PertanikaJ. Trap. Agric. Sci. 20(2/3): 147-155(1997) ISSN: 0126-6128© niversi ti Putra Malaysia Press

Phytotoxicity of Phenolic Acids Extracted from Pahn Oil Dry Solids

O. RADZIAH, M. RAHMANP and AZIZAH HASHIMDepartment of Soil SciencelDepartment of ChemistryUniversiti Putra Malaysia

43400 UPM Serdang, Selangor, Malaysia

Keywords: palm oil dry solids (PODS), phytotoxicity, bioassay, phenolic acid, vanillic acid

ABSTRAK

Pengekstrakan ke atas pepejal kering kelapa sawit (PODS) telah dilakukan bagi mengenalpasti kompaunterlarutkan air yang terlibat dalam kefitotoksikan PODS. Ekstrak akueus PODS telah dilakukan sekatanberturutan dengan beberapa pelarut organik. Setiap ekstrak kering telah dibiocerakinkan untuk aktivitiperencatan terhadap pertumbuhan radikel tomato. Perencatan maksimum ke atas pertumbuhan radikel diperolehipada ekstrak dietil eter, yang menghasilkan 53.3 % pertumbuhan berbanding kawalan. Pemisahan pecahan etermenggunakan kromatografi turus menghasilkan satu pecahan toksik, RM10, yang hanya dapat menampung30 % pertumbuhan radikel. Perbandingan pecahan tersebut dengan 14 kompaun fenolik sintetik ke ataskromatograf lapisan nipis menunjukkan persamaan antara empat kompaun tersebut. Analisis lanjut menggunakankromatografi cecair berkeupayaan tinggi menunjukkan pecahan RM10 terdiri dari asid vanilik. Namundemikian, pecahan RM10 lebih merencatkan pertumbuhan radikel tomato berbanding asid vanilik sintetik.

ABSTRACT

A study on the extraction of palm oil dry solids (PODS) was conducted to identify the water-soluble compoundsinvolved in the phytotoxicity of PODS. The aqueous extract of PODS was sequentially partitioned using variousorganic solvents. Each of the dried extracts was bioassayed for inhibitory activity on the growth of tomato radicles.Maximum inhibition ofradicle growth was observed in the diethyl ether extract, resulting in 53.3 % growth comparedwith control. Further separation of the etherfraction using column chromatography resulted in a single toxic fraction,RM10, which caused only 30% radicle growth. The fraction was compared with 14 synthetic phenolic compoundsusing thin-layer chromatography and was observed to be similar to four of the compounds. Further analysis by highperformance liquid chromatography revealed that the RM10 fraction comprised vanillic acid. However, the RM10fraction was more inhibitory to the growth of tomato radicles than synthetic vanillic acid.

INTRODUCTION

The increase in palm oil production has gener­ated abundant waste effluent from the palm oilmills. Due to the pollution problem from theeffluent, several treatment methods have beendeveloped, producing different types of by­products. Palm oil dry solids (PODS) is one ofthe by-products generated from mills equippedwith a decanter drier system. It contains sub­stantial amounts of plant nutrients and is cur­rently being applied to soil as an organic ferti­lizer. However, PODS must be properly decom­posed before it can be applied to soil. Growthof plants, especially vegetables, has been shown

to be adversely affected by undecomposed PODSor other forms of palm of mill effluent (POME).Application of raw or partially decomposedPODS to sandy tailing soil has been shown toinhibit growth of tomato and spinach seedlings(Radziah et al. 1997). Zulkifli and Rosmin (1990)observed low yields of cowpea and mustardgreens grown on sandy tailing soil withundecomposed POME added. The inhibitoryeffect of raw effluent has been associated withthe presence of lipid and volatile substances(Lim 1986). However, such effects are reducedwhen the material is completely decomposed.Growth of vegetable seedlings increased when

O. RADZIAH, M. RAHMANI AND AZIZAH HASHIM

Extract with hexane

PODS aqueous filtrate

ri Extractwithdiethyl ether

Bioassay +- '" Diethyl ether extract Insoluble

PODS (Dry)

IMix 100 g with 500 ml watershake for 6 hours,centrifuge and filterrepeat with 250 ml water

InsolubleBioassay +- ... Hexane extract

ri Extractwithchloroform

Bioassay +- ... Chloroform extract Insoluble

ri Extractwithethyl acetate

Bioassay +-... Ethyl acetate Insolubleaqueous

material, previously described by Radziah et al.(1997), contained 19.4% C, 1.44% N, 0.32% P,1.32% K, 1.46% Ca, 0.32 Mg, 12.0% lipid andthe pH was 5.0 (1:5 in H

20). It was kept dry in

the cold room at 8 ± 1°C before use to preventmicrobial decomposition. The method of ex­tracting the water-soluble compounds wasadapted from that of Weston et al. (1989). Theflow diagram of the extraction and partitioningprocedures used is shown in Fig. 1. One hun­dred gram batches of dried PODS were ex­tracted for 6 hours with 500 ml distilled waterin a 1-1 conical flask on an orbital shaker, andthe suspension was left to stand in the refrig­erator for one hour at 4 ± 1°C. The supernatantwas decanted and centrifuged at 5,000 rpm for15 min and the clear brown solution obtainedtransferred into a clean flask, and the residuereturned to the original flask and re-extractedusing 250 ml water. A total of 750 ml of waterwas used to extract every 100 g PODS. The fine

grown on soil containing decomposed PODS(Radziah et al. 1997).

It has been shown that decomposed PODScontains higher quantities of plant nutrients,especially nitrogen, and low lipid contents. In­teractions of nutrients and improved physicaland biological properties finally lead to im­proved plant growth. However, there is littleinformation which explains the inhibitory ef­fects of undecomposed PODS or other formsof POME.

The phytotoxicity of plant residue whichreduces growth of plants has been associatedwith the presence of various organic compoundsincluding phenolic compounds (Rice 1984)which are widely distributed in various plantspecies. These compounds are either leachedfrom plant residues or are formed as by-prod­ucts during residue decomposition in soil. Sev­eral phenolic acids such as ferulic, p-coumaric,p-hydroxybenzoic, syringic, vanillic acids and p­hydroxybenzaldehyde have been identified inextracts of wheat mulch (Lodhi et al. 1987), riceresidues (Chou and Lin 1976) and sorghum­sudan grass hybrid (Weston et al. 1989). Theactivities of various compounds in soil are oftenattributed to the water-soluble fractions. Theallelopathic effects of phenolic compounds couldalso be due to both the water-soluble and freeform compounds (Whitehead et al. 1983). Growthis adversely affected as these water-soluble com­pounds are taken up by plant roots which in­hibit nutrient uptake and affect various physi­ological processes (Rice 1984). Currently, thepotential phytotoxicity of soluble compounds inraw PODS has not been properly characterized.The soluble compounds in PODS could be simi­lar to those in other crop residues since PODSis a by-product originating from plant materiali.e. oil palm fruit. Identification of these water­soluble compounds is essential in order to bet­ter understand the nature of phytotoxicity andmechanisms involved in its formation, thus ena­bling proper management of the effluent. There­fore, the following studies were conducted toextract and identify the water-soluble compoundspresent in raw PODS which are inhibitory toplant growth.

MATERIALS AND METHODS

Extraction of Water-soluble Phytotoxic Compounds

Raw PODS was collected from the Rantau palmoil mill, Negeri Sembilan. The dark granular

Bioassay

Fig. 1. Flow diagram of the extraction and partition­ing procedure of PODS

148 PERTANIKAJ. TROP. AGRIe. SCI. VOL. 20 0.2/3,1997

PHYfOTOXICITY OF PHENOLIC ACIDS EXTRACTED FROM PALM OIL DRY SOLIDS

particulates in the aqueous extract were re­moved by vacuum filtration through Whatman

o. 4, 1 and 42 filter papers sequentially. Theextraction procedure was repeated until suffi­cient aqueous extract was collected for furtheruse. A total of 2.2 kg PODS was used during theextraction process, giving 16.5 1 of aqueousextract.

Partitioning of Aqueous PODS Extract

The aqueous PODS extract was sequentially par­titioned with hexane, diethyl ether, chloroformand ethyl acetate. The solvents were used toseparate the polar and non-polar compoundspresent in the aqueous PODS. The separationprocedure was conducted in batches using 1 1ofaqueous extract each time. Each litre of theaqueous extract in a 2-1 separatory flask waspartitioned six times with 200 ml of the respec­tive four solvents. The extracts were transferredto clean flasks, dried with anhydrous MgSO4 andfiltered. The solvent was subsequently removedfrom each extract by rotary evaporation at 35­40°C. The degree of inhibitory properties ofeach extract was then evaluated on growth oftomato radicles as described below.

Bioassay of the Solvent Soluble Fractions

Each extract (yellowish brown residue) wasweighed and redissolved in chloroform to forma solution with a concentration of 1.0 mg resi­due ml- l chloroform. Ten millilitres of the re-

maining aqueous solution were then freeze-driedand redissolved in water to form a similar con­centration of 1 mg ml-I

. One millilitre of eachextract was then placed in separate sterile glasspetri dishes (90 x 15 mm), lined with a doublelayer of Whatman No. 1 filter paper. The chlo­roform was allowed to evaporate overnight be­fore adding 3 ml sterilized distilled water, to afinal concentration of 334 Jlg residue mP. Thecontrol dish was given 1 ml chloroform whichwas allowed to evaporate overnight before theaddition of 3 ml distilled water. Ten uniformpregerminated tomato seeds were then placedequidistantly in each dish and incubated in thedark at 30°C. Radicle length of each seedlingwas measured 72 h after placement in the dish.The activity of the extracts was expressed aspercentage radicle growth of control. Thebioassay for each extract was replicated threetimes.

Separation of the Active Fractions l7y ColumnChromatography

The bioassay conducted on the different ex­tracts showed that the diethyl ether extract wasthe most toxic to growth of tomato radicles(Table 1). This extract was further separatedusing column chromatography. The ether ex­tract (4.4 g) was precoated with silica gel (60,Baker, 200-250 mesh) and loaded into a silicagel column (30 cm x 6 cm). The column waseluted with petroleum ether, gradually increas-

TABLE 1Quantity of solvent extracts partitioned from 2.2 kg PODS and their effects on growth of tomato radicles

Solvent extract

Hexane

Diethyl ether

Chloroform

Ethyl acetate

Remaining aqueous

LSD (0.05)

ND - not determined

Quantity Recovery Radicle length Radicle growthextracted (%) ± standard error (% of control)

(g)

7.26 0.33 35.8 ± 5.0 115.0

4.40 0.20 16.6 ± 1.3 53.3

1.54 0.07 24.0 ± 1.4 77.1

1.98 0.08 30.7 ± 1.9 98.7

D ND 41.4 ± 3.1 132.1

31.1 ± 2.8 100.0

8.4 20.9

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 0.2/3,1997 149

O. RADZIAH, M. RAHMANI AND AZIZAH HASHIM

ing the amount of chloroform and methanol.Two hundred fractions of 100 ml each col­lected from the column were monitored usingthin-layer chromatography (TLC) (Kieselgel60 F

254by Merck, 0.2 mm layer) and the pro­

files were visualized under UV light at 254 nm.Fractions with similar profiles under the UVlight were combined to form 11 fractions.During the combination process, precipitationwas observed in fractions 9, 10 and 11. Thesefractions were then separated further into chlo­roform and methanol soluble fractions. Therespective fractions at the concentration of334 ~g ml-1 was bioassayed for their inhibitoryactivity using two controls. Chloroform wasused as control for fractions soluble in chloro­form and methanol for fractions soluble inmethanol. There was strong inhibitory activityin one of the fractions (Table 2), which gavea single spot on the TLC, was active under UVlight and was hence selected for further char­acterization

Comparison of the Unknown Fraction with Stand­ard Phenolic Compounds

The active unknown fraction obtained (desig­nated as RM10) was tentatively identified bycomparing its migration profile with the other

synthetic (standard) phenolic compounds. TheRMIO fraction and the standards were subse­quently dissolved in methanol, spotted on TLCplates using a mobile phase of 6:4 acetone­chloroform, and their profiles visualized underUV (254 nm) light. The 14 standard compoundsused were: 4-hydroxybenzaldehyde, vanillin, ac­ids of p-hydroxybenzoic, ferulic, p-coumaric,vanillic, caffeic, gentisic, p-hydroxyphenyl acetic,sinapic, gallic, salicylic, trans-cinnamic andsyringic (Fluka Chemical Company).

HPLC Profile of the Unknown Fraction

The identity of the unknown fraction RM10was further confirmed by using high perform­ance liquid chromatography (HPLC). TheRM10 fraction and the four standard samples(ferulic, p-hydroxybenzoic, vanillic and p­coumaric acid) were dissolved singly in metha­nol to form a concentration of 1 mg ml-1, andfiltered through 0.45 ~m Millipore membrane.Ten microlitres of the sample was injected intothe Waters HPLC system fitted with an absorb­ance detector model 460 set at 254 nm, a pumpmodel 510 and a reverse-phase ~ Bondapak CI8

column (300 mm x 3.9 mm). The isocraticelution solvent used consisted of a mixture ofmethanol, ethyl acetate and acetic acid in the

TABLE 2Recovery of different fractions from column chromatography of diethyl ether extract and their effects on

growth of tomato radicles

Fractions Quantity recovered(mg/4.4 g)

Recovery(%)

Radicle length (mm)± standard error

Radicle growth(% of control)

R1-R3R4R5R6R7R8R9 CHCIg

R9 MeOHRIO CHCIg

RIO MeOHRll CHCIg

R11 MeOHControl (CHCIg)'

Control (MeOH)bLSD (0.05)

126488752

1271869158194

4527

1843

31

2.911.117.128.919.8

3.64.41.00.64.20.10.7

D37.0 ± 3.831.5 ± 4.438.2 ± 2.534.0 ± 1.335.6 ± 2.215.7 ± 0.430.0 ± 2.613.9 ± 1.713.2 ± 4.339.0 ± 1.024.9 ± 4.836.6 ± 3.341.7 ± 4.6

ND92.578.795.785.188.939.268.734.830.297.556.9

100.00100.00

18.8

D - not determineda for fractions dissolved in CHCIg

b for fractions dissolved in MeOH

150 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 0.2/3,1997

PHYTOTOXICITY OF PHENOLIC ACIDS EXTRACTED FROM PALM OIL DRY SOLIDS

ratio of 35:1:2, at a flow rate of 2 ml min-! (Blumet al. 1984).

Effect of Various Concentrations of RM10 Fractionand Standard Phenolic Acids on Growth of Tomato

Radicles

The bioassay technique described previously wasused to compare the toxicity of RMI0 fractionwith standard phenolic acids. The activity ofRMI0 was assessed with four standard phenolicacids of similar TLC profiles. The standardsused were: ferulic, p-hydroxybenzoic, vanillic andp-coumaric acids. The concentrations used were;0, 250, 500, 750 and 1000 ~g ml-!. Growth re­sponse curves were then drawn to determine theconcentration of each compound responsiblefor causing 50% inhibition (150) on elongationof tomato radicles.

RESULTS

Extraction of Water-soluble Phytotoxic CompoundsVarious amounts of water-soluble organic frac­tions were extracted from PODS. The highestamoun t of dry fraction recovered from thepartitioning procedure was the hexane extract(7.26 g), followed by diethyl ether extract (4.4g) (Table 1). The hexane extract probablyconsisted of lipids and non-polar compoundswhich were present in PODS in substantialamounts. The hexane extract made up 0.33%and diethyl ether extract 0.20% of the totaldried PODS used. Smaller amounts of residueswere obtained from the chloroform and ethylacetate extracts.

Results from the bioassay studies showedthat different solvent extracts significantly (P ::::;0.01) influenced elongation of tomato radicles.The maximum inhibitory activity of radiclegrowth was observed in the diethyl ether extract

where radicle elongation was significantly re­duced to 53.3% of the distilled water control.This indicated that the ether had extracted outmost of the inhibitory compounds present inthe aqueous PODS. The chloroform and ethylacetate extracts exhibited lesser inhibition onradicle growth. The hexane extract as well as theremaining crude aqueous extract, however, stimu­lated growth of tomato radicles. Improvementin radicle elongation, especially in the remain­ing aqueous fractions, indicated absence of thetoxic compounds.

Isolation and Characterization of Phytotoxins

Since the greatest inhibitory effect was obtainedfrom the diethyl extract, this extract was furtherseparated by column chromatography and eachnew fraction recovered was bioassayed for in­hibitory activity. Two hundred fractions werecollected from the separation process and weremonitored by using TLC. Fractions with similarTLC profiles were then combined to form 11distinct fractions. Fractions R9, RIO and Rllwhich formed precipitations were further ex­tracted by dissolving the individual fraction inchloroform and methanol. Three additional frac­tions were obtained from this extraction (Table2). The flow chart of the separation procedureis shown in Fig. 2.

Results of the bioassay of all fractions (R4­Rll) showed significant (P ::::; 0.01) differencesin their inhibitory activities (Table 2). Highinhibition on radicle growth was concentratedin three fractions, R9 which is soluble in CHCI

3,

RIO which is soluble in CHCl3

and RIO which issoluble in MeOH. Among these three fractions,the RIO in MeOH (designated as RMIO) hadthe lowest radicle growth of 30.2%. This brown,viscous fraction was found to produce a single

Diethyl ether extract (4.4 g)

R)-.R-8---'lr--9

-------Jr--10

------JI

I __~I__ __.J..I _

R9(CHCl l ) R9 (MeOH) RIO (CHCl l ) RIO (MeOH) RIl (CHCl) Rll (MeOH)

Radicle growth 39.2% 68.7% 34,8% 30,2% 97.5% 56.9%

Fig. 2. Separation of the diethyl ether extract and the effect on growth of tomato radicles

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 0.2/3,1997 151

O. RADZIAH, M. RAHMANI AND AZIZAH HASHIM

spot on the TLC and was strongly visible underUV light (254 nm). Although the other twofractions (R9-CHCIg and RIO-CHCIg) showedalmost similar activity to RMIO, they did not givea distinct spot on the TLC, and thus requiredfurther separation for identification. No attemptwas made to identify them in this study.

The amount of RMIO fraction recoveredfrom the crude diethyl ether extract using thecolumn chromatographic procedure was verysmall (only 184 mg/4.4 g or 4.2%) (Table 2).This indicates that the active RMIO fractionwhich could be toxic to plant growth was only0.0084% (dry weight) of the original raw PODS.

The unknown RM10 fraction was tenta­tively identified by comparing its TLC profilewith the standard phenolic acids. The resultsshowed that this fraction was similar to 4 of thestandards: ferulic, p-hydroxybenzoic, p-coumaricand vanillic acids (Fig. 3). The identity of RMIOwas further confirmed by injecting the sampleinto HPLC using the reverse-phase J..l BondapackCIS column. A single peak at the retention timeof 2.89 min was obtained (Fig. 4). Furthercoelution with known standards indicated that

Solvenl Front

0013 14

Fig 3. Thin-layer chromatography Profiles of frac­tion RMlO and standard phenolic acidswith mobile phase of acetone : chloroform(6:4)

*Key: 1) p-hydroxybenzoic acid; 2) ferulic acid;3) p-coumaric Acid; 4) vanillic acid; 5)sinapic acid; 6) s)'ringic acid; 7) caffeicacid; 8) p-hydroX')'phen)'l acetic acid; 9)gentisic acid; 10) saliC)·lic acid; (11)trans-cinnamic acid; 13) 4-hydroxy­benzaldehyde; 14) vanillin

RM10 was similar to vanillic acid. The reten­tion times of the respective standards are shownin Table 3.

VA fAI 8.00'&.00

4.0.0 6.00...t +-

'0g 3.00 >N 4.00-

I '0~ 2.00

X 2.00X

1.00

0.000.00'

00 2 4 6 8Minutes

RMIO4.00I C I

3.00III 3.00VI.... ....

"0 '0> >

I 2.00 I 2.00

~ ~

X X1.00 1.00

0.00 0.000 4 6 8 0

Minutes

PC

4 6Minutes

RMIO t VA

4 6

Minutes

8

a

191

101

Fig 4. HPLC chromatograms of (A) vanillic [VA], (B) p-coumaric [PC], (C) RM10 and (D) RM10 + VA, using C}s columnwith flow rate of 2 ml min'} and solvent of 35 % methanol, 1 % ethyl acetate and 2 % acetic acid

152 PERTANIKAJ. TROP. AGRIC. SCI. VOL. 20 NO. 2/3,1997

PHYTOTOXICITY OF PHENOLIC ACIDS EXTRACTED FROM PALM OIL DRY SOLIDS

TABLE 3Retention times of the unknown and standard

phenolic compounds

Growth Response of Tomato Radicles to VariousConcentrations of RM10 and Standard Phenolic

Acids

The length of tomato radicles was found to besignificantly (P ~ 0.01) affected by the concen­trations of RM10 and the standard phenolicacids (ferulic, vanillic, p-coumaric and p­hydroxybenzoic acids) (Fig. 5). Radicle elonga­tion was reduced with increase in concentrationof RM10 and phenolic acids. This indicated thatthe toxicity of these compounds increased withincrease in concentration. The percentage ofradicle growth showed negative liner responsesto increasing concentration of the compounds.

p-coumaric acid was found to be the mosttoxic to growth of tomato radicles of all thecompounds. Total inhibition (no growth) on radi­cle elongation was observed at 500 J..lg ml-! p­coumaric acid concentration. Ferulic acid wasleast toxic to the growth of tomato radicles. Atlow concentrations, the activity of RM10 was closeto that of p-coumaric acid. However, the activitiesdiffered as the concentration of compounds in­creased to 500 J..lg mt!. Total lack of growth of

Phenolic acid

Ferulicp-coumaricp-hydroxybenzoicVanillicExtract RMIO

Retention time(min)

5.404.643.022.892.89

tomato radicles was observed as the concentra­tion of these compounds increased to 1000 J.lgml-!, i.e. the highest concentration studied.

The 50% radicle growth inhibition (Iso)' i.e.the compound concentration at which 50% ofthe radicle elongation was inhibited, was alsocalculated from the growth response curves inFig. 5. The RM10 fraction was more toxic then p­hydroxybenzoic, vanillic and ferulic acids. Thedescending order of toxicity of the compoundswith increasing Iso values recorded was p-coumaricacid (194 J..lg ml'!) > RM10 (292 J..lg mI'!) > p­hydroxybenzoic acid (434 J..lg ml-!) > vanillic acid(466 J..lg ml-]) > ferulic acid (524 J..lg mI-l).

DISCUSSION

Results from the extraction procedures usedshowed that most soluble toxic compoundspresent in raw PODS were concentrated in thediethyl ether extract. This extract was found toreduce the growth of tomato radicles by 46.7%of control (Table 1). Most of the earlier findingshave also shown that the toxic compounds foundin other crop residues are soluble in polar sol­vents such as ether (Weston et al. 1984). It wasalso observed that the toxicity of the remainingaqueous PODS was completely eliminated afterremoval of the toxic extracts, where growth oftomato radicles was found to be stimulated.Further separation of the ether extract usingcolumn chromatography recovered a more toxicRM10 fraction. The amount of this phytotoxiccompound present in raw PODS was small(0.0084%). Hence, under normal conditions theapplication of a low amount of raw PODS to soilwould probably cause minimum inhibition on

0--0 FA (Y-l04.22-0.1X; r2-0.90··; n-15)•....• VA (y-1l1.1l- O.OllX; ,2-0.85··; n-15)

t:r---l:l PC (y-67.45 - O.09X; ,.:z..0.62"; n-15).... -A PHS (Y-102.0~ - 0.12)(; ,2-0.96··; n-15)

0- .. -0 RM10 (y-71.0J-0.072X; ,2-0.61··; n-15)

1000750

80

60

40

20

o-20+----+----+-----+----~

o

160,.-----------------,

140

~120

~100 ...ioi;,Gl'0'6IIIa:

250 500Concentration (ug mr1)

Fig 5. Growth of tomato radicles in relation to concentration ofphenolicacids: ferulic (FA), vanilic (VA), p-coumaric (PC), p­hydroxybenzonic (PRB) and fraction RM10

PERTANlKAJ. TROP. ACRIC. SCI. VOL. 20 NO. 2/3, 1997 153

O. RADZIAH, M. RAHMANI AND AZIZAH HASHIM

growth of plants. Degradation of these com­pounds is rapid in well-drained soils. However,continuous application of raw PODS to clayeysoil with long periods of water saturation couldprobably lead to accumulation of thesephytotoxic compounds.

Although RM10 was confirmed to be vanillicacid on the HPLC, its inhibitory effect on growthof tomato radicles was higher than that causedby the synthetic vanillic acid. The higher toxiceffect of RM10 was probably a result of beingbound to other compounds which have highertoxic effect. No attempt was made to purifyRM10 in the present study.

The activity of RM10 was also compared tothe activity of the other toxic phenolic com­pounds (besides vanillic acid) commonly foundin soil: ferulic, jrcoumaric and p-hydroxybenzoicacids (Lodhi et at. 1975). The inhibitory effectexhibited by RM10 was found to be intermediatebetween that of jrcoumaric and vanillic acid (Fig.5). This indicates that the phytotoxic compoundin PODS is present in mixed form. This is to beexpected since under natural conditions, phe­nolic acids usually occur in mixed rather than insingle form. The phytotoxic effects of organicresidues as observed is probably the result of thestrengthening effect of complex organic mixtures(Rasmussen and Einhellig 1977; Blum 1996).

Results of the present study showed that theRM10 fraction was more toxic then standardferulic, vanillic, and p-hydroxybenzoic acids, butless toxic than p-coumaric acid on the growth oftomato seedlings. However, the inhibitory activ­ity of RM10 extracted from PODS was low com­pared to the activity of some other standardphenolic compounds. The concentration ofRM10 which caused 50% reduction in radiclegrowth (1

50) of tomato was 292 ~g ml-I

• Thisvalue was higher than other compounds re­ported to be phytotoxic. Vanillic and p-coumaricacids at as low a level as 100 ~g ml-I werereported to inhibit growth of rice seedlings,seed germination and growth of ryegrass, lu­cerne and wheat (Hartley and Whitehead 1985).Weston et al. (1989) observed that p­hydroxybenzoic acid and p-hydroxybenzaldehydeat concentrations of 70-140 ~g mI-l reducedradicle growth of curly cress, lettuce and radishby 50%. This indicates that the degree of toxic­ity of compounds differs with plant species. Thetomato seeds used in the study were more toler­ant to the presence of phenolic acids than other

seeds. The choice of plant species in the bioassaytechnique is crucial in determining the inhibi­tory activity of phenolic compounds.

The amount of the inhibitory compoundextracted from PODS in the present study wasonly an estimate and could not therefore reflectthe actual amount present in the entire PODS.This study could only focus on compounds thatare soluble in water and not on the total com­pounds present. The amount of toxic compoundsmay also vary with the type of POME. There arevariations in the physical, chemical and biologicalproperties of POME generated from differentpalm oil mills which use different methods ofeffluent treatment (Lim 1986). Raw POME needsto be decomposed in order to eliminate theinhibitory effect on plant growth. The propermanagement of POME applied to soils is impor­tant in order to reduce its phytotoxic effect, whileobtaining optimum benefits for plant growth.Decomposition is important to reduce thephytotoxicity of various types of POME in orderto convert them to valuable organic fertilizer.

ACKNOWLEDGEMENTSAppreciation is extended to all technicians inthe Soil Microbiology Laboratory for their assist­ance. This project was funded by the MalaysianGovernment through IRPA Programme 0: 1­07-05-048.

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(Received 12 June 1997)(Accepted 6 December 1997)

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