ethanol, water and chloroform. Table II shows C-extractable residues after treatment with 0.4 M EDTA. The levels of ETU and EDA are shown in 14 Table IIItota e Tabln th I . lV eI C-residues (extractabld ean non-extractable) are shown.
50 Tabl Residue- eI solvent"f so 1" extractable in fruits, stem d leavesan s
Residues (ppm) Time of Fruits Stems Leaves sampling 1 2 applicatiot 1s n 0,081 0,093 — —
2nd application 0,240 0,520 — —
3rd application 0,320 0,378 1,572 8,920
Day 1* 0,540 0,620 2,340 12,840
Day 2 0,540 0,440 2,490 8,860
Week 1 0,460 0,280 2,760 5,740
Week 2 0,113 0,158 1,000 6,944
Week 3 0,220 0,204 0,620 4,320
Week 4 0,428 — 1,620 11,420
+ Ethanol-water-chloroform Afte* r last application
TablResidue- I eI s of EDTA- extractable l^fruitsn ci , stems d leavean s
Residues (ppm) Timf eo Fruits Stems Leaves sampling 1 2
1st application — 0,085 — — 2nd application 0,240 0,280 — —
3rd application 0,156 0,102 0,220 2,220
Day 1 0,048 0,062 0,590 3,920
Day 2 0,006 0,112 0,660 1,840
Week 1 0,240 0,102 0,660 0,774
Week2 0,107 0,045 0,186 0,783
Week 3 0,067 0,062 0,179 0,503
Week 4 0,149 — 0,410 1,649
51 Table III - Concentrations of 14C-ETU and 14C-EDA in fruits, stems and leaves
Residues (ppm) Time of Fruits Stems Leaves sampling 1 2 ETU **EDA ETU EDA ETU EDA ETU EDA
1st application 0,000 0,062 0,000 0,051 — — — — 2nd application 0,000 0,352 0,009 0,176 — -- — — 3rd application 0,000 0,234 0,002 0,326 0,038 1,254 0,113 2,880 Day 1 0,002 0,459 0,009 0,366 0,146 2,486 0,258 5.244 Day 2 0,000 0,419 0,006 0,364 0,188 3,056 0,260 4.132 Week 1 0,003 0,344 0,011 0,318 0,108 2,802 0,183 3.654 Week 2 0,028 0,094 0,002 0,163 0,062 0,928 0,138 7,835 Week 3 0,019 0,122 0,000 0,220 0,002 0,381 0,854 2,739 Week 4 0,000 0,297 — — 0,117 2,673 0,140 5,331
Table IV - Total -^C in samples of fruits, stems and leaves
Residues (ppm) Timf eo Fruits Stems Leaves sampling 1 2
1st application 0,013 0,035 —
d applicatio2n n 0,727 0,262 —
3rd application 1,390 0,705 0,886 1,80
Day 1 0,235 0,300 0,132 3,00
Day 2 0,359 0,727 1,310 14,00
Week 1 0,192 0,835 2,678 8,57
Week2 — 0,861 0,495 0,22
Week 3 0,930 0,086 0,16
Week 4 _ — — 18,32
52 In conclusion, ETU residues in the fruits of large pepper were very low and these are unlikely to present any measurable hazard to consumers. However, total residue fruin so t wer week3 e m approxspp afte9 lase .0. rth t application othee th rn O hand. , appreciable quantitie manef so manebd ban - derived residues were found in steins and leaves.
REFERENCES
(1) Only, J.H. and G. Yip. "Determination of Ethylenethiourea Residues in Foods, Using Thin Layer and Gas Chromatography", J. Assoc. Off. Anal. 54^ (1) 165 (1971).
) Rhodes(2 , R.C. "Studies with Manganese (14C)-Ethylenebis(dithio- carbamate). J. Agric. Food Chem. 25 (3) 528 (1977).
Next page(s) left blank 53 RADIOTRACER STUDY OF MANCOZEB RESIDUE TOMATN SI O PLANTS
Lianzhong ZHANG*, Huafang WANG**, Hanhong MO* *Research Centre for Eco-Environmental Sciences, Academia Sinica **Beijing Forestry University Beijing, China
Abstract
A persistenc e studth f yo d residuean e distributio mancozef no n bi tomato plant-soil syste conductes mwa d using radiotracer techniques. Half-life value totar sfo l mancozeb residue tomatn so o soin leavei ld wersan e found to be 9.5 and 7.6 days, respectively. Mancozeb residues in soil were readily takeplanty b p d leachenu san soio t d l withim layerc 8 2 dees n sa s a p two week sresiduee wit th layerm c f h o 6 oves.% - remainin90 r0 e th n i g Translocatio mancozef no b residues, especially water soluble metabolites including ETU, throughou plante tth s coulmajoa e db r factor responsiblr efo the presenc residuef eo tomatn i s o fruits. Deposit sprayef so d mancozen bo the fruit skin was another important factor. Fruit skin contained higher levels of residues than the pulp. Washing with water could remove more than 50% of the residues on the skin. It was also found that 20 - 30% of the residual mancozeb degraded to ETU during cooking resulting in a highly significant increas contenU fruite ET th f n eo i t.
1. INTRODUCTION Ethylenebisdithiocarbamates (EBDCs) have long been use fungicides a d o st protect food plants from destructive pathogen their fo s r high efficiencd yan low mammalian toxicity. Mancozeb is one of the EBDC fungicides which has been used in China in recent years on vegetables, fruit trees and, in some cases, on Chinese medical herbs. Considerin face th gt that ethylenethiourea (ETU), maie th nf o breakdow e on n product EBDCsf so goitrogenie s founb , wa o t d d can tumorigeni experimentan i cenviron e stude th th f yo , -4) , l3 animal, 2 , (1 s mental behaviou mancoze f residuers o it d foon ban i s d plant becoms sha e essentia fungicidee evaluato lt th f o saf e e eth e us . This paper reporte sth results of a study on the persistence, uptake by tomato plants, leaching in soieffectd an l cookingf so .
55 2. MATERIAL METHODD SAN S
2.1 Materials 14 C-ethylene-labelled mancozeb mad Hungarn i e provides ye wa th y b d International Atomic Energy Agency. Cold formulated mancozeb (80% W.P.) with ETU content less than 0.5 obtaines %wa d from Nantong Chemical Plan Chinaf to . C-labelled and cold mancozeb were mixed to a specific activity of 14 132 dpm/vg and extracted three times with methanol to obtain ETU-free C- mancozeb. A working suspension was prepared by mixing l g and 300 ml water. The solvents and the Na.EDTA used in the extraction procedure were analytical grade reagents. wer) HF25cm e 0 2 4 plate C x silic TL 0 l (1 sage obtained from Jiangsu Fushan Bio-Reagen Chinaf o . .tCo
2.2 Equipment 14 C-activity in liquid samples was determined in a Beckman liquid scintillation counter LS 9800. A Harvey sample oxidizer OX 300 was used for cumbustion of solid samples prior to LSC determination.
2.3 Treatment of plants and soil Tomato plants were planted in the field in late April. In the middle of May, tomato seedlings were transplanted into plastic buckets and left to grow igreenhousea n plante Th . s receive rainfalo dn necessart lbu y watering. After the plants had begun to bear fruits, they were sprayed with suspensions 14 of C-mancozeb at the rate of 0.1 g a.i./10 pCi per plant once or three times at weekly intervals (for fruits). Plant samples were collected for analysis at 0, 1, 4, 9, 16, 23 and 30 days after treatment. Soil samples layerm c 3 )- wer0 ( e als days6 o1 collected .an 9 , d4 , afte1 , r0
2.4 Extraction and analysis 14 Soilead lan f samples were combuste totad dan l C-activits ywa determined. Fruit samples were frozen and then sectioned into skin, outer pulp (3 mm thick) and inner pulp. Part of the sectioned samples was subjected to oxidation and LSC analysis for total radioactivity. The rest of the samples was extracted with methanol and 0.4 M Na EDTA. The methanol extracts were concentrate thin-layed an d r chromatographe chloroforn i d : m butane : methanol : water (100:5:1:0.5). The TLC radioactive zones and 14 Na EDTA eextract: s were analysed for C. Boiled samples were similarly analysed.
56 2.5 Effects of cooking on mancozeb residues Part fruif so t samples were min0 boilethed 2 an .r n fo danalyse e th s a d raw fruit. Analytical results for boiled and raw fruit samples were compared to determine the effects of cooking on the residues.
2.6 Uptak planty eb d leachinsan soin i g l Soil was packed in 28 x 3.5 cm ID PVC columns which were inserted into soil contained in plastic buckets. Three weeks old seedlings were trans- planted into the columns. A week later, the 0 - 2 cm upper layer of soil was 14 fortified with a.i./4g C-mancoze 1 rate 0. 9th f eo yCi/columnt ba e Th . columns receive amounn a d watef to r correspondin e averagth o gt e local precipitation. After two more weeks, tomato plants were collected and subjected to radioautography. The plant samples were also sectioned into roots, stems and leaves, and the sectioned samples were anlaysed for 14C by oxidation-LSC techniques. Soil columns were cleft to form vertical soi.1 profiles which were subjected to radioautography. Soil columns were also vertically sectione d soian dl sample eacn i s h section were analyser fo d 14 C-activity.
3. RESULT DISCUSSIOD SAN N
1 Persistenc3. mancozef eo plann bi soid tan l Table I shows the decline of mancozeb residues in tomato leaves and soil. The half-life of mancozeb residues was calculated to be 9.5 days for leaves and 7.6 days for soil. The studies of the persistence of mancozeb and zine plantn i b soid ) suggessan (5 l t that mancoze less i b s stable than zineb in the agricultural environment.
Table I - Mancozeb residues* in tomato leaves and in 0 - 3 cm top soil layer
Sampling time (days) 0 1 4 9 16 23 30
Leaf residues (ppm) 3323 2540 1563 1563 1833 1567 15 5
Soil residues (ppm) 67.1 32.20 24.1 13.8 12.6 treatmene On * t
57 3.2 Mancozeb residues in tomato fruits Table II shows that mancozeb residues in fruit skin are much higher than in the outer and inner pulp. Washing with water could remove more than half of the residues on the fruit skin, suggesting that a major part of the residues was physically adsorbed to the fruit skin.
Tabl I showII e s mancozeb-derived residues befor d afteean r cooking. After boiling, ETU associated with the skin increased 4-fold, while ETU in the pulp increased 24 times, apparently related to decomposition of mancozeb. ETU totae th contribute lf o residu e % ski 6 pulpd th nd an n abouo i ean ,t d % t3 respectively. During the process, about 20 - 30% of extractable residues degrade ETUo t d , resultin significana n i g t increas U contenET f eo cooken i t d fruits.
Table II - Mancozeb residues in tomato fruits harvested 3 weeks after last application
Skin Outer pulp Inner pulp Unwashed Washed
ppm 18.4 8.22 4.38 2.52
Three treatments
Mancozeb-derive- Tabl I II e d residue tomatn i s o fruits (ppm)
Skin Pulp Raw Boiled Raw Boiled
ETU 0.183 0.718 0.07 0.167
Mancozeb- extracted 4.03 3.22 0.66 0.46 residues*
Non-extracted 13.6 12.9 2.51 2.46
Other metabolites 0.29 0.29 0.076 0.038
Wit+ h Na2EDTA * Expresse mancozes a d b equivalent
58 3.3 Uptak planty eb d leachinsan soin i g l Mancozeb residue soin i s l were readily takeplanty b p nu s growine th n i g soi determines a l radioautographyy b d . Analysi plante th f so showed that mancozeb residues were presen leavesn ti , root d stemssan , wit highese hth t concentratio leavesn i n . This suggest translocatioe sth mancozef no b residues and/o e factorrth metabolitef o s e leadine on th e b o st gy ma includin d an U gET presence of residues in the fruit.
Autoradiography of a soil column profile also showed that mancozeb residue e sois th hav ln i columne m leachec 3 depta 1 . o f t dho Result f so oxidation-LSC analysis indicate leachina d . Howevercm g 8 dept2 f ,ho more residuee layem th c f soif 6 o ro tha - s% l 0 remainen90 wit e hth lesn i d s than 1% leaching beyond 5 cm. Compared with zineb (5), leaching of mancozeb in soi mors i l e profound.
Mancozeb residues in the young tomato plant accounted for more than 20% totae oth f l mancozeb residue plant-soie th n i s l system e amounTh . f to mancozeb residues taken up by tomato plants was much higher than the amount of zineb residues take p (2%previouna u n )i s study (5).
The difference in the plant uptake and soil leaching behaviour of mancoze d zine ban y relat bma differenceo et o stabilitn tw i s e th f yo fungicides.
REFERENCES
(1) Graham, S.L., and W.H. Hansen. Bull. Environ. Contam. Toxicol. 7 (1972) 19.
(2) Graham, S.L., W.H. Hansen, K.J. Davis and C.H. Perry. J. Agric. Food Chem (19731 .2 ) 324.
(3) Innés, J.R.M. et al. J. Nat. Cancer Inst. 42 (1969) 1101.
(4) Graham, S.L., K.J. Davis and W.H. Hansen. Fd Cosmet. Toxicol. 13 (1975) 493.
(5) Zhang, L., et al. "Radiotracer study of zineb residues in egg-plant - soil systems". This issue.
Next page(s) left blank 59 RESIDUE MANCOZEF SO ETHYLENETHIOURED BAN A IN CUCUMBER TREATED WITH C-MANCOZEB 14 UNDER FIELD CONDITIONS
S.M.F. CALUMPANG*, M.J.V. BARREDO*, N.P. ROXAS**, E.D. MAGALLONA* ^Pesticide Toxicology and Chemistry Laboratory, National Crop Protection Center **Department of Animal Nutrition, Institute of Animal Science College of Agriculture, Universit Philippinee th f s Banosyo Lo t a s, College, Laguna, Philippines
Abstract 14 Potted cucumber plants sprayed four times with C-mancozeb ETU-free Dithane M-45 were expose fielo t d d conditions harvestt A . , onlleavee yth s and stem contained mancozeb. Ethylenethiourea (ETlf), 0.26 yg/g, was found e leavestemie th nth ,n si frui onlyU soir ET o t. ldetectee b coul t no dy b d high pressure liquid chromatography. These results were confirmey db radiotracer techniques. Washin fruitf go s reduced radiocarbon level 46%y sb . Peeling practically removed all the radiocarbon, while boiling in water and vinegar (pickling) also reduced thes 67%d an e, 4 e fruilevel6 th y tn b i s respectively. ETU levels found in commercial formulations rose rapidly and reached a peak after 2.4 months before declining. An initial concentration of 440 yg/g in sealed packages increased to 2450 ppm after 10 weeks and declined to 450 yg/g after 70 weeks. In open packages, ETU substantially declined to 30 yg/g afher 70 weeks.
1. INTRODUCTION evee Th r increasing world population deman foor d intensifiefo dan d d driv foor fo ed production often necessitate greateagrochemicalsf o e rus . Agrofungicides like ethylenebisdithiocarbamates (EBDCs) have been used to control various fungal diseases on fruits, vegetables and ornamentals. These fungicides include zineb, mane mancozed ban causin s i theid ban e rus g concern fro toxicologicama l standpoint. Previous studies have shown that food plants treated with EBDCs resul terminan ti l residues containing ethylenethiourea (ETU) (1-4), a toxicologically significant decomposition product. ETU has been reporte posseso t d s carcinogenic, goitrogeni mutagenid can c potentian i l test animals (5-7).
61 Some workers reported that terminal residue werU detectet ET eno f so n i d tomatoes, potatoes, cucumber, squash and cantaloupes even in presence of 4 ppm maneb (8). ETU was reported not to accumulate or persist in tomatoes or bean plants treated with maneb (9).
It, therefore, becomes necessary to study the fate and magnitude of terminal residues of EBDG fungicides and ETU in food plants under local conditions using farmer practices especiall ybees sincha na U knowe eb ET o nt decomposition product of commercially formulated products (10-11). The formation of ETU from EBDC fungicides is hastened by elevated temperatures moisturd an (12) e (13), conditions whic prevalene ar hPhilippinese th n i t .
This stud s thereforywa e conducte o examint d e fat magnitudd eth an e f o e mancozeb and ETU residues in cucumber plants and soil, using radiotracer techniques and to evaluate the effect of processing on mancozeb and ETU residues e extenformatioU Th ET . f to n fro commerciaa m l product, Dithane M-45 (Rohm and Haas Co.), in sealed and open packages under storage conditions was also investigated.
2. MATERIALS AND METHODS
2.1 Chemicals Ethylene- 14C-mancozeb, specific activity 64.16 yCi/mgd ,an ethylene- 14C-ethylenethiourea, specific activity 117.72 yCi/mg, were obtained from Institute of Isotopes of the Hungarian Academy of Science, Budapest, Hungary.
Dithane M-45, a widely used EBDC fungicide formulation in the Philippines, was used in this study. It contains 80% mancozeb which is a coordination produc zinf (2%n to manganesio d c )an e (16%) ethylenebis- dithiocarbamate.
14C-mancozeb and ethylacetate-washed Dithane M-45 (Rohm and Haas Co.) were mixe distillen i d d wate yielo rt suspensiona d . Three dropf so Triton X-100 were added with sonication to further improve 14 suspensibility. C-ETU content of the suspension was determined by thin-layer chromatography (TLC liquid )an d scintillation counting (LSC).
62 Analytical grade mancozeb and ethylenethiourea were obtained from the United States Environmental Protection Agency (Research Triangle Park, othel Al r NC)chemical. s used were analytical grade.
2 Experimenta2. p u t lse Cucumber plants were grown in pots exposed to field conditions. These were sprayed alternately with Dithane th e f M-4o witd l 5m an h5 14 C-fungicide mixture (specific activity 9.90 yCi/mg) from the flowering to the fruiting stage at the rate of 0.13 kg product/ha following the schedule shown in Table I.
14 Iseparata n e experiment suspensiol m 0 ,1 f no C-mancoze Dithand ban e M-45 in water (specific activity =5.96 yCi/mg) were carefully sprayed omaturn5 e cucumber fruits fruite Th . s wer aftey edr allower ai o t d one spraying and the solution was reapplied until it was all used up. plante Th s were expose sunligho t d protectet tbu d from rain.
Processing of the fruits was as follows:
—washed (1) -vinega) (2 r
fruit — i— cooken i d
— unwashed -wate) (3 r
—uncooked -whole (4)
_pee) (5 l ! Lflesh (6)
2.3 Sampling Leaf, stem, fruit and soil samples were taken 1 hr. after first spraying 14 wit e hth C-mixtur upod ean n harvest e treateTh . d fruite th n i s second experiment were harveste day5 1 d s after application. These were sectioned longitudinally compositA . e sampl mads ewa combininy eb e on g longitudinal sample from each fruit.
63 Table I - Spraying schedule followed for application of l^c-mancozeb/Dithane M-4 cucumben 5i r
Day Treatment Plant Stage
0 Cold Dithane M-45a) Flowering
4 l^C-mancozeb/Dithane M-45 Early fruiting/ flowering
7 Cold Dithane M-45
11 14C-mancozeb/Dithane M-45 Mature fruit
12 Harvest
a> 0.13 kg ai/ha
2.4 Methods and analysis
2.4.1 Extraction and clean-up of ethylenethiourea. A 10-40 g chopped sample was extracted by blending with 100 ml methanol and 20 g anhydrous sodium sulfat min2 d filterer an .fo e d (Whatman //I) with mild vacuum. e filtratTh s drieewa d over anhydrous sodium sulfat d concentrateean o t d about 10 ml on a rotary evaporator at about 55-60°C. Twelve grams of florisil (3% deactivated) topped with 1 cm of anhydrous sodium sulfate were placed in a glass column (2 x 26 cm), pre-wet with 50 ml chloroform. Afte sample th rbeed eha n added, this eluteswa dl m wit 0 h10 chloroform. The eluate was then concentrated to near dryness and the solvent changed to methanol by repeatedly concentrating the solution. The final extract was concentrated to 3-5 ml and analysed immediately by HPLC.
2.4.2 Analysis of mancozeb and ETU. Total ETU content was analysed by HPLC using a Tracer 950 Chromatographie pump combined with a Tracor 970A variable wavelength detector. Column: Partisil 10-ODS, 4.6 x 25 cm, reversed phase microns0 ,1 ; mobile phase: 95:5 wate methano: r l (for cucumbe soid an rl samples 80:2d )an 0 wate methano: r l (for formulation samples); flow rat ml/mine1 wavelengtd .an . nm 3 h23
The amount of intact parent fungicide in each sample was determined using -evolutioS C e th n metho Keppef o d l (14) equipmene .Th shows ti n i n Figure 1 and the experimental conditions are shown in Table II.
64 A-CS2 Trap (CuAc2 soin) B-NaOH Trap vacuum C-Condenser D-Dropping Funnel E-Sample Flask F-Heating Mantle
FIG. 1. Set-up for CS2 analysis.
Table II - Experimental conditions for CS2~evolution analysis
Condition Cucumber Formulated Plant Material
Boiling time (min.) 30 30
1 (ml3NHC ) 60 60
NaO% 10 H (ml) 30 30
Cu(Ac)2 (ml) 15 30
SnCl2.2H) 20(g 4 4
Sample weigh) (g t 5 g m 1 - 5 0.
Mean recovery (%) 76 100
14 C-ET othed Uan r metabolites were determineprecoaten o C TL y ddb Silica gel 60 F 254 plates, 0.2 mm thickness (Merck 5749), using chloroform-butanol-methanol-water (100:5:1:0.5 iodind )an e vapor rfo 14 visualization (15). C-ETU spots were scrape counted dan n i d Insta-gel II.
65 2.4.3 Analysis of commercial preparations. Two-gram samples were taken from sealed and partially opened packages of Dithane M-45. Freshly distilled anhydrous ethyl acetat s adde emixturd wa an d e shaker nfo 30 min. Ethyl acetate was decanted and filtered. Further washings (15 min.) were made until ETU could not be detected. The combined filtrates were evaporate methanon d takei an dp nu r analysi U lfo ET f o s content by HPLC. The analysis was repeated approx. every 2 months up to 18 months.
3. RESULTS AND DISCUSSION
1 Plan. 3 t Studies harvest A t time, mancozeb residue analyses a s -evolutioS C y b d n could onl e detecte yb e leave th sted n an si dm (Table III). Mancozeb residues represented 35.6% of the total EBDC applied. Residues could not be detected in the fruit or soil. Cucumber fruits have a waxy covering and fungicidw lo e e reasoth th r ee nfo b e fruilevelthi th y s sta ma n i s compared to the hairy leaves and stem. EBDCs can easily be degraded in the presence of moisture, oxygen and elevated temperature (10, 1.1, 15).
Mancoze- TablresidueU I ET eII d cucumben ban i s r plan d soitan l
Residues, Substrate 2 sprayings3__ 4 sprayings*3 ETUC Mancozeb" ETU Mancozeb
Leaves 0.84 164.42 0.26 78.74 Stem n.d. 14.52 n.d. 12.20 Fruit n.d. n.d. n.d. n.d. Soil n.d. n.d. n.d. n.d.
o Three days after first spraying Eleven days after first spraying C By HPLC -evolutioS ByC n n.d. Non-detectable
66 It. is therefore not surprising that residues in leaves declined rapidly from 164.42 to 78.74 yg/g within 8 days despite additional sprayings within this interval. Mancozeb appear persiso t s t longe tomatn i r o fruit than in cucumber. Results from our experiment on tomatoes showed significant mancozeb residue level fruin i s t harveste day7 1 d s aftee rth last spraying. Likewise, ETU did not persist on the cucumber leaves. Residue levels decreased from 0.84 to 0.26 yg/g in the same time span. When sprayed on lettuce leaves under greenhouse conditions, ETU was rapidly losvirtualld an t y undetectable afte hrs8 r4 . (16). Similar results were found in other crops (3, 9). The small amount of ETU present in cucumber leaves at harvest time is most likely due to the conversion of mancozeb to ETU. A 0.5 - 1.0% conversion of maneb to ETU has been reported by Pease and Holt (8) while other workers have reported higher values (13). Residues of mancozeb and ETU were not detected in soil. These finding agreemenn i e sar t with previous studie , 17)(4 s . radiotracen I r studies (Table IV) representeU ,ET d only 0.2-2e th f %o methanol extractable radiocarbo s detectewa d cucumben an i d r leaves only. Other polar metabolites could account for the radiocarbon detected in the methanol extract. ETU is not a final product but a relatively
Tabl Radioactivit- V eI y level cucumben i s r plan d soitan l after spraying with •'•^C-mancozeb3
Residues, yg/g Substrate 0 days after 1st treatment 8 days after 1st treatment ETUb Methanol ETU Methanol Extractable Extractable
Leaves 0.07 3.29 0.006 2.43 Stem n.d. 0.02 n.d. 0.24 Fruit n.d. 0.006 n.d. 0.04 Soil n.d. n.d. n.d. 0.003
Specific activit 12.4y= 8 yCi/mg b Determined by TLC/LSC n.d Non-detectabl= . e
67 stable intermediate in the ultimate degradation of the RBDC fungicides easils i (13) t I y. oxidize ethyleneureo t d biologican i a l systemy b d san photolytic reactions especiall presence th n i y photosensitizer f eo s like acetone, riboflavin, chlorophyll, tryptopha tyrosind nan e (18, 19).
Sample f cucumbeso r fruit from plants spraye time8 d s with Dithane M-45 in the field were analysed for mancozeb and ETU. Soil under these plants s alswa o analysed. e detecte Mancozeb Ü coul ET t no dd cucumben i dban r frui r soilo t . These results agree wit e dathth a generated froe th m 14 C-mancozeb/Dithane M-45 experiment.
r studOu y showmancozef o e s us than cucumbere bo tth , sprayed twicea e rat 0.1th f wee eo t product/hka g 3k weeks4 r fo a, doeproduct sno e terminal residues of mancozeb or KTU in cucumber fruit or soil at harvest time.
3.? Effect of processing 14 Analysis showed that ETU was present in the Dithane M-45/ C-mancozeb spray solution at a concentration of 0.0343 mg ETU/ml. Assuming that all s retaine U concentratiowa e fruitET th U e ET y ,th db e oth f n shoule b d 0.5446 vg/g fruit.
RTU content of the fruit before and after processing was well below the minimum detectable limit of the HPLC method, which is 0.006 yg/g. The 14 14 use of C-mancozeb, however, allowed us to detect C-ETU residues down to 0.0002 vg/g.
Washin whole th g e fruit reduce initiae th d residuU % (TabllET 50 y eb e mainls wa U yET locate peee d residue. th an l V) n i d peelen i s d cucumber fruit wer t detectableeno .
Boiling cucumber frui waten i t r reduce e initiath d l radiocarbon content by 64% with no detectable residues of ETU. Since ETU is water soluble, residues would be most likely in the broth. Boiling of field-sprayed tomato in water for 10 min. produced a 38-48% (molar basis) conversion of mancozeb and other EBDC fungicides to ETU (2). Boiling unwashed cucumber fruit in vinegar did not produce detectable amounts of ETU. The radiocarbon methano e leveth f o l l extractable portio seemingle b y nma y high (0.13-0.61 yg/g). Wor TLC/LSy kb C showe leveU d ET thale tth produced from mancoze belos recommendebwa e wth d temporary maximum
68 Table V - Effect of processing on weathered residues of •"•^C-mancoze cucumben i b r fruit
Treatment equivalents ETUb yg/g dry wt. . wt pg/y gdr
Uncooked fruit Unwashed whole 0.61 0.002 Unwashed peel 0.60 (2)c 0.002 Unwashed flesh n.d. n.d. Washed whole 0.33 (46) 0.001 (50)
Boile waten i d r (Unwashed) Fruit with broth 0.36 Fruit 0.22 (64) n.d. Broth 0.14
Boile vinegan i d r (Unwashed) Fruit with broth 0.47 Fruit 0.20 (67) n.d. Broth 0.27
a Determine LSCy db , minimum detectable leve 0.000= l 2 vg/g
b Determined by TLC/LSC, minimum detectable level = 0.0002
c Percentage reduction based on initial residue level (unwashed whole fruit)
n.d Non-detectabl= . e
residue limit set by the Joint FAO/WHO Meeting on Pesticide Residues (0.002 mg/kg bw) (20). An important limitation in this study was that it gave data only on ETU residues formed in the field and during processing, since the originally present quantity was removed by ethyl acetate.
3 Stabilit3. commerciaf yo l formulations The initial ETU level in a sealed package of Dithane M-45 was found to be 0.44 mg/g representin formulatee gth 0.04f o % d product. This valus ei withi U (0.02-2%range ET nth f eo ) initially founsurvea 8 1 n i df yo commercial formulations contenU ET .highess twa maner tfo b (2%d )an mixtura leas r tfo manef eo b plu zin% (~0.5%n s1 cio ) (11).
69 z o © SEALED PACKAGE H f£. A OPEN PACKAGE H Z UJ u z O u
MJC OCT NCXf
TIME
levelU ET Dithann FIGsi . 2 . e M-45 packages whil storagen ei .
U levelET botn i s h oped sealean n d packages rose rapidly, reachin peaa g k at abou month4 2. t s before declining (Fig . Thi2) . s rapid generatiof o n U suggestET s formatioU thaET rate th f teo s initiallnwa y faster thas it n degradation. Bontoya Looked nan r (11 d observe)ha d thisuggested san d that the formation of ETU may be the result of two decomposition reactions; first, the degradation of EBDC and second, the decomposition of other EBDC degradation products. Thi s furtheswa r elucidatey b d Marshall who proposed a decomposition scheme for nabam in aqueous medium at high temperature showing the degradation of several intermediates to ETU (12). ETU after 17 months represented 93% decrease from the original ETU level in open packages.
ACKNOWLEDGEMENTS
e authorTh s gratefully acknowledg financiae eth l suppore th f to International Atomic Energy Agency under Research Contract 4003.
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(11) Bontoyan, W.R. and Looker, J.B. Degradation of commercial ethylenebis- dithiocarbamate formulation to ethylenethiourea under elevated temperature and humidity. J. Agric. Food Chem. 21. (1973) 338.
(12) Marshall, W.D. Thermal decomposition of ethylenebis(dithiocarbamate) fungicide o ethylenethiouret s aqueoun i a s media . AgricJ . . Food Chem_ 25 . (1977) 357.
(13) IUPAC. Ethylenethiourea. Pure and Applied Chem., Pergamon Press, New York 49 (1977) 675-689.
(14) Keppel, G.E. Collaborative study of the determination of dithiocarbamate residue modifiea y sb d carbon disulfide evolution method Assoc. J . . Off. Anal. Chem. 54 (1971) 528-532.
71 (15) Czegledi-Janko, G. Determination of the degradation product of ethylene bisdithiocarbamates by thin layer chromatography and some investigations of their decompositio vitron ni Chromatog. J . 1 (1967.3 ) 89-95.
(16) Smith, R.M., Madahar, K.C., Salt, W.G. and Smart, N.A. Degradation of ethylenethioure n lettuceso a . Pestic. Scienc (1988> e23 ) 337-349.
(17) Calumpang, S.M.F., Barredo, M.J.V. and Magallona, E.D. Radiotracer studies on -^C-zineb in cabbage and soil. Philippine Agriculturist TL 2 (1988) 139-147.
(18) Ross, R.D Crosbyd an . , D.G. Photolysi ethylenethioureaf so Agric. J . . Food Chem (19731 .2 ) 335.
(19) Cruickshank, D.A Jarrowd an . , H.C. Ethylenethiourea degradation. J . Agric. Food Chem(19733 1 .2 ) 333-335.
(20) FAO/WHO. 1987 Evaluation of pesticide residues in food. FAO Plant Productio Protectiod nan n Pape4 (1987)8 r# .
72 RESIDUES OF MANCOZEB AND ETHYLENETHIOUREA IN BEANS
M.R. MUSUMECI, M.C.D. SILVA, E. FLORES RUEGG . ROOP , M Radioisotope Center, Biological Institute, Sâo Paulo, Brazil
Abstract
Mancozeb and ethylenethiourea (ETU) residues were determined in bean 14 plants after 5 field sprays with C-mancozeb at 12-day intervals. The highest residues were found on leaves. After 14 days from last application, mancozeb residues declined to 10 vg/g from 79 vg/g of the 0-day sample. Mancozeb concentration in pods was 2.5 yg/g, while ETU in pods at the sampling time was 0.15 yg/g. Baking of homogenized pods resulted in partial mancozeb conversio ethylenethioureao nt , correspondin t increasne a o f get o ETUf o .% 25 Onl radiocarboe y th 0.5 f o % n detecte 0-dan o dfouns ywa seedn i d s at harvest day5 ,3 s after last spray. Seed extracts were ETU-free.
1. INTRODUCTION Ethylenebisdithiocarbamate (EBDC) fungicides are used extensively on a number of food crops to control plant pathogenic fungi. These compounds are subject to decomposition, and they yield ethylenethiourea (ETU) as one of the degradation products. Reports indicate that ETU is carcinogenic (1). Analysis of commercial formulations of these fungicides showed that a considerable quantity of ETU was present as a degradation product (2). Becaus concerf eo n about possibl residueU eET foon i s d crops enhancey db tropical and semi-tropical conditions in Brazil, this investigation was undertaken to obtain information about the levels of mancozeb when applied to bean plants and to monitor ETU residues as a product of the fungicide degradation.
2. MATERIA METHODD LAN S
1 Chemical2. s 14 C-mancozeb, specific activity 64.16 yCi/mg froInstitute mth f eo Isotopes, Budapest, Hungary. Formulated mancoze W.P% b80 . ethyleneureETd Uan a (EU).
73 2.2 Carbon-14 mancozeb formulation 14 C-mancozeb mixed with 1.0 g of formulated mancozeb was washed with 20 ml freshly distilled ethyl acetate. After stirring for 15 min., the ethyl acetate layer was removed, the fungicide dried in a dissecator and then dilute df formulate o wit g h9 d mancoze d store dissecatoa ban n i d r at 4°C. Mancozeb concentratio e formulateth n i n d materia s determinelwa d S evolutioC e b th yU concentratio ET nd methoan C (4)) TL (3 d.y nb
2.3 Application and sampling Beans (Phaseolus vulgariCarioc. cv ) aL. s were e fiele th grow th t a dn i n Institute Biologico in 4 rows with 6 plants in each row. The field experimen s conductetwa 198n i d 8 during springtime, accordine th o t g local agricultural practice. An aqueous suspension of 14C-mancozeb (1000 mg/50 waterl 0m sprayes plante )wa th treatmend n san o d s twa repeate time5 d 12-dat a s y intervals. Total rainfall durine th g experiment period was 531 mm. Sampling was first made immediately after the last application of mancozeb and at various intervals up to 28 days. Three plants were collected at each sampling time; foliage and pods were separately analysed. At the last sampling time, seeds already dry were analysed instea podsf o d . Soil samples aroun e plantsth d depth m c ,5 , were collected at the end of the experiment.
4 Extractio2. d analysinan s For total radioactivity, 100 g samples were homogenized in a blender at higaliquotg m h 0 speed 10 triplicatn i sd an , e were subjectey dr o t d combustion in a Harvey oxidizer. The 14CO collected was measured t liquid scintillation counting (Beckma 5801)S nL .
U analysisET r Fo , samples fro plant3 m s werduplicateg 0 e 10 mixe d an ds extracted in a blender with 200 ml ethanol, 100 ml chloroform and 10 g celite (4). Clean-up of extracts for TLC followed the procedures described with maneb studie tomatn o s o (5) o solven.Tw t systemC TL r fo s
were used: ethyl acetat NH0 : e(90:6:62 H 4 O: H ethed )an MeO: r H (9:1). Reference compound swer) (ETEU e d Uchromatographean d wite hth extracts. The plates were developed for 15 cm and ETU and EU spots visualized by nitroprusside-ferricyanide spraying reagent (6). Radioactive spots were determined by counting 1 cm zones on the TLC plates.
74 To examine the effect of cooking on mancozeb degradation to ETU, 100 g pods froe 14-damth y samples were autoclavn min0 heatea 3 n r .i fo dd ean
extracted (4). After clean-u A1n po 2 03 column eluate ,th s ewa concentrate developed an d silicn o d platel ge a s (Merc F-2540 k6 ethyn )i l acetat NH.O: e H_: H 0 (90:6:6). Non-heated pods were similarly extracte d chromatographean d same th e n plateo d determino T . e radiocarbo tissuen i n s remaining after extraction triplicatn i g m 0 ,10 e e drieoth f d tissue were subjecte y combustiondr o t d , followeC LS y b d counting. Soil samplewer) kg e3 ( divides d int equa6 o l portiond san triplicateg m 0 10 eacf so h were analyse radiocarbor fo d combusy dr y nb - tion and LSC.
Table I - Residues of mancozeb and ETU in bean plants after treatment with l^C-mancozeb
Days after ETU last Tissues yg/g % of vg/6 treatment extractables
Foliage 79 17.2 13.4 0 Pods 10.6 12.1 1.2
Foliage 29 13.1 3.8 3 Pods 4 7.0 0.2
Foliage 10.4 12.1 1.2 8 Pods 3.2 + •f
Foliage 10.2 9.7 0.09 14 Pods 2.5 6.0 0.15
Foliage 1.1 19.9 0.2 28 Pods nd nd nd a - Based on total radiocarbon determined by dry combustion
75 Table II - Products detected by TLC in extracts of bean plants treated with ^C-mancozeb
Relative distribution of extractable radiocarbon Rf Compounds (estimated froC developemTL systen i d m b) y da 0 values y da h 8t 14th day Foliage Pods Foliage Pods Foliage Pods
a b 8 3 6 1 0.03 0.0 A ED 0 38 13 33 28 0.13 0.26 EU 18 16 9 42 10 30 7 1 7 2 0.46 0.5 U ET 4 18 Trace 13 4 0.90 0.99 2 0 9 Unknow3 n 3 4 5 3 44 38
a - Solvent system: ethyl acetate : NH4OH : H2O (90:6:6) Solven- b t system: ethe methano: r l (9:1)
Distributio- Tabl I eII radiocarbof no leaven i n podd san s
Days Leaves Pods after Total 14C Extracted Non- Total 14C Extracted Non- treatment (dpm) extracted (dpm) extracted
0 49,346 39 61 16,583 63 37
8 29,150 62 38 7,012 73 27
14 21,385 79 21 5,775 62 38
TablEffec- V I eresidueU bakinf o tET n o gbean i s n pod 4 day1 s s after treatment with -^C-mancozeb
Treatment Mancozeb ETU (VS/6) (Vg/g)
Non-baked 4.6 0.15
Baked 4.2 1.13
76 3. RESULTS AND DISCUSSION originae th n i totae lTh U lET C-mancozeb suspensio 0 yg.18 s nwa 14 Tabl showeI s that mancozeb residue bean si n plants decreased sharply with time. Most of the activity was found in the leaves. The decline of mancozeb agreemenn ii s lettucn i ) kaled t(4 e an wit . ,resulte al hth t e sp founYi y db beann i tomatod ) san rapie (7 anNewsom y Th b d. . d al dissipatiot e f no mancozeb (Ti/sequenca e days7 ab y heavf )eo ma y rainfall between sprays.
extractf o C TL s showe presence th d ethylenediaminef eo , ethyleneurea, ethylenethiourea together wit unidentifien ha d metabolite (Table II). Only small amounts of ETU were detected 2 weeks after the last application. At harvest, seeds were analysed (35 days after last spray) and only 0.5% of the graif o founds radiocarboC wa n TL y extract. da 0 n no s showed that activity
relates wa f 0.90dzonR e f th onle o ; o yt therefor ETUr EDAo o en .U ,E Becaus heavf eo y rainfall activitw ,lo y (2700 dpm/g soil founs )wa soin i d l samples 35 days after the last spray.
Combustio leavef no podd san s after extractio ) showe (4 npresence dth f eo 14 substantiaantial amount non-extractablf so of non- e C-activity which decline leaven i d s afte weekr2 s (Table III).
Baking homogenized pods increase contenU ET e tth d from 0.15 vg/o gt 1.13 VS/S» while mancozeb concentration decline % (Tabl9 y b de IV)termn I . s of absolute quantities, ETU increased by 25%.
Analysis of commercial mancozeb formulations showed an appreciable decline from 83 to 70% over a period of 4 months.
REFERENCES
) Ulland(1 , B.M., Weisburger, J.H., Weisburger, E.K., Rice, J.M Cypherd .an , R. Thyroid cance ratn ri s from ethylenethiourea intake Nat. J .. Cancer Inst (19729 .4 ) 583-584.
) Bontoyan(2 , W.R., Looker, J.B., Kaiser, T.E., OliveGiangd an . ,,P B.M. Surve ethylenethiouref yo commercian ai l ethylenebisdithiocarbamate formulations Assoc. J . . Off. Anal. Chem 5 (1972.5 ) 923-925.
(3) Keppel, E.G. Modification of the carbon disulfide evolution method for dithiocarbamate residues. J. Assoc. Off. Anal. Chem. 52 (1969) 162-167.
(4) Yip, G. and Onley, Y.H. Determination of ethylenethiourea residues in foods, using thin-laye chromatographys ga d ran Assoc. J . . Off. Anal. Chem (19714 .5 ) 165-169.
77 ) Thi(5 s issue.
(6) Vonk, J.W. and Sijpesteijn, K. Tentative identification of 2-imidezoline as a transformation product of ethylenebisdithiocarbamate fungicides. Pestic. Chem. Physiol (19711 . ) 163-165.
(7) Newsome, W.H., Shields, J.B. and Villeneuve, D.C. Residues of maneb, ethylenethiuram monosulfide, ethylenethiourea and ethylenediamine on bean d tomatoesan s field treated with maneb Agri. J . . Food Cheman d3 .2 (1975) 756-758.
78 MANCOZEB RESIDUE TOMATN SI O PLANTS
Johar RAMLIn bi i , Juzu H.B. ARSHAD, Maliki bin ISMAIL, Samsuddin bin ABDUL WAHAB Department of Biochemistry and Microbiology, Agricultural Universit f Malaysiayo , Serdang, Selangor, Malaysia
Abstract
Three different samples of commercial formulations were analysed for ETU during a storage period of 6 months. Depending on storage conditions and the source of the formulation, ETU in the mancozeb formulation increased after 6 months by 70 to 180%; ETU in the maneb formulation increased from 0.035% to 14 0.04 50.080%- . When C-mancoze sprayes bwa plantn o d field an s d weathered under conditions of local practice, ETU concentration in fruit was 16.8 ppm 14 after 4 weeks. These values are considered extremely high. When C-ETU was applied directly on fruits and field weathered, the percentage remaining after 4 weeks was 1.43% of the total applied. Washing and cooking removed residueU morfruitse ET th f e o n tha i s% . n50
. 1 INTRODUCTION The vegetable growing sector in Malaysia provides about 90% of the country's needs. Ethylenebisdithiocarbamate (EBDC) fungicides, commercially availabl manebs a e , zine mancozer bo usee r controbar fo d diseasf lo n ei vegetable crops, especially in the highlands.
EBDCs degrad foro et varietma residuef yo e fiel durind th an dn i s g processing. One highly toxic product, ethylenethiourea (ETU), is known to induce thyroid cance Swiss-albinn i ralss i ot I know . on2) rattha, (1 st EBDCs stored under condition higf so h temperatur humiditd ean convern yca o t a variet productf yo U formee sleveET th includin f d lo an usuall U gET y depends on the storage time of the fungicide and the manufacturing procedure. ETs alsUwa o foun varyinn i d g amount cookind vegetablen i san ) s 4 i g , (3 s know produco nt e increase dpartially b amountU ET f syo degradin parene th g t . 6) EBD, (5 C
Many techniques have been used for the determination of ETU and other residues. These include high pressure liquid chromatography (HPLC), gas liquid chromatography (GLC) and thin layer chromatography (TLC). A technique whic s provehha n valuabldetectioe th levelw r lo efo residuef f o sno s i s radiolabelling.
79 Studie thin i s s laboratory have shown tha sprayine tth tomatf o g o plants with mancozeb at doses used locally will result in high residue levels in the leaves and stems and much lower levels in the fruit, root and soil. This report will presen tstabilite th dat n ao commerciaf o y l EBDC formulationse ,th formatio U froET mfielo t f n o EBD e d Cdu weathering e effec,th cookinf to n go the levels of EBDC in tomato fruits and the degradation of ETU in the field.
2. METHODOLOGY
2.1 Analysis of ETU in commercial mancozeb and maneb
Manzat 0 (mancozee20 b 80%), Trimanzone yellow (mancozeb 79% Maned )an b (maneb 80%) were obtained from DuPon Eastr tFa , Hoechst Malaysia Bhdn d nSd .an Choon Huat Sdn Bhd., respectively. As in practice, storage conditions were simulated as follows: Open container root sa m temperature (25°C) - Closed containers at room temperature (25°C) Open containers at elevated temperature (33°C) Closed containers at elevated temperature (33°C)
At bimonthly intervals, samples were processed in the following manner:
Commercial formulatio g2 ( )n
Extracte freshlf o d l witm y0 2 hdistille d ethyl acetate
Shaken for 15 min. at 150 rpm usin flasa g k shaker
Centrifuged at 3,000 rpm
Extraction repeate A timed s
Precipitate filtered Supernatant rotary evaporated and dried to dryness and resuspended in 10ml of methanol
CS2 analysis Residues analysed using HPLC
80 parene Th t fungicid analysi„ determines CS ewa e th s y db metho d describey b d Keppel (7) and ETÜ determined by HPLC (8). TLC was also used for the determin- ation of degradation products (9).
2.2 Effects of washing and boiling on 14C-mancozeb residues in tomatoes
14 2.2.1 Preparation of radioactive formulation. C-mancozeb was obtained froInstitute th m Isotopesf eo , Budapest, Hungary throug Internationae hth l Atomic Energy Agency totae Th .l activit s 1.00ywa 2 mCi/15.6 . Eac3mg h fruit was painte solutiof do l witm 1 hn containin colf o dg m formulatio 3 g x 2 n+ 10 dpm of radioactive fungicide (or 34.45 yg C-mancozeb).
2.2.2 Preparatio fruitsf no . Fruits weighing approx werg 0 e .10 treate d with the1 4C-chemica d lef drylo an t e fruit .Th s were then wraped individually in aluminium foil and stored in the refrigerator at 4°C for 48 hrs. Washing effectes wa watef o l dippiny rm b d thre0 fruie 40 th gen i ttimes cookinn I . g experiments, fruits were sliced into four part min.0 cooked 1 san r s ,a fo d normally practiced. Radioactivit tomatn i y o washing water, cooking wated ran painting brushe determineds swa .
2.2.3 Extraction and analysis. Total residues were determined by combustion using a Harvey biological oxidizer and LSC. Samples were extracted with CHC1- : CH-OH (1:1) and analysed by TLC to characterize/identify organic extractables remainine Th . g residu analyses ewa parenr fo d t compound using thS eevolutioC n method (7).
3 2. Determinatio tomatn i U oET fruitf no s
14 14 2.3.1 Preparation of C-formulation. 1.56 mg C-mancozeb (100 jiCi) were mixed colg wit 0 dh1 mancozemixture th d bean waterl suspendem 0 .50 n i d Each plan s sprayetwa thif o d sl wit m suspension0 h3 e amounTh . origf to - inally present ETU was determined by extracting 10 g mancozeb with 25 ml x 4 of ethyl acetate. The four extracts were combined filtered and rotary evaporated to dryness. The sample was dissolved in 10 ml methanol and ETU determined by HPLC (8).
2.3.2 Applicatio analysisd nan fruitinx Si . g tomato plants bearing twenty mature fruits were used for this experiment. The plants were covered with 14 plasti l m C-formulatioc0 3 bag d san sprayes nwa d ontplante oth s through bagse holeplante th Th .n si s received three treatment weeklt sa y intervals.
81 Each sample consisted of two fruits. Each fruit was weighed and sliced into two roughly equal halves hal e s storeOn .fwa r determinatio fo d totaf o n l radioactivit e othe th processes rwa d e followin an yth n i d g manner:
The sample was blended in a Waring blender with 50 ml of chloroform : methanol mixture (1:1) homogenate Th . s filterefiltrate ewa th d an de rotary evaporated to dryness. The sample was then dissolved in methanol
and chromatographed by TLC in chloroform : butanol : methanol : HyO (100:5:1:0.5). The spots were visualized by iodine vapour and the band which corresponded to ETU was scraped and counted in a liquid scintilla- tion counter. e effecTh f fiel to 4 2. d weatherin e levelU painteth ET n f fruito gn so o d s In this experiment, 10 fruits were painted with ETU-free mancozeb 14 containing C-ETU e plantTh . s were subjecte o fielt d d weatherine th d gan fruits analysed at intervals. 14C-ETU, specific activity 120 pCi/mg, was obtained from the International Atomic Energy Agency. Each fruit was painted wit- C suspensioha m mancozeg containinl m dp m 3 1 0 f 1 n no i b x 2 g ETU. Sampling and analysis were made as described.
3. RESULTS AND DISCUSSION
3.1 Analysis of commercial fungicides Data obtaine e CS_th evolutioy b d n method were errati d coult an c no d be validated. ETU concentrations generally increased during storage. For manzate-200, ETU increased from 0.065% to 0.11 - 0.18% depending on storage conditions ove 0 periodaysa r 18 f .o d Parallel increases were also observed with trimanzone-yellow (from 0.01 0.02%)o %t deriveU ET . d from maneb increased from 0.035% to 0.045 - 0.080%. Analysis by TLC (9) showed that all formulations contained ETU, EDA, ETD, ETM and sulfur.
3.2 Effect washinf so boilind gan residuen go tomatoen si s 14 Tabl showI e e effecth s washinf o t cookind an g n o gC-residue e th n i s various fruit sections. washin, Frovg 8 totaa m3 g f alonlo e removed only 4 pg while washing and cooking removed about 22 pg equivalents. The water 14 used for boiling contained generally 14 - 53% of the applied C.
3 Determinatio3. fieln i U d ET weathere f no d tomato fruits During the experimentation period, the rainfall was unusually heavy. Tabl I showeI e concentratiosth fruitn i U sET ovef no periora weeks4 f o d .
82 Table I - Effect of washing and cooking on l^C-mancozeb residue tomatoen i s s
Treatment (yg 14C-mancozeb* (wt.of fruit) Skin section Seed section Middle section
Washed/cooked 10.13 4.21.86 9 (104.0 g)
Unwashed /cooked 23.08 14.45 9.06 (109.) 5g
Washe 4 5 . d9 /uncooke2 2.6d 9 1.92 (108.) 5g
Unwashed /uncooked 30.57 4.67 2.57 (103.7 g)
* Values are mean of four determinations.
Table II - ETU concentration in fruits
Weeks pg ETU/g fruit*
0 15.2
1 13.6
2 14.2
3 16.8
* Data are mean of two determinations.
Compared wit initiae hth l concentration, little changes were observede Th . amounU initiallET f to y deposite whole th en o dplan s calculatetwa e b o t d 180 iag/plant. These concentrations are considered extremely high.
3.4 ETU levels painted on fruits Ove periora weeks4 f percentage o d ,th remaininU ET f eo fruitn go d sdi not exceed 1.43% of the dose applied. Also small fluctuations were observed from one sampling time to another (range 0.91 to 1.43%).
83 REFERENCES
(1) Innés, J.R.M., Ulland, B.M., Valérie», M.G., Petrucelli, L., Fishboin, L., Hart, E.R. and Palotta, A.J. J. Nat. Cancer Inst. 41 (1969) 1101.
) Ulland(2 , B.M., Weisburger, JH.H., Weisberger, E.K., Rice, J.M Cythend .an , R.J. Nat. Cancer Inst 9 (1972.4 ) 583.
(3) Vonk, J.W. and Sijpesteijin, A. Kaars. Ann. Appl. Biol. 6_5 (1970) 489.
) Boutoyan(4 , W.R Lookerd .an , J.B Agr. J . . Food Chem (19731 .2_ ) 338.
) Gustafsson(5 , K.H Fahlgrend .an , C.H Agric. J . . Food Chem (19831 .3_ ) 463.
) Ankumah(6 , R.O Marshalld .an , W.D Agric. J . . Food Chem (1984_ 32 . ) 1194.
) Keppel(7 , E.G Assoc. J . . Off. Anal. Chem 2 (1969.5 ) 162.
) Onley(8 , J.H., Guiffrida Ives, WattsStorherr, L. d ,N. an . ,R . ,R J. Assoc. Off. Anal. Chem. 60 (1977) 1105.
(9) Onley, J.H. and Yip, G. J. Assoc. Off. ZAnal. Chem. 54_ (1971) 165.
84 PERSISTENCE AND METABOLISM OF MANCOZEB IN BRINJAL (EGG PLANT)
H.C. AGARWAL . KUMAU , R Departmen f Zoologyo t , University of Delhi, Delhi, India
Abstract
Brinjal (Solanum melongema) or egg plants in flowering and fruiting stage were sprayed with ETU-free formulation containing 45 mg mancozeb and 9.71 14 \iCi C-mancozeb per plant. Spraying was repeated twice every 7th day. Leaves, stem, fruit rootd san s were collecte extracted an d d with chloroform- methanometabolitee th d lan s identifie quantified an d TLC y db parene Th . t fungicid determines ewa CS.-methoy db radiocountingd an d . Morf eo tha% n90 mancozeb residues were detected in the leaves, while the fruits contained only weeko Tw abous . afte t2% las e rth t treatment fruite ,th s containe ppr1 2 da mancozeb which declined to 4 ppm 1 week later. The half-life of mancozeb was 6 day 5 day8. fruitn s9. o n leavess o d san . Four productsd ETU, an ,EU D ,ET ETM were found generally in all samples. Five more unknown products were detecte thet dbu y accounte vera r y fo dsmal l proportio residuese th f no U E . was the major metabolite and its concentration was about 1.3 ppm in the fruits 2 weeks after the final spray. ETU accounted for 0.12 ppm at that time. In storage, commercial formulations of mancozeb decomposed by 25% after 390 days.
1. INTRODUCTION Discoveries of Penicillin (1929), Dithiocarbamate fungicides (1934), DDT (1939) and 2,4-D herbicide (1942) within a short span of 13 years brought revolution in the field of disease and pest control and marked the beginning eraw otheine fn si r respective fields dithiocarbamatee Th . s which represent the third generation fungicides after sulfur and copper, are the most important group of organic fungicides yet discovered. Ethylenebisdithio- carbamates (EBDCs) are widely used as agricultural fungicides and are registere againse us r fo td 1,296 disease crop1 27 sn si (1). Maneb, mancozeb and zineb are the three EBDCs registered for use as fungicides in India with mancozeb consumption reaching 1,500 mt per year. Mancozeb is marketed as Dithane M-45 or Dhanuka M-45 as a wettable powder containing 75% of the active ingredient and has been recommended for use against many plant diseases like brow blacd nan k rust, leaf spot, leaf blight, dowry mildew etcvarieta n .o y of plants including brinjal (egg plant), tomat potatod oan .
85 The use of these compounds is, however, associated with an undesirable presence th o effect ethylenethioure f o e du t a e fungicid(ETUth n i ) e formula- tions (2). ETU may also be formed from EBDC under field conditions and as a resul cookinf o t potena g s (3)i tU .neuroteratogeET ratr nfo s (4).t I induces perinatal toxicity in rodents (5) and is a potential carcinogen and goitrogen (6). For safe use of compounds like mancozeb it is essential that residues it dat n metabolisd o a san made mb e available. Several reports have been published on residues in crops and grain (7, 8). However, data in India are very scanty and this work was undertaken to examine residues in brinjal (egg plant) under field conditions.
2. MATERIAL METHODD SAN S
2.1 Chemicals 14 C-mancozeb, specific activity 64.16 yCi/m s obtainewa g d froe th m Institut Isotopesf eo , Budapest, Hungary. Dithane M-45 (W.P. a.i% ,75 . 14 mancozeb) of Indofil Chemicals, Bombay, India was purchased locally. C- mancozeb was mixed with Dithane M-45 and the mixture extracted with freshly distilled ethyl acetate three time obtaio t s n ETU-free formulation. This swa suspende waten i d suspensiol tham o rs 0 t2 n containe mancozeg m 5 4 dd ban 9.75
2.2 Application and sampling Brinjal (Solanum melongena) plants in the flowering and fruiting stage 14 grown in baked clay pots were sprayed with 20 ml aqueous suspension of C- mancozeb formulation per plant using an all glass sprayer. Care was taken to minimize losd crossan s contamination during spraying e sprayinTh . s gwa repeated twice at 7-day intervals. Zero-hour samples were collected within firse th tf o spraying . hr 1 . Three samples were collected. Fruits, leaves and stem were collected separately from each plant. The second and third sets of samples were taken just before the second and third mancozeb treatment, i.e. on days 7 and 14 from the initial spray. Sampling was continued weekly week6 o ut ps afte e lasrth t (third sample)e sprayth l sAl . were storen i d polythene bags in a deep freezer until analysed.
2.3 Analysis Fresh fruits were weighed, finely chopped and mixed thoroughly. Small samples were weighed and dried at 70°C for a few hours and weighed after
86 coolin rooo t g m temperature. Only whe weighe nth t became constans wa t ti taken as the dry weight. Samples (1.5 - 5 g) were used for the estimation of mancoze carbondisulfidy b e evolution method (9)originae th . f o Abou l% 70 t sampl s extracteewa describes a d p (10 Yi Onley b d)d methano l yan m usin 0 g20 l / l chloroformm 0 an10 d filtrate Th . s cleaneewa accordinp du methoe th f o o dgt Rhodes (11). The residue was extracted with 200 ml of IN BDTA (disodium salt) for determinatio mancozebf no . Leave sted san m samples were similarly processed.
Radioactivit s determineywa Packara y db d Model 200 TricarA 0C b liquid scintillation spectrometer using aquasol-2 universal LSC cocktail. The extrac s examinetwa thin-layey b d r chromatography (TLC) using glass plates coated with 250 nm thick Silica gel G. Two solvent systems were used: chlorofor butano: m methano: l wate: l r (100:5:1:0.5, v/v) (12 ethyd )an l acetate : ammonia : water (15:1:1, v/v) (13). The spots were visualized by exposure to iodine vapours or Grote's reagent (14) and marked on the back side platesC TL e o.th f Identificatio mads nwa comparisoy eb n with reference chemicals ethyleneured (ETan U a [EU]). Other products were identifiey b d comparing e literaturevalueth n i s . Visible spots were scraped into scintil- latio radioactivitne th vial d an s y determined.
3. RESULTS AND DISCUSSION
1 Mancoze3. b residue brinjan i s l plants The data on mancozeb residues estimated by CS evolution method are presente totae TablTh n i dl . mancozeeI b residue recovere zert a d o tims ewa about 33 mg (74% of the mancozeb applied). This declined gradually after the third treatment to 3.3 mg, 8 weeks after the first treatment. The leaves initially contained > 90% while fruits accounted for only 2.4%. Table I also shows residue concentrations in leaves, stems and fruits. Fruits contained a maximum concentratio zert a om pp tim 1 e4 whicf no h declined graduallo yt 1.4 ppm after 8 weeks. By comparison, apple contained 1.7 ppm mancozeb 6 weeks after the last spray (15). Under comparable conditions tomato contained 0.03 - 0.80 ppm (16).
14 The percent recovery of C-mancozeb in different parts of brinjal plant shows i s Tabln ni . WithieII weekna , recovery sharply dropped from 84.820.3%o t %fruitn I . s only abou totae tth 0.05lf o residu% lefs ewa t afte weeksr6 .
87 Table I - -^C-mancozeb residues in brinjal plants
Residues (mg/plant) Weeks 0 1 2 3 4 5 6 8
Leaves 31.79 10.03 24.03 11.74 9.29 5.90 3.23 2.61 ±1.25 ±1.13 ±1.07 ±0.94 ±0.46 ±0.41 ±0.04 ±0.17
Stem 0.84 0.34 0.26 1.38 1.08 0.41 0.57 0.68 ±0.066 ±0.02 ±0.012 ±0.069 ±0.10 ±0.02 ±0.04 ±0.04
Fruits 0.79 0.75 0.81 0.91 0.79 0.25 0.66 0.04 ±0.11 ±0.02 ±0.02 ±0.09 ±0.03 ±0.17 ±0.08 ±0.0003
Total 33.42 11.12 25.10 14.03 11.16 6.56 4.46 3.33
Residues (ppm)
Leaves 2130 610 1170 2500 1950 1530 650 510 ± 80 ± 70 ±50 ±200 ± 95 ±110 ± 4 ± 30
Stems 107 18 36 101 130 111 56 46 ± 8 ± 1 ± 2 ± 5 ± 13 ± 6 ± 3 ± 3
Fruits 41 15 20 18 21 4 12 1 ± 6 ± 2 ± 1 ± 2 ± 1 ± 0.1 ± 2 ± 0.7
Values are 3 replicates each from 3 samples (± standard error),
r cenPe tTabl - recover I I e f mancozeo y b afte weekl3 r y applications of l^C-mancoze o brinjat b l plants
% Weeks after last treatment t 1/2 0 1 2 3 4 6 (days)
Leaf 80.9 17.0 13.5 8.6 4.7 3.8 9.5
Stem 1.6 2.0 1.6 0.6 0.8 0.99 36 .5
Fruit 2 .3 1.3 1.5 0.4 0.96 0.05 8.6
Total 84 .8 20.3 16.6 9.6 6.46 4.84 11.1
88 The half-life of mancozeb in whole brinjal plants was found to be 11.1 days. Corresponding values for leaves, stem and fruits were 9.5 days, 36. 56 daysdays8. d , ,an respectively . Previous studier laboratorieou n si s gave T values of 15 and 19 days for the whole plant and fruits, respectively. This difference may be attributed to environmental conditions earliee ath s r experiments were conducted during winter months whereae sth present work was done in summer.
3.2 Mancozeb metabolite fruitn si s Thin-layer chromatography using the ethyl acetate-ammonia water solvent syste s capablmwa resolvinf eo ETÜd an usiny B .u e secon E g th g d solvent system, 4 spots including mancozeb at the origin were identified. The spot nearest to the origi s ETUn wa ETMd ,an followe .D FivET y b ed spots remained unidentified but they accounte vera r y fo dsmal l proportio radioactivitye th f no . Quantif- ication of the major products is shown in Table III. In most cases the parent fungicide accounted for over 90% of the total fungicide residues detected. Ethyleneurea (EU) was the predominant product showing a high concentration of ppm3 1. ,wee1 k afte lase rth t spray 0.0U ,ET 7 ppm date Th .a available th n i e literature are generally based on methods which often do not separate EU from
Table III - Mancozeb and degradation products in brinjal fruits
Days after Mancozeb ETU ETD ETM EU last MS/6 ng/g treatment
7+ 30.111.5 73.210.9 38.611.9 49.912.3 1326.61127.7 (95.3) (0.2) (0.1) (0.16) (4.2)
14 16.811.1 205.810.03 92010.01 40.82118 1184.81310 (87.7) (1.1) (4.8) (0.2) (6.2)
21 6.310.38 7.6212.28 5.014.3 18.14113.6 336.5141 (94.5) (0.1) (0.07) (0.27) (5.1)
28 10.110.16 18.2913.0 6.4411.93 29.9113.6 139.6132.46 (98.1) (0.18) (0.06) (0.29) (1.4)
42 0.9210.05 4.5710.61 5.4110.86 (98.9 (0.5) (0.6)
Values estimated from radioactivity. Value parenthesen i s s shopercentage wth e (total residu 100H)e= .
89 r cenPe tTabl - mancoze V I e commercialn i b * samples
Condition % mancozeb of package Days of storage 0 72 44 210 300 390
Closed 69.50 68.80 67.70 66.10 66.05 65.94 ±1.94 ±1.31 ±0.96 ±3.07 ±2.81 ±3.03
Open 69.70 58.80 54.10 51.70 52.01 52.0 +1.25 ±0.78 ±0.42 ±0.67 ±1.23 ±4.17
* Dithane M-45, 75% WP.
ETU and thus the values for ETU are likely to include also EU. The data presented here indicate that EU is essentially a predominant product. It shoul notee b d d that results presente f thin o i d e s us pape e basee th r ar n o d ETU-free formulation wit hviea quantifo wt y formatio produceU ET f no d from the parent mancozeb. Under practical conditions presenU ,ET t initialle th n yi formulation will constitut extrn ea a burde residuef no foodn i s .
3.3 Concentrations of mancozeb and ETU in commercial formulations Analysi S mancozef evolutioC so HPLy e b th CU y ET b nd methoan ) (9 d (14) gav resulte eth s show Tabln ni . MancozeeIV b decompose closen i d d package open i n% packageonly 25 sb % aftey y8 b day0 d 39 ran s ovesame th re period. It is noted that the major degradation (17%) occurred in the first 72 daysU datET a. wer t represent consistenno e no d di d tspecifia tan c trend. This probably relates to varying climatic conditions and the inclusion of EU, as the HPLC method was not specific for ETU and did not separate both closen i d m an dpp products 4 41 - .2 10 ETU/Ed an m U pp value 0 50 s- wer9 e10 open packages, respectively.
ACKNOWLEDGEMENTS
This work was partly supported by the Agrochemicals and Residues Section of the Joint FAO/IAEA Division, Vienna, Austria under RC/4000/RB.
REFERENCES
(1) Brandes, G.A. "Acreage for crops for which EBDC fungicides are registered" 0959c ,Do 6 RohHaasd man , Philadelphia (1977).
90 (2) Bontoyan, W.R., Looker, J.B., Kaiser, T.E., Giang, P., and Olive, B.M. Survey of ethylenethiourea in commercial ethylenebisdithiocarbamate formulations Assoc. J . . Off. Anal. Chem5 (1972 5 .5 ) 923.
(3) Newsome, W.H. Residues of four ethylenebis(dithiocarbamates) and their decomposition product fieln so d sprayed tomatoes Agric. J . . Food Chem. 24 (1976) 999.
(4) Khera, K.S. Ethylenethiourea: A review of teratogenicity and distribution studies and an assessment of reproduction risk. CRC Critical Review Toxicologn i s (19872 8 y1 ) 129.
(5) Chernoff, N., Kavlock, R.J., Rogers, E.H., Carver, B.D. and Murray, C. Perinatal toxicit manebf yo , ethylenethioure ethylenebisisothiod aan - cyanate sulfide in rodents. J. Toxicol. Environ. Health 5_ (1979) 821.
) Graham(6 , S.L., Hansen, W.H., Davis, K.J.Perryd an , , C.H. Effecte on f so year administration of ethylenethiourea upon the thyroid of the rat. J. Agr. Food Chem. 21 (1973) 324.
(7) Cheah, U.B. Mancozeb residues in highland and lowland tomatoes. MARDI Res. Bull. 13 3 (1985) 279.
) Rosenberg(8 Sutanend an . ,C . ,ResidueH mancozef so ethylenethioured ban a in grain samples. Bull. Environ. Contam. Toxicol. 2g (1979) 475.
(9) Keppel, G.E. Modification of the carbon disulfide evolution method for dithiocarbamate residues. J. Assoc. Off. Anal. Chem. 52 (1969) 162.
(10) Onley, J.H., Yip . DeterminatioG , ethylenethiouref no a residuen si foods, using thin-laye chromatographys ga d ran Assoc. J . . Off. Anal. Chem 4 (1971.5 ) 165.
(11) Rhodes, R.C. Studies with manganese [-^C] ethylenebis(dithiocarbamate) U14C] Maneb) fungicide [d 14san C ] ethylenethiourea ([14C] ETUn )i plants, soiwaterd lan Agric. J . . Food Chem 5 (1977.2 ) 528.
(12) Czegledi-Janko . ,DeterminatioG degradatioe th f no n productf so ethylenebis(dithiocarbamates) by thin-layer chromatography and some investigations of their decomposition in vitro. J. Chromatogr. 31 (1967) 89.
(13) Ankumah, MarshallR.Od an . , W.D. Persistenc fatd eethylenethioan f eo - ure tomatn i a o sauc pasted ean Agric . J . . Food Chem (19842 3_ . ) 1194.
(14) Onley, J.H., Giuffrida, L., Ives, N.F., Watts, R.R. and Storherr, R.W. s liquiGa d chromatograph d liquiyan d chromatograph ethylenethiouref yo a in fresh vegetable crops, fruits, milk and cooked foods. J. Assoc. Off. Anal. Chem (19770 .6 ) 1105.
(15) Ross, R.G., Wood, StarkF.Ad an . . ,EthylenR dithiocarbamats ebi d ean ethylenethiourea residues in apples and apple products following sprays of mancozeb and metiram. Can. J. Plant Sei. 58 (1978) 601.
(16) Vonstryk, F.G Jarvisd .an , W.R. Residue mancozebf so , mane ethylened ban - thioure fungicidn ai e treated fiel greenhousd an d e tomatoes. Can. J . Plant. Sei (19788 .5 ) 623.
Next page(s) left blank EFFECT OF WASHING, BAKING AND COOKING ON AGED RESIDUES OF MANCOZEB IN BRINJALS (EGG PLANTS)
H.C. AGARWAL, U, KUMAR Department of Zoology, University of Delhi, Delhi, India
Abstract
Twelve brinjal fruits (unplucked) were treated eac hl aqueoum wit 5 h0. s 14 suspension of ETU-free C-mancozeb containing 1.81 mg mancozeb/1.09 pCi. e fruitTh s were plucke day5 1 d s late d subjecteran washingo t d , bakinr o g cooking unwashee Th . d brinjals containe mancozef o dm aboupp b8 residuet4. s showin e initiallth losga f o abouf o s% y 70 tapplie d mancozeb. Washine th f go fruits reduced the residues further to about 0.8 ppm and baking led to a further reduction. Boilin fruite th g waten si r gave similar result termn si s of residue losses revealeC TL . presence th d tracf eo d e an amount M ET f so ETD. ETÜ (and possibly EU) was the major degradation product.
. 1 INTRODUCTION Fruit vegetabled san oftee sar n processe washingy db , bakin cookinr go g before consumption bees ha n t observeI . d that surface washing removesa significant quantit mancozef yo cookind ban g further degrade largsa e propor- remainine th tio f no g residu ) int(1 evarietoa degradatiof yo n products including ethylenethiourea (ETU). It is essential that the levels of EBDCs and ETU in food and food products should be within acceptable levels. This work examines the effects of baking and cooking on brinjal content of mancozeb residues, using radiotracer techniques.
2. MATERIALS AND METHODS
14 1 r_2. C-mancozeb 14 ETU-free C-mancozeb suspension was prepared as previously described (2); 3.62 mg/2.18 pCi/ml suspension.
2.2 Application, sampling and processing Twelve brinjal (Solanum melengena) plants in the post-bloom stage were 14 used and each fruit was treated with 0.5 ml of the C-mancozeb suspension which was applied by a pipette all around the fruit.
93 e brinjaTh l fruits were plucke 5 day1 d s after treatmen d dividetan n i d o equatw l group bakinr fo sboilingd an g .fruite th Hal f so f from each group s analysewa suchs a d , whil othee th e r min3 hal traa s kepr , wa fn fo yi t unde r running water. All fruits were weighed individually. Baking was done at a temperature of 185°C to 260°C for 25 min, in a gas oven. In boiling tests, fruits were individuall min5 2 watel m .y r 0 boilerfo 25 n i d
2.3 Sample preparation and analysis The unbaked fruits were finely chopped and mixed while the baked fruits were thoroughly mixed. Boiled fruits and water were mixed into a uniform paste and analysed as one sample. Extraction, clean-up, Chromatographie techniques and other analytical methods were the same as previously described (2).
3. RESULT DICUSSIOD AN S N
3.1 Effect of washing and baking The result e recoverth n o s f totao y l mancozeb residue n fruiti s day5 1 s s 14 after treatmen e showar t s n e Tablappliei nth wa . f I eC o Aboud% 29 t recovered. Washing reduced residues to about 5% indicating that brief surface
14 Tabl - Effec I e f washingo t , bakin cookind gan n go C-mancoze b residue n fruiti s s
Residues (ug/g fresh weight)
Unwashed Washed
Control Baked Control Baked
Applied Recovered Applied Recovered Applied Recovered Applied Recovered
«) ______ill______(*)______(%}
16.17 4.75±0.57 15.72 1.8910.06 15.88 0.7710.06 17.01 0.4110.02 (29.4) (12.0) (4.9) (2.4)
Boiled Boiled
28.39 10.6410.35 30.82 4.910.39 31.52 5.6110.84 31.21 1.8610.13 (37.5) (15.9) (17.8) (6.0)
Residue day5 1 s s after treatment.
94 TablRelativ- I eI e percentage mancozef metabolites so it d ban fruitn i s s
as affecte washingy b d , bakin cookind an g g
Relative percentages Treatment Mancozeb ETU/EU ETD ETM (ppm) (ppm)
Unwashed Control 81.8 9 0. 16.6 0.6 Baked 82.3 1 1. 15.5 1.1
Washed Control 77.0 20.3 0.8 1.9 Baked 76.0 18.2 3.4 2.4
Unwashed Control 66.3 33.4 0.14 0.2 (3.08+21) (1.55±17)
Boiled 82.0 6 0. 16.8 0.7 (1.67119) (0.34210.07)
Washed Control 89.0 9.9 0.5 0.6 (1.94129) (0.2210.03)
Boiled 75.3 6 0. 23.5 0.6 (0.5810.01) (0.1810.01)
washing can remove substantial quantities of the residue. Baking of brinjals further reduce residue th damoune th halo et f o tf present (Tabl . CheaeI) h ) reporte(1 d that heat treatmen highlane th f to d tomatoes reduced mancozeb levels to 0.27 mg/kg from 0.58 mg/kg. For lowland tomatoes, the corresponding figures were 0.5 0.7d 0an 9 mg/kg, respectively.
In addition to mancozeb, thin-layer chromatography revealed the presence of three compounds (Table II). These were ETU, ethylenethiouram disulfide (ETD ethylenethiourad )an m monosulfide (ETM). Sinc solvene eth t system used
95 did not separate ethyleneurea (EU) from ETU, both compounds would be super- imposed e relativTh . e percentage f identifieso d products show that mancozeb 14 contributed about 80% of the total C-activity (Table II). ETU possibly containin contributeU E g majoa knowe s b i 20%r- o U metabolit5 nt 1 d. ET f eo field-sprayen EBDi % C conversioe hig49 s fungicide a th s a he d b an dy ) nma (3 s tomatoes (4). Ross et al. (5) reported about 34% conversion of mancozeb to n apple i resula U s a sET cookingf o t .
2 Effec3. washinf o t cookind gan g Table I shows a similar trend for cooking as for baking. Boiling reduced bakee e residu th case n thirdth i don f e o s o A sampleset . , three productf so mancozeb were identified (Table II). ETU was a major metabolite (10 - 33%). e absolutTh e quantitie U showeET f continuoua dso s reduction starting froe th m unwashed controls which contained about 1.6 v>g/g of ETU to 0.18 yg/g in washed/boilee th d brinjals.
e abovTh e results show that washin brinjalf o g s remove substantiasa l amoun agef to d mancozeb residues whic e reducehar d furthe resula s ra f to bakin boilingr o g .
ACKNOWLEDGEMENTS
This wor s partlkwa y supporte Agrochemicale th y b d Residued an s s Section e Joinoth f t FAO/IAEA Division, Vienna, Austria under RC/4000.
REFERENCES
) Cheah(1 , U.S., Mancozeb residue highlann i s d lowlanan d d tomatoes MARDI Res. Bull. 13_ (3) (1985) 279.
) Agarwal(2 Kumar. ,U H.Cd .an . This issue.
) Marshall(3 , W.D. Thermal decompositio ethylenebis(dithiocarbamatef o n ) fungicides to ethylenethiourea in aqueous media. J. Agric. Food Chem. 25 (1977) 357.
) Newsome(4 , W.H. Residue fouf so r ethylenebis(dithiocarbamates theid )an r decomposition products on field sprayed tomatoes. J. Agric. Food Chem. 24. (1976) 999.
(5) Ross, R.G., Wood, F.A. and Stark, R. Ethylenebisdithiocarbamate and ethylenethiourea residues in apples and apple products following sprays of mancoze metiramd ban . Can Plan, J . t Sei 8 (19785 . ) 601.
96 RADIOTRACER STUDY OF ZINEB RESIDUES IN EGG PLANT-SOIL SYSTEMS
Lianzhong ZHANG, Guangshun WANG, Hanhong MO, Fengchun AN, Jianguo QIAN Research Centre for Eco-Environmental Sciences, Academia Sinica, Beijing, China
Abstract
widespreae vien th I zinef f o wo n fooe bo dus d toxicit e plantth d an sy metabolitess it f o e oon f , ethylenethiourea (ETÜ) ,studa f thiyo s agrofungicide in plant-soil systems was conducted. The half-life values for total zineb residues on egg-plant leaves and in soil were calculated to be 22.4 and 16.0 days, respectively. Egg-plant skin contained higher amounts of zineb residues than the fruit pulp. Washing with water could greatly reduce zineb residue levels, presenfruie th tn to skin . Compared with zinebs i U ,ET muc egg-plane h th o easieleac t takee y b b d ho p n rt an u into deeper layerf so wates it ro t solubility soie ldu soil-plann I . t studies using artemisia plant uptake ,th e froafteU exceet ET m no weeksr3 % soi zine5 % d d3 ld di .ban
1. INTRODUCTION Zineb, a member of an important group of fungicides, ethylenebisdithio- carbamates (EBDCs), has been widely used to control diseases of agricultural crops. The discovery, that ethylenethiourea (ETU), one of the degradation product EBDCsf so , possesses goitrogeni tumorigenid can c propertie, 3) , 2 , (1 s has caused great concern due to contamination of food plants with EBDC residues and ETU. Because zineb is extensively used in China on food plants and therlaca f informatios o kei n concernin e fat d behaviouth g ean zinef ro b residues, the Research Centre for Eco-Environmental Sciences, Academia Sinica, with the technical and financial aid of the Joint FAO/IAEA Division undertook a study on zineb residues in egg-plant - soil systems.
2. MATERIALS AND METHODS
2.1 Materials 14 C-ethylene-labelled zineb produced in Hungary was provided by the Joint FAO/IAEA Division. Cold zineb (80% W.P.) wit contenU hET t less than 0.3% was obtained from Shenyang Pesticide Plant. 14C-labelled and cold
97 zineb were mixed and extracted three himes with methanol to remove ETU. An experimental suspensio s prepare nwa waterl m 0 y suspendinb d;30 n i g l g specific activity 0.43 yGi/ml.
14 C-ETU used in the study was also supplied by the Joint FAO/IAEA Division. An aqueous solution of ETU with specific activity of 2.0 pCi/ml
was prepared. All the solvents and Na??EDTEDTjA used in the extraction procedures were analytical grade reagents.
2.2 Equipment A Harvey oxidizer e OX-30combustioth uses r 0wa fo d f solio n d samples 14 14 for determination of total C-content. C-activity in liquid samples was determined in an FJ-2100 liquid scintillation counter produced in China.
2.3 Zineb residue egg-plantn i s s Young egg-plants were planted in the field and then transferred to buckets in a greenhouse. The plants received no rainfall, only necessary watering. After having begu blossomo t n e plant,th s were divided into tw o 14 groups. One group was sprayed once with the C-zineb suspension (0.1 g 14 a.i. and 13.5 pCi C/plant). Leaf samples were collected at 0, 2, 5, 9, 14 1 days3 e tota Th s d determine.wa l an C amoun7 2 , 16f y samplto b d22 , e combustio d liquian n d scintillation counting e seconTh . d grou plantf o p s swa treated three times witsame th he suspensio t intervalna week1 f o s. Egg- 14 plant fruits e fruitwerth s edeterminedn i swa collecte C e frud Th an id. samples were also analysed for zineb, metabolites and bound residues.
2.4 Uptake of zineb and ETU residues from soils by plants columnC PV D I sSoim packes c whicwa l 5 dh3. werx int 8 e2 o inserted into soil containe plastin i d c buckets uppee Th e column.r th e soi layen th i l s f o r 14 14 was treated with C-zineb (7.2 mg, 2.15 yCi/column) or C-ETU (0.22 mg, 2.0 |aCi/column) . xhe treated layers were then covered with 1 cm layers of fresh soil. Artemisia plants were planted in the columns and the columns received waterin e amounth t ta g correspondin e averagth o t ge local precipitation. Plant samples were collecte t intervala d d analysean s r fo d 14C. Some samples were also radioautographe d soian dl columns were sectioned and analysed to examine the leaching behaviour of zineb and ETU.
98 Table I - l^C-zineb residues in egg-plant leaves (one treatment)
Sampling time (days) 0 2 5 9 16 22 27 31
Residue (ppm)* 1 10433 6 570 1326 89 8 3 73 129 5 88 1
determination5 Mea* f o n s
Table II - ^C-xineb residues in 0 - 1 cm top soil layer (one treatment)
1 3 8 2 2 2 5 1 2 1 7 Samplin 4 g time 0 (days)
4 10 5 12 5 15 5 17 5 19 4 21 6 36 Residu7 42 e (ppm)*
determinations3 Mea* f no .
Table III - Total zineb residues in egg-plant fruit (three treatments)
Fruit Skin Pulp Sample base Water- Non- Water- Non- washed washed washed Washed
Residue (ppm)* 304.2 52.8 160.4 26.8 37.1
* 2-3 weeks after last application.
Tabl Relativ- V eI e distributio 14f C-residueno n si artemisia plants
Residum pp n i e 14C-chemical (% of total plant uptake) Roots Stems Leaves
14C-zineb 25.0 7.89 12.5 (66.0) (3.4) (30.6)
14C-ETU 36.6 12.9 28.2 (30.2) (6.6) (63.2)
99 3. RESULTS AND DISCUSSION
1 3. Zineb residue egg-plantn i s d soisan l 14 Table I shows the decline of C-residues on egg-plant leaves with time and the half-life was calculated to be 22.4 days, which is little longer than a half-life of 2 weeks previously determined for zineb on plant leaves (4).
Table II shows the behaviour of zineb residues in the 0 - 1 cm top layer of soil. The half-life was estimated to be 16 days, which is longer than a half-lifday6 - s 4 previousl f eo y determine ziner fo dsoin bi l (4).
The data in Table III confirmed previous results (4) which indicated that zineb residues mainly deposiskie f fungicide-treateth no n to d fruits. The removal of residues from the fruit pulp as a result of washing suggests 14 thapule tth p contains water soluble C-compounds sequentian I . l extraction of the fruit, methanol extracted 39% of the radioactivity and Na EDTA extracted 42%; 12% remained bound.
3.2 Behaviour of zineb and ETU in plant-soil systems
3.2.1 Uptake of zineb and ETU by arteroisia plants. Table IV shows the distributio residueU ET zinef n o d artemisin ban i s a plants harveste day2 2 d s after planting. ETU with a high water solubility is more readily taken up by plants than zineb. With elapsing time, ETU gradually moved from roots to leaves termn I .totaf so l residues plane ,th t too onlp ku y very small 14 14 quantities of C-zineb and C-ETU which did not exceed 2.8% and 4.4% of e radioactivitth soiln i y , respectively.
3.2.2 Leaching of zineb and ETU residues in soil columns. Table V shows a limited leachin zinef o g b residues whic hdetectee b coul t dno d beyone th d sixth centimetre. The half-life of zineb residues in the 0 - 1 cm upper layer s calculate wa days6 1 e factore .b th o t Leachindf o e regardes b e n on ca gs a d responsible for the disappearance of zineb residues from the upper layer of soil. Leaching of ETU residues in soil columns was also limited. However, unlike zineb, traces of ETU could be detected in soil layers as deep as uppem c 1 r - laye0 soif e estimates ro th lwa half-lifn e i Th U dET . 2f cm 5eo to be 10.5 days which is shorter than that of zineb residues in the same layer, suggestin highea g r leaching ratETUr efo .
100 Table V - 14C-zineb and 14C-ETU residues (ppm) in soil columns
Zineb
Day2 s1 7 4 0 15 22 28 31
Soim lc laye1 0- r 6 6 36 19 214284 175 155 125 104
Soi6 1 l laye 6 r1 1-3 c0 m1 9 6. 16 21 18 21
Soil laye6 1. r 3- 67 3. cm D N NO 0.7 2.7 3.2 1.5
ETU
Days 0258 13 20 63 90
Soilm c laye1 0- r 14.1 4 5. 12. 4 88. 4.1 4.3 4.0 3.1
Soil layer 1-3 cm 0.36 2 1. 3.1 4 33. 1.4 0.5 0.9 0.3
Soilm c 3 aye5 3- r ND 0.06 0.07 0.2 0.1 0.03 0.1 ND
Soil layer 5-8 cm ND ND ND 0.03 ND ND 0.03 ND
Data are mean of 3 determinations.
3-4 Bound residues of zineb in soil A bound residue is normally defined as chemical residue which can not be extracte conventionay db l extracting procedures n casI .zinebf e o , bound residues may be regarded as residues remaining in samples which have been
exhaustively extracted with methanol and Na?EDTA. Under these conditions, a fortifying concentration of 7.2 mg/column resulted in 11.3 ppm bound residue after 4 days which progressed to 19.6 ppm after 22 days.
REFERENCES
) (! Innés, J.R.M al.t .e Nat . J , . Cancer Inst (19692 .4 ) 1101.
) (2 Graham, S.L W.Hd .an . Hansen. Bull. Environ. Contain. Toxicol (19727 . ) 19.
) (3 Graham, S.L., W.H. Hansen, K,J. Davi C.Hd san . Pery Agric. J . . Food Chem (19731 .2 ) 324.
. "Th) al (4 t Zhanee stabilit . gL somf yo e EBDC preparations during storage and zineb residue egg-plann si tomatd tan o plants". Report presentet a d the Second FAO/IAEA Research Coordination Meeting on Radiotracer Studies of Fungicide Residues in Food Plants, Munich, Germany F.R. (1987).
Next page(s) left blank 101 PERSISTENC 14F C-ETHYLENETHIOUREEO A BRINJAN I L PLANT SOID SLAN (Abstract)
H.C. AGARWAL, U. KUMAR, O.K. SINGH Departmen f Zoologyo t , Universit f Delhiyo , Delhi, India
14 C-ethylenethiourea, ETU, (8.6 mg) applied to 12 leaves on the plant dissipated quickl% afte28 daysr3 o t y . Afte weeks4 r treatee ,th d leaves contained only 0.25%. Residues in all fruits were only 0.47 v6 after 3 days, increasing to 3.31 pg after 2 weeks and declining to 0.69 yg after 8 weeks. Ethyleneurea (EU) was undetectable in fruits after 3 days. Seven-day samples containe increasin, vg 0 1. d 3.1 o t g 5 ng afte weekr2 declinind san o t g 14 1.2 vig after 8 weeks. The persistence and leaching of C-ETU was studied in soil-filled PVC columns under irrigated and non-irrigated conditions. Each column was treated with 3.27 mg ETU/1.365 pCi in 10 ml water and radio- activit s determineywa variour fo d s depths ove periora days2 5 f .o d Under non-irrigate d irrigatean d d conditions majoe ,th r C-activit s retaineywa d14 in the top 10 cm of soil with only < 2% and < 4% leaching to lower depths, respectively e absencth n moistureI f e.o , soi lappliee containeth f o d % 75 d ETU after 22 days. Tn irrigated soils, 54% was found in soil after 25 days; this declined to 30% after 52 days.
Next page(s3 ) lef10 t blank STUDIE 14F C-MANCOZESO B FUNGICIDD EAN C-ETHYLENETHIOURE SILTN AI Y CLAY LOAM SOIL
14 (Summary)
S.M.F. CALUMPANG*, M.J.V. BARREDO*, . ROXAS**N.P , E.D. MAGALLONA* *Pesticide Toxicology and Chemistry Laboratory, National Crop Protection Center **Departmen Animaf to l Nutrition, Institute of Animal Science College of Agriculture, University of the Philippines at Los Banos, College, Laguna, Philippines
persistence Th movemend ean mancozef degradatios to it d ban n products, ethylenethiourea (ETÜ) and ethyleneurea (EU) were studied in soil columns under outdoor conditions using conventiona radiotraced lan r techniques. There was a rapid loss of mancozeb in non-sterile soil with a half-life of 2.9 days; afte days5 r1 , mancoze virtualls bwa y undetectable, Mancozeb residues were p layeto re founonlth n yi d (2.5 cm)rapie Th . d los mancozef so s similabwa r to the results reported previously for Dithane where residues could not be detected 12 days after application (1).
Ethylenethiourea levels initially presen appliee th n ti d suspension (1.59 yg/g) degraded rapidly showing a 78% reduction 3 days after applica- tionsoif U leache o day4 ET maximul1 m a .c s o 8 t d afte f mo r applicatiof no mancozeb. Other workers have found movetha" tETU d5 inche 2. dow o snt (6.3) 5cm day5 1 s after treatment with maneb (2). Althoug wates i U r hET soluble d di t ,i appeat mobile no b o siltn rt ei y clay loam soil. TraceU (0.00ET f 4so yg/g) e soith lf o colum m c day1 n5 2 2. s coulp afte to detectee b dre th n i d application. This represents only 0.2% of the initial ETU level found at the time of application of Dithane M-45. Degradation of ETU in soil had a half-life of 2.5 days. A short half-life of less than 1 week was reported by Rhodes (3).
Ethyleneurea (EU) was also detected in the soil. Its concentration increased fro initian a m l leve 0.4f lo 7 yg/ 1.7o t g 6 yg/g day3 , s after application. This represents an increase of 1.29 yg (EU)/g soil which parallel decrease sth U (1.2 ET (ETU)/g 5n y ei g soil) durin same th ge time span. ETU has been reported to degrade to EU in soil (3).
105 After the initial increase, 3 days after application, EU degraded steadily with a half-life of 4.8 days which is almost twice as long as the half-life of ETU. EU is considerably more stable than ETU and can be considere majoa s a dr metabolite (4). Degradatiobees ha n U reporteET f no o t d be microbial (3) and photochemical (4). EU was detectable in the top 8 cm of the soil column up to 21 days after application of mancozeb.
REFERENCES
) Blazquez(1 , C.H. Residue determinatio ethylenethiouref no a (2-imi- dazolidinethione) from tomato foliage, soil and water. J. Agric. Food Chem (1973L 2J . ) 330-332.
) Newsome(2 , W.H .Shields, , J.B Villenueved .an , D.C. Residue Manebf o s , Ethylenethioram Monosulfide, Ethylenethiourea and Ethylenediamine in Beans and Tomatoes Field Treated with Maneb. J. Agric. Food Chem. 23 (1975) 756-758.
) Rhodes(3 , R.C. Studies with manganes ) ethylenebis(dithiocarbamate(C e ) [14C]-maneb) fungicide and (14C) ethylenethiourea [14C] -ETU) in plants, soiwaterd an l Agric . J . . Food Chem3 (1977 _ .25 ) 528-533.
(4) Kauf man, D.D. and Fletcher, C.L. 1973. Degradation of ethylenethiourea in soil. In: Abstracts, 165th National Meeting Amer. Chem. Soc. Div. of Pest. ACS1 .# : Dallas, Texas, USA, April 8-13, 1973.
106 AN ATTEMPT TO SEPARATE ETHYLENETHIOUREA AND ETHYLENEUREA, DEGRADATION PRODUCTF SO ETHYLENEBIS(DITHIOCARBAMATES), USING HPLC
P.N. MOZA, K. MUSTERT Institut für Ökologische Chemie, Gesellschaf r Strahlenfü t Umweltforschund un - g mbH, Freising, Federal Republic of Germany
Abstract
Ethylenethiourea (ETU ethyleneured )an aprimare th (EU e )yar biotid can abiotic degradation products of ethylenebis(dithiocarbamate) fungicides. The analytical methods useidentificatior fo d quantificatiod nan involvU ET f no e extraction, clean-u derivatizationd an p e derivatizatioTh . n methodt no o sd give quantitative recoveries. In recent years, HPLC has been extensively used for analysis of polar compounds and this technique has been tried to determine the chemicals in water and soil. The limit of detection of ETU/EU was shown to be 0.1 ppm using a silica gel or chemically modified silica gel as a stationar detectorV yU phasa d ean . HPLe th NonC f ecolumno s like silica gel, RP-18 reverse phase, Hucleosil (CN cyclobonr )o d using different eluting solvent mixtures could effect satisfactory separation of ETU and EU in water or soil extracts. Because of their close structural resemblance, HPLC-based methods tested in this research have failed to satisfactorily separate the two chemicals.
1. INTRODUCTION Ethylenethiourea (ETU) and ethyleneurea (EU) are the primary metabolites in environmental (1, 2) degradation and contaminants in the fungicides maneb, zineb, nabam, amoba mancozed man b whic widele har y usecontrollinr fo d g plant diseases bees ha n U suspecteET . causo t d e various toxic. effect6) - 3 ( s
In recent years, concern has been expressed about the presence of ETU in food and drinks (7). Various analytical methods have been reported for ETU determination e analyticaTh . l methods involve extractio clean-ud nan n o p various columns, derivatization 2-(butylthio)-2-imidazolineo t ) 11 - 8 ( s , 2-(benzylthio)-1-(pentafluorobenzoyl)-2-imidazoline, 2-(benzylthio)-1-(tri- fluoro-acetyl)-2-imidazoline, 2-[m-(trifluoromethyl)-benzylthio]-l-(tri- fluoroacetyD-2-imidazoline. These derivatization methods do not give consistan quantitativr to e recoveries.
107 £ c O CM
Fig. 1: a. UV-spectrum of EU . UV-spectrub U ET f o m
In our previous work (12), a simple clean-up method for the determination of ETU and propylenethiourea (PTU) in some food items using a capillary GLC was reported. During recent years, high performance liquid chromatography (HPLC) has been extensively used for analysis of polar compounds in environ- mental samples. ETU and EU, both being polar, soluble in water, and would be feasibl o identifet quantifd an y y using HPLC analysis absorptioV U e Th . f no m (Fig n lieU . E 5 Thi. 1) s21 d ET sbetweeUan d woulan 0 dn23 enable identific- atio d quantificationan thesf no e products simultaneously, provided thet yge well separate HPLn a Cn o dcolumn .
2. EXPERIMENTAL AND RESULTS For achieving separation of ETU and EU, a variety of column packings were used. The specifications of these columns are listed in Table I. The stationary phase of these columns is either silica gel or chemically modified silica gel. Silanol hydrogen of silica gel is substituted by octadecyl (RP-18), alkylamino (Shedon hypersi alkylnitril, ) H lN e (Nucleosil SCNr )o dextrin (cyclobond) moiety leadin differeno t g t substituted silicl age molecules for specific and efficient separation of diverse classes of chemicals.
Fig. 2 is a typical HPLC chromatogram of ETU and EU on a silica gel column. The retention time of ETU is not sufficiently different from EU. Variatio solvenf no t mixturt improvno d e separatioedi eth n capacite th f yo
108 Table I - HPLC columns and their specifications
No. Column Length x Corn gross diameter
1 Silicl age 250 x 4 mm 5 pro 2 RP-18 m m 4 x 0 25 5 pro 3 Shedon hypers il (NH,) 250 x 4 mm 5 pro 4 Nucleosil SCM m m 4 x 0 25 5 pro 5 Cyclobond m m 6 4. x 0 10 5 pro
Fig. 2: HPLC chromatogram of ETU (a) and EU (b) on silica gel
column. Solvent CH2C12:MeOH:H20 - 98:1:1 at 230 nm (5 ng each).
column. RP-18 reverse phase colum unquestionabls ni colume yth choicf no r efo separatio polaf n o wate d ran r soluble compounds. This colum differend nan t combinations of methanol - water or acetonitrile - water mixtures as a mobile phase did not improve separation. Variation of pH between 2.5 and 7 was also ineffective worthwhils i t I . mentioo et n that RP-18 colum2 stabl s nH i p t ea highestationart e a th ; 7 -H rp y phas hydrolyseds ei . Salts like NH.Cd lan 4 helt effectinno n alsF pi K d odi separatione gth .
Ion pairing reagents like tetrabutylamoniumbromide (1% in methanol:H O 60:4- 80:20r 0o ) N-cetyl N*,N',N'-tetramethylamoniumbrotnide (0.5% tetrad )an - amoniumhydrogensulphate (1%) valueo alsn f oo . provee b o t d
109 knows Ii t n that modifie fore Si-0-(Cf dth mo n silici H N l ) Hge a 2 n 2 or Si-0-(CH ) CN are good stationary phases for HPLC analysis of amino- 2 n acids. Considering the similarity of ETU and aminoacids, common moiety the amide group, Shedon hypersi H basis(N l Wucleosid )an N (CNSO l ) columns were tried for separating ETU and EU. Both EU and ETU elute at the same time. Changing the composition of the eluting mixture also failed to change their retention volumes r smalFo .l molecular weight, compound chirad san l chemicals, cyclobond (Si-0-Dextrin) have been usetheir fo d r effective havU separationE ed smalan U lET .molecula r6 8 weights d an 2 ,10 respectively. Therefor s thoughwa t i ef interes to teso t t these columnr sfo their separation. This column also showed no effectiveness in changing the retentio. EU d n an volume U ET f so
All the five columns tested are suitable for detection and estimation of ETU and EU separately. 0.1 ppm levels of ETLJ and EU can be detected in soil anO irrespectivH d elutine colume th th r f no eo g solvent.
REFERENCES
(1) Engst, R., Schnaak, W. Residue Rev. 52 (1974) 45.
) Graham(2 , S.L., Davis, K.J., Hanson, W.H., Graham, C.H. Food Cosm. Toxicol. 13 (1975) 493.
) Seifer(3 Ehrlich, ,J. , W.E.J. Pharmacol. Exp. Ther 3 (1948.9 ) 303.
(4) Graham, S.L., Hanson, W.H., Davis, K.S., Perry, C.H. J. Agric. Food Chem. 21 (1973) 324.
) Innés(5 , J.R.M., Ulland, B.M., Valerio, M.G., Petrucelli Fishbein, ,L. , ,L. Hart, E.R., Pallota, A.J., Bates, R.R., Falk, H.L., Cart, J.J., Klein, M., Mitchell, I., Peters, J.J. Nat. Cancer Inst. (U.S.) 42 (1969) 1101.
) Khera(6 , K.S. Teratolog (19737 y ) 243.
(7) Nitz, S., Moza, P., Jutta, K., Freitag, D., Behechti, A., Körte, F. J. Agric. Food Chem. 32 (1984) 600.
(8) Otto, S., Keller, W., Drecher, N.J. Environ. Sei. Health, Part B, B12 3 (1977) 179.
) Onley(9 , S.H. Assoc,. J Yip . .G , Off. Anal. Chem 4 (1971.5 ) 165.
(10) Onley Guiffrida, ,Y. Ives, Watts,L. , , N. Storherr , ,R. Assoc . J . .,R Off. Anal. Chem 0 (1977.6 ) 1105.
(11) Nash, R.G Assoc. .J . Off. Anal. Chem 7 (1974.5 ) 1015.
(12) Nitz Moza, AgrKörte,S. . , J ,P. .. ,F Food Chem 0 (1982.3 ) 593.
110 APPRAISA CO-ORDINATEE TH F LO D RESEARCH PROGRAMME
1. INTRODUCTION
e growinTh g challeng securo et e wholesome foo quantitien i d s sufficient to fee even a d r increasing world populatio s resultenha continuinn i d g search fomeanw rne enhancinf so g agricultural production essentiae On . l inpus ti pesticides. Unfortunately, the use of pesticide chemicals is not without problems. Without continuing surveillance and intelligent control, some of those that persis r foodstuffou n i t s could timest ,a , conceivably endanger public health. To ensure safety-in-use of these many chemicals is a dynamic challenge. One such example is the theme of this international programme on fungicide residue foon i s d plants. Chemical control procedures continuo et hav e advantageth effectivenessf eo , simplicity cosw d immediat,lo tan e availability. Unfortunately resula s ,a pesticidf to e application, substantial residues may remain on food.
Agricultural fungicides are chemicals used on seeds, crops and in soils throughout the growing season. Fungicide treatments may lead to various level chemicaf so l residue foon i s d commodities. Primary emphasi bees sha n placed on ethylenebisdithiocarbamates (EBDCs), an important group of agro- fungicides used in preparations for spraying or dusting major crops such as apples, pears, broccoli, cabbages, egg plants, cauliflower, grapes, lettuce, peppers, celery, cucumbers and tomatoes. Treatments with EBDCs result in terminal residues containing ethylenthiourea (ETU). toxicologicallThia s si y significant decomposition product which has attracted considerable attention in recent indicationo yeart e potentias du it f so l goitrogeni carcinod an c - genic properties produceU ET . s thyroid carcinom d liveaan r tumorn si experimental animals and has also proven to be teratogenic and mutagenic.
There is also ample evidence that higher ETU levels are likely to form in tropica subtropicad lan l regions tha temperatn ni e climates since EBDCe sar known to be unstable in the presence of moisture and ETU yields increase with elevated temperatures. In addition, it has been shown that cooking of commodities of plant origin containing EBDCs can produce ETU to an extent corresponding to 25% of the weight of the original residues. EBDCs and ETU residue data are virtually lacking in most developing countries and problems have been encountered in the development of specific assay methods for ETU determination.
Ill n recognitioI coordinatea neee r th fo d f no d examinatio U levelET n f i nso food, particularly under tropical conditions, this programme was initiated in programme Th 198. 4 e would generate data whic importane har nationao t l food quality programme whicd san assisn hca evaluatinn i t possible th g e impacn to consumere th thin I .s context, radiotracer techniques constitut powerfuea l too studyinn i l g fungicide residue problem foodn so .
primare Th y objective currene th f so t programme were: determino T - radiotracey eb r technique fate magnitudd sth ean residuef eo s of agro-fungicide theid san r product majon i s r crops following fungicidal applications under conditions of local practice. Emphasis was placed on ethylenebisdithiocarbamtes . o evaluatT potentiae eth r appearanclfo fungicidaf eo l residue foon i s d commodities. - To develop and validate appropriate analytical methodologies.
2. OBJECTIVES OF THE MEETING - To review and discuss progress within the coordinated programme; To prepare experimental protocols for use as guideline for relevant research; preparo T finaea l programmee reporth n to .
3. PREVIOUS HIGHLIGHTS Preliminary investigations have provided consistent trend residuen so s and behaviour of EBDCs . Several research groups have reported the presence of ethylenethiourea 0.5%o (ETUt ethylenebisdithiocarbamatn p i )) (u e (EBDC) preparations whic beed hha n imported, formulated and/or manufactured locally. Only minimal decompositio EBDf no C formulation s observeswa d under typical local storage conditions quantite Th . activf yo e ingredient measures ,a y db the CS evolution method, in formulated maneb, zineb or mancozeb which had been stored for 6 to 16 months was reduced by less than 4% relative to fresh formulation. Formulations in stored open packages decomposed appreciably faster tha closen ni d packages.
Zineb residue foliage plantg th eg n so tomatf r es o o o plants were characterized by a half-life (t ) of 14 to 15 days in Central China. Zineb was appreciably less persistent in soil at this locale (t = 4 days). persistence Th mancozef eo brinjan bo l plants (egg plant Indin )i vers awa y i5 similasi r (t1/= 2 days) whereas mane less bwa s persisten thin to s plant (tl/2
112 In China, residues, at harvest, of ETU on brinjal and tomato fruits from plants which have received up to 3 successive sprays of ETU-free EBDC were low detectablt bu e ranging from 2-20 ppb. Residue EBDf so harvesten Co d fruit were higher and variable (1-16 ppm).
At harvest, the major EBDC residues were confined to the foliar portions and fruits of brinjal and tomato plants. The roots contained only negligible activity. Activit soile th ,n i y surroundin g treated plants s als,wa o negligible at harvest.
Cookin bakinr go contaminatef o g d brinjal frui cabbagr to e leaves resulted in appreciable conversion of EBDC residues to ETU. The conversion appeared to be approximately 20% to 37% based on the quantity of EBDC present on the fresh produce at harvest.
4. HIGHLIGHTS OF THE THIRD RESEARCH CO-ORDINATION MEETING
1 4. Analysi commerciaf so l formulations The most extensively used organic fungicides worldwide are the ethylenebisdithiocarbamates (EBDCs). In many instances, these materials are the only successful counteragent for controlling fungal diseases of major food crops mose Th .t important member thif so s group are: - Zineb (Zn salt of nabam), a light colured powder introduced in 1943, almost insolubl waten ei ppm0 (1 rused )an d specifically against downy mildew. - Maneb (manganese salt of nabam), introduced in 1950, a yellow crystalline solid, slightly solubl waten ei d insolublran mosn i e t organic solvents, effective against foliage diseases, particularl blighte yth tomatf so o and potato. Mancozeb, a complex of Zn ion and maneb, containing 20% manganese and 2.5% zinc, a greyish powder introduced in 1961 for use against potato blight. Concern oveEBDCe safete bees th th r sha f nyo expresse resula s a drenewef to d awareness that commercial fomulations contain significant amountf so ethylenethiourea (ETU bees ha ) n U (1)showET . caus o t n e thyroid hyperplasia (2) and at high doses thyroid carcinoma (3).
formes i byproduca U s a dET t durin manufacture th g EBDCy f eb o r so decomposition of the parent chemical during storage or shipment and thus may influenc efficace eth increasr yo hazare eth d potentia pesticidee th f lo r Fo .
113 Tabl - Quantities I e f activ*o e ingredien ETd an Utn commercia i l formulation f EBDCo s s
Period Concentrations EBDC Country Manufacturer of storage Sealed package Open package (months) Initial Final Initial Final
Maneb Ecuador Delemarck Equatoriana 7 a.i%. 66.3 67.8 66.3 61.3 W.P. 80% ETU (ppni) 2.6 3.4 2.6 4.2
Maneb Turkey Bayer 24 a.i%. 76.5 68.9 79.5 47.2 W.P. 80%
Maneb Malaysia Choon Huat Sdn. Bhd . 6 a.i%. 45.9 33.4 45.9 43.0 W.P. 80% ETU (ppm) 32.0 51.0 32.0 52.0
Maneb Brazil Du Pont 17 a.i% . 68.0 60.0 W.P% 83 . ETU (ppm) 2.0 0.01
Zineb China Shenyang Pesticide Plant 18 a.i%. 78.5 74.4 80.6 76.6 W.P. 80% ETU (ppm) 2850 7500 2240 6400
Zineb China Tianjin Renmin 1 1 a.i. % 69.1 62.5 69.7 54.8 W.P. 70% Pesticide Plant ETU (ppm) 34600 43000 28700 40000
Zineb China Shenyang Pesticide Plant 18 a.i% . 89.5 79.8 90.0 76.1 (technical) ETU (ppm) 5550 19200 4670 13600
Mancozeb China Laboratory Formulated 18 a.i% . 78.9 55.2 76.6 30.0 W.P% 80 . Shenyang Research Inst. ETU (ppm) 21700 26300 18100 16200 of Chemical Engineering
Mancozeb India Indofil Chemicals 13 a.i%. 69.5 65.9 69.7 52.0 W.P. 75% Bombay ETU (ppm) 239 449 220 413
Mancozeb Malaysia Ponu r D EasFa tt 6 ETU (ppm) 50 180 50 160 Manzate 200
*For methodologies, the original papers should be consulted. these reasons, pesticide formulations are routinely analysed for concentrations of the active ingredient and presence of acceptable levels of impurities. Analysi sample5 3 f so manef so b produce t varioua d s location showeA US n di s average mg/kth 3 81 contenU ge ET b wito t rangha 249-140f eo 8 mg/kg (4). Samples less tha yearn2 frod sol m various formulations containing 72-80% EBDC showe U levelET d 0.0f so 2.02o 2t % (1) U leve.ET l could reac dependin» h57 n o g d agformulationean e rat Th EBDf .eo C degradatio s influencei n y temperaturb d e and more importantl moistury yb e (5). Under controlled experimental conditions days)9 3 lever U ,fo ET founs lwa H R d % 14.5% 80 49° t basid w (a ,Can w/ s (5).
In current studies, three EBDCs, maneb, zineb and mancozeb from different manufacturer differenn i s t countries were analyse storagn i d e (Table I). In general, the concentration of EBDCs declined at slower rates in sealed packages than in open packages. A mancozeb preparation made in a laboratory in China declined to 40% of the initial concentration over a period of 18 months termn I . stabilityf so mos,e maneth t s stablbwa mancozed ean e bth least.
ETU was detected in all samples examined. Other products, such as EU and some unidentified compounds were also found. No profound differences U conteniET n t were observe oper d sealefo dnan d packages. Wit exceptioe th h n of formulation Chinan i s U concentration,ET a s a d an s m variepp 9 d44 o frot 3 m rule they tend to increase during storage. The exceptionally high ETU quantitie Chinese th n i se formulations probably relatprocese th o et f s o manufacturing. In addition; the analyses could have included EU which does not separate from ETU in the HPLC procedure. It is, therefore, necessary to C systemTL e sus whic separatn hca froU suc, eET ethys mEU ha l acetat: e ammonia : water (15:1:1, v/v) (6).
The data obtained show that EBDCs stored in sealed packages in the dark remain reasonabl years2 1/ y1 . stabl- 1 periodr o efo t p su
REFERENCES
(1) Bontoyan, W.R., Looker, J.B., Kaiser, T.E., Giang, P. and Olive, B.M. J. Assoc. Off. Anal. Chem 5 (1972.5 ) 923.
) Graham(2 , S.L Hansend .an , S.H. Bull. Environ. Contain. Toxicol. 7_ (1972) 19.
) Graham(3 , S.L., Hansen, W.H., Davis, K.J Perryd .an , C.H Agr. J . . Food Chem (19731 .2 ) 324.
115 (4) Keoler, D.R. Unpublished report, DuPont de Nemours & Co. Inc., Debware, USA (1977).
(5) Bontoyan, W.R. and Looker, J.B. J. Agr. Food Chem. 21 (1973) 338.
(6) Czegledi, G. J. Chromatography 31 (1967) 89.
TablSom- I eeI selected dat residue n fooo an i dU plantET f EBDCso d san s
Country Food Fungicide Application Weeks Resdidues rate (mg/plant) after (vg/g) last EBDC ETU app] ication equiv.
Brazil Beans l^C-mancozeb 50 2 2.5 0.15 (5x 12-day inter- vals)
India Egg l^c-mancozeb 45 4 10.12 0.12 plants (ETU-free) (3x, weekly)
Malaysia Tomato -^C-mancozeb 600 4 ___ 16.8 (3x, weekly
China Tomato l^C-mancozeb 100 3 5.22 0.08 (ETU-free) (once) Egg 14C-zineb 100 3 160.4 (skin) plants (3x, weekly) 37.1 (pulp)
Ecuador Tomato ^ C-maneb 60.72 8 7.8 0.28 (ETU-free) (4x, weekly) 11 3.2 0.038 12 3.5 0.058
Egypt Bean -^C-maneb 4.01 4 (green) 0.056 0.007. (Vicia (ETU-free) (3x, weekly) 9 (dry) 0.057 0.010 faba) Soya 14C-maneb 2.15 8 0.041 0.009 bean (ETU-free) (3x, weekly) 11 0.037 0.007
Panama Large l^C-maneb 3.08 1 0.17 0.007 pepper (3x, weekly) 2 0.12 0.015 3 0.06 0.009
Turkey Tomato 14C-maneb 5 3 0.12 0.01 (3x, weekly)
Brazi1 Tomato 14C-maneb 100 2 ' 3.0 0.03 (ETU-free) (4x, weekly) 10 0.6 N.D.
116 4.2 Residue data in food and soil
4.2.1 Residue foodn si majoA . r objectiv currene th f eo t studieo t s swa investigate the residues of EBDCs in food under conditions of local agricultural practices with emphasi highle th n yso toxic degradation product ETU. Studies were also conducted in soil to evaluate the persistence of BBDCs and ETU.
14 Investigations in nine countries used C-labelled fungicides (maneb, zineb and mancozeb) on a variety of commodities (Table II). In most studies, EBDCs were freed from ETU and therefore data presented reflected only ETU quantities formed biologically and/o decompositioy rb parene th f tno chemical frotime mapplicatioth f e o n till analysis, i.e concentrationU .ET s represen minimue tth m value casesw s fe likel founde a b , o n terminayt I . U lET residues included ETU derived from the original formulation. Table I shows that concentration terminaf so l residues correlate with application rates. EBDC residues have exceeded the temporary maximum residue limits in egg plants (India and China) and in tomato (Ecuador). The ETU guideline value has been exceeded in tomato (Malaysia, China and Ecuador).
4.2.2 Behaviou soiln ri soin I .l column experiments majoe ,th r portion of chemicals remained in the top 5-10 cm. Table III shows a summary of residues remaining after various incubation period EBDCf so ETUd san . Undera
ResidueTabl- soin i l lU eTi ET EBDCf so d san
Period of Amount % remaining Country l^C-chemical incubation applied 14C-activity (mg/column)
India 14C-ETU day2 5 s 3.27 6.6
Brazil l^C-maneb 35 weeks 74 22 14C-ETU 35 weeks 100 1.9
Philippines l^C-mancozeb 21 days 3.93 0 0.2 ETU 9.4 EU
China 14C-zineb 31 days 7.2 24.3 14C-ETU 90 days 0.22 21.5
117 variet f climatiyo d soian cl conditions, EBDC persistence showed different patterns, e.g. complete disappearance of mancozeb after 3 weeks while 24% of zineb remaine manef o d % afteb 22 afteday1 d r3 weeks5 an s3 r .
ETU also showed different behaviour patterns in different soils and residues range % afted22 ro frot incubatiom2 24o nt 5 2 timedays5 n f I so . the laboratory, it was demonstrated that 27-42% of ETU was mineralized over a period of 5 weeks and binding to soil progressed to 91% of the terminal residue afte weeks3 1 r .
3 4. Effect foof so d processin fungicidn o g e residues Several research groups have reported the presence of ethylenethio- urea (ETU) as one of the degradation products of EBDCs in different parts of plants treated with these fungicides. Cooking either during processing or at home can result in the conversion of EBDC to ETU (1, 2). The effect of cooking may be monitored according to the procedure of Watts et a_l. which used GLC techniques (3). Several studies were conducted to examine changes in the magnitude and nature of the residues present in food after processing (e.g. baking, cooking). Emphasi parene places th swa n to d fungicid ETUd an e .
Table IV - Effect of food processing on EBDC and ETU residues
Type of Percentage Percentage Country Food Fungicide processing decrease in increase in EBDC ETU
China Tomato Mancozeb Washing 50 Tomato Mancozeb Cooking 70-80 390 (skin)** 2390 (pulp)**
Turkey Tomato Maneb Cooking 28 40
Brazil Tomato Maneb Washing-cooking 65 70
Ecuador Tomato Maneb Cooking -- 31-50
Brazil Beans Mancozeb Baking 10 65
India Egg plants Mancozeb Baking 47-60 Cooking 49-66
For application rates, initial residues, and methodologies original papers should e referreb . to d ** Initial residues extremely high.
118 14 In current tests, fruits were sprayed with the C-fungicide and residues were examined after processing (Table IV). Washin removn gca s ea much as 50% of the initial residue (China). Cooking fruits with water resulted in a substantial decrease of EBDC and a significant increase in ETU. tomatoesr Fo , this increas eboilinn o range % fruite 70 th gd o t fros 1 witm3 h water after 7-15 days following treatment with the fungicide (Brazil, Ecuador and Turkey). Data from selected studies (Tabl clearl) eIV y indicate degradation of EBDCs and formation of ETU by some processing procedures. This would add to the residue burden of ETU. These results are in agreement with those reported by other authors. A linear increase was reported for ETU content for carrots at a rate of 0.01 mg/kg-food/minute during cooking for up to one hour after addition of 50 mg zineb/kg-food (4).
Like cooking, blanching prio freezino rt cannind gan g usually decrease e amounsth EBDCf to thermay sb l degradation, which increasee sth amount of ETU present. Increased levels of ETU were observed in processed products from grapes (5). Washed grapes, containing 1.3-2. EBDC/kg-foog 7m d bu o detectabltn residueU eET s were processed residueU ET . 0.15-0.1f so 9 mg/1 wer egrape founth en i djuic e after processing.
REFERENCES
) Newsome(1 , W.H Agr. J . . Food Chem 4 (1976.2 ) 999.
(2) Ripley, B.D. and Simpson, C.M. Pestic. Sei. 8 (1977) 487.
) Watts(3 , R.R., Stroberrr, R.W Onleyd .an , J.H. Bull. Environ. Cont. Toxicol (19742 2 .1 ) 224.
) Newsorae(4 , W.H Laverd .an , G.W. Bull. Environ. Contam. Toxicol (1973) .1C ) 151-154.
) Gordon(5 , C.F. Human exposur EBDCo et theid san r residues. Rah Haam& s Co. RPAR, 23 Dec. 1977, Vol. I, Section 3, 65 pp.
5. STATE-OF-THE-AR ANALYTICAF TO L METHODOLOGIEU ET R SFO This part of the report seeks to briefly appraise the methods that have been publishe determinatioe th r fo d ethylenethiouref no a (ETU foodstuffn )i s and other substrates suc sois hwatera d lan . Analytical methods, using thin- layer chromatography (TLC), gas chromatography (GLC) and high-performance liquid chromatography (HPLC) are given particular consideration.
119 Table V - Structures of various EBDC pesticides and their alternative names
Common name Other name(s) Chemical names Chemical structure Molecular formula (M.W.) .
1. None Amobam -d1 ammonium ethylenebisdithlocar hamate CHjNHCSSAH,, C^H14N„S^(246.21 ) -carbamod1th1o1c acld.l ,2-ethaned1ylb1s- CHjNHCSSAH,, diammonlum salt
2. Mancozeb Dlthane H-45. -coordination product of maneb contalnina Manzate 200, 16 to 2ÛÏ Mn and 2.0 to 2.5% Zn Carmazlne, Farmanz, Fore, -[[1 ,2-ethaned1ylb1s[carbamod1th1oato]i s ] s r Moromate (2-)] manganese mixture with [[1 ,2- n »! ethanediyibis[carbamod1th1oato]](2-)] |SCNHCH2CH2NHCSM j n (Zn) manganese y x
3. Maneb Manzate, Man- -manqanous ethylene-1 ,2-b1 sd1 thlocarbam- a) Monomer C H N S Mn(265.3) zate D, Dlthane ate 1| 6 2 1( M-22, HEB, HN-CH CH2— 2— NH MnEBO -[ethyleneb1s(d1thiocarbamato)]manqanese J S 1 -[[ 2-ethanediylbis[carbamod1, oato]i h t i ] \ S C- \ " (2-)]manganese Mn / \ S'" V
) Polymer(sb ) (C„H6HnN2SJx
r-SCNHCHjCH2NHC-S-Mnl
[s S Jx
4. Metlram Polyram, Poly- -mixture of [ethyl enebi s (dl thiocarbamato) ] [-CH2NH-CS-S-Zn-S-CS-NH-CH2]x ram 80-W, Poly- zinc ammonlates with ethyl enebi s[d1th1o- Combom ra , NIA- carbamlc add] anhydrosul fides [-CH2NH-CS-S-S-CS -NH-CH2] 9102 * where x = 5.2 y
5. Nabam Parzate, Dlthane -disodlum ethyl enebi sdithlocarbamate S D-14, OSE, D-14, | + Dlthane A-10 -disodiu ,2-ethaned1ym1 l bis[carbamodi thlo- CH2NHCSNa C^H6N^Na^S^(256) ate] |
CH2NHCSNa 1 S
6. Z1neb ethyl zlmate, - [et hylenebl s (d1 thiocarbamato) -zinc ) a Monomer C^HjNjS,, ,Zn(275.8) Dlthane Z-78. Lomacol , Par- - zinc ethylene-1 ,2-bisd1 thlocarbamato HN — CH2 — CH2 — NH zate ,2-ethaned1ylbis[carbamod1 [[ - i thioato]] 1 »v S 1 (2-)]z1nc \ */sv C ^ sZ n \ / C s s
) b Polymer(s) (CuHtN,SuZ. n). r s s i f.
SCNHCH2CH2NHCSZnJ Thin-layer chromatograph bees yha n successfully usedeterminatior fo d f no ETÜ residues especially with radiotracers rather than colour visualization reagents. Prior to TLC analysis, the extracts of the sample requires considerable clean-up and decomposition of ETU on the TLC plate may occur if it remains for a long time on the plate. A major advantage, however, is the efficien witU tE hseparatiod an specia U ET f lno solvent systems.
Some workers have used GLC for direct analysis of ETU. Some difficulties have been experienced using this method because of the polarity of ETU and the possibilit breakdowf yo EBDCf no co-extracted san d intermediate products. More extensively, derivatize residueU ET d s have been determined using packed columns in GLC. However, these derivatization techniques have the disadvantage of prolonged analysis time and do not give reproducible quantitative recoveries with some substrates. In recent years, capillary column GLC has been used with success in some cases for ETU and its derivatives.
HPL moss Ci t usefu determininr lfo U residueET g n i EBDCn i sd san foodstuffs without derivatization. This method is less sensitive than GLC analysis. It is also observed that the most commonly used HPLC columns fail to separate ETU from EU.
Against this background, the TLC emerges as the technique of choice, particularl. EU r d separatioyfo an U ET f no
6. CHEMISTR TOXICOLOGD YAN U ET EBDCF Y O D sAN
6.1 EBDCs. The structures of the various EBDCs and their alternative names (1) are shown in Table V.
Degradatio2 6. metabolisd nan ETUf mo . Ethylenethiourea (2-imidazoli- dinethione whita s )i e crystalline substance, m.p. stabls i 204° d eCan under normal conditions solubls i (1) U .waten ET i e r (20,00 30°Ct a d m )an 0pp slightly solubl ethanon ei ethyd lan l acetate. EBDCs degrad presence th n ei e of moistur d oxygee an biologican i wels n a s la l systems int numbeoa f ro products includin finaa t lno g products relativela i ETU s U i .ET t ,bu y stable intermediate in the ultimate degradation of EBDCs. It is easily oxidized to ethyleneurea (EU), primarily in biological systems (1).
121 ^NHc 0 C- ^WH H N-C-NH Ethylene urea '(""H 2 2 7 2-imidazoline Urea {2'- Imidazolidine) ^XfclL. CHj-NHg
CH2-NH2 Hydantoin Ethylene diamine
NH •c" Unknown II metabolites Jaffe's base CH£COOH
NH2 Natural substances Glycine C02 (Protein,Fat) Oxalic acid
= photodecompositioa n = chemicab l oxidation c = plants d = animals soi= e l Letters in parentheses () indicate proposed pathways.
FIG. 1. Reaction products of ETU in biological and non-biological systems (1).
ÜV-irradiation of ETU converts ETU mainly into EU and photo-oxidation might be expected to be the major degradation reaction of ETU occurring as surface deposit EBDC-treaten o s d plant water so r (1).
In biologically active soils, ETU is oxidized to CO , hydantoin and Jaffe's base and two unknown products (Fig.l). In plants and animals, a variet productf o y formee ar s d (Fig.l)environmente th n I burdeU . ET e n,th and the dynamic behaviour is determined by: ETU amount formed before EBDC's application U formatio,ET n sequenca rat s ea EBDC'f eo s degradatioU ET d an n degradation rate.
Toxicolog3 6. ETUf o y . Ethylenethiourea (ETU) ,toxicologicalla y significant decomposition product, is formed during the chemical or biological degradation of the EBDCs, an important group of agrofungicides used on seeds and crops throughou growine tth g season. Major crops spraye duster o d d with preparations containing EBDCs include apple, grapes, broccoli, sprouts, cabbages, cauliflower, egg plants, lettuce, tomatoes, cucumber, peppers, and green onions.
122 In recent years, the goitrogenic, teratogenic and carcinogenic properties of this chemical have attracted considerable attentiont no s i . , se ETU r ,pe particularly toxic in a lethal sense when exposure is short term. The acute toxicity of single doses of ETU to mature, non-pregnant rats, mice and hamsters appear slighe b o moderatst o t ETU's ei (2)t sI .effect a s sa carcinoge teratoged nan nf concern o tha e tar .
ETU is only slightly mutagenic, but oncogenic effects (primarily thyroid carcinomas) have been observed when rats were fed 60 mg ETU/kg-diet for two years (2). As little as 5 mg ETU/kg-diet can cause a significant degree of hypothyroidism, leading probably to hyperplasia (2). ETU has repeatedly been reported to induce severe teratogenesis in rats, affecting mainly the nervous system (2). Existing studies sho cleao wn r relationship between thyroid dysfunction and the teratogenic action of ETU.
6.4 Acceptable daily intake (API) and maximum residue limit (MRL) API: In 1988, the Joint FAO/WHO meeting on pesticide residues in food (3) could not fully evaluate the temporary ADI because the available data base was incomplete and therefore the temporary ADI was extended; (0.05 mg/kg bw. [EBDC] of which no more than 0.002 mg/kg bw. may be present as ETU)
MRL: Temporary MRLs for EBDCs have been recommended (4) at 0.2 ppm on wheat up to 3 ppm on peaches and apples. Only guidelines have been set for ETU (4) :potaton i 0.0m 1pp , cord lettucenan tomatoen ;i 0.0m d 5pp san 0.1 ppm in beans.
REFERENCES
(1) Ethylenethiourea. Pure and Applied Chemistry 4j9 (19/7) 675-689.
(2) ETU criteria for the assessment of its effects on man. National Research Council of Canada, NRCC No. 18469 (1980).
) Pesticid(3 e Residue Foon i s Evaluation- d s 1988PlanO FA .t Productiod nan Protection Paper, 93/2, FAO, Rome, Italy.
(4) Pesticide Residues in Food - Evaluations 1980. FAO Plant Production and Protection Paper FAO, ,26 , Rome, Italy.
7. FUTURE LINES OF RESEARCH It should be pointed out that some of the programme participants are extending their researc topie th cn i harea . Studie mancozen so b residuen si various commodities will continue in India, Malaysia, Panama and Turkey.
123 Maneb residue tomatn i s o wil furthee lb r examine Ecuadorn i d . Participants from Brazil, Egypt Philippinee , th Indi d an a s will emphasize investigationn so the fate and binding of ETU in soil and its uptake by plants.
8. CONCLUSIONS notes i 8.1t dI .wit h satisfaction thaprogramme s th it mostt f me to s eha objective provides ha d san d evidenc unique th f eo role that nuclear techniques pla studyinn i y g fungicide residue foon i s d commoditied san processed food, and behaviour in soil including mineralization and binding.
8.2 The programme has generated important worldwide residue data in food and processed products which wer t availableno e before initiatioe th f no programme. These also make a significant contribution to national food quality programmes.
3 Severa8. l local practice treatmenf so residuto t hav d le ee levelf so ethylenebisdithiocarbamates (EBDCs) and ethylenethiourea (ETU) which are withi Temporare nth y Maximum Residue Limits (TMRLs Guideliner )o s (GL) recommended by the FAO/WHO Joint Programme on Pesticide Residues. There some ar e exceptions exceedewhere b TMRL e n eth ca s d s and/oGL e rth (China, India and Malaysia).
4 Commercia8. l formulation EBDCf so s Data indicate that ETU is present in commercial preparations in varying quantitie) dependin 4% suppliere o t th p n o gquantite (u s Th . y increases storagn o e particularl open i y n packages under warhumid man d conditions. e stabilitTh formulatione th f yo sordee werth rn ei mane zineb> mancob> - zeb.
5 Residue8. foon si d plants Studies have confirmed the frequent presence of EBDC residues in/on raw agricultural commodities, treated according to local practices. ETU is formed as a primary reaction product of EBDCs and environmental degrada- tion of the EBDC fungicides also leads to ETU formation. ETU is detected as a metabolite in food plants. Other metabolites (EU, ETM, ETD) were als oplantsg founeg n i d, soybean bead san n plants.
124 6 Foo8. d processing The local custom of washing commodities prior to processing can remove substantial quantities of residues and is an invaluable practice in reducin finae gth l residu poine th consumptionf t o ea e losseTh . f so EBDC residues observed in frying or cooking are very variable. ETU increases during cookin confird consistene an gar 400%o t )m p previou(u t s observations that cooking invariably leads to increased ETU residues.
8.7 Residues in soil Most of EBDC and ETU residues do not leach beyond the top 10 cm of soil. ETU mineralize non-steriln si weeks5 n i bindd )% an s42 eo t soil p (u s strongly (with other degradation product EBDCsf so soio )t l matrices.
8.8 Analytical methods radiotracee Th r techniqu bees eha n very usefu detectinn li g residue levels as low as 0.0002 ppm. For quality control purposes, the TLC metho particularls i d y usefu separatinn li HPLe s th Ci d froU an gET U m E highly efficien quantificatior tfo ETUf no .
8.9 The Coordinated Research Programme The close contact established between the FAO/TAEA Secretariat and research scientists in various countries as a result of participation in Coordinatee th d Research Programm represenn eca significanta t contribu- tion to a country's development. It aims to assist scientists to identify and solve their own problems under local conditions, to use nuclear and related technologies effectively and to maintain the closest possible contact with their counterpart more th e f sadvanceo d countries. This sha been demonstrated to be beneficial to all concerned.
9. RECOMMENDATIONS
Due to the wide use of mancozeb, maneb and zineb, work should continue to investigate their residues in food plants. - Shoul becomt i d e necessar storo yt e EBDC formulation lonr sfo g periodf so time, these should preferably be kept in sealed packages and in the dark. Analytical procedures used for determination of ETU in formulations or in food should includ systeC TL ea m capabl separatinf eo froU gE m ETU.
125 Since washing fruits with wate n removrca substantiaea l portiof o n residues, this practice should be included as a part of processing procedures whenever possible. Participating institutes should continu o investigatet e local chemical residue problem foon soild si an d , using nuclea associated ran d techniques.
126 MODEL PROTOCOLS FOR THE DETERMINATION OF ETHYLENEBIS-DITHIOCARBAMATE RESIDUES
A comprehensive assessment of fungicide residues and their significance i'.un best be made with data obtained with techniques that have been standardized to a sufficient degree to allow for valid comparisons. Data on residues are most useful when they can be compared with similar information from other laboratories or when they are obtained from other commodities treated unde same differenth r eo t conditions e analyticaTh . l procedures should hav e capabiliteth f identifyino y d quantifiyinan g parene gth t material as wel breakdows a l n and/or reaction products e samth e t timA e . eth procedures shoulsimpls a d straighe db ean t forwar possibls a d thao e s tth e.ssential informatio obtainee b n nca d under conditions that may ,timest a e ,b less than ideal.
e firsth t A Research Coordination meetin 198n i g 5 membere th f so committee developed model protocol e det«rminatioth r fo s EBDf o n C residues using radiotracer techniques broae Th .d objective programme th f so e wero et develop expertis d capabilitean acquirinr fo y g residue data that woule b d uefu participatino lt g countrie thein i s n foo processind row an d g systems. e procedureTh s given here have been teste d adapteparticipante an dth y b d d san much useful data have been obtained on residues of RBDC's on different commodities under different environmental and local conditions.
e currenTh t report describe modee sth l protocols IIId (Annexean )I I , sI as they were set up for détermination of residues in commodities and soil, using radiotracer and conventional Chromatographie techniques.
127 Annex I
TREATMENT OF FOOD PLANTS WITH l^C-EBDC FUNGICIDES AND ANALYSI RESIDUEF SO S
1. Introduction
This metho proposes i dResearce th y db h Co-ordination Coromiten eo Radiotracer Studie Fungicidf so e Residue Foon i s d e testinPlantth r f o gfo s EBDC fungicides applied to food plants in conditions simulating actual practice. It draws on the experience of many of the participants in the co-ordinated research programm havo ewh e overcom probleme eth e sb than tca imposed by such non-ideal conditions.
The objective of this protocol is to provide guidelines under which a fungicid e testeb n deca whilst adoptin practicee th g s prevailiny an n i g particular country, such that meaningful comparative residue b datn ca a produced. It is recommended that the protocol as described be used and amended onl necessars ya suio yt t requirement f locaso l practice. Modifications might include the method of application of fungicide, conditions of treatmen methoe th samplingf d o d tan A complet. e descriptio sucl al h f no modifications should be included when the data are evaluated.
It must be emphasized that the measurement of radioactivity cannot necessarily be equated with the presence of the parent compound due to degradation processes occurring withi plane nthereford th tan e further chemical analysis is required to establish the nature of the residue itself.
2. Treatmen tesf to t material
1 Typ2. labellinf eo s
14 widele th r y Fo used maneb, zine mancozebd ban , C-labelling shoule db in the ethylene carbons.
2 Preparatio2. labellee th f no d formulation
14 Transfer quantitatively the C-chemical into a vessel containing a known quantity of the cold formulated fungicide, using approx. 10 ml dry
128 freshly distilled ethylacetate. A concentration of 1 mCi/500 mg may be taken as a guideline. Stir cautiously for 10 min. and remove the ethylacetate layer by careful aspiration. Repeat washing twice, dry the residue under vacuum and store in a disiccator at 4 C. This material is ETU-free and may be directly preparatioe th user fo d aqueouf no s suspensions o stud,t y formation ratef so KTU.
Alternatively practicar fo d ,an l purposes e initially-presen,th y ma U tET removee b t permio no t d inclusios it te termina th n i n l residue thin I .s case, 14 sprayine th g solution wil preparee lb dilutiny b d e th C-EBDg f o C g wit 0 h1 cold EBDC formulation, essentially eliminatin ethye th g l acetate extraction. dilutee th n i de conten U formulationTh ET f to determinee b y ,ma HPLCy db C ,G or TLC (Annex III). The specific activity of ETU must be also determined.
3 Selectio2. Foof no d Plant Fungicided san s
To be made according to national priorities and economic importance as ethylenebisdithiocarbamatf o e relevanus e th o t e chemicals. Experimenty sma e conducteb greenhousa n i d e and/or experimental field plot, usin plant0 2 g s for each experiment.
2.4 Method of Application
Amounts, concentrations, and frequency of application should be as close as possibl thoso et e use locan i d l agricultural practice e all-glasUs . s sprayer similar to that used for spraying chromatograms, observe safety precautions (fume hoods, gloves, etc. d avoi)an d cross contaminationn Te . mirocuries per plant per application would be adequate.
Condition treatmenf so t (temperature, humidity, etc.) shoulcloss a e eb d as possible to those used in practice.
3. Sampling
(a) Plant
Sampling times should correspond to zero hour (within the first hour after the first application), to time immediately before second application antimo t d e immediately before third application, etc n additio.I n (anf di
129 applicable) 3 sampling,2- s shoul made b ddifferent a e t intervals aftee th r last treatment according to different ripening stages of the fruit, e.g. tomato.
Three plant r eacsfo h sampling time shoul e collecteb d d storean d t a d -20 C if analysis cannot be made immediately. The edible parts must be analysed separately. It is also desirable to separately analyse the remaining parts of the plant (leaves, stem, root).
) Soil-wate(b r
Samples should be collected, only from pot experiments, whenever plant sampling is being made. Soil samples should be thoroughly mixed before storage and analysis.
. 4 Analysi Chemicaf so l Residue Plann i s t Samples
14 ) Determin(a totae eth l C-activity e sampldr parth a a f y n eto b i y (Packard sample oxidizer combustiot we r )o n procedure (1). Determinations shoul made b dt leasea duplicaten i t . sample th parA extractes f i eto ) (b d (accordin b (2> OnleYi o t g)d yan to remove organic degradation products including ETU. Use 20 ml solvent per one gram sample. Remove supernatant carefully after centrifugation and reduce the extract volume under vacuum. This concentrated extrac usee r b fo dy tma 14 quantificatio( f no of C-organic products and for identification by TLC of individual products.
) Extrac(c remainine th t g residue twic eEDTM wit 4 A h0. (eac ml/g)0 h2 . After centrifugation, remove supernatant (combined extracts) and determine total radiocarbon by direct LSC counting or by combustion and counting if therstrona s i e g quenching effect EDTe Th A. extract would contai parene nth t fungicide (and possibly some conjugates).
) Determin(d radiocarboe eth n remaining afte previoue rth s extractions.
(e) Determine the amount of the parent fungicide in the original sample
by CS2-metho Keppef do l (3).
130 (f) For characterization/identification of degradation products, samples should be subjected to clean-up procedures described by Onley and Yip (2) and cleanee th d extracts analyse C (4)TL .y b d
In the absence of a radioscanner, 1 cm zones of the thin-layer chromatograms may be scrapped and counted and Rf values compared with those of 14 authentics including C-ETU reference. To provide positive confirmation of metabolites, particularly ETU, GLC analysis is recommended (Annex III).
5. Effect of Cooking and Processing on Fungicide Residues
Changes in the magnitude and nature of fungicide residues present in food should be examined, if appropriate, after cooking, roasting, frying and other processes. Emphasis should be placed on the parent fungicide and ETU. Effect of cooking may be monitored according to the procedure of Walts et al. (5), using GC techniques. Alternatively, the food substrate may be spiked with the 14 C-fungicide and processed as in practice. When spiking, a reasonable degre homogenitf o e y shoul ensurede b d . Studies involving frying procedures will requir elaboratn ea e pla includo nt e solvent-partitioning techniques.
6. Problems encountered in residue evaluation
e developmenTh assaf o t y methods specifiU determinatioET r fo c s nha claimed specifit d variouan y s detection limits. These have been questioned due to background interference (6). Detection limits are furthermore affected by possible EBDC conversio durinU ET o gnt work-up (6) e field-weathere.Th d residues of mancozeb appeared more stable toward conversion than did fortified samples during residue analysis, possibly because mancozeb and/or its metabolites had become bound. It can be seen that the extraction efficiency of the detection methods poses a problem because of the potential for bound residues to be toxicologically significant.
In the light of these limitations, the use of 14C-labelled EBDCs and ETU seemed highly desirable, since it enables tracing and sheet-balancing of the quantity applied, quantification of transformation products, and determinatio terminaf no l residue harvest sa d througtan h processing.
131 REFERENCES
(1) Smith, G.N. et al., J. Agr. Food Chem. 12 (1964) 172
) Onley(2 , S.HAssoc. J Yipd ., an . G. ,Off . Anal. Chem 4 (1971.5 5 )16
) Keppel(3 , G.E. Assoc. ,J . Off. Anal. Chem 2 (1969.5 2 )16
) Czegledi-Janko(4 Chromatograph. J , ,G. 1 (19673 y 9 )8
) Walts(5 , R.R,, Storberr, R.W Onleyd an . , J.H., Bull. Environ. Contam. Tox. 12_ (1974) 224
) Pease(6 , H.L Holtd .an , R.F. Agr. ,J . Food Chem 5 (1977.2 ) 561.
132 AnneI xI
TREATMENT OF SOIL WITH 14C_EBDC FUNGICIDES AND ANALYSI RESIDUEF SO S
1. Introduction normae Th l spraying procedure fungicider sfo s usually resule th n ti deposition of only a part of the chemical on the plant with a substantial quantity being released intsurroundine oth g environment, mainly soile Th . fungicide and/o metabolites it r plantstakee y b b y p nu sma , leac binr ho o t d soile th . Therefore becomet ,i s necessar studo yt movemene yth e th f to chemicals in soil and their fate to determine the potential for environmental contamination.
2. Soil treatment and sampling
2.1 Preparation of the labelled formulation - (Annex I)
2.2 Application to soil Drive into the soil Polyvinylchloride (PVC) cylinders of uniform length into the ground leaving about 1-3 cm above the soil surface. Leave undisturbed for 2-4 weeks prior to application of fungicide. Apply fungicide formulation to soil by pipet (about 10 yCi/column). Shake the suspension well before each application. Care mus takee tb o nt ensure adequate homogenity of the EBDC suspension. An emulsifier may be added with sonicatio improvo nt e suspensibility.
2.3 Sampling pipeC freezd PV horizontasa an e n th ei Pul t lou l positio least a r tnfo 1 hr. Cut and weigh each section. Analyse immediately or freeze until analysis. This would determin extene eth downwarf to d migration into soil.
3. Analysis Total radiocarbo determinee b y nma d usin biologicaga l oxidizet we a r ro combustion procedure followed by liquid scintillation counting (Annex I).
parene Th t fungicid determinee b y CSe ema th ? evolutioy b d n method. Fo rothed analysian rU metaboliteET f so sextractee b soi y lma d with various organi c. Clean-u3) solvent concentratee , 2 th , f po (1 s d necessare b extract t no somr y yfo e sma soil samples. Extracte b y sma used directl TLCr yfo .
133 14C-ETU and other degradation products may be chromatographed on silica gel plates and developed in the following solvent systems:
a. EtOAc : NH^OH : H20 (90:6:6) b. Chlorofor n-butanom- methanol- O (100:5:1:0.5H - l ) )(4 c. Ethano chloroforl- benzenm- e (1:5:10) )(2 d. Ether - methanol (9:1) e. Méthylène chloride - acetone (3:1) (5)
Care should be exercised in identification and quantification of ETU residues since ethyleneurea (EU) possesses similar R,. values in many solvent systems. Authentic material mus alwaye tb alongsiden sru . Spots may be made visible by exposure to iodine vapor, nitroprusside- ferricyanid usiny eb reagenr go AgNO) (6 t „ impregnated silicl ge a plates. Zone spotsr (o s ) correspondin o t gC-ET d othean U r 14 14 C-products may be scraped and counted in a liquid scintillation counter.
4. Soil-bound residues This exercise is an attempt to demonstrate whether regular use of EBDCs would resul "persistentn i t " residue soin i s l whic y poshma s ethreaa it o t quality. Bound pesticide residues, by definition, are those residues which cannot be extracted by methods commonly used for pesticide residue analysis (7).
Soil samples (25 g), preferably those collected towards the end of the 14 C-fungicide experiment Soxhlee ,ar t extracted with methano hrs2 1 r .lfo Radioactivity measured in the extract represents the extractable radiocarbon (degradation products of EBDC). The remaining residue is then dried, 14 combusted and counted. C-activity measured would represent the methanol non-extractable actvity.
Following methanol extraction, some samples are further extracted with e sodiuth f mo salM colethylenf 4 o t 0. d e diamine tetra-acetic acid (Na.EDTA) to remove the free parent EBDC. Radioactivity left behind represents the bound residue.
If bound fungicide residues are appreciable it may be worthwhile to examine uptak radiocarbof eo plana y nb t cultivate extractee th n i d d soile B . sure to restore the microflora in the soil by adding fungicide-free soil to the extracted soil.
134 5. Mineralization studies Placsoig 0 le1 samples into wide necked screw-capped jars wated Ad .r 14 to 2/3 field capacity and incubate for 1 week. Add the C-fungicide (e.g. ppm5 placd )an scintillatioea n vial containin ethanolaminl m 0 g1. e th n ei jar. Collect traps weekly for analyses of 14CO , New traps with fresh ethanolamin introducee ear d weekly into jars.
Control measuro st e non-biologica O evolutioC l n mus14e tb autoclaved at 120°C for 3 days prior to treatments.
REFERENCES
(1) Onley J.H. and Yip, G., J. Assoc. Off. Anal. Chem. 54 (1971) 165.
(2) Rhodes, R.C., J. Agr. Food Chem. 25 3 (1977) 528.
) (3 Smith, R.M al.t e . , Pestic. Sei 3 (19882 . ) 337.
(4) Czegledi-Janko, G., J. Chromatog. 11 (1967) 89.
) (5 Bontoyan, W.R al.t .e Assoc. J , . Off. Anal. Chem 5 (1972.5 ) 923.
(6) Vonk, J.W. and Sijpesteijin, A.K., Pestic. Chem. Physiol. 1 (1971) 163.
(7) Radiotracer studies of bound pesticide residues in soil, plants and food. IAEA TecDoc 306, IAEA, Vienna (1985).
135 AnneI xII
METHODS FOR THE DETERMINATION OF EBDCs AND DEGRADATION PRODUCTS
EBDCs undergo bioti d abiotican c degradatio y leavma d e an nresidue f so the parent compound and/or its degradation products, primarily ethyleneurea (EU) and ethylenethiourea (ETU) on crops. ETU has been suspected to cause a variet detrimentaf o y l effects mose Th .t common methods usedetermininr fo d g residuesummarizee ar U ET EBDCf so d d san below .
A. Analysis of commercial formulations
parene Th t fungicid formulatioe th n determinei e b y CSe nma .th y b d evolution metho Keppef o d l (1)e analyticaTh . l metho bases i d n o d decomposition by hot HCl to the amine and carbon disulphide. The CS, 2 evolve trappes i d d int colouoa r reagent (mixtur f cuprieo c acetate monohydrat d diethanolamineean d colorimeteri)an c measuremen -compleS C f to x s i iS sC madee weigh e e absorbancth th Th d .f to an n ru measures ei 5 43 t a d calculated from a standard curve. The amount of dithiocarbamate is calculated s equivalena f CS_to .
Fifty millilitre of dry (over CaOxide) freshly distilled ethyl acetate are added to 5 g of the commercial fungicide and the suspension stirred with a magnetic stirremin5 1 e supernatanr .Th rfo decantes i t washind an d g with ethyl acetate repeated (3-4 times) till free from ETU. This is checked by TLC. Quantification of ETU in the combined washings may be made according to p (2) monitoo Yi OnleT .d effece yan th r storagf o te stabilit th e n eo th f yo commercial preparation above ,th e analysis shoul repeatee b dmont3 2- ht a d intervals (up to 18 months) on closed and open (partially used) packages. Storage conditions shoulcloss a possibls e eda b thoso et e use practicen i d .
B. Analysi EBDCf so foon si d plants The procedure used is essentially the same as described under A.
C. Identification and quantification of ETU The method (2) for determination of ETU involves extraction and clean-up extracte oth f sample Th analysir . fo e homogenizes i s d with CHC MeO1: H (2:1) and celite. The mixture is filtered, the filtrate is reduced to a minimum volume and subjected to column chromatography on Al-0„ (memtral).
136 The column is eluted with petroleum ether (60-80°C)(fraction 1) and methanol:acetonitrile:benzene (3:15:82) (fraction 2). This fraction (No. 2) reduces i minimua o t ddeterminationU mET uses volumr i fo dd ean simplA . e extraction and clean-up method has also been developed (3). The method involves extraction of a sample with cold methanol, filtration and volume reductio reducea t na d pressure residue Th . dissolves i e waten i d r whics hi passed through an Extralut column. The column retains water and ETU is eluted with méthylène chlorid ethylacetatr eo analysir efo HPLCr TLCy o sb C .,GL
TLC analysis e cleaneTh d extrac silicn o subjectes i tl ) 5 C analysiage TL , 4 o t d, (3 s
plates. Among other solvent systems, CHCl3:BuOH:MeOH:H20 (100:5:1:0.5s )i use resolvo t d degradatioe eth n compound EBDCsf so : ETU ethylene, ,EU - thioramonosulphide (ETM), ethylenethiuramdisulphide (ETD) and ethyleneamine (EA)compounde Th . visualizee sar d eithe exposiny rb platee gth iodino st e vapour sprayinr so g wit alkalinn ha e solutio sodiuf no m nitroprussidd an e potassium ferrocyanide weighe e identifieTh th . f to d produc calculates i t d 14 fro standarma d curve usiny B . g C-ethylene-labelled EBDCs producte ,th s may be identified and quantified by radioscanning.
GLC analysis knows i polaa t ns I i rtha U compountET d difficulan d analyso t e directl C analysisGL y yb orden I .mako t rnon-polat ei o increast d re an eth efficienc detectionf yo , various derivatization methodhav) 6 e, bee(4 s n used. The common derivatives are 2-(butylthio)-2-imidazoline, 2-(benzylthio)- l-(pentafluorobenzoyl)-2-imidazoline, 2-(benzylthio)-l-(trifluoroacetyl)-2- imidazoline, 2-(m-(trifluoromethyl)-benzylthio)-l-(trifluoroacetyl)-2- imidazoline. These derivatives carry a number of halogen atoms in the molecule which enable a high detection efficiency on GLC using an EC detector. A simple capillary gas chromatography method (7) has also been developed for the estimation of ETU in beer, grapes and hops without derivatization e detectioTh . n limit reporte 0.00s i d 5 ppm, usin sulphuga r detector.
HPLC analysis HPLC is the technique of choice for assaying polar substances. ETU in bee wind ran e were detecte levea t 0.025-0.0f a dlo 1 ppm, using alumind aan
sherisorb CN columns and eluting with CH2Cl2:MeOH (98:2) or hexane:EtOH (2:1) (8, 9). Recovery of ETU from soil and river water has been reported to be 99% on a cation exchange resin (10), eluting the column with 1.0 M aqueous ammonium sulfate.
137 REFERENCES
) Keppel(1 , AssocG.E.. J , . Off. Anal. Chem (19692 .5 ) 162.
(2) Onley, S.H., Yip, G.J., Assoc. Off. Anal. Chem. 54 (1971) 165.
) Rhodes(3 , R.C.Agr. J ., Food Chem 5 (1977.2 ) 528.
) Onley(4 Guiffrida, ,Y. Ives, Watts,L. , ,N. Storherr, . Assoc. ,R J , .,R. Off. Anal. Chem. 60 (1977) 1105.
(5) Czegledi-Janko, G.J., Chromatograph/ 3JL (1967) 89.
(6) Nash, R.G., J. Assoc. Off. Anal. Chem. 57. (1974) 1015.
(7) Nitz, S., Moza, P., Körte, F., J. Agr. Food Chem. 30 (1982) 593.
, ) ChimLazzariniA. (8 , .Re Ind l Rossi, De , C. ., ,E. (Milan 2 (1980)£ ) 923.
(9) Massey, R.C., Key, P.E., McWeeny, D.J., J. Chromatography 240 (1982) 254.
(10) Hashimoto A., Anal. Chem. 51 (1979) 385.
138 LIST OF PARTICIPANTS
Prof. H.C. AGARWAL University of Delhi Departmen Zoologf to y Delhi - 110 007 India
Ms. S.M.F. CALUMPANG National Crop Protection Center university of the Philippines at Los Banos Pesticide Chemistry and Toxicology Laboratory College, Laguna Philippines
Prof. A.H.E. HARHASH universit Cairf yo o Facult Sciencf yo e Department of Chemistry Giza Egypt
KOLANKAY. D . Dr A Hacettepe University Facult Sciencf yo e Dept f Zoolog.o y Beytepe - Ankara Turkey
Dr. P.M. MOZA Institu Oekologisch. tf e Chemie der Gesellschaft für Strahlen- und Umweltforschung, München Schulstrasse 10 D-8050 Freising-Attaching Federal Republi Germanf co y
DrRaphael. .M a MUSUMEGI Institute Biologico Radioisotope Center Av. Cons. Rodrigues Alves, 1252 04014 Sao Paulo Brazil
Dr. O. PAZMlSO MORALES Comision Ecuatoriana de Energia Atomica Research Division P.O251x .Bo 7 Quito Ecuador
Dr. Johar RAMLn ibi I Department of Biochemistry & Microbiology Universit Agriculturf yo e Malaysia 43400 UPM, Serdang Selangor Malaysia
139 Prof RAMO. .F N Centro de Investigaciones con Tecnicas Nucleares Faculta Cienciae d s Naturalesy Exacias Universida Paname d a Panama City Panama
Dr. Lian-zhong ZHANG Pesticide Laboratory Research Centre for Eco-environmental Sciences Academia Sinica P.O. Box 934 Beig jin People's Republi Chinf o c a
Secretariat: Dr. A. HASSAN International Atomic Energy Agency Joint FAO/IAEA Division 0 10 x P.OBo . A-1400 Vienna Austria
Other participant o collaborateswh programmee th n o d : Dr. J.W. HYLIN P.O 632x .Bo 3 Incline Village, NV 89450 USA
W.D. Dr . MARSHALL Macdonald Colleg McGilf eo l University Faculty of Agriculture 21111 Lakeshore Road St. Ann Bellevuee ed , P.Q. Canada H9X ICO
140 LIS PUBLICATIONF TO S PRODUCED WITHIN THE AGROCHEMICALS AND RESIDUES PROGRAMME SINCE 1982
STI/PUB/623 Agrochemicals :Environmente Fatth Foon d ei an d . Proceeding Symposiua f so m (including Summary Reporn to Consultants' Meeting on Persistent Pesticides and Herbicides Tropice inth s Using Isotope Technique Thirn o d d san FAO/IAE A Research Co-ordination Meetin Agriculturan go l Chemical Residue-Biota Interaction Soin Aquatisd i lan c Ecosystems), IAEA, 1982.
IAEA TECDOC-283 Agrochemica Biotl- a Interaction Soin Wated si lan r Using Nuclear Techniques. Report of a Research Coordination Meeting. Technical Document, 1983.
STI/DOC/10/225 Laboratory Trainin Nucleaf o e gUs r Manuae Techniqueth n lo s in Pesticide Research. Technical Reports Series, 1983.
IAEA- TECDOC-306 Radiotracer Studies of Bound Pesticide Residues in Soil, Plant Foodd san . Repor Researca f to h Coordination Meeting, Technical Document, 1984.
STI/PÜB/724 Quantification, Nature and Bioavailability of Bound pesticide Residue Soiln si , Plant Foodd san . Proceedings and Repor Researca f to h Coordination Meeting, IAEA, 1986.
IAEA-TECDOC-404 Research and Development of Controlled-release Technology for Agrochemicals Using Isotopes. Report of a Seminar, Technical Document, 1987.
IAEA-TECDOC-405 Radiotracer Studie Pesticidf so e Residue Storen si d Products. Repor Researca f to h Coordination Meeting, Technical Document, 1987.
IAEA-TECDOC-406 Radiotracer Studie Agrochemicaf so l Residue Meatn i s , Milk and Related Products of Livestock and Poultry. Report of a Research Coordination Meeting, Technical Document, 1987.
STI/PUB/764 Pesticides: FooEnvironmentad an d l Implications. Proceedings oSymposiuma f , Neuherberg, Germany F.R., 24-27 November 1987 (including Summary Report on Second FAO/IAEA/GSF Research Co-ordination Meetin Controlled-releasn go e Formulationf so Pesticides to Reduce Residues and Increase Efficacy Using Radioisotopes), IAEA, Vienna, 1988.
IAEA-TECDOC-475 Improved Biomass Utilizatio Nucleaf no Througe Us r e hth Techniques. Report of a Final Research Coordination Meeting on Improved Rural Methane Production from Biomass by Utilizing Nuclear Techniques, IAEA, Vienna, 1988.
141 lAEA-TECDOC-476 Isotope Techniques for Studying the Fate of Persistent Pesticide Tropicse th n si . Repor Finaa f to l Research Coordination Meeting on the Fate of Persistent Pesticides in the Tropics, Using Isotope Techniques, IAEA, Vienna, 1988.
IAEA-TECDOC-494 Radioactive Fallou Foon d Agricultureti an d backgrounA . d review prepared by F.P.W. Winteringham, IAEA, Vienna, 1989.
STI/PUB/822 StudieMagnitude th f so d Naturan e f Pesticideo e Residuen i s Stored Products Using Radiotracer Techniques. Proceedings of the Final Research Coordination Meetin Isotopin o g c Tracer- aided Studie Pesticidf so e Residue Storen i s d Products, Ankara, Turkey, 30 May to 3 June 1988, IAEA, Vienna, 1990.
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