DIASpø Production

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(1)DIAS. December 1999. p ø. klCn. P = c„-. Production. + Ct,(1 - e-'^3 t). (k^ + k 2C^e'^1^ - k 2 Cn. Inge S. Fomsgaard. Ph.D. dissertation The mineralisation of pesticides in surface and subsurface soil - in relation to temperature, soil texture, biological activity and initial pesticide concentration. M in istry o f Food, A g ric u ltu re and Fisheries. Danish Institute o f Agricultural Sciences.

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(3) Ph.D. dissertation The mineralisation of pesticides in surface and subsurface soil - in relation to temperature, soil texture, biological activity and initial pesticide concentration. Inge S. Fomsgaard Research Centre Flakkebjerg D ep a rtm en t o f Crop Protection Flakkebjerg DK-4200 Slagelse D enm ark (e-m ail; lnge.Fom sgaard@ agrsci.dk). DIAS rep ort Plant Production no. 19 • Decem ber 1999 Publisher:. Danish Institute o f Agricultural Sciences Tel. +45 89 99 19 OO Research Centre Foulum Fax +45 89 99 19 19 P.O. Box 50 DK-8830 Tjele. Sale by copies: (ind. VAT). up to 50 pages up to 10O pages more than 1OO pages. Subscription:. Depending on the number o f reports sent but equivalent to 75% o f the price o f sale by copies.. 50,- DKK 75,- DKK 10O,- DKK.

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(5) Table of contents Table o f contents............................................ . .. . 3 Foreword......................................................... ....4 The structure o f the th esis........................... . .. . 5 1. Background................................................ . . . 6 1,1 Pesticides in the environment.......... ...6 1.2. M odelling............................................ ....6 1.3. Degradation kinetics......................... ....9 1.4. Degradation rate................................ .. 11 2. Purpose........................................................ ..13 3. SuiTunary o f the scientific papers I-V I ., .. 14 4. Synopsis o f the investigated pesticides . ..22 5 Synopsis o f the results and the discussions..................................................................... ..2 6 5.1. The mineralisation kinetics in relation to geo-environmental factors................ ..2 6 5.1.1. The experiments........................................................................................................ ..2 6 5.1.2. The choice o f kinetic m odels................................................................ .................. ..28 5.1.3. Mineralisation kinetics in plough layer at low concentrations........................ ..35 5.1.4. Mineralisation kinetics in subsoil at low concentrations................................... ..3 9 5.1.5. Extended kinetic models used in experiments from tropical clim ate............. ..4 0 5.1.6. Extended kinetic models used in experiments with varying pesticide concentrations ..41 5.1.7. The development o f a general mineralisation m odel......................................... ..4 8 5.1.8. The application o f the general kinetic m o d el...................................................... ..5 2 5.2. The mineralisation rate in relation to geo-environmental factors............................................. ..5 6 5.2.1. The mineralisation rate in relation to varying pesticide concentrations and soil depth ..5 6 5.2.2. The mineralisation rate in relation to temperature, concentration, soil depth and content o f organic m atter.................................................................................................................................................................................... 57 5.2.3. A model describing the mineralisation rate in relation to microbial activity, depth, content o f organic matter and soil texture....................................................................................................................................................... 62 5.2.4. The causality o f the mineralisation m odel......................................................................................................... 64 5.2.5. The future development o f the m od el................................................................................................................ 69 6 . Synopsis o f the conclusions................................................................................................................................................. 70 7. Resumé (dansk)....................................................................................................................................................................... 74 8 . Abstract (E nglish)...................................................................................................................................................................75 9. Resumen (espaflol)................................................................................................................................................................. 76 10. References.............................................................................................................................................................................. 78 11. Enclosures.............................................................................................................................................................................. 84 I. I S. Fomsgaard, 1995. Degradation o f pesticides in subsoil - a review o f methods and results. Intern. J. Environ. Anal. Chem. 231-245.................................................................................................................................85 II. I.S, Fomsgaard, 1997. Modelling the mineralisation kinetics for low concentrations o f pesticides in surface and subsurface soil. Ecol, Mod., 102. 175-208......................................................................................................... 101 III. I.S. Fomsgaard, H. Johannesen, J. Pitty & R. Rugama, 1998. Degradation o f '“'C-maneb in sediment from a Nicaraguan estuary. Intern. J, Environ. Stud. B, 55, 175-198............................................................... 135 IV. A. Helweg, I.S. Fomsgaard, T.K. Reffstrup & H. Sørensen, 1998. Degradation o f mecroprop and isoproturon in soil influence o f initial concentration. Intern. J. Environ. Anal. Chem. 7 0 (1 -4 ), 133-148.......................................................................................................................................................... 159 V. I.S. Fomsgaard & K. Kristensen, 1999. ETU mineralisation in soil under influence o f organic carbon content, temperature, concentration, and depth. Toxicol. Environ. Chem. 70, 195-220................................ 175 VI. I.S. Fomsgaard, & K. Christensen, 1999. Influence o f microbial activity, organic carbon content, soil texture and soil depth on mineralisation rates o f low concentrations o f “*C-mecoprop - development o f a predictive model. Ecol. Mod. 122, 45-68........................................................................................................... 201. Papers I-VI are reproduced with the kind permission from the pubhshers..

(6) Foreword. T he w o rk b ehind this Ph. D. thesis w as perform ed at the R oyal D anish S chool o f P harm acy, Institute o f A nalytic C hem istry w ith Dr. Eng., Dr. Scient. Sven E rik Jø rg en sen as m y p rincipal tutor. I have been an external Ph.D . student, as I have b een em ployed at the D anish Institute o f A gricultural S ciences (form er the D anish Institute o f P lant and S oil Science) during the w hole period. D r. A gro. A rne H elw eg has been m y co-tutor. T he Ph.D . p ro ject w as financed b y the D anish M in istry o f A griculture, the D anish A gricultural and Veterinciry R esearch C ouncil and th e D anish E nvironm ental P rotection A gency. I w ish to express m y p articu lar g ratitude to Dr. Sven E rik Jørgensen, w h o se in spiration in relation to the use o f m athem atical m odels as a scientific tool has b een inestim able, and to Dr. A rne H elw eg, a p io n e er in the field o f d egradation o f pesticid es in soil, w h o g enerously allow ed m e to draw on his experience. I ow e a debt o f gratitude to laboratory technician H elle Priess, w h o jo in e d m e during the w hole period w ith h er clev er effort in the technical field. M y thanks are also d u e to laboratory technicians A lice B in d er and M arianne N ielsen for th eir clev e r efforts in parts o f the project. O ur laboratory techn ician students w ere engaged in the p ro ject durin g th e ir p erio d o f training. W ith th eir sym pathy and kindness, all m y o th er colleagues at the lab o rato ry created an environm ent, w hich to a h ig h extent pro m o ted m y project. Jette Jep p esen , E llen M arie B entsen, S on ja G raugaard, M aria L ange L ehm ann, P hyllis R asm u ssen and M ariaim N aundrup assisted m e w ith th eir linguistic qualifications. H enny R asm u ssen drew all the figures I asked for, b o th in the papers and in th e synopsis w ith p atience and creativ ity , and K ristian K ristensen w as a fabulous sparring partn er in the area o f statistics and m odelling. L ast b u t not least very special than k s to m y husband, G unnar, w h o se love and p atien ce w as and is - m o st essential to m e..

(7) The structure of the thesis T he present Ph.D . thesis co n sists o f a synopsis and 6 scientific papers. T he th esis beg in s w ith the B ackground (C h ap ter 1) for the p erfo rm ed research and a d ecla ratio n o f th e P urpose (C hapter 2). A fter that com es chapter 3, S um m ary o f the included scien tific p a p e rs: ch ap te r 4, Synopsis o f the investigated p e sticid es: chapter 5, S ynopsis o f the resu lts an d th e d iscu ssio n s: and finally chapter 6 , Synopsis o f th e co n clu sio n s. A short d escrip tio n o f th e u sed m ethods can be seen in the sum m aries o f th e included publications. A th o ro u g h d escrip tio n o f the m ethods can b e seen in the publications. In chapter 5, S ynopsis o f the resu lts and the discussions, the m ost im portant results from the included scientific p ap ers are p resen ted and discussed in relation to each o th er and to o th er current research in th e area. E ach o f the scientific papers com prises results and discussion b o th o f the p esticid e m in eralisatio n k inetics and o f the pesticide m ineralisatio n rate. T he synopsis w as th erefo re d iv id ed into tw o su b chapters 5.1, T he m ineralisatio n kin etics in relation to geo-environm ental factors and 5.2, The m ineralisation rate in relation to geo-environm ental facto rs. In b o th su b-chapters, I discuss the results o f the p u blications in relatio n to each other. In ch ap te r 6 , S ynopsis o f th e conclu sio n s th e results are connected to the purpose o f the p ro ject and th e n eed s in future research in the area is discussed. T he scientific papers, w hich are included in the thesis, are n u m b ered I-VI and enclosed in th eir full length in chapte r 11, E n clo su res. I had the m ain resp o n sib ility for th e w o rk in papers. I, II, III, V and VI and the resp o n sib ility for th e collation o f d ata in p a p er IV. W ith the p resentation o f a sum m ary (ch ap ter 3) o f the included scien tific p ap ers I h o p e the reader w ill b en efit from reading the synopsis even w ith o u t read in g th e w h o le papers. It is o f course im possible to express in a short sum m ary w hat w as describ ed in a p a p e r o f i.e. 23 pages. T he read er w ill therefore on ly have the full b en efit o f th e thesis, i f the inclu d ed scientific papers are read firstly..

(8) 1. Background 1.1 Pesticides in the environment F o r a num ber o f years th e ten d en cy o f the d evelopm ent o f D anish a g ricu h u re has b een to raise the efficiency and th e yield. C onsequently, a total am ount o f 184,011, 622 o f k g o f p esticides (m easured as active ingredient) o f the 200 m ost-sold co m p o u n d s w as u sed in th e y ears 19561993 (M iljøstyrelsen, 1997a). T he approval o f pesticides for use in D an ish agriculture is undertaken by the D an ish E n vironm ental Protection A gency acco rd in g to the guidelines given in ”R am m er for vurd erin g a f planteb esk y ttelsesm id ler” (M iljøstyrelsen, 1994). B eyond a broad to xicological assessm ent, the p ersistence in and sorption to soil o f th e co m p o u n d s are evaluated. C om pounds considered to b e leachable to gro u n d w ater are n o t approved.. It w as thus against m ost p e o p le’s expectations that p esticide resid u es above 0.1 |ig l ' w ere detected in ground w ater in the extensive ground w ater m o n ito rin g p ro g ram m es p erfo rm ed in the begirm ing o f the ‘90s (G E U S , 1997). Y et H elw eg (1984) already p o in ted o u t th e risk. T he num erous finds o f p esticide residues in ground w ater raised p ublic co n cern and p rom pted the pop ulation to dem an d reduction o f ground w ater contam ination. A su bstantial need for investigating the fate o f p esticides in soil w as built up. T he guidelines fo r approval o f p esticides (M iljøstyrelsen, 1994) co ncerning d egradation rates o f pesticid es, d em and that degrad ation studies m ust be p erfo rm ed in three different p lo u g h layer so ils and that in n one o f the three soils the h a lf life tim e, D T 50, m ust exceed 90 days. A n u m b er o f indicators have been developed w here the p urpose is to ran k th e risk for p esticides leaching into ground w ater on the b asis o f few param eters for each com pound. The G U S— index ranks pesticides exclusively on the basis o f inherent p ro p erties, degrad ab ility (m easured as half-life tim e, D T 50) and sorption (m easured as K oc), and thus, giv es a m easure o f the leaching potential. L indhardt et al. (1998) show ed that ran k in g o f 11 pesticides according to the G U S -index o n the b asis o f the h alf-life tim es and K oc v alu es rep o rted to the D anish E nv ironm en tal P rotection A gency, resulted in a high deg ree o f uncertainty, caused by the great variation in the data m aterial. 1.2. M o d e llin g M odels have been applied in natural science as long as natural science has existed. N e w to n ’s law s i.e. are m odels w hich d escribe the influence o f g ravity on b o d ies (Jørgensen, 1994). A m athem atical d escrip tio n o f the k inetics according to w hich a p esticid e is deg rad ed is also a m odel, like the m athem atical d escription o f the p rocess that takes p lace w h en a pesticid e is sorbed to soil is a m odel. Such m odels have been used in pesticid e research for decades. W ith the progress o f advanced co m p u ter techniques, it has b eco m e p o ssib le to develop dynam ic m odels, w hich can sim u late tran sp o rt and degradation o f x en o b io tic co m p o un ds in the environm ent. D ynam ic m odels, used to sim ulate p esticide leaching to ground w ater, co n sist o f.

(9) a n u m b er o f subm odels, all o f them containing m ath em atical d escrip tio n s o f the processes w hich are relevant to transport pesticides in soil into th e actual com p artm en ts. Such subm odels could be: a soil m odel d escribing the structure o f th e soil layers, a h y drological m odel describing the transport o f w ater through the soil layers, an ev ap o -tran sp iratio n m odel, a ru n -o ff m odel, a m odel for p esticide sorption, a m odel for p esticid e d eg rad atio n , a m odel for p esticide application and a p lan t grow th m odel. O f 9 frequently used d y n am ic pesticid e leaching m odels, PR Z M -2, P R Z M , PE L M O , G L E A M S , PE S T L A , V A R L E A C H , L E A C H M , M A C R O and PL M , 8 o f them use a subm odel for p esticide d eg radation w h ich assum e that the degradation follow s first o rd er k inetics (B oesten et al., 1995). T h ey all assum e th at the degradation rate depends on tem perature and/or soil depth an d /o r soil w ater content..

(10) Concentration and Total i^C02-evolution. F IG U R E 3 Genera] diagranune o f m odels for pcslicide degradation. A. first-order reaction kinetics*^* R ate equation:. - ^ =kc. Disappearance: c=Coe**“ . Form ation: P=Co( l-e"*“ ).. Concentration and Total '^COj-evolution. B. Zero-order reaction kineiics*^’^ R ate equation:. _. D isappearance : c=Co-kol. Form ation: P=kot.. Concentration and TotaM^COj-evolution. C . D egradation with grow th R ate equation, log. growth*^’ ':. Disappearance. log. grow th“” ':. - ^ = k | C ( c o + X o “ C). ^. Form ation, log. growthl^^':. Rate equauon, exp. grow th'^’ ^:. Disappearance, exp. grow th“^^^:. - ^ =ke". c=C q -. Form ation, exp. growth*^^':. Figure 1.1. D iagram s describ in g d egradation o f p esticides, w here d isap p earan ce o f p esticide as w ell as form ation o f m in eralisatio n p roduct ‘‘'C O 2 is show n. A. F irst o rd er k inetics, B. Z ero ord er kinetics and C. K inetics w ith grow th o f m icro-organism s. (F ig u re 3 from IV ).. 8.

(11) 1.3. D e g ra d a tio n k in e tic s D epend ing on experim en tal conditions, d egradation rates are rep o rted d ifferently. W hen the am ount o f m ineralisation p roduct ' “*C02 evolved from ' ‘’C -lab elled p esticid es durin g tim e is m easured, either the am ount o f evolved '''C O 2 after a certain n u m b er o f day s, o r the rate constant from the kinetic p ro cess describ in g the m in eralisatio n is reported. W hen th e am ount o f residual pesticide durin g tim e is m easured, the kinetic process is analysed, and th e rate constant for the k inetic p ro cess is reported. F igure 1. 1 show s th ree ex am p les o f a graphical presentation o f a degradatio n process, w here the am ount o f residual p aren t com p o u n d as w ell as the form ation o f m ineralisatio n product (i.e. '''C O 2) is m easured. In th e follow ing text, I w ill d istin guish betw een m easurem ent o f degradation, degrad atio n studies and m easurem ent o f m ineralisation, m ineralisatio n studies both for m y ow n studies as for studies from references. Y et in m ore general d iscussions I w ill use the ex pression degradation. In the literature, the kinetics for the degradation o f xenobiotic c o m p o u n d s in th e en vironm ent has been described w ith tw o different bases. O ne b asis is the po w er-rate m odels; the o th er is the hyperbolic rate m odel, as d escribed b y H am ak er (1972), w h o se p u b licatio n still is used by m any authors as a reference. T he p o w er rate m odel, w hen residues o f p aren t com p o u n d are m easured, is expressed as. --= kc" dt. (1.1). w here c is the concentratio n o f pesticide, k is the rate constant, and n is the o rd er o f reaction. T he hyperbolic rate m odel, w hich is founded on M ichaelis-M en ten en zy m e k inetics, is expressed as (1.2). dt. (* 2 + c ). w here c is the concentratio n o f pesticide, ki is the m axim um reaction rate obtain ed w ith grow ing concentrations, and k2 is a p seudo-equilibrium constant, also called th e h a lf saturatio n constant. T he h yperbolic rate m odel w as used to d escribe th e d eg rad atio n o f pesticides in aquatic solutions b y for instance Sim kins & A lex an d er (1 9 8 4 ) and S chm idt et al. (1985). H am aker (1972) and P ark er & D ox tad er (1982) used th e h y p erb o lic rate m odel to describe the deg rad atio n o f pesticid es in soil, w hile Scow et al. (1 986), B ru n n er & F ocht (1984) and Jacobsen & P edersen (1992) excluded th e use o f the h y p erb o lic rate m odel, either based on the resu lts o f em pirical trials o r on the th eoretical c o n sid eratio n s that in th e very com plex soil environm en t an equilibrium situation w ould n ev er occur. T he p o w er rate m odel w as often u sed to d escribe the deg rad atio n o f p esticid es in soil. K em pson-Jones & H ance (1 9 7 9 ) and M oorm an & H arper (1989) d eterm in ed b o th th e rate constant k and the reactio n o rd er n in th eir d egradation studies, w h ere the reactio n o rd er w as * 1..

(12) In m any published studies it w as show n that the d egradation follow ed first o rd er kinetics w here «=1 in the pow er rate m odel. In o th er published studies it w as assum ed th a t the degradation process should follow first o rd er kinetics, the d eg rad atio n o n ly depen d ing on the pesticide concentration. W ith b asis in a first o rd er d egradation, the d egradation rate can b e g iven as h a lf life tim e (DTso o r t./J. ~^2=kc at. (1.3). In integrated form it is w ritten as. c (0 = c „ e - * '. (1.4). w here. c(t) = co ncentration o f p esticid e at tim e t, co = start concentration o f pesticide, k = rate constant In the pesticide approval p ro ced u re (M iljøstyrelsen, 1994) degrad atio n rates are m ain ly given as half-life tim e, w hich traces back to first o rd er kinetics. F om sgaard (1 9 9 8 ) w ent thro u g h all the d egradation studies reported to the D anish E n vironm ental P ro tectio n A gen cy for 12 com pounds and stated that in m any cases the d egradation d id n o t follow first o rd er kinetics. Pseudo first o rd er kinetics, em pirical one and a h a lf o rd er k inetics, h a lf o rd e r kin etics o r p o w er rate kinetics w ith n-*\ w ere reported. In several cases, b o th in the p esticid e approval d ocum ents (F om sgaard, 1998), in H ill & Schaalje (1985) and in G u stafso n & H olden (1990) it w as show n that the deg rad atio n reaction took place in several com p artm en ts, for w hich reason the m athem atical d escription consisted in several first o rd er term s and th en no longer w as a sim ple first o rd er process. A nother im portant reason for no t b ein g able to anticipate a first o rd er degrad atio n , is that a lag-phase can o ccu r in the degrad atio n process. In th e lag-phase the m icro -o rg an ism s adapt to the presence o f the pesticide, w hereupon the m icro -o rg an ism s achieve en erg y from the degradation process. A ch iev in g energy, the m icro-organism s grow and th e d eg rad atio n rate increases (T orstensson, 1988). L inders et al. (1994) ex am ined reports for 243 pesticid es and corrected the reported h alf-life tim es, leaving out th e lag-phase. Figure 1.1 show s three th eoretical exam ples o f degrad atio n and m in eralisatio n , a) according to first o rd er kinetics, b) according to zero k inetics and c) according to kin etics w ith grow th. T he last exam ple, kin etics w ith grow th, could for instance be logistic o r exponential grow th, as explained in the text o f the figure.. 10.

(13) N o m atter how pesticide deg rad atio n is m easured, quan tify in g resid u es o f p aren t com pound or form ation o f a m ineralisatio n product, i.e. ' “C O 2 from ' “'C -lab elled pesticid es, it is essential to analyse and describe the kin etics o f the process, to get to know th e p aram eters n eeded for com paring d egradation rates.. 1.4. Degradation rate In I, I exam ined publish ed degrad atio n studies in subsoil and concluded th at for m any pesticides, results from subsoil w ere very lim ited and th at the g reat variatio n in techniques, used for the studies, m ade it d ifficult to com pare the results. A lm ost all th e pub lish ed studies w ere p erform ed w ith pesticid e concentrations, w hich w ere un realistic com p ared to theoretical concentrations o f p esticid es in subsoil after norm al agricultural u se o f the pesticid es. A t the sam e tim e, the studies w ere n o t p erfo rm ed in co n centrations high en ough to sim ulate situations, w here the pesticides could be present in subsoil becau se o f poin t-so u rce contam ination. M any studies show ed th at soil depth influenced the degrad atio n rate o f the p esticid es due to different ch em ical and biolo g ical conditions at varying d epth (D icto r et al., 1992; M u eller et al., 1992; M inton et al., 1990; M oorm an & H arper, 1989; P othuluri et al., 1990). T he effect o f tem peratu re on th e d egradation rate o f p esticid e w as also w ell described (H elw eg, 1993; H elw eg, 1987; M atoba et al., 1995; Ism ail & L ee, 1995; W alk er et al., 1996). W alker et al. (1996) rev iew ed a h ig h nu m b er o f degrad ation studies and calcu lated m ean Qio values. W ater content o f th e soil w as also often described as h av in g im portance for the degrad ation rate (Ism ail & Lee, 1995; H elw eg, 1993; H elw eg, 1987), as w ell as th e initial concentration o f pesticid e (H elw eg, 1993; H elw eg, 1987; R effstrup et al., 1998; Jaco b sen & P edersen, 1992; P ark er & D oxtader, 1982; M ueller et al., 1992). T em perature, soil w ater content and soil depth are the factors that are considered to have an effect on th e p esticide degrad ation rate in the 9 frequently used dynam ic leaching m odels, P R Z M -2 , PR ZM , PE L M O , G L E A M S , P E S T L A , V A R L E A C H , L E A C H M , M A C R O and P L M (B o esten et al., 1995). M icrobial activity /b io m ass w as often m easured and related to th e d eg rad atio n rate o f pesticides, either directly or through the v ariation in soil depth (A nderson, 1984; T orstensson & Stenström , 1986; M o n ro zier et al., 1993; D icto r et al., 1992). T he am ount o f o rganic m atter - also frequently related to soil depth - and its influence on th e d eg rad atio n o f x enobiotic com po unds w as also investigated (R eddy et al., 1995; D uah-Y entum i & K uw atzuka, 1980; G reer & S helton, 1992; K naebel et al., 1994). W alker (1976a, 1976b, and 1976c) stu d ied the effect o f tem perature, soil w ater con ten t and p esticide con cen tratio n in soil from p lo u g h layer and developed sim ulatio n m odels. FO C U S (F O rum for the C o -ordination o f p esticide fate m o d els and th e ir U S e) - a w orking party under the E U (B o esten et al., 1995) com pared 9 dynam ic leach in g m o d els and concluded that a b etter descrip tio n o f subsoil d egradation is n ecessary, to im prove the predictive value o f the m odels. A s regards d egradation rates, th e m o d els on ly include the. 11.

(14) dependence on soil d epth an d /o r tem perature and/or soil w ater content, and 8 o f 9 m odels assum e that the degrad atio n follow s first o rd er kinetics. A b etter description o f the d egradation k inetics not only in subsoil, b u t also in p lo u g h layer, is required for the further dev elo p m en t o f the dynam ic leaching m o d els in o rd er to o b tain a b etter sim ulation o f actual conditions. K now ing the great variatio n in d eg rad atio n rates and the high nu m b er o f factors w hich influence the degradation, m akes it n ecessary to study the concurrent in fluence o f th ese factors and develop m odels, w hich describ e this influence.. 12.

(15) 2. Purpose. T he aim o f this Ph. D. pro ject w as to describe the m in eralisation k in etics for p esticid es in soil and to develop and validate a predictive m in eralisatio n m odel, w h ich co u ld d escribe the effect o f external geo-environm ental factors on the m ineralisation. A com plete validation o f th e m odel w o u ld entail a study o f all relev an t p esticid es in com bination w ith all relevant external geo-environm ental factors. T h at is o b v io u sly n o t p ossible w ithin a realistic tim e scale. F o u r ch aracteristic pesticid es w ere th erefo re selected on the grounds o f consum ption, the risk o f leaching and the form ation o f m etabolites. The degradation o f these pesticid es w as investigated for various co m b in atio n s o f external geoenvirorunental factors, w h ich support the m odel. T he follow ing stu d ies w ere carried out: ❖ the m ineralisation o f 4 ch aracteristic pesticides; m ecoprop, ben tazo n , iso p ro tu ro n and m aneb, and 1 m etabo lite o f m aneb, E TU ❖ the effect on m ineralisatio n o f >. the d epth o f soil (0-75 cm ). >. the b io lo g ical activity. >. the concentration o f the pesticide. >. the content o f organic carbon in the soil. >. the tem perature. >. the texture o f the soil. >. the content o f nutritiv e salts in the soil. 13.

(16) 3. Summary of the scientific papers I- V I. I. I.S. Fomsgaard, 1995. Degradation of pesticides in subsoil - a review of methods and results. International Journal of Environmental Analytical Chemistry 58, 231-245. A s the starting point o f the w hole project, in the present paper, I ex am ined the pubH shed degrad ation studies in soil from the unsaturated zone. D uring the search for pub lish ed studies it becam e clea r that th e n u m b er o f studies perform ed in subsoil w as su b stan tially sm aller, than the nu m b er o f studies from the p lo u g h layer. O nly fo r the p esticides: m eco p ro p , 2,4-D , atrazin, alachlor, aldicarb, carbofiiran, linuron, oxam yl, m ethom yl, M C P A , dichlorprop, m onochlorprop, dichlo rp h en o l, T C A , parathion, m etribuzin, m eto lach lo r and fenam iphos, subsoil studies w ere reported. G oing th rough the publications, I firstly focused on the used m eth o d s to b e able to m ake a clear decisio n on w hich m ethod to use m y s e lf T he m ain p art o f th e p u b lish ed studies w as perform ed as laborato ry studies, w here the soil w as dried and sieved p rio r to th e studies. In alm ost all the exam in ed studies, p esticides w ere added to soil in co n cen tratio n s o f 0,5-5 n g g"' dry soil. In part o f the studies the d egradation w as follow ed b y m easu rin g the concen tratio n s o f residual pesticide durin g tim e. In another p art o f the studies, '"'C -Iabelled co m p o u n d s w ere used and the degradatio n (m ineralisation) w as follow ed b y m easu rin g the dev elo p m en t o f ' ‘’C O 2. In the last-m entioned studies the m ineralisation rate w as reported as % ' ‘'C O 2 dev eloped after a certain nu m b er o f days, w hich m ade a co m p ariso n o f m in eralisatio n rates betw een studies difficult. I concluded that degrad atio n studies in the laboratory should b e p erfo rm ed u n d er conditions that are as close to n atural circum stances as possible. D isturbance o f th e m icro -o rg an ism s that cause the d egradatio n o f m o st p esticides is avoided, by using u n d istu rb ed soil sam ples from subsoil. D egradation studies should be p erform ed in co n cen tratio n s c lo se to the actual probable co n centratio n s in soil, since the concentration o f th e p esticid e can effect the m icro organism s and th u s th e d egradation rate o f th e pesticide. F u rth erm o re, I h av e co n clu d ed that studies w hich include b o th the d evelopm ent o f '“C O 2 from ' “'C -lab elled pesticid e and those w here residual co n cen tratio n s o f the p esticide are d eterm ined, should b e p referred. M easuring ’“C O 2, th e total m ineralisatio n o f the com pound is m easured. S ince soil is a very heterogeneous environm ent, it is furtherm ore o f high im portance th at d eg rad atio n studies are perform ed w ith replicates. S econdly, w hile going thro u g h the p ublished studies I focused on the d escrip tio n o f degradation kinetics and the influence o f the soil en vironm ent on th e d eg rad atio n rate. P art o f the studies show ed that th e d egradation follow ed first o rd er kin etics, w h ile an o th er part. 14.

(17) sim ply assum ed that the deg rad atio n follow ed first o rd er kinetics. T hose studies rep o rted the degradation rate by m ean s o f the h alf-life tim e. H ow ever, som e o f th e studies show ed th at the d egradation did N O T follow first o rd er kinetics, b u t could b etter b e d escrib ed b y m eans o f em pirical equations or by m ean s o f a p o w er rate m odel w ith a reactio n o rd er d ifferen t from first order.T he factors m en tio n ed as b ein g o f im portance for the d eg rad atio n rate o f the pesticid es w ere b iological activity, soil tem perature, soil w ater content, o x ygen con ten t in soil air, pesticide concentration, soil type and adaptation o f the m icro -o rg an ism s after rep eated use o f a pesticide. T he influence o f all the m entioned factors on the d eg rad atio n o f th e pesticid es w ere all described separately. T he reading o f the publish ed studies m ade m e give a high prio rity to th e follow ing a) to study the degrad ation in plough layer as w ell as in subsoil b) to p erform m y o w n studies in subsoil w ith undisturbed soil sam ples c) to p erfo rm the d egradation studies at v ery low co n centrations (w hich w as only p o ssib le b y using ' ‘'C -lab elled com pounds) w h en th e fate o f the pesticid es after norm al agricultural use w as to be exam ined and d) to find a stan d ard ised w ay b o th to perform the studies and to d escribe the results.. II. I.S. Fomsgaard, 1997. Modelling the mineralisation kinetics for low concentrations of pesticides in surface and subsurface soil. Ecological Modelling, 102. 175-208. In the present publication I described m ineralisation studies for m eco p ro p , E T U and b entazon in concen trations as low as 0.04 n g g"‘, 0.07 )ig g’’ and 0.08 jig g '', respectively. S uch low concentrations o f p esticid es could typically b e presen t in subsoil after n orm al agricultural use o f the com pounds. T he low concentrations w ill n aturally be p resen t in p lo u g h layer too, at a certain tim e after the application. T he experim ents w ere p erfo rm ed w ith th e ad d itio n o f ‘‘Re labelled co m pounds to th e soil sam ples, incubation at 10°C, and the m in eralisatio n w as follow ed b y m easuring the evolved ’''C O 2. T h e data w as sh o w n as m in eralisatio n curves, d epicting the accum ulated am ount o f ' “*€02 as a function o f days. T he ex p erim en tal set-up m ade it possible to follow the m ineralisation at very low co n cen tratio n s and to follow the m ineralisation in each single soil sam ple, w ith o u t the need for tak in g o u t aliq u o ts (w hich is not possible w hen the soil sam ples are incubated w ith a n atural w a te r content). T he experim ents w ere p erform ed in sandy soil, sam pled tw o differen t y ears in th ree d ifferent fields in D enm ark and in soil w ith varying clay content from G erm any, S pain and Italy. D egradafion studies w ere perfo rm ed in soil from p lough layer (0-15 cm ) as w ell as in soil from varying depths, determ ined b y the ground w ater level at each site. T h e deg rad atio n studies in soil from p lo u g h layer w ere p erfo rm ed in distu rb ed sam p les (m ix ed and sieved), w hile undisturbed soil sam ples w ere u sed for the studies in subsoil. In m o st o f th e pub lish ed studies, in w hich ' “C O 2 w as m easured, the results w ere p resented as % '''C O 2 evolved after a certain nu m b er o f days. Such resu lts are difficult to com pare. T h erefo re th e p u rp o se o f the w ork in the p resen t p ap er w as to find a m ath em atical m odel, w hich could describ e the. 15.

(18) m ineralisation. A nu m b er o f m athem atical descriptions o f tran sfo rm atio n k inetics, used by o th er authors either to d escribe d egradation o f p esticides o r degrad atio n o f o th er xenobiotic com pounds, w ere tried out w ith the m ecoprop, E T U and ben tazo n m in eralisatio n data. 18 different m odels w ere u sed o f w hich som e w ere a) m odels w ith o u t g ro w th o f m icro organism s, expressing com etabolic d egradation (first order, zero order, tw o -co m p artm en t first order, com bined first + zero order, sequential first order, sim ple M on o d kin etics) b) m odels w ith grow th o f m icro -o rg an ism s (linear grow th, logarithm ic grow th, logistic grow th, exponential grow th) and c) em pirical m odels. M odels, w hich w ere used in th e literature to d escribe the disappearan ce o f added pesticide, w ere converted to express th e form ation o f m ineralisation produ ct ' ‘'C O 2 co m in g from ' “C -labelled p esticides. T he m o d els w ere fitted to the data using n on-lin ear regression.. % '*C as ’*COj. Figure 3.1. M ineralisatio n o f 0.08 n g g ' ' “C -bentazon in Spanish soil. D epth (0 and 45 cm ), replicate n u m b er and m odel equation from p ap er II show n at the end o f each curve. (F igure 2b from II).. T he w ork show ed th at a n u m b er o f m athem atical m odels w ere useful for th e d escrip tio n o f the m ineralisation o f the investigated pesticides. T here w as a clear d ifferen ce betw een m ineralisation kin etics w ith and w ith o u t grow th. W ith few exceptions, th e m in eralisatio n kinetics in plo u g h layer soil sam ples show ed to b e w ith o u t grow th o f m icro -o rg an ism s (com etabolic m ineralisation ). In subsoil - w ith o n ly a few exceptions as w ell - the m ineralisation kinetics show ed to be w ith grow th o f m icro -o rg an ism s (m etabolic). The com etabolic m ineralisatio n is seen in the d epicted m in eralisatio n cu rv e as a gradual rise in the. 1 6.

(19) accum ulated am ount o f '''C O 2 follow ed b y a deflection w h ereu p o n th e cu rv e turns flat (F igure 3.1. - 0 cm ). T he m etabo lic m in eralisatio n results in m in eralisatio n cu rv es h av in g a sigm oidal form , w ith a slow evolutio n in the b eginning (lag-phase), follow ed b y a h eav y increase in the form ation o f ''‘C O 2 for a perio d w hereu p o n the m in eralisatio n cu rv es turns flat (F igure 3 .1 .45 cm ). M ecoprop can be deg rad ed bo th through com etabolic and m etab o lic processes according to literature. It w as th erefore n o t a surprise that co m etab o lic d eg rad atio n occurred in ploug h layer w here a h ig h am ount o f o th er organic m atter is presen t, w h ich can serve as a nutrient for the m icro-org an ism s that carry o u t the d egradation o f m ecoprop. B en tazo n and E T U have been reported as com pounds w hich can on ly go thro u g h co m etab o lic degradation. T he m etabo lic m ineralisatio n seen in the presen t study co u ld b e due to th e form ation o f m etabolites, w hich could g ive rise to p ropagation o f m icro-organism s. A n o th er explanation could be that d egradation o f low co n centrations o f ben tazo n and E T U do follow kin etics w ith grow th o f m icro-organism s becau se o f the special living con d itio n s for m icro -o rg an ism s in subsoil (e.g. presen ce o f d orm ant m icro-organism s).. III. I.S. Fomsgaard, H. Johannesen, J. Pitty & R. Rugama, 1998. Degradation o f ' ‘'C-maneb in sediment from a Nicaraguan estuary. The International Journal of Environmental Studies B, 55,175-198.. In a jo in t p ro ject in N icarag u a th e influence o n an estuarine envirorm ient o f the use o f pesticide in the drainage basin w ere to b e investigated. A s p art o f th e pro ject, m in eralisatio n studies w ith m aneb in sedim ent from the estuary w ere perform ed. M an eb is u sed as a fungicide in N icaragua in the cu h iv atio n o f onions, beans, m aize, tobacco and tom atoes. T he m in eralisatio n studies w ere p erfo rm ed w ith a concentration o f m an eb o f 0.08 |ag g"' sedim ent (dry w eight), covered by 2 cm w ater from the sam pling site. T he stu d ies w ere p erfo rm ed b o th in July and S eptem ber 1994. S edim ent sam ples w ere taken at five sites, site 1 closest to the m outh o f the riv er and site 5 in the u p p er p art o f the river. A n u m b e r o f m ath em atical m odels taken from II w ere fitted to the m ineralisation data. T he b est fit to th e m in eralisatio n experim ents from the m onth o f July w as obtained w ith m ath em atical m o d els describ in g kinetics w ith grow th. T he experim ents from S eptem ber could b e describ ed w ith b o th m odels describing kin etics w ith grow th as w ell as w ith n o -grow th k inetic m odels. T h e m odel. Kcn. P = C o - (/c, „ -+k^c^)e‘ . _ ‘'' -k^c^. (3.1). w here P = am ount o f pesticid e m in eralised at tim e t (% '''C as '''C O 2 ). Co = total am ount o f pesticid e converted to ' ‘'C O 2 according to th e m o d elled process ki = rate constant. 17.

(20) k2 = rate constant t = tim e in days w as fitted to all the d ata curves, and a tw o-w ay analysis o f varian ce (A N O V A ) w as applied to com pare co and ki. A significant difference in co b etw een stations w as seen w h ere th e highest am ount o f ' “'C -m an eb w as converted to ' “C O 2 at the sites 4 and 5 in the u p p er p art o f th e river, probably because o f a hig h er b iological activity. A fter 150 days o f incu b atio n all the soil sam ples from th e ex p erim en ts from S eptem ber w ere ex tracted and the am ount o f residual ' “CE T U in the extract w as m easured. L ess than 2.72 % w as found. T h erefo re it m u st be concluded that the form ation o f E T U as a m etabolite, after the u se o f m an eb in the drainage basin, is not a problem .. IV. A. Helweg, I.S. Fomsgaard, T.K. Reffstrup & H. Sørensen, 1998. Degradation of different pesticide concentrations in soil. International Journal of Environmental Analytical Chemistry, 70(1-4) 133-148. P esticides can appear in the soil enviro n m en t w ith a w ide range o f co n cen tratio n d epending on w hich source they com e from . N orm al agricultural u se o f p esticid es (ex cep t the new low dose products) can lead to concentrations in the p lough layer o f about 1 |ig g “' and to concentrations in subsoil several tim es low er. D irect contam in atio n , w aste d isposal by b u rying etc. can lead to extrem ely high co n centrations o f pesticid es in th e soil environm ent. M any published studies have show n th at the d egradation rate o f p esticid es is in fluenced by th e initial concentratio n o f the com pound. In the present study, m in eralisatio n studies in concentrations from 0.0005 to 5000 n g g ' for ' “'C -m ecoprop and from 0.001 to 5000 n g g ' '■’C -isoproturon for iso proturon w ere perform ed. A ll studies w ere p erfo rm ed in soil from plough layer as w ell as in soil from 40 -6 0 c m ’s depth. T he ex p erim en ts w ere perfo rm ed by ad ding the ' “'C -lab elled com p o u n d to soil sam ples, w here each soil sam p le w as m ixed th oroughly w ith the com pound. T he m ineralisation w as m easured by co llectin g '''C O 2 and m easuring it in a scintillation counter. T he m ineralisation curves, total am o u n t o f ' ' ‘C as '''C O 2 in function o f n u m b er o f days, w ere fitted to a n u m b er o f m athem atical m odels, m odels describing kinetics w ith grow th as w ell as m odels d escribing kin etics w ith o u t grow th T he m ineralisation o f m ecoprop at 0.0005 n g g ' follow ed first o rd er kin etics b o th in p lo u g h layer and in subsoil. T he rate constant for the m ineralisation p rocess w as sig n ifican tly h ig h er in plough layer than in subsoil, pro b ab ly due to the low er biolo g ical activ ity in subsoil. K inetics w ith grow th w as seen at the concen tratio n o f 5 \ig g ' in bo th p lo u g h layer and subsoil and o f 50 fig g’' in plough layer. A t the co n centrations o f 50 and 500 n g g ”' in subsoil and o f 5000 Hg g ' in plo u g h lay er (concentrations w hich p robably have been toxic to th e m icro organism s) the m in eralisatio n w as very slow. F o r that reason the cu rv es could n o t b e fitted w ith any m odel. T he m in eralisatio n o f isoproturon follow ed kin etics w ith o u t grow th in all concentrations. A t th e h ig h est initial concentration o f iso proturon th e m in eralisatio n w as slow , but m easurable. A clea r d ifference b etw een m in eralisatio n rates in soil from d ifferent. 18.

(21) depths w as seen. T he m ineralisation in plo u g h layer w as faster than in soil from 40-60 c m ’s depth, probably because o f lo w er b iological activity in subsoil.. V. I.S. Fomsgaard and K. Kristensen, 1998. ETU mineralisation in soil under influence of organic carbon content, temperature, concentration and depth. Toxicological and Environmental Chemistry 70, 195-220. E T U is a toxic w ater-solu b le m etab o lite o f the E B D C fungicides. In the p resen t study the m ineralisation o f '^'C-ETU w as investigated in soil from tw o differen t dep th s (15 and 75 cm ), w ith tw o different concen tratio n s o f '''C -E T U (0.07 and 2.0 |ig g '') , at tw o tem p eratu res (5 and 20°C ), and w ith tw o differen t am ounts o f soluble carbon in the soil (a) natural; o n ly w ater w as added to obtain 50% W H C and b) added: an extract o f solu b le so il-carb o n w as added to obtain 50% W H C ). U nd istu rb ed soil sam ples w ere used, and th e m in eralisatio n w as follow ed by collection and quantify in g th e m ineralisation p roduct ''‘C O 2. In th e rev iew in p a p er I it w as said that in m ost p u blish ed subsoil degradation studies, the influence o f g eo-environm ental factors on the d egradatio n w as investigated, o n e factor at a tim e. C o n trary to this, th e present study w as designed as a 2“* factor study, w here the effect o n the m in eralisatio n rate o f '"'CE T U w as investigated for all the com binations o f th e tw o levels o f all 4 factors. A s described earlier, oth er pub licatio n s have show n th at is has n o t b een p o ssib le to find a m athem atical expression, w hich could d escribe the m in eralisatio n o f x en o b io tic com pounds under all circum stances. M y co nclusions in the papers II, III, and IV w ere th at one m athem atical m odel w hich could d escribe all types o f m in eralisatio n cu rv es d id n o t exist. Y et w ith different m athem atical expressions it w as p o ssib le to describ e all ty p es o f m ineralisation curves. O ne o f the m athem atical m odels, used in p ap ers II, III and IV to d escribe m in eralisatio n w ith grow th o f m icro-organism s, w as fiirther dev elo p ed in the p resen t study to. P =. ^ -+ c ,( l-e - * 0 (A:, +k.^c„)e ' -k^c^. (3.2). w here. P = total am o un t o f evolved m in eralisatio n pro d u ct ( ’‘'C O 2), equ iv alen t to the total am ount o f m ineralised ' ‘'C -p esticid e at tim e t (m easured as % '^’C evolved as ' “'C 0 2 ). c„ = total % ’“'C -p esticid e converted to ' ‘'C O 2 according to the L iu & Z h an g -m o d e l (L iu & Z hang, 1986). Cb = total % '^'C-pesticid converted to ' ‘’C O 2 according to th e first o rd er m odel ku ki = rate constants k i = k ( n io + A c J k 2 = -k Å. k} = rate con stan t for the first order process X = grow th rate o f the m icro-organism s mo = the initial am ount o f d egradation m icro-organism s. 19.

(22) T he m odel consists o f tw o term s, in w h ich the first term d escribes the m in erah sa tio n o f the '^'C-ETU that w as available for im m ediate d ecom position, w h ile th e seco n d term describ es the first o rd er m ineralisatio n o f o rganic m aterial, in w hich ' ‘'C from the p esticid e had been built in. T w o variants o f the m odel w ere used. M odel A , in w hich c„ + c* = 100% and m odel B, in w hich c„ + Cb< 100%. T he d eveloped m odel show ed to fit all m in eralisatio n curves, bo th w hen a long lag-phase follow ed b y a v igorous rise w as seen and w h en th e insp ectio n o f the curve resulted in dou b ts w h eth er a lag-phase w as presen t or not. A v ery u seful m ineralisation m odel w as thus developed, w hich p robably w ould b e useful for the d escrip tio n and the com parison o f the m in eralisatio n curves o f oth er x enobiotic com pounds. S ince the study w as b u ilt up as a 2'' factor study, it w as p o ssib le to in v estig ate the interaction effects betw een the ex am in ed factors. A three-w ay interaction effect depth*concentration * tem p eratu re w as seen for b o th c„, ki, k2 and V w». T h e interaction betw een tw o o f those factors (depth*concentration, d epth*tem perature, concentration*tem peratu re) thus depended o n the level o f the third factor. T h e three-w ay interaction effect dep th * co n cen tratio n * su sp en sio n w as only seen for c„, w h ile a tw o-w ay interaction effect concen tratio n * su sp en sio n w as seen for ki and k2. It w as th u s co n clu d ed that an investigation o f th e interactiv e effects o f the factors w hich influ en ce th e m in eralisatio n rate, is im portant w hen the m in eralisatio n o f'''C - E T U is to be described. S uch in vestigations w ould pro b ab ly be im portant for o th er co m pounds as w ell.. VI. I.S. Fomsgaard and K. Kristensen, 1999. Influence of microbial activity, organic carbon content, soil texture and soil depth on mineralisation rates of low concentrations o f '‘*Cmecoprop - development of a predictive model. Ecol. Mod. 122, 45-68. T his p u blication con tin u es the m o d ellin g w ork carried out in p ap er I. W e w o rk ed w ith all the m ecoprop m in eralisatio n studies from D anish soil and used the m o d el w h ich w as d eveloped in p ap er V:. P = c „ -------------- ------------------+ c , ( l - e - ‘>') (A:, + k ^ c j e ■ -k^c„. (3.2). w here. P = total am o un t o f evo lv ed m ineralisation p ro d u ct ( ‘''C O 2), eq u iv alen t to th e to tal am ount o f m ineralised '''C -p e stic id e at tim e t (m easured as % ' “’C evolved as ''*C02). c„ = total % '''C -p e sticid e converted to ' ‘'C O 2 according to the L iu & Z h an g -m o d el (L iu & Z hang, 1986). Cb = total % ' “'C -p esticid e converted to '''C O 2 according to the first o rd e r m odel ki, k2 = rate constants ki = k(mo + Åc„). 20.

(23) k2 = -kÅ ks = rate co nstant for the first o rd er process X = grow th rate o f the m icro-organism s mo = initial am ount o f degrad in g m icro-organism s T he param eters c„, c*, kj, k2 and k} w ere estim ated. T h e m odel gave u seab le fits for the m ecoprop m ineralisation curves from p lo u g h layer as w ell as from subsoil and th u s fulfilled o u r expectations after h av in g seen the ap p licability o f the m odel in p a p e r V. T h e relation betw een p aram eters c„, ct, ki, k2, k3 and the follow ing g eo-environm ental factors: b iological activity, M P N -num ber, % h um us, % clay, % sand, % silt, pH , solu b le C (m g k g '') N O 3 -N (m g kg '), N H 4 -N (m g k g ''), soil d epth w as determ ined. T he b io lo g ical activity w as d eterm in ed as the rate constant ki.„aac for th e m ineralisation o f '^'C-Na-acetate. It w as concluded, that the m ineralisation o f m ecoprop at the sam e tem perature and initial co n cen tratio n depends b o th on hum us content, clay content, b iological activity and soil depth. A full m odel describ in g the param eters c„, Cb, k/, k2, k 3, as a function o f soil depth, % hum us, b io lo g ical activ ity and % clay w as constructed and sub seq u en tly validated w ith m ecoprop m in eralisatio n resu lts fi-om G erm an soils. T he used functions w ere:. . lo g , k, = a , + ß , - lo g ,. %humus. „ , ,, --------+ A ■ploughlayer. 1 0 0 -Vohumus. (3.3). k^ = Ü 2. + ß , ■ploughlayer. (3.4). ÄTj = «3. + y?4 • ploughlayer. (3.5). 100-c „ lo g ,. \0 0 -c b. = « „ + A - l o g e ^ i_ « a « ^ ^. = a , + ß , - lo g ,. \ 0 0 -% c la y. (3-6). + ß i ■ploughlayer. (3.7). T he pred ictio n o f the initial lag-phase resu ltin g fi-om the m odel w as n o t optim al, h ow ever, the m odel w as able to p red ict the tim e, w hen no m ecoprop w as left. It w as thus sh o w n th at it is po ssib le to develop a m in eralisatio n m odel for m ecoprop, w ith w hich th e m in eralisatio n rate can be predicted on the b asis o f easier m easurable param eters.. 21.

(24) 4. Synopsis of the investigated pesticides. T he ch oice o f w hich p esticid es to investigate w as based on th eir use, leachabiU ty and/or existence o f im portant m etab o h tes. F orm erly m ecoprop w as used in h ig h am o u n ts in D enm ark in the autum n. D egrad atio n at low tem peratures is th erefo re p articu larly interesting.. NHCON(CH3)2- ^. NHCONH2 T7TTT. NH2. ?. /7 ^. .. ^. SE Proposed pathway of photodecomposiiion (P) and degradation of isoproturon in soil(s) I s N-(4*isopropyl phenyl) -N ’.N’-dimcthyl urea II s N-(4-isopropyI phenyl) -N ’-meihyl urea III » N-<»sopropyI phenyl) urea IV ss 4-(isopropyl) aniline V = 4,4'-diisopropy1 azobenzene V I s 4,4'-diisoprbpy] azoxybenzene V II s N-(4-(2-Kydroxyisopropyl) phenyl) -N ’-methylurea V ill a 4-(2-hydroxy isopropyl) phenyl urea IV s 4-(2-hydroxy isopropyl phnyl) aniline. Figure 4.1. P roposed m etabolic p athw ays for isoproturon in soil (S) and b y pho to ly sis (P) (K ulshrestha & Singh, 1995. (C opyright G ordon and B reach P ublishers. R ep ro d u ced w ith perm ission).. B entazo n has proved to be leachable in several countries, am ong them S w eden (K reuger, 1997), for w hich reaso n th e d egradation rate m ust be know n both in p lo u g h layer and in subsoil. A ccording to th e literature, the fungicide m aneb is read ily d eg rad ed to th e m etabolite E T U , w hich is suppo sed to be carcinogenic (N ational R esearch C ouncil, 1987). T he am ount o f isoproturon, used in D enm ark, has increased during the last years, becau se iso proturon in m any crops replaced th e phenoxyacids, o f w hich the use w as restricted y ears ago.. T able 4.1 show s the chosen p esticides, the sales figures, to x icity and p h y sical-ch em ical properties. P roposed d eg rad atio n pathw ays for the first steps o f the d eg rad atio n o f isoproturon and b entazon are show n in F igures 4.1 and 4.2. Figure 4.3 show s th e total m in eralisatio n o f m ecoprop and F igure 4.4 show s p roposed p athw ays for the total m in eralisatio n o f the E B D C fungicides m aneb and m ancozeb.. 22.

(25) Table 4.1. Summary o f properties o f investigated pesticides. M ecoprop. Bentazon. Maneb. Ethylene thiourea (ETU). Isoproturon. Systematic name lUPAC. 2-(4-chloro-otolyloxy)propionic acid. m anganese ethylenebis(dithiocarbam ate). 4,5-dihydroim idazole-2(3H )thione. 3-p-cum enyl-1, 1-dim ethylurea. Herbicide(H), Fungicide(F), M elabolite(M ) Sales figures (kg a.i.) Denmark 1994 Sales figures (kg a.i.) Denmark 1995 Sales figures (kg a.i.) Denmark 1996 Sales figures N icaragua Vapour pressure Solubility w ater (25°C) Use. H. 3-isopropyl-1H -2,1,3benzothiadiazin-4(3H )-one 2 ,2 -dioxide H. F. M. H. 2 9 1 .4 0 2 ”. 69 .3 5 2 '’. 256.072". 313.287 '. 9 3 .3 2 6 '. 2 5 1 .2 4 6 '. 210 .8 3 8 ''. 80.577'*. 0. Name Chemical formula. LD 50 m am m als m g kg ' LD 50 birds m g kg'. 0.00031 Pa* 0.62 g r ' ^ Cereals/ grass for seed p ro d u ctio n '' 1166 * 5000“. LD 50 w orm s mg kg '. .. 4 5 3 .1 6 8 '. .. 523.547“' .. .. 9. .. 0.00046 Pa“ 0.5 g i ' “ cereals/grass. neg“ 0.16 g r ' “ onions, beans, maize, tobacco. to m a to e s') 750“ 5000“. -. 1710“ 5000“ 10 0 0. “. “. 346.767'’. *. “. -. . -. g l '*. 0.055 g r ' * c e re a ls '' 1800* 10 0 0 * *. “. -. 10 0 0. *. -. 9* 10 0 0 0 0. 10 0 0. .. 20. LC50 fish mg r '. 10 0. LC 50 daphnia mg r '. 420“. LCso algae m g l '. 2 2 0. Cancerogenity. -. References. '’)M iljøstyrelsen, 1995; ‘)M iljøstyrelsen, 1996; ‘‘)M iljøstyrelsen, 1997; “)PC-Planteværa: “N a tio n a l Research Council, 1987; ®) lU PA C, 1977. ) In D e n m ark ,') In Nicaragua. “. 10 0. 0 2 2. 125*. 0.52*. -. 47’. 0.43*. -. -. 0.03“ cancerogenic and teratogenic in laboratory animals*^. *.

(26) c^ 6. H. Bentazon. B s "ortho-quinotd structural elements". F igure 4.2. P roposed degrad atio n p athw ays for b entazon in soil (H u b er & O tto, 1994). (C opyright Springer-V erlag. R eproduced w ith perm ission). CH3. I. 0-CH. - COOH. C. I. c M CPP Figure 4.3. M ineralisatio n o f m ecoprop in soil.. 24.

(27) ETD eihyleneihhiramdisulfide EDI ethylene diisoihiocyanaie F igure 4.4. D egradation p athw ays for E B D C fungicides in soil. A dap ted from W H O (1988) and IU P A C (1977). (F igure 12 from III).. 25.

(28) 5.. Synopsis of the results and the discussions. 5.1.. The mineralisation kinetics in relation to geo-environmental factors. 5.1.1. The experiments T he pesticide m ineralisation experim ents in soil w ere p erfo rm ed b y follow ing the ev olution o f '''C O 2 from ' ‘*C-labelled pesticide. Soil sam ples from the p lo u g h layer w ere m ixed w ith the added ''*C-labelled pesticid e and incubated in E rlenm eyer flasks. S ubsoil sam ples w ere taken as u ndisturbed sam ples in m etal tubes and the '''C -lab elled p esticide w as added to the soil by injection or b y drippin g before incubation. T he use o f ' “C -labelled p esticid e assured th at the com pound could be qu an tified in very low concentrations. Inv estig atio n s o f th e degrad atio n o f pesticides in very low concen tratio n s are im portant, since o th er studies earlier show ed th at the degradation o f x enob io tic com pounds in the soil en vironm ent d ev elo p s d ifferen tly at d ifferent initial concentrations o f the com pound (H elw eg, 1993; S tenström , 1988; Jaco b sen & Pedersen, 1992). C om m on agricultural use o f a p esticide leads n o rm ally o n ly to low concentrations o f the p esticid e in soil below the p lough layer. A rep resen tativ e aliquot o f a soil sam ple can n o t be taken durin g incubation. T he u se o f ' “'C -lab elled co m p o u n d can therefore furtherm ore assure th at the m ineralisation in each soil sam ple can b e follow ed during tim e, by m easu rin g the evolved '''C O 2. Figure 5.1, 5.2 and 5.3 show th e m ineralisatio n o f m ecoprop in G erm an soil, ben tazo n in G erm an soil and E T U in D an ish soil. %'‘C as ’"CO 2. Days. Figure 5.1. M ineralisatio n o f 0.04 p,g g '' ' ‘'C -m eco p rop in G erm an soil. D epth (0 and 75 cm ), replicate and equatio n nu m b er from p ap er II is show n at the end o f each cu rv e (F igure Ih from II).. 26.

(29) Figure 5.2. M ineralisatio n o f 0.08 (xg g ' b entazon in G erm an soil. D epth (0 and 75 cm ), replicate and equation nu m b er from p ap er II is show n at the end o f each cu rv e (F igure 2c from II).. % - C a s ’- C O ,. Figure 5.3. M in erah satio n o f 0.07 |ig g"' E T U in D anish soil (FB 3_II). D epth (15, 45 and 75 cm ), rep licate and equatio n n u m b er from p ap er II is show n at th e end o f each curve (F igure 3b from II).. 27.

(30) 5.1.2. The choice o f kinetic models B ru n n er & F ocht (1984), Jacobsen & P edersen (1992), S tenström (1988), Scow et al. (1986) and R effstrup et al. (1998) all stated th at under v arying circum stances (several soil depths, different concentratio n s) a one and only m athem atical m odel d escrib in g all th e m ineralisation curves did not exist. B ru n n er & F ocht (1984) declared that w ith th eir d ifferen t m o d els at least one o f them fitted to th eir m ineralisation curves. Jaco b sen & P ed ersen (1992), S cow et al. (1986) and R effstrup et al. (1998) p ointed out that even i f th ey used the m o d els g iven by B ru n n er & F ocht (1984) or the further d eveloped m o d els p resented b y F o ch t & B runner (1985) cases w ere seen in w hich no m odel at all could fit the m in eralisatio n curves. T he existence o f a m athem atical d escription o f the m ineralisation is o f d ecisiv e im p o rtan ce for a trustw orthy com pariso n o f m in eralisatio n rates. T herefore, I ex am ined w h e th e r a n u m b er o f theoretically as w ell as em pirically founded m athem atical expressions, used in th e literature to describe the d egradatio n k in etics o f x enobiotic com pounds in soil and w ater (T ab le 5.1), w ere useful for d escribing th e m in eralisatio n k inetics for m ecoprop (II), E T U (II), b en tazo n (II) and m aneb (III) in extrem ely low concentrations, and o f m eco p rop and iso proturon in a w ide range o f concentratio n s (IV). T able 5.1 furtherm ore contains th e m o d els w hich w ere subsequently developed (V and VI). W hen the d egradatio n is follow ed by m easu rin g the evolved ’‘'C O 2 from ''’C -labelled pesticide, the results co v er the total m ineralisation o f the added, as already explained. H ow ever, the m in eralisatio n is likely to proceed through several steps o r to o ccu r in various com partm ents. T he very sim ple m athem atical expressions w ill th erefo re seld o m b e useful for the d escription o f m in eralisatio n results.. 28.

(31) Table 5 .1 . M ath em atical m odels used to describ e the m in eralisatio n o f. C -lab elled p esticid es in p ap er II, I I I , IV , V , and V I.. Equation. First order. Equation no. in Growth/no-growth paper. P=c„(l-e-*'). (5-1). II-6 , IV-2. no growth. References. Knaebel et al., 1994; Simon eta!., 1992; Mueller et al., 1992;. P = amount of pesticide mineralised at time l (% '*C as '^CO2 ) Co = total amount of pesticide converted to ‘^CO2 k = rate constant for the mineralisation I - time in days. First order (co=100). no growth. (5.2). P = amount of pesticide mineralised at time i (% '^C as '‘‘CO^) k = rate constant for the mineralisation I - time in days Two-compartment first order (c/+c2 < 1 0 0 ). P = c,{\-e-. (5.3). II-8 , III-l, IV3. no growth. Scowet al.. 1986; Hill & Schaalje. 1985;. (5.4). 11-9, III-2. no growth. II, III. P - amount of pesticide mineralised at time i (% ''*C as ‘^CO2 ) Cl = total amount of pesticide converted to 'V O 2 through one first order process Ci = total amount of pesticide converted to '*C0 2 through another first order process k,. k: = rate constant for the two first order processes I = time in days Two-compartment first order (a+(l-a))=l). P - amount of pesticide mineralised at time t (% ‘^C as '*C0 2 ) fl = fraction of the total amount of pesticide converted to '*C0 2 through one first order process. kt. k: = rate constants for the two first order processes. N> VO. / = time in days (can be replaced with eq. (3) with c/+c: =100).

(32) U) o. Model. Equation no. in Growth/no-growth paper. Ek|uation. Three half order without growth. no growth. Brunner & Focht, 1984; Scowet al-, 1986; ICnaebel etal., 1994;. 11-10, III 5. growth. Brunner & Focht, 1984; Scowet al., 1986; Knaebel etal., 1994;. (5.7). II 12. no growth. Simkins & Alexander, 1984. (5.8). IM 4, III-6 , IV-5. growth. Simkins & Alexander, 1984; Albrechtsen & Winding, 1992. (5.5) IV-4. P = amount of pesticide mineralised at time t (% '*C as '^CO2 ) Ca= total amount of pesticide converted to '*C0 2 through the first order process ki - rate constant for the first order process ko = rale constant for the zero order process t - time in days Three half order with linear growth. References. (5.6). P = amount of pesticide mineralised at time t (% ‘^C as '^COj) Co= total amount of pesticide converted to '*CÖ2 through the first order process ki - rate constant for the first order process ko - rate constant for the zero-order process k: = the growth rate constant for the micro-organisms / - lime in days. de. Simple Monod without growth. dt. k, (_c„ - c ) + (<^0 - c ). c= amount of pesticide at time / C(7= initial amount of the pesticide ki= rate constant for the degradation km= the half samration constant t = time in days Logistic growth. P = c „ --. P = amount of pesticide mineralised at time t (% '*C as **C0 2 ) Co- total amount of pesticide converted to '^CO2 through the first order process xo - the amount of substrate (pesticide) necessary to produce the initial population density k - rate constant for the mineralisation t = time in days.

(33) Model. Equation. P =c , -. Logistic growth + zero order. k\(C(,+Xo)l. ■+ V. (5.9). Equation no. in paper. Growth/no-growth. References. IV- 6. growth. IV. 11-15. growth. Simkins & Alexander, 1984. 11-13, IV-1. no growth. Simkins & Alexander, 1984 Schmidt et al., 1985. 11-16. growth. Schmidt et al., 1985. 11-18« I1I-8. growth. Schmidt etal., 1985. Co P = amount of pesticide mineralised at time I (% '*C as '^CO2 ) Co- total amount of pesticide converted to ‘‘COj through the first order process xo = the amount of substrate (pesticide) necessary to produce the initial population density k = rate constant for the mineralisation ko - rate constant for the zero order process I ~ time in days (5,10). Logarithmic growth. P = amount of pesticide mineralised at time i (% '^C as ‘^CO2 ) xo= the amount of substrate (pesticide) necessary to produce the initial population density maximum specific growth rate t= time in days. P = Kt. zero order. (5.11). P = amount of pesticide mineralised at time t (measured as % '*C as '^CO2 ) ko =rate constant / = time in days Logistic growth. (5.12). P = amount of pesticide mineralised at time l (% '^C as ’^CO2 ) Co= total amount of pesticide converted to '*C0 2 through the modelled process k = rate constant ^ relation between initial population density and maximum population density r= maximum specific growth rale t= time in days Linear growth, low concentration of pesticide. (5.13). P = artKJunt of pesticide mineralised at time I (% '*C as '^CO2 ) Co= total amount of pesticide converted to '*C0 2 through the modelled process k, =rate constant for the first order process k: =linear growth rate constant for micro-organisms.

(34) U) K). Model. Equation. Equation no. in paper. Growth/no-growth. References. 11-17, m-7,. growth. Schmidt etal.,. 1985;. 1985;. t — lime in days Exponential growth, low concentration of pesticide. P = Co-Coe-'*'^X'"-'>. (5.14). IV-9. P - amount of pesticide mineralised at time t (% '*C as '^CO2 ) Co ~ total amount of pesticide converted to ‘*C0 2 through the modelled process k = rate constant for the exponential mineralisation r - maximum specific growth rate I - time in days Exponential growth -♦ zero order, low concentrations of pesticide. (5.15). IV-10. growth. IV- 1 1. growth. Schmidt et al... IV-12. growth. IV. P = amount of pesticide mineralised at time t (% '*C as '*COi) Co = total amount of pesticide converted to ' V O 2 trough the modelled exponential process k = rate constant for the exponential process ko = rate constant for the zero order process r - maximum specific growth rate I = time in days. Exponential growth, high concentration of pesticide. p =k. (5.16). P - amount of pesticide mineralised at time i (% '^C as '^CO2 ) k = rate constant for the exponential process r = maximum specific growth rate I ~ time in days Exponential growth + zero order, high concentration of pesticide. Empirical. (5.17) r P - amount of pesticide mineralised at time t (% '*C as '^COj) k - rate constant for the exponential process ko - rate constant for the zero order process r = maximum specific growth rate t = time in days. P = kt'' + a P - amount of pesticide mineralised at time / (% ‘V as '^CO2 ). (5.18). 11-20. Stenström, 1988.

(35) Model. Eiquation no. in paper. Equation. Growth/no-growth. References. A - constant a = constant I = time in days Empirical. P = k,t + k / ^ + a P = amount of pesticide mineralised at time / (% k, ^constant k: = constant. (5.19). 11-21. Stemström, 1988. as '*CÖ2 ). fl - constant t = time in days Stenström, 1988. Empirical + exponential growth. P = k / ' + ‘^^^ ( e ‘= '- l ) K. (5.20). 11-21. growth. (5-21). 11-23, III-4. no growth. Jandell Scientific, 1994. (5.22). 11-19, III-9, IV-7. growth. Liu & Zhang, 1986; Liu et al., 1988;. P = arrrøunt of pesticide mineralised at time t (% ‘*C as '^CO2 ) k, =constant k: = rate constant for growth of micro-organisms q - maximum specific rate of metabolism No = initial amount of micro-organisms I - time in days First order sequential. k. -. k. P = amount of pesticide mineralised at time t {% '^C as '^COz) Co = total amount of pesticide converted to through the first order process ki. k: = rate constant for the two first order processes / = time in days Logistic growth. P = c,-. U) U). (A:, +k^c^)e '' -k^c,,. P = amount of pesticide mineralised at time t (% '*C as '^CO:) Co= total amount of pesticide converted to '^COj through the modelled process ki - rate constant k: = rate constant / = time in days.

(36) U) -p^. Equation no. in. Equation. Model. Growth/no-growth. References. growth. IV. V-1,2,3, VI2,3,4. growth. V,VI. V-1,2,3, VI2,3,4. growth. V,VI. paper Logistic growth order. h. IV-8. P = c ----------------— -------------- + (Ä:, +. (5.23). “ ^2^0. P = amount o f pesticide mineralised at time / (% '^C as '^C O :) Co - total amount o f pesticide converted to '^CO2 by the modelled process ki = rate constant k: = rate constant ko = rate constant for the zero order process t = time in days Logistic growth • first order (c„ + c/,= 100). P = c {k ,+ k ,cy" -k ,c_ P - total arrK)unt o f mineralisation product ('^CO2 ), equivalent to the total amount of mineralised '^C-pesticide at time / (measured as % ‘^C evolved as '^CO:). c„ = total % '^C-pesticide converted to '^CO2 according to the Liu & Zhang-model Ch - total % ‘^C-pesticid converted to '^COj according to the first order model ki. k: =rate constants k, = k(m„ + k: = -kX ki = rate constants for the first order process X = growth rate o f micro-organisms. fti(^ initial amount o f degrading micro-organistns Logistic growth - first order. P = C -. c„ + Cft< 100. {k, +k^c„)e^'' - k ^ c , P = total amount o f mineralisation product ( '* C O i ), equivalent to the total amount of mineralised '*C-pesticide at time ( (measured as % '*C evolved as '‘CO 2) c„ = total % '*C-pesticide converted to "C O 2 according to the Liu & Zhang-model ct = total % ''C -pesticid converted to '‘CO 2 according to the first order model ki. k; =rate constants ki k; ki X. = k(mo + ÅcJ = -kX = rate constants for the first order process - growth rate o f micro-organisms. mo= initial amount o f degrading micro-organisms.

(37) B runner & F ocht (1984), S cow et al. (1986), S tenström (1988), K n aeb el et al. (1 9 9 4 ) and Sim on et al. (1992) developed an d /o r used th eir m athem atical m o d els for ' ‘'C O 2 m ineralisation curves, the rest o f the m ath em atical expressions in T ab le 5.1 are m y conversions o f the m athem atical expressions that o riginally w ere p resen ted b y the authors for the description o f d eg rad atio n curves, in w hich residual co n cen tratio n s w ere m easured. The m odels w ere fitted w ith n o n -lin ear regression using the pro ced u re N L IN from SA S (SA S, 1989; SA S, 1990; SA S, 1996). In som e cases the fit resulted in asy m p to tically correlation coefficients betw een p aram eter estim ates w hich w ere so h igh th at it w as im p o ssib le to estim ate the param eters o f th e m odel. In other cases, p aram eters, w hich w o u ld o n ly have m eaning w ith p ositive estim ates, turned out w ith neg ativ e estim ates. B o th situ atio n s lead to the conclusion that the m odel could not b e em ployed. C onseq u en tly no m ean square values are show n in the S um m ary tables in papers II, III and IV. T he co m p ariso n o f various m odels, resulting in u seable fits for one data set, w as carried out b y co m p arin g th e m ean square values. T he best m odel cam e out w ith the low est m ean square value. T able 5.2 show s a sum m ary o f all m y experim ents, giving info rm atio n about the sam pling site, the pesticide, the in cubation technique, the con cen tratio n o f th e p esticid e, th e sam pling depth, the soil texture, the incubation tem perature and g row th/no grow th. G row th/no grow th indicates w h eth er the n o n -lin ear regression resulted in useable fits w ith m o d els, w h ich include grow th o f m icro-organism s. F urtherm ore the num ber o f p aper, II, III o r IV, w h ere th e results w ere presented, is show n in th e table. S om e o f the results from p a p er II w ere used again in p ap er VI.. 5.1.3. Mineralisation kinetics in plough layer at low concentrations O N L Y m odels not including grow th o f m icro-organism s gave u seab le fits for E T U (0.07 |jg g ') in all soil sam ples in p lo u g h layer from Fladerne B æ k, D enm ark, for b en taz o n (0.08 |ig g' ‘) and m ecoprop (0.04 |ig g ') in p lo u g h layer from Italy, S pain and G erm any, and for m ecoprop (0.04 \\.g g ’) at 3 o u t o f 5 sam pling sites/tim es in D enm ark (F lad ern e B æ k) (II) (T he sam ples in p a p er II are identified as for exam ple m c fb l l w h ich m ean s m ecoprop. F laderne B æ k, field 1, tim e o f sam pling I). T he m ecoprop ex p erim en ts w ere late r used in p ap er VI, in w hich they w ere only identified by site/tim e o f sam pling, for ex am p le F B I I (T able 5.2). V in ter (1998) counted the n u m b er o f m icro -o rgan ism s in soil from v arying depths from F laderne B æ k b y staining w ith acridin o range and found th at the n u m b er o f m icro-organism reduced from 10® to 10^, m oving from p lo u g h lay er to 1 m e te r’s depth. In soil from all sites/depths and tim es o f sam pling from Fladerne B æ k, I su b seq u en tly d eterm ined the nu m b er o f m ecoprop-deg rad in g m icro-organism s b y a '''C -M P N -m eth o d (VI). N o significant d ifference betw een depth s (0 ,4 5 and 75 cm ) w as seen for the M P N num bers. M ecoprop has been reported as deg radab le m y m etabolism (L appin et al., 1985). W hen a com etabolic. 35.

(38) T able 5.2. S um m ary o f experim ents from papers II, I I I and IV sho w in g sam p lin g site, the pesticide, the concen tratio n o f th e pesticide, th e in cubation technique, th e co n cen tratio n o f the pesticide, the soil texture, th e in cubation tem perature and gro w th /n o gro w th for th e applied m odels. Paper. 36. Site. Pesticide. Incubation. Cone.. Soil depth Humus. Clay. technique. ng g '. cm. %. %. pH. Inc.. FBI J. mecoprop. disturbed. 0.04. 15. 3.1. 5.0. 7.1. F B IJI. mecoprop. disturbed. 0.04. 15. 2.8. 3.6. 6.9. FB3_I. mecoprop. disturbed. 0.04. 15. 2.7. 3.2. 6.6. FB3_1I. mecoprop. disturbed. 0.04. 15. 2.8. 4.0. 6.7. FB4-I. mecoprop. disturbed. 0.04. 15. 4.7. 4.6. 5.2. 10 10 10 10 10. F B IJ. mecoprop. undisturbed. 0.04. 45. 0.9. 3.0. 6.2. 10. F B IJI. mecoprop. undisturbed. 0.04. 45. 0.3. 2.5. 6.3. 10. FB 3J. mecoprop. undisturbed. 0.04. 45. 0.8. 2.3. 6.1. 10. FB3_II. mecoprop. undisturbed. 0.04. 45. 0.9. 3.5. 5.6. 10. FB4-I. mecoprop. undisturbed. 0.04. 45. 5.1. 3.6. 5.2. 10. F B IJ. mecoprop. undisturbed. 0.04. 75. 2.5. 5.9. 10. F B IJI. mecoprop. undisturbed. 0.04. 75. 2.1. 6.4. 10. FB 3J. mecoprop. undisturbed. 0.04. 75. 0.2 O.i 0.2. 1.4. 6.1. 10. F B 3 J1. mecoprop. undisturbed. 0.04. 75. 0.3. 3.0. 5.5. 10. FB4-I. mecoprop. undisturbed. 0.04. 75. 0.5. 2.1. 5.6. Italy. mecoprop. disturbed. 0.04. 3.6. 16.6. 7.2. 3.5. 30.5. 8.1. 2.1 0.6 0.6. 7.9. 7.4. 10 20 20 20. 20.9. 7.5. 15. 21.1. 7.1. 15. 3.7 ♦. 30.1. 8.2. 15. 0.2 0.1. 9.7. 6.6. 6.9. 7.1. 3.6. 16.6. 7.2. Spain. mecoprop. disturbed. 0.04. Germany. mecoprop. disturbed. 0.04. 0 0 0. Italy. mecoprop. undisturbed. 0.04. 50. Italy. mecoprop. undisturbed. 0.04. 50. Spain. mecoprop. undisturbed. 0.04. 45. Spain. mecoprop. undisturbed. 0.04. 45. Germany. mecoprop. undisturbed. 0.04. 75. Germany. mecoprop. undisturbed. 0.04. 75. Italy. bentazon. disturbed. 0.08. 30.5. 8.1. 2.1 0.6 0.6. 7.9. 7.4. 20.9. 7.5. 15. 21.1. 7.1. 15. 3.7 *. 30.1. 8.2. 15. 9.7. 6.6. 10. 7.1. 10. 6.9 6.7. 10. 15. 0.2 0.1 2.8 2.8. 0.04. 45. 0.3. 6.3. 10. undisturbed. 0.04. 45. 0.9. 5.6. 10. ETU. undisturbed. 0.04. 75. 0.1. 6.4. 10. FB 3JI. ETU. undisturbed. 0.04. 75. 0.3. 5.5. 10. Nicaragua. maneb. dist. sed.. 0.08. 25. maneb. dist. sed.. 0.08. 0.2 1.1. 9.0. Nicaragua. 0-10 0-10. 8.9. 25. Nicaragua. maneb. dist. sed.. 0.08. O-iO. 1.7. 7.6. 25. Nicaragua. maneb. dist. sed.. 0.08. 0.9. 7.7. 25. Nicaragua Flakkebjerg Flakkebjerg Flakkebjerg. maneb mecoprop mecoprop mecoprop. dist. sed. disturbed disturbed disturbed. 0.08. 0-10 0-10. 0.4. 7.9. 25. 0.0005 0-30 0-30 0-30. Flakkebjerg. mecoprop. disturbed. 5000. 0-30. 2.9. 6.1 6.1 6.1 6.1. 15. 5.0 50. 2.9 2.9 2.9. bentazon. disturbed. 0.08. bentazon. disturbed. 0.08. Italy. bentazon. undisturbed. 0.08. 50. Italy. bentazon. undisturbed. 0.08. 50. Spain. bentazon. undisturbed. 0.08. 45. Spain. bentazon. undisturbed. 0.08. 45. Germany. bentazon. undisturbed. 0.08. 75. Germany. bentazon. undisturbed. 75. F B IJI. ETU. disturbed. 0.08 0.04. FB 3JI. ETU. disturbed. 0.04. F B IJI. ETU. undisturbed. FB3_II. ETU. F B IJI. 15. no fit. no fit. 10. 3.5. Spain Germany. Growth. 15. 10 20 20 20. 0 0 0. No growth. temp. °C. 15. 10. 15. 15 15.