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Journal of Environmental Management 84 (2007) 134–140

www.elsevier.com/locate/jenvman

Polyphenols dynamics and phytotoxicity in a soil amendedby olive mill wastewaters

Ali Mekki, Abdelhafidh Dhouib, Sami Sayadi�

Laboratoire des Bioprocedes, Centre de biotechnologie de Sfax, , Route Sidi Mansour BP: ‘‘K’’, 3038 Sfax Tunisie

Received 2 July 2005; received in revised form 18 March 2006; accepted 4 May 2006

Available online 20 September 2006

Abstract

The effects of unprocessed olive mill wastewaters (OMW) on soil characteristics were investigated. Phenolic compounds levels in the

treated soil were compared to those of a control soil profile. Results showed that OMW infiltration caused a modification of soil

physicochemical characteristics. Phenolic compounds were detected at a depth of 1.2m four months after the last application of OMW.

A moderate phytotoxic residual phenolic fraction (F) was extracted from the superficial soil layer 1 year after the OMW application. This

residual F had a phytotoxic potential comparable to that of 25-fold diluted OMW.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Olive mill wastewaters; Soil; Phenolic compounds; Phytotoxicity

1. Introduction

One of the sources of pollution of the soil and water iscaused by industrial, agricultural and urban wastewaterswhich cannot be sent to ordinary systems of wastewatertreatment. These include olive mill wastewaters (OMW)(Spandre and Dellomonaco, 1996), which have the follow-ing chemical properties: an enormous content of organicmatter (COD between 60 and 185 g l�1; BOD5 between 14and 75 g l�1), a low pH, and high polyphenols, potassiumand phosphorus contents (Tomati and Galli, 1992; Rinaldiet al., 2003). These wastes constitute a pollution constraintfor environmental and aquatic bodies (Rodriguez et al.,1988; Ramos-Cormenzana et al., 1996; Yesilada et al.,1999). The disposal of OMW is a serious problem for theproducers, millers, and for the whole community due to itspotential pollution risk (Vazquez et al., 1974; Martinezet al., 1986; Rinaldi et al., 2003). Although the observedtoxicity of OMW has been generally related to theconcentration of phenols, nonphenolic-related toxicitywas also reported by Capasso et al. (1992).

e front matter r 2006 Elsevier Ltd. All rights reserved.

nvman.2006.05.015

ing author. Tel./fax: +216 7444 0452.

ess: sami.sayadi@cbs.rnrt.tn (S. Sayadi).

To solve the problems associated with OMW, differentelimination methods based on physicochemical and biolo-gical treatments have been proposed (Levi-Menzi et al.,1992; Ehaliotis et al., 1999; Paredes et al., 2000; Marques,2001; Kissi et al., 2001; Casa et al., 2003). However, themost frequently used methods nowadays are the directapplication to agricultural soils and storage in evaporationponds (Fiestas and Borja, 1992; Martinez Nieto andGarrido, 1994). Agriculturally, these wastewaters can beused as soil biofertilizers (Solinas et al., 1975; Ranalli,1989; Lopez et al., 1993). Moreover, positive effects onchemical fertility have been generally reported, but lessattention has been paid to the effect on the undergroundwater resource quality. D’acqui Luigi et al. (2002) reportedthat OMW can constitute a serious organic pollution riskfor the water table and the deep underground waters.According to Levi-Menzi et al. (1992), the high COD

value (150–200 g l�1) and the presence of phytotoxic andantibacterial polyphenols (1–10 kg t�1) in this waste can bea serious pollution risk for superficial and undergroundwaters. Furthermore, the presence of phenolic compoundsin OMW makes them highly toxic and ecologically noxious(Gonzalez et al., 1994; Aggelis et al., 2003). Angus (1983)reported that the river fish Gambusia affinis and thecrustacean Daphnia magna are severely intoxicated on

ARTICLE IN PRESSA. Mekki et al. / Journal of Environmental Management 84 (2007) 134–140 135

exposure to phenol derivatives at concentrations of40mg l�1 for only 15min. OMW proved to have an almostimmediate toxic action on gold fish Carassius auratus at aconcentration of 10% (v/v) (Bellido, 1989), on fish and ray-finned fish Cyprinus carpio and Chondrostoma polylepis atconcentrations of 6.8% (v/v) and 8.8% (v/v), respectively(Fiesta Ros de Ursinos, 1977) and on marine Branchiopoda

Artemia sp., the freshwater Branchiopoda Daphnia magna

and freshwater sediments Ostracoda Heterocypris incon-

gruens (Aggelis et al., 2003).Negative effects were also recorded on soil properties

(Paredes et al., 1999; Mekki et al., 2005). Jelmini et al.(1976) and Ben Sassi et al. (2005) observed phytotoxiceffects in plants when OMW is used directly as an organicfertiliser. The toxicity of OMW and its phenolic contentagainst the seeds of Lepidium sativum was well detailed inthe study of Aggelis et al. (2003). However, worksconcerning the evolution at the real scale of OMWphenolic compounds on the soil and their consequenceson the groundwater are rare (Spandre and Dellomonaco,1996). The aim of this work was to characterize a soilwhich was amended with raw OMW and to demonstratethat in spite of the considerable microbiological activity ofthe soil to degrade and to attenuate the toxic effect of theOMW phenolic compounds, these chemicals persist orchange and keep their toxicity even long after thespreading period. They infiltrate in soil and can pollutethe groundwater.

2. Materials and methods

2.1. OMW characterization

The fresh OMW was taken from a three-phase dis-continuous extraction factory located in Chaal-Sfax south-ern Tunisia. This OMW was first recovered in a storagebasin for 1 week then characterized before application.

pH and electrical conductivity (EC) were determinedaccording to Sierra et al. (2001) standard method. Organicmatter (OM) was determined by combustion of the samplesin a furnace at 550 1C for 4 h. Total organic carbon wasdetermined by dry combustion (TOC Analyser multi N/C1000). Total nitrogen was determined by Kjeldahl (1883)method. COD was determined according to Knechtel(1978) standard method. BOD5 was determined by themanometric method with a respirometer (BSB-ControllerModel 620 T WTW) and phenolic compounds (ortho-diphenols) were quantified by means of the Folin–Ciocal-teu colorimetric method (Box, 1983) using caffeic acid asstandard. The absorbance was determined at l ¼ 765 nm.Phosphorus, iron, magnesium, potassium, sodium andchloride were determined by atomic absorption.

2.2. Study area and soil sampling

The study area consisted of a field of olive trees locatedin Chaal at 60 km to the South-West of Sfax, Tunisia,

North latitude 341 30, East longitude 101 200. The meanannual rainfall is 200mm (Ben Rouina et al., 1999).Annually, since 1994 three experimental plots P1, P2 and P3

had been respectively amended with 50, 100, and200m3 ha�1 of unprocessed OMW. This amendment wasrealised in February in one application (Ben Rouina, 1994).The plot PC was not amended and served as control. Soilsamples (Sc, S50, S100 and S200) were collected monthlyfrom different parts and at variable depths (0–120 cm) ofeach plot, using a soil auger. All soil samples, taken fromeach plot were mixed and the water content wasimmediately determined. Then they were air dried andsieved with a mesh size of 450 mm and stored at 4 1C priorto use.

2.3. Soil analysis

pH, electrical conductivity, organic matter and phenoliccompounds content in all soil samples were determined.

2.4. Soil phenolic compounds determination

For soil phenolic compounds, only water solublesubstances were determined. A 1/25 (w/v) soil/aqueousmixture was shaken for 12 h in a mechanical shaker. Thesupernatant was extracted 3 times with ethyl acetate. Thecollected organic fraction was dried and evaporated undervacuum. The residue was extracted twice with dichlor-omethane in order to remove the non-phenolic fraction(lipids, aliphatic and sugars). The liquid phase wasdiscarded while the washed residue was analysed by theSize exclusion-HPLC technique (Allouche et al., 2004).Using this same extraction and characterisation method,

a residual phenolic fraction (F) was extracted from theupper layer (0–10 cm) of P3 plot in January 2001, one yearafter the last application of OMW (February 2000). Thisyear was the last year of the 7 years of OMW application.The molecular-mass (MM) of the soil extract or OMWphenolic compounds was estimated by the comparison oftheir retention time to the retention time of standardcompounds having a known molecular-mass and injectedin the same conditions.

2.5. Phytotoxicity tests

Phytotoxicity of OMW phenolic compounds wasassessed by the determination of the germination index(GI) of a very sensitive plant belonging to the familyof Cruciferae Brassicaceae (Brassica cernua) according toZucconi et al. (1981) standard method. MS mediumdescribed by Murashige and Skoog (1962) was usedfor control germination index. For germination indexof (F) fraction and OMW, the water in MS is replacedby the 1/25 (w/v) soil/aqueous mixture extraction sol-ution of (F) or OMW diluted in distilled water 4, 25 and 50times.

ARTICLE IN PRESSA. Mekki et al. / Journal of Environmental Management 84 (2007) 134–140136

2.6. Statistical analysis

For soil analysis, three replications were used for eachparameter. For the germination test, three replicationswere used in each issue. Data were analysed using theANOVA procedure. Variance and standard deviation weredetermined using Genstat 5 (second edition for windows).

3. Results

3.1. OMW characterization

The physicochemical characteristics of crude OMW aresummarized in Table 1 and correspond to the mean valuesof 3 analysis. The high pollutant load of OMW (72 g l�1 ofCOD) and its elevated salinity (6.75 g l�1) could beobserved. The OMW C/N ratio was unfavourable for thebiodegradation and humification processes. OMW con-tains an enormous supply of organic matter very rich inphenolic compounds (9.2 g l�1) which are toxic.

3.2. Soil characterization

The soil of the study area was described in a previousstudy (Mekki et al., 2005). It had an important content ofactive calcareous (0.6%w/w) at the surface, and wascomposed of sand (89.82%w/w), clay (7.44% w/w) andsilt (2.74%w/w). It had an alkaline pH and a weakelectrical conductivity. The soil was very poor in nitrogen(0.5 g kg�1 dry soil), and in organic matter (0.16%w/w).The levels of potassium and phosphorus were 0.014% w/wand 0.002%w/w, respectively. As it was discussed in thatprevious study (Mekki et al., 2006), the soil water content

Table 1

Physico-chemical characteristics of unprocessed olive mill wastewater used

in ferti-irrigation (values after7represent standard deviation)

Characteristics Data

pH (25 1C) 5.170.2

Electrical conductivity (25 1C) (dSm�1) 8.970.1

Chemical oxygen demand (g l�1) 7272.8

Biochemical oxygen demand (g l�1) 1370.9

COD/BOD5 5.5370.72

Salinity (g l�1) 6.7570.66

Water content (g l�1) 948717.2

Total solids (g l�1) 5272.98

Mineral matter (g l�1) 870.43

Volatile solid (g l�1) 4471.85

Total organic carbon (g l�1) 25.5271.18

Total nitrogen Kjeldahl (g l�1) 0.670.06

Carbon/nitrogen 4378

P (mg l�1) 3673.6

Na (g l�1) 0.9470.09

Cl (g l�1) 1.670.15

K (g l�1) 8.870.8

Ca (g l�1) 1.270.11

Fe (mg l�1) 3272.9

Mg (mg l�1) 187718.8

Ortho-diphenols (g l�1) 9.271.8

was very low and it varied between 0.8% and 1.15%w/w.The water-holding capacity, the salinity and the content oftotal organic carbon, humus, total nitrogen, phosphate andpotassium increased when the spread amounts of thetreated or untreated OMW increased. Soil irrigated withraw olive mill wastewater showed also a significantinhibition of some enzymatic activities such as ureases,ammonium oxydases and nitrate reductases while someother enzymatic activities such as xylanases and cellulaseswere slightly stimulated (data not shown).

3.3. Analytical results of soil profiles

For soil profiles, pH, electrical conductivity, organicmatter and polyphenols contents were analysed 4 monthsafter OMW spreading. Taking into consideration thedeviation of the pH determination, it can be said that thepH does not vary as a function of depth. However, a veryweak decrease was observed when OMW spreadingquantity increased. The good buffering capacity of the soilrelated to its calcareous content neutralized the relativelyacidic pH 5.12 of OMW (Table 2). This table shows thatelectrical conductivity increased proportionally with theincrease of the quantity of unprocessed OMW anddecreased with depth in all analysed soils. The electricalconductivity increased in all soil depths compared to thecontrol soil which suggests the leaching of OMW from thesurface to the soil deeper layers.The analysis of the organic matter content of the soil

showed an increase in all soil depths compared to thecontrol soil. This organic matter increase is proportional tothe OMW spread quantities (Table 2).

3.4. Phenolic compounds dynamics

This part of the study proposed to investigate thedynamics of the phenolic compounds in a very porous andpermeable sandy soil irrigated by increasing doses ofunprocessed OMW 50, 100 and 200m3 ha�1 yr�1 and totrace the change of the phenolic compounds content as afunction of time and depth. Results showed that theapplication of increasing OMW doses increased the totalphenolic compounds content in all soil layers (Table 2).Besides, Table 2 shows that the majority of phenoliccompounds are kept in the soil upper layers. The phenolicconcentration decreased rapidly from 0 to 25 cm thencontinued to decrease weakly with depth but remainedeven detectable at 120 cm. The control soil showed veryweak detectable phenolic substances.Comparison of phenolic compound spectra shows that

especially the high molecular-mass compounds decreasedwith depth while the low molecular-mass polyphenolsremained more abundant.According to the HPLC principle, polymers were eluted

with low retention time, while monomers needed higherretention time. Fig. 1 shows that phenolic compounds withlow retention time (22.8–25.8min) were detected in upper

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Table 2

Evolution of pH, electrical conductivity, organic matter and phenolic compounds as function of soil depth and OMW quantity spread (data determined 4

months after OMW spreading, and in the 7th consecutive year irrigation by OMW)

Soil depth 0 cm 25 cm 50 cm 120 cm

. SC 8.170.2 8.270.2 8.370.2 8.2670.2

pH S50 8.170.2 8.170.2 8.370.2 8.270.2

. S100 870.2 870.2 8.170.2 8.170.2

. S200 7.970.2 7.770.2 7.870.2 870.2

Electrical SC 19870.1 15470.1 11270.1 8570.1

conductivity S50 24870.1 20570.1 18370.1 12670.1

(mS cm�1) S100 37570.1 31570.1 28470.1 19670.1

. S200 74370.1 69570.1 54870.1 45270.1

Organic SC 0.1670.003 0.1470.002 0.0870.002 0.0270.0004

matter S50 0.3470.007 0.270.004 0.1470.002 0.0970.0018

(%, w/w) S100 0.6270.012 0.5470.01 0.1270.002 0.0870.0016

. S200 1.0770.02 0.9570.019 0.3270.006 0.1170.002

Phenolic SC 0.02070.0004 0.00570.0001 0.00470.0001 0.00370.0001

compounds S50 0.06070.0012 0.01570.0003 0.01270.0002 0.00870.0002

(g kg�1) S100 0.17870.003 0.04570.0009 0.03470.0007 0.02370.0005

. S200 0.31370.006 0.05770.001 0.04970.001 0.04270.0008

A. Mekki et al. / Journal of Environmental Management 84 (2007) 134–140 137

layers (0–25 cm), while higher retention time phenoliccompounds (28.8–31.9min) infiltrated in deeper layers ofthe soil (50–120 cm). The HPLC analysis realised onphenolic compounds of the surface layers (0–10 cm) of P3

plot treated with 200m3 ha�1 y�1 (S200), 1 year after OMWapplication showed that only a residual phenolic fraction(F) persisted. This fraction which had a molecular-massbetween 17 and 25 kDa (Fig. 2) neither originated from thesoil nor from the OMW.

3.5. Phenolic fraction (F) phytotoxicity

The residual phenolic fraction extracted from the soiltreated annually with 200m3 ha�1 yr�1 of OMW (S200)proved to be phytotoxic to the plant B. cernua. Fig. 3illustrates the germination index of B. cernua seeds incontrol medium (CM), phenolic fraction (F), OMW diluted4 times (OMW/4), 25 times (OMW/25) and 50 times(OMW/50). In the CM, the germination index reached100% after 24 h of incubation. In the medium containingthe soil phenolic residual fraction (F), the seed germinationindex reached 100% after 72 h of incubation. In themedium containing 4, 25 and 50 times diluted freshOMW, the germination index showed a delay or even atotal absence of the B. cernua germination.

4. Discussion

The unprocessed OMW pH seemed to be neutralized bysoil alkalinity. This can be explained by the fact that theOMW acidity was neutralized by carbonates present in soilhorizons thus generating soluble calcium bicarbonate aswas shown in earlier works (Sierra et al., 2001). Theincrease of the soil salinity involved the alteration of thecation exchange capacity (CEC) and could affect the soil

fertility. The salinity decrease at the lower horizons couldbe explained by the phenomena of adsorption and evapo-transpiration which concentrate ionic species (especiallyNa, Cl and K) in the upper horizons (Zanjari andNejmeddine, 2001). According to the sandy compositionand the porous property of the studied soil, we observed anincrease of the OM in all layers of soil with the increase ofthe dose of OMW used in the irrigation. This suggests animportant leaching of OMW from the surface to the soildeeper layers and therefore of phenolic compounds. Theincrease of the OM content was not accompanied with anincrease of the respiration potential. On the contrary, itwas found that soil respiration decreases when the OMWamendment becomes higher than 50m3 ha�1 yr�1 (Mekki etal., 2005). This finding seems to differ with the previousfindings by Cox et al. (1998) who reported that themineralization of the OMW organic matter in the soilupper layers could be explained by the proportion ofbiodegradable OM in OMW. The beneficial effect of wasteon soil organic matter depends rather on the quality ofwaste, the type of soil and the environment.The study of the dynamics of the polyphenols showed

that phenolic compounds of low molecular-mass migratedmore in depth than those of high molecular-mass. Thedetection of monomers in depths can have two explana-tions. First, the polyphenols can be adsorbed by the soiland monomers can be mobilized and transported byinfiltrating rain water. Second, the monomers are easierto degrade by the aerobic microorganisms at the soilsurface. This is in line with previous findings of acorrelation between places of olive oil mill waste spreadingand wells with high phenolic concentrations (Spandre andDellomonaco, 1996).The residual phenolic fraction (F) extracted 1 year after

OMW application in the soil annually treated with

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22.8

0323

.61

22.8

625

.76

22.9

625

.78

25.6

26.3

22.7

424

.19

29.6

3

22.6

225

.69

23.1

37 25.6

9333

.543

34.8

32

28.1

7230

.922

42.9

87

0.21

5 27.3

6529

.32

30.7

27.2

3829

.41

30.9

32

27.3

3529

.62

31.0

72

27.6

1329

.18

31.2

43

28.8

1831

.917

S50 S100 S200

S50 S100 S200

S50 S100S200

S50 S100 S200

OMW/25

0 m

0.25 m

0.5 m

1.2 m

Fig. 1. HPLC spectrum of phenolic compounds detected at different soil depths 4 months after OMW application and in the 7th consecutive year

irrigation by OMW (the numbers associated to the peaks correspond to the retention time in min).

A. Mekki et al. / Journal of Environmental Management 84 (2007) 134–140138

200m3 ha�1 yr�1 of OMW was not detected in control soil.Therefore, it was neither originated from the soil nor was itfound in the OMW. Hence, it might have been newlysynthesized during the incubation time of OMW. Indeed,Saiz-Jimenez et al. (1987) observed the simultaneousdegradation of high molecular-mass fractions to producefractions of a smaller molecular-mass and the synthesis ofcompounds which were more resistant to degradation.Feria (2000) reported that polyphenols residual levels canbe important even 6 years after OMW spreading.

The phytotoxicity effect of the OMW phenolic com-pounds on vegetative production of olive trees wasevaluated by Ben Rouina (1994) and Ben Rouina et al.(1999). The authors showed no apparent effects on thephysiology and growth of olive trees after 5 successiveyears of irrigation with OMW whereas better oliveproduction yields were reported. However, olive trees cannot be used for monitoring phytotoxicity since they are

very resistant to stresses such as drought, salinity, etc. Inour study, the model sensitive plant B. cernua was used anddemonstrated that spreading OMW, even in one time ayear, may modify the soil structure and composition whichcould affect olive trees after long-term application. Thephenolic fraction (F) extracted from soil 1 year after OMWapplication is not very phytotoxic and it only increased thetime required to obtain 100% of germination of Brassica

cernua seeds. Until now, nothing has proved that long-termaccumulation of this fraction in soil makes it much morephytotoxic. Moreover, this fraction could serve as pre-cursor for the formation of humic acids in soil. Morestudies should be investigated to understand the role andevolution of this fraction in the soil. On the other hand, thesuccessive load of acidic and salt effluent would necessarilylead to the acidification and salinisation of soil which, inthe long term, could impair its cation exchange capacityand affect its fertility.

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Fig. 2. HPLC spectrum of the crude OMW and of the residual phenolic

fraction (F) extracted from the upper layers (0–10 cm) soils amended with

200m3 ha�1 yr�1 one year after OMW application and in the 7th

consecutive year irrigation by OMW (the numbers associated to the

peaks correspond to the retention time in min).

0

20

40

60

80

100

120

0.5 1 1.5 2 2.5 3 3.5

CM OMW/4 OMW/25 OMW/50 F

Time (day)

GI (

%)

Fig. 3. Germination index (GI) of Brassica cernua determined on the

phenolic fraction (F) in comparison with the untreated OMW and the

control medium (CM). (OMW/4: OMW diluted 4 times, OMW/25: OMW

diluted 25 times and OMW/50: OMW diluted 50 times).

A. Mekki et al. / Journal of Environmental Management 84 (2007) 134–140 139

5. Conclusion

OMW amendment without pre-treatment seems to affectthe structure and the composition of the soil. This workshowed the presence of phenolic compounds at differentdepths of all the OMW amended soils 4 months afterOMW spreading. A new specific phenolic fraction (F) notdetected in control soil nor originated from OMW wassynthesized and persisted in the soil 1 year after OMWapplication. This fraction displayed a certain ecotoxicity.The practice of OMW spreading in agricultural soil hasuntil now been subject to a great controversy in thescientific communities. Our survey showed that thephytotoxic phenolic monomers tend to disappear fromthe upper layer of soil after some time due to degradation

or polymerization or to their important leaching to the soildeeper layers that may provoke the contamination of theground water in the long term. Nevertheless, the highmolecular weight polyphenol (fraction F) formed in the soilis moderately phytotoxic. Therefore, if a biological pre-treatment of OMW is conceived for reducing its toxicityprior to its soil spreading, this would allow an ecologicaluse of this waste in agriculture. However, this practiceshould take into account the cumulative effect of soilsalinization, which would transform little by little the soilinto an unproductive one.

Acknowledgements

This research was funded by E.C contract ‘‘ICA3-CT2002-10034’’ and contracts programmes (MRSTDC,Tunisia). The authors acknowledge the help of Dr. BechirBen Rouina from ‘‘Institut de l’Olivier de Sfax, Tunisie’’.

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