sdarticle10

Uptake of soil applied paclobutrazol in mango

(Mangifera indica L.)and its persistence in fruit and soil

Debi Sharma *,Mahesh D.Awasthi

Soil Science and Agricultural Chemistry,Indian Institute of Horticultural Research,Hessaraghatta,

Hessaraghatta Lake P.O.,Bangalore 560089,Karnataka,India Received 26August 2004;received in revised form 9December 2004;accepted 24December 2004

Available online 3February 2005

Abstract

Paclobutrazol is a plant growth regulator that is used to counter alternate bearing habit of mango (Mangifera indica L.).The uptake and persistence of paclobutrazol residues in mango fruits and soil respectively,was studied following its application at tree basin soil @5and 10g a.i.per tree for three consecutive years.Residues of paclobutrazol were found in unripe mango fruits at levels below permissible level while the same was generally not detected in fully mature mango fruits ready for harvest.There was no e?ect of consecutive yearly applications on the amount of paclobutrazol residues in mango fruit.However,the residues of paclobutrazol were found in tree basin soil (0–15cm)at the end of each season and there was a small increase in the amount of residues corresponding to the number of yearly applications that had been made.GC–MS analysis con?rmed that the sample peaks obtained corresponded to paclobutrazol residues present in mango and soil.

Keywords:Uptake;Dissipation;Mango;Soil;Paclobutrazol

1.Introduction

Mango (Mangifera indica L.)is the most important fruit crop of India and Alphonso,is one of the most popular mango cultivars grown here for domestic market as well as export.However,the production of Alphonso mango is low due to its distinctly alternate bearing habit.Paclobutrazol or (2RS,3RS)-1-(4-dichlorophenyl)-4-di-methyl-2-(1,2,4-triazole-1-yl)pentan-3-ol,is a plant growth regulator that has been very successfully used to counter this alternate bearing habit of Alphonso mango (Negi,

1999).Soil application of paclobutrazol has been found to be more responsive in regard to suppressing the vege-tative growth and enhancing the reproductive growth in mango than foliar application (Burondkar and Gunjate,1991;Singh,2000).Studies have shown that paclo-butrazol is needed to be applied annually to increase mango fruit yields (Yadava and Singh,1998).However,the paclobutrazol treatments to the tree basins (soil under the canopy drip area within a radius of 1.5m of the tree trunk)may result in its uptake into the trees and thereby result in persistence of its residues in mango fruit and also in the soil at tree basin.Such persistence of paclobutrazol residues in mango fruit may lead to ad-verse e?ects on human health.Persistence of paclo-butrazol residues in soil may result in contamination of nearby water bodies,which in turn may also be a

Corresponding author.Tel.:+918028466420;fax:+918028466291.

E-mail address:dsharma@iihr.res.in (D.Sharma).

Chemosphere 60(2005)164–169

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hazard to human and animal health.Persistence of pac-lobutrazol residues in soil may in?uence the soil micro-bial activity too.Soil microbial count of a mango orchard soil where paclobutrazol was frequently applied was shown to be reduced by up to58%(Silva et al.,2003). Although physiological persistence of paclobutrazol has been investigated in mango on the basis of?owering, fruit set etc.(Salazar-Garcia and Vazquez-Valvidia, 1997),the same has not been determined analytically from the perspective of environmental contamination. Similarly soil residues of paclobutrazol have been esti-mated by indirect bioassay method(Jacyna and Dodds, 1995),but the same have not been analyzed at trace levels by analytical techniques.

The objectives of the present investigations therefore, were(1)to estimate the uptake of paclobutrazol into mango fruits following its application at the tree basins,(2)to estimate the possible accumulation of pac-lobutrazol residues in mango tree following its yearly applications in soil for three consecutive years and(3) to estimate the paclobutrazol residues in the tree basin soil at the time of harvest.

2.Materials and methods

Paclobutrazol,analytical standard was obtained from M/s.Riedel-de Hain,https://www.360docs.net/doc/4016937760.html,anic solvents were obtained from M/s.Qualigens Fine Chemicals, India.Florisil was obtained from M/s.Sigma Chemical Co.,USA.

2.1.Experimental details

Field experiments were conducted at I.I.H.R.Farm, Bangalore,India.A small16-year old orchard with28 mango trees(Cv.Alphonso)in7rows of4trees each (30ft.row to row spacing)was selected for experimenta-tion.Paclobutrazol was applied as Cultar25SC(Pac-lobutrazol25%w/v)at the tree basins@20ml i.e.5g a.i.mixed in5l water per tree at lower treatment level and@40ml(10g a.i.)in5l water per tree at higher treatment level.The paclobutrazol treatments were poured uniformly in circular trenches(30cm wide and 30cm deep)around the trunk base at a radial distance of45cm from it.Five trees were treated with lower(re-commended)dose of paclobutrazol while another?ve trees were treated with the higher concentration of the same,each tree representing one replication of a treat-ment.The treatments were given on the last week of Sep-tember for three consecutive years,starting on September,2000.The trees were irrigated for at least 10days after application of paclobutrazol if there were no rains.The soil belonged to the textural class of sandy clay loam(taxonomic classi?cation—Kandic Hap-lustalfs).It had a pH(1:2.5)of7.09,EC of0.19dS mà1,CEC of13.88c.mol(p+)(kgà1)and organic carbon con-tent of0.5%.During the time of paclobutrazol applica-tion,the average temperatures were29.5°C(max)and 19.3°C(min),the average relative humidity at1.30pm was62.7%,average evaporation4.2mm and the average rainfall was138.9mm.

2.2.Sampling

2.2.1.Soil

Soil samples were collected from tree basins at the time of fruit harvest,i.e.,June?rst or second week, starting June2001using a soil auger.A homogeneous soil sample was drawn from each of the treated or con-trol tree basins.Soil was collected from three spots of each tree basin at0–15cm depth.Samples from control tree basins were collected prior to collecting samples from treated tree basins.The soil collected from each tree basin(%1kg)was pooled together,air-dried,clods broken,mixed thoroughly and sieved through a2mm sieve.5·50g soil was taken from the above for extraction.

2.2.2.Plant

Mango fruits were sampled from the treated trees at three stages of fruit development.Stage-I—pre-stone formation,Stage-II—stone formation and Stage-III—fully mature fruits ready to harvest.The fruits from Stages-I,II and III were sampled in the?rst week of April,May and June respectively in all the three years. About2kg of evenly sized fruits from each of the trea-ted trees were picked at each sampling,chopped into100 pieces,mixed,quartered and5·50g sample withdrawn for further processing.

2.3.Sample storage

All the samples were processed immediately and ana-lyzed within7days.

2.4.Analytical method

The analytical method followed was modi?ed from that of Stahly and Buchanan(1986).A50g soil sample was taken in an Erlenmayer?ask and extracted with 70ml of chilled methanol using a rotary mechanical sha-ker for1h at200rpm.The extract was?ltered using a Buchner funnel.The soil was re-extracted along with the?lter paper with2·50ml of chilled methanol.The extracts were?ltered and all the?ltrates combined. The combined?ltrate was diluted with30ml water, evaporated to aqueous phase in a rotary vacuum evapo-rator at35°C.The pH of the extract was adjusted to11 with1N sodium hydroxide solution.

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2.5.Partition

The above aqueous layer was partitioned against 3·50ml of dichloromethane in a500ml separating fun-nel.The lower dichloromethane layers were collected each time,passed through anhydrous sodium sulphate and combined.The combined extract in dichlorometh-ane thus obtained was evaporated under vacuum to 5ml.

2.6.Clean-up

A glass column(50cm·1.3cm)was packed with5g ?orisil in diethyl ether.The above extract was added slowly to the column.The column was eluted with 10ml of diethyl ether+methanol(97+3,V/V),the elu-ate collected and evaporated to dryness in a rotary evap-orator.The residues were immediately re-dissolved in 5ml of double distilled AR grade acetone and analyzed by gas liquid chromatograph(GLC).

2.7.Plant samples

Mango fruit samples,cut into%1cm cubes,mixed and quartered were homogenized using a high speed Waring Blender for1min,50g of the homogenized sam-ple was drawn and processed as per the same procedure as described for soil samples.

2.8.Instrumentation

2.8.1.GLC

Paclobutrazol was estimated quantitatively using a varian gas liquid chromatograph,Model-3800equipped with a thermionic sensitive detector(TSD).A capillary column Varian CPSil8CB(30m·0.53mm,df= 0.25l)was used.The temperature of the column oven, injector oven and detector were210°C,250°C and 300°C respectively.The gas?ow rates were7.6ml/min (nitrogen),4ml/min(hydrogen)and175ml/min(zero air).All gases used were high purity gases passed through moisture and hydrocarbon traps before passing through the GLC column.The carrier gas nitrogen was also passed through an oxy trap in order to block traces of oxygen from entering the column.The TSD bead current was set at3.0A.The retention time of pac-lobutrazol at the above parameters was13.2min.The limit of detection of paclobutrazol at the signal to noise ratio of4:1was0.001l g/g,the limit of quanti?cation (LOQ)of paclobutrazol residues in mango and soil was 0.01l g/g.

2.8.2.Gas chromatography–mass spectrometry

(GC–MS)

Paclobutrazol peak was further con?rmed using a Hewlett-Packard GC–MS,Model-6890.The column used was HP-5MS,5%,phenyl methyl siloxane,capil-lary30m·250l m·0.25l m(?lm thickness),carrier gas was helium(He)at a pressure of9.76psi,?ow rate of 1.0ml/min.The column oven temperature was 50°C(initial,hold time 2.0min),ramp rate—10°C/ min,260°C?nal temperature,hold time—20min). The injector was in splitless mode and the injection vol-ume was1.0l l.The injector and detector temperatures were260°C and280°C respectively,auxiliary tempera-ture was280°C.The emission current was34.6l A and amu gain was2324counts.Ionisation mode was electron impact at70eV.The GC–MS spectrum of paclobutrazol standard is shown in Fig.1.The possible fragments of paclobutrazol which result in the above spectrum are de-picted in Fig.2.

3.Results and discussion

3.1.Recovery

Stock solution of paclobutrazol(100l g/ml)was pre-pared in double distilled AR grade acetone.This stock solution was further diluted to obtain standard solutions of0.1,0.5,1.0and2.0,4.0,6.0and8.0l g/ml.Recovery study of paclobutrazol was carried out in soil and mango fruit.To50g soil0.5or1ml of5l g/ml pac-lobutrazol standard solution was added to obtain forti?-cation levels of0.01and0.1l g/g respectively.To this ?ask70ml of chilled methanol was added and left undis-turbed for1h.The analysis of paclobutrazol residues in the above forti?ed soils was carried out as per the proce-dure described in Section2.Fifty gram chopped mango fruit sample weighed in an Erlenmayer?ask was also similarly forti?ed with standard paclobutrazol solution and recovery of residues estimated.At each level of for-ti?cation in soil and fruit,samples were analyzed in trip-licate.The results of the recovery studies are given in Table1.The per cent mean recovery was83%from soil and79%from mango fruit.No substrate interferences were observed in any sample as compared to control at the detection level of0.001l g/g.

3.2.Uptake and dissipation of paclobutrazol in mango

In season-I,the paclobutrazol residues in mango (Stage-I)were0.011and0.089l g/g from the trees trea-ted with20and40ml/tree Cultar respectively.This, however,reduced in Stage-II and further reduced at?nal harvest(Stage-III)when paclobutrazol residues present in whole fruit or pulp of mango fruits were found to be below detectable limits(Table2),in all cases except 0.02l g/g in mango pulp after?rst year of application. In the subsequent years,i.e.,Seasons2and3,although, the residues increased only marginally in mango fruits at

166 D.Sharma,M.D.Awasthi/Chemosphere60(2005)164–169

the ?rst two stages,the same were not detected at the ?nal harvest stage of mango fruit.Thus,although pac-lobutrazol residues were present in raw mango fruit in all the three seasons,the same were not detected at the fully mature fruits at harvest.While no maximum resi-due limit (MRL)has yet been assigned to paclobutrazol residues in mango the same in apples (Codex Alimen-tarius,1993)is 0.5l g/g.Considering the same MRL here it can be concluded that paclobutrazol tree basin treatments to mango trees at the speci?ed dosages did not result in residues above permissible levels at any stage of mango even after three consecutive years of the treatments.It has been reported earlier (Singh and Ram,2000)that paclobutrazol residues in mango were higher in seed than in mesocarp of the harvested fruits.Paclobutrazol residues were also detected in ?Tommy Atkins ?mango at very low levels after two consecutive

years of application (Osuna-Garc?

′a et al.,2001).How-ever,the present study is the ?rst to document the persis-tence of paclobutrazol residues in ?Alphonso ?mango fruits at di?erent stages of crop growth following its application in tree basin soil.3.3.Persistence of paclobutrazol in soil

Paclobutrazol was applied at the soil at tree basins in the month of September every year.The persistence of paclobutrazol residues in this soil a little over 8months after application of same i.e.,at the time of fruit

harvest

Table 1

Recovery of paclobutrazol residues from mango fruit and soil

Mango fruit

Soil Recovery (%)79±2.283±3.4RSD (%)

2.3–2.5

3.5–3.8

Forti?cation level—0.05–1.00l g/g,limit of detection:0.01l g/g.

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was estimated.It was seen that paclobutrazol residues accumulated in the surface soil (0–15cm)at the tree ba-sins (Table 3)and that the soil residues at Season-I were 0.065and 0.302l g/g from the lower and higher treat-ments respectively.At the end of second and third sea-sons,the level of paclobutrazol residues increased progressively from both the treatment combinations (Table 3)indicating a cumulative e?ect of the residues in soil.The results of GC–MS analysis con?rmed the identity of paclobutrazol peaks in soil and plant samples and established that the residues recorded were indeed of paclobutrazol alone and not of any interfering substrate matrix component.The GC–MS spectra of pac-lobutrazol standard and the possible mass fragments are depicted in Figs.1and 2.While there is no reference in literature regarding the persistence and cumulative accumulation of paclobutrazol residues in soil at mango tree basins at trace levels,it is generally known that pac-lobutrazol residues have a high persistence in soil.Soil residues of paclobutrazol have been shown to be present 20months after its application in sweet cherry orchards (Jacyna and Dodds,1999)and for almost 3years after its application as trunk drench in apricot trees (Jacyna and Dodds,1995)using indicator plant bioassay.The extent of growth suppression of indicator plants was in direct relation to the treatment rate.The paclobutrazol residues in soil may therefore be carried into ground water by leaching and nearby water bodies through sur-face run-o?.

Thus,this study indicated that use of paclobutrazol to counter alternate bearing in mango continuously at recommended doses may not result in its residues in mango fruits at harvest at levels which may pose any

risk to human health.However,in areas where pac-lobutrazol is applied regularly,there may be risk of envi-ronmental contamination due to its residues persisting in soil for a very long time.There is,need,therefore to study the leaching potential of paclobutrazol in soil and methods to reduce the accumulation of residues at the tree basins.

Acknowledgment

The authors are grateful to the Director,IIHR for the necessary facilities used in the study and Dr.V.Par-dhasarathy of Rallis Research Centre,Bangalore,India for assistance in GC–MS analysis.

References

Burondkar,M.M.,Gunjate,R.T.,1991.Regulation of shoot

growth and ?owering in Alphonso mango with pac-lobutrazol.Acta Hortic.291,79–82.

Codex Alimentarius—Pesticide residues in food,1993.Joint

FAO/WHO food standards programme,second ed.F.A.O.,Rome,p.144.

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paclobutrazol on performance of ?Sundrop ?apricot (Prunus armeniaca L.)trees and on residue in the soil.New Zealand J.Hort.Sci.23(3),323–329.

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Table 3

Persistence of paclobutrazol residues in tree basin soil Season Residues in l g/g 5g 10g Season-I 0.0650.302Season-II 1.211 2.362Season-III

2.720

2.954

Table 2

Uptake and persistence of paclobutrazol residues in mango fruits Stage of sampling

Mean residues of paclobutrazol (l g/g)2000200120025g

10g 5g

10g I—Pre-stone formation 0.11(±0.03)0.089(±0.01)0.053(±0.01)0.141(±0.02)0.054(±0.00)0.160(±0.02)II—Stone formation BDL 0.050(±0.00)0.010(±0.00)0.012(±0.00)0.011(±0.00)0.017(±0.01)III—Full maturity (WF)

BDL BDL

BDL BDL BDL BDL (Pulp)

BDL

0.023(±0.01)

BDL

BDL—below detectable limits,limit of detection—0.001l g/g.

168 D.Sharma,M.D.Awasthi /Chemosphere 60(2005)164–169

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