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Polyphenols dynamics and phytotoxicity in a soil amended by olive mill wastewaters
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Journal of Environmental Management 84 (2007) 134–140 www.elsevier.com/locate/jenvman
Polyphenols dynamics and phytotoxicity in a soil amended by olive mill wastewaters Ali Mekki, Abdelhafidh Dhouib, Sami Sayadi Laboratoire des Bioproce´de´s, 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 1.2 m 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 is caused by industrial, agricultural and urban wastewaters which cannot be sent to ordinary systems of wastewater treatment. These include olive mill wastewaters (OMW) (Spandre and Dellomonaco, 1996), which have the following chemical properties: an enormous content of organic matter (COD between 60 and 185 g l1; BOD5 between 14 and 75 g l1), a low pH, and high polyphenols, potassium and phosphorus contents (Tomati and Galli, 1992; Rinaldi et al., 2003). These wastes constitute a pollution constraint for 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 the producers, millers, and for the whole community due to its potential pollution risk (Vazquez et al., 1974; Martinez et al., 1986; Rinaldi et al., 2003). Although the observed toxicity of OMW has been generally related to the concentration of phenols, nonphenolic-related toxicity was also reported by Capasso et al. (1992).
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E-mail address: [email protected] (S. Sayadi). 0301-4797/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvman.2006.05.015
To solve the problems associated with OMW, different elimination methods based on physicochemical and biological 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, the most frequently used methods nowadays are the direct application to agricultural soils and storage in evaporation ponds (Fiestas and Borja, 1992; Martinez Nieto and Garrido, 1994). Agriculturally, these wastewaters can be used as soil biofertilizers (Solinas et al., 1975; Ranalli, 1989; Lo´pez et al., 1993). Moreover, positive effects on chemical fertility have been generally reported, but less attention has been paid to the effect on the underground water resource quality. D’acqui Luigi et al. (2002) reported that OMW can constitute a serious organic pollution risk for the water table and the deep underground waters. According to Levi-Menzi et al. (1992), the high COD value (150–200 g l1) and the presence of phytotoxic and antibacterial polyphenols (1–10 kg t1) in this waste can be a serious pollution risk for superficial and underground waters. Furthermore, the presence of phenolic compounds in 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 the crustacean Daphnia magna are severely intoxicated on
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exposure to phenol derivatives at concentrations of 40 mg l1 for only 15 min. OMW proved to have an almost immediate toxic action on gold fish Carassius auratus at a concentration of 10% (v/v) (Bellido, 1989), on fish and rayfinned fish Cyprinus carpio and Chondrostoma polylepis at concentrations 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 incongruens (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 phytotoxic effects in plants when OMW is used directly as an organic fertiliser. The toxicity of OMW and its phenolic content against the seeds of Lepidium sativum was well detailed in the study of Aggelis et al. (2003). However, works concerning the evolution at the real scale of OMW phenolic compounds on the soil and their consequences on the groundwater are rare (Spandre and Dellomonaco, 1996). The aim of this work was to characterize a soil which was amended with raw OMW and to demonstrate that in spite of the considerable microbiological activity of the soil to degrade and to attenuate the toxic effect of the OMW phenolic compounds, these chemicals persist or change and keep their toxicity even long after the spreading period. They infiltrate in soil and can pollute the groundwater. 2. Materials and methods 2.1. OMW characterization The fresh OMW was taken from a three-phase discontinuous extraction factory located in Chaaˆl-Sfax southern Tunisia. This OMW was first recovered in a storage basin for 1 week then characterized before application. pH and electrical conductivity (EC) were determined according to Sierra et al. (2001) standard method. Organic matter (OM) was determined by combustion of the samples in a furnace at 550 1C for 4 h. Total organic carbon was determined by dry combustion (TOC Analyser multi N/C 1000). Total nitrogen was determined by Kjeldahl (1883) method. COD was determined according to Knechtel (1978) standard method. BOD5 was determined by the manometric method with a respirometer (BSB-Controller Model 620 T WTW) and phenolic compounds (orthodiphenols) were quantified by means of the Folin–Ciocalteu colorimetric method (Box, 1983) using caffeic acid as standard. The absorbance was determined at l ¼ 765 nm. Phosphorus, iron, magnesium, potassium, sodium and chloride were determined by atomic absorption. 2.2. Study area and soil sampling The study area consisted of a field of olive trees located in Chaˆal at 60 km to the South-West of Sfax, Tunisia,
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North latitude 341 30 , East longitude 101 200 . The mean annual rainfall is 200 mm (Ben Rouina et al., 1999). Annually, since 1994 three experimental plots P1, P2 and P3 had been respectively amended with 50, 100, and 200 m3 ha1 of unprocessed OMW. This amendment was realised in February in one application (Ben Rouina, 1994). The plot PC was not amended and served as control. Soil samples (Sc, S50, S100 and S200) were collected monthly from different parts and at variable depths (0–120 cm) of each plot, using a soil auger. All soil samples, taken from each plot were mixed and the water content was immediately determined. Then they were air dried and sieved with a mesh size of 450 mm and stored at 4 1C prior to use. 2.3. Soil analysis pH, electrical conductivity, organic matter and phenolic compounds content in all soil samples were determined. 2.4. Soil phenolic compounds determination For soil phenolic compounds, only water soluble substances were determined. A 1/25 (w/v) soil/aqueous mixture was shaken for 12 h in a mechanical shaker. The supernatant was extracted 3 times with ethyl acetate. The collected organic fraction was dried and evaporated under vacuum. The residue was extracted twice with dichloromethane in order to remove the non-phenolic fraction (lipids, aliphatic and sugars). The liquid phase was discarded while the washed residue was analysed by the Size exclusion-HPLC technique (Allouche et al., 2004). Using this same extraction and characterisation method, a residual phenolic fraction (F) was extracted from the upper layer (0–10 cm) of P3 plot in January 2001, one year after the last application of OMW (February 2000). This year was the last year of the 7 years of OMW application. The molecular-mass (MM) of the soil extract or OMW phenolic compounds was estimated by the comparison of their retention time to the retention time of standard compounds having a known molecular-mass and injected in the same conditions. 2.5. Phytotoxicity tests Phytotoxicity of OMW phenolic compounds was assessed by the determination of the germination index (GI) of a very sensitive plant belonging to the family of Cruciferae Brassicaceae (Brassica cernua) according to Zucconi et al. (1981) standard method. MS medium described by Murashige and Skoog (1962) was used for control germination index. For germination index of (F) fraction and OMW, the water in MS is replaced by the 1/25 (w/v) soil/aqueous mixture extraction solution of (F) or OMW diluted in distilled water 4, 25 and 50 times.
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2.6. Statistical analysis For soil analysis, three replications were used for each parameter. For the germination test, three replications were used in each issue. Data were analysed using the ANOVA procedure. Variance and standard deviation were determined using Genstat 5 (second edition for windows). 3. Results
was very low and it varied between 0.8% and 1.15% w/w. The water-holding capacity, the salinity and the content of total organic carbon, humus, total nitrogen, phosphate and potassium increased when the spread amounts of the treated or untreated OMW increased. Soil irrigated with raw olive mill wastewater showed also a significant inhibition of some enzymatic activities such as ureases, ammonium oxydases and nitrate reductases while some other enzymatic activities such as xylanases and cellulases were slightly stimulated (data not shown).
3.1. OMW characterization 3.3. Analytical results of soil profiles The physicochemical characteristics of crude OMW are summarized in Table 1 and correspond to the mean values of 3 analysis. The high pollutant load of OMW (72 g l1 of COD) and its elevated salinity (6.75 g l1) could be observed. The OMW C/N ratio was unfavourable for the biodegradation and humification processes. OMW contains an enormous supply of organic matter very rich in phenolic compounds (9.2 g l1) which are toxic. 3.2. Soil characterization The soil of the study area was described in a previous study (Mekki et al., 2005). It had an important content of active calcareous (0.6% w/w) at the surface, and was composed of sand (89.82% w/w), clay (7.44% w/w) and silt (2.74% w/w). It had an alkaline pH and a weak electrical conductivity. The soil was very poor in nitrogen (0.5 g kg1 dry soil), and in organic matter (0.16% w/w). The levels of potassium and phosphorus were 0.014% w/w and 0.002% w/w, respectively. As it was discussed in that previous study (Mekki et al., 2006), the soil water content
For soil profiles, pH, electrical conductivity, organic matter and polyphenols contents were analysed 4 months after OMW spreading. Taking into consideration the deviation of the pH determination, it can be said that the pH does not vary as a function of depth. However, a very weak decrease was observed when OMW spreading quantity increased. The good buffering capacity of the soil related to its calcareous content neutralized the relatively acidic pH 5.12 of OMW (Table 2). This table shows that electrical conductivity increased proportionally with the increase of the quantity of unprocessed OMW and decreased with depth in all analysed soils. The electrical conductivity increased in all soil depths compared to the control soil which suggests the leaching of OMW from the surface to the soil deeper layers. The analysis of the organic matter content of the soil showed an increase in all soil depths compared to the control soil. This organic matter increase is proportional to the OMW spread quantities (Table 2). 3.4. Phenolic compounds dynamics
Table 1 Physico-chemical characteristics of unprocessed olive mill wastewater used in ferti-irrigation (values after7represent standard deviation) Characteristics
Data
pH (25 1C) Electrical conductivity (25 1C) (dS m1) Chemical oxygen demand (g l1) Biochemical oxygen demand (g l1) COD/BOD5 Salinity (g l1) Water content (g l1) Total solids (g l1) Mineral matter (g l1) Volatile solid (g l1) Total organic carbon (g l1) Total nitrogen Kjeldahl (g l1) Carbon/nitrogen P (mg l1) Na (g l1) Cl (g l1) K (g l1) Ca (g l1) Fe (mg l1) Mg (mg l1) Ortho-diphenols (g l1)
5.170.2 8.970.1 7272.8 1370.9 5.5370.72 6.7570.66 948717.2 5272.98 870.43 4471.85 25.5271.18 0.670.06 4378 3673.6 0.9470.09 1.670.15 8.870.8 1.270.11 3272.9 187718.8 9.271.8
This part of the study proposed to investigate the dynamics of the phenolic compounds in a very porous and permeable sandy soil irrigated by increasing doses of unprocessed OMW 50, 100 and 200 m3 ha1 yr1 and to trace the change of the phenolic compounds content as a function of time and depth. Results showed that the application of increasing OMW doses increased the total phenolic compounds content in all soil layers (Table 2). Besides, Table 2 shows that the majority of phenolic compounds are kept in the soil upper layers. The phenolic concentration decreased rapidly from 0 to 25 cm then continued to decrease weakly with depth but remained even detectable at 120 cm. The control soil showed very weak detectable phenolic substances. Comparison of phenolic compound spectra shows that especially the high molecular-mass compounds decreased with depth while the low molecular-mass polyphenols remained more abundant. According to the HPLC principle, polymers were eluted with low retention time, while monomers needed higher retention time. Fig. 1 shows that phenolic compounds with low retention time (22.8–25.8 min) 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
. pH . .
SC S50 S100 S200
8.170.2 8.170.2 870.2 7.970.2
8.270.2 8.170.2 870.2 7.770.2
8.370.2 8.370.2 8.170.2 7.870.2
8.2670.2 8.270.2 8.170.2 870.2
Electrical conductivity (mS cm1) .
SC S50 S100 S200
19870.1 24870.1 37570.1 74370.1
15470.1 20570.1 31570.1 69570.1
11270.1 18370.1 28470.1 54870.1
8570.1 12670.1 19670.1 45270.1
Organic matter (%, w/w) .
SC S50 S100 S200
0.1670.003 0.3470.007 0.6270.012 1.0770.02
0.1470.002 0.270.004 0.5470.01 0.9570.019
0.0870.002 0.1470.002 0.1270.002 0.3270.006
0.0270.0004 0.0970.0018 0.0870.0016 0.1170.002
Phenolic compounds (g kg1) .
SC S50 S100 S200
0.02070.0004 0.06070.0012 0.17870.003 0.31370.006
0.00570.0001 0.01570.0003 0.04570.0009 0.05770.001
0.00470.0001 0.01270.0002 0.03470.0007 0.04970.001
0.00370.0001 0.00870.0002 0.02370.0005 0.04270.0008
layers (0–25 cm), while higher retention time phenolic compounds (28.8–31.9 min) infiltrated in deeper layers of the soil (50–120 cm). The HPLC analysis realised on phenolic compounds of the surface layers (0–10 cm) of P3 plot treated with 200 m3 ha1 y1 (S200), 1 year after OMW application showed that only a residual phenolic fraction (F) persisted. This fraction which had a molecular-mass between 17 and 25 kDa (Fig. 2) neither originated from the soil nor from the OMW. 3.5. Phenolic fraction (F) phytotoxicity The residual phenolic fraction extracted from the soil treated annually with 200 m3 ha1 yr1 of OMW (S200) proved to be phytotoxic to the plant B. cernua. Fig. 3 illustrates the germination index of B. cernua seeds in control medium (CM), phenolic fraction (F), OMW diluted 4 times (OMW/4), 25 times (OMW/25) and 50 times (OMW/50). In the CM, the germination index reached 100% after 24 h of incubation. In the medium containing the soil phenolic residual fraction (F), the seed germination index reached 100% after 72 h of incubation. In the medium containing 4, 25 and 50 times diluted fresh OMW, the germination index showed a delay or even a total absence of the B. cernua germination. 4. Discussion The unprocessed OMW pH seemed to be neutralized by soil alkalinity. This can be explained by the fact that the OMW acidity was neutralized by carbonates present in soil horizons thus generating soluble calcium bicarbonate as was shown in earlier works (Sierra et al., 2001). The increase of the soil salinity involved the alteration of the cation exchange capacity (CEC) and could affect the soil
fertility. The salinity decrease at the lower horizons could be explained by the phenomena of adsorption and evapotranspiration which concentrate ionic species (especially Na, Cl and K) in the upper horizons (Zanjari and Nejmeddine, 2001). According to the sandy composition and the porous property of the studied soil, we observed an increase of the OM in all layers of soil with the increase of the dose of OMW used in the irrigation. This suggests an important leaching of OMW from the surface to the soil deeper layers and therefore of phenolic compounds. The increase of the OM content was not accompanied with an increase of the respiration potential. On the contrary, it was found that soil respiration decreases when the OMW amendment becomes higher than 50 m3 ha1 yr1 (Mekki et al., 2005). This finding seems to differ with the previous findings by Cox et al. (1998) who reported that the mineralization of the OMW organic matter in the soil upper layers could be explained by the proportion of biodegradable OM in OMW. The beneficial effect of waste on soil organic matter depends rather on the quality of waste, the type of soil and the environment. The study of the dynamics of the polyphenols showed that phenolic compounds of low molecular-mass migrated more in depth than those of high molecular-mass. The detection of monomers in depths can have two explanations. First, the polyphenols can be adsorbed by the soil and monomers can be mobilized and transported by infiltrating rain water. Second, the monomers are easier to degrade by the aerobic microorganisms at the soil surface. This is in line with previous findings of a correlation between places of olive oil mill waste spreading and wells with high phenolic concentrations (Spandre and Dellomonaco, 1996). The residual phenolic fraction (F) extracted 1 year after OMW application in the soil annually treated with
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OMW/25
S100
22.62 25.69
S50
22.74 24.19 29.63
0m S200
S50
27.335 29.62 31.072
0.5 m S100
27.365 29.32 30.7
S100
27.613 29.18 31.243
0.215
42.987
S50
28.172 30.922
0.25 m S200
23.137 25.693 33.543 34.832
S200
27.238 29.41 30.932
S100
S200
28.818 31.917
22.86 25.76
22.803 23.61
S50
25.6 26.3
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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).
200 m3 ha1 yr1 of OMW was not detected in control soil. Therefore, it was neither originated from the soil nor was it found in the OMW. Hence, it might have been newly synthesized during the incubation time of OMW. Indeed, Saiz-Jimenez et al. (1987) observed the simultaneous degradation of high molecular-mass fractions to produce fractions of a smaller molecular-mass and the synthesis of compounds which were more resistant to degradation. Feria (2000) reported that polyphenols residual levels can be important even 6 years after OMW spreading. The phytotoxicity effect of the OMW phenolic compounds on vegetative production of olive trees was evaluated by Ben Rouina (1994) and Ben Rouina et al. (1999). The authors showed no apparent effects on the physiology and growth of olive trees after 5 successive years of irrigation with OMW whereas better olive production yields were reported. However, olive trees can not be used for monitoring phytotoxicity since they are
very resistant to stresses such as drought, salinity, etc. In our study, the model sensitive plant B. cernua was used and demonstrated that spreading OMW, even in one time a year, may modify the soil structure and composition which could affect olive trees after long-term application. The phenolic fraction (F) extracted from soil 1 year after OMW application is not very phytotoxic and it only increased the time required to obtain 100% of germination of Brassica cernua seeds. Until now, nothing has proved that long-term accumulation of this fraction in soil makes it much more phytotoxic. Moreover, this fraction could serve as precursor for the formation of humic acids in soil. More studies should be investigated to understand the role and evolution of this fraction in the soil. On the other hand, the successive load of acidic and salt effluent would necessarily lead to the acidification and salinisation of soil which, in the long term, could impair its cation exchange capacity and affect its fertility.
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or polymerization or to their important leaching to the soil deeper layers that may provoke the contamination of the ground water in the long term. Nevertheless, the high molecular weight polyphenol (fraction F) formed in the soil is moderately phytotoxic. Therefore, if a biological pretreatment of OMW is conceived for reducing its toxicity prior to its soil spreading, this would allow an ecological use of this waste in agriculture. However, this practice should take into account the cumulative effect of soil salinization, which would transform little by little the soil into an unproductive one. Acknowledgements
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 200 m3 ha1 yr1 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).
CM
120
OMW/4
OMW/25
OMW/50
F
100 GI (%)
80 60 40 20 0 0.5
1
1.5
2 Time (day)
2.5
3
3.5
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).
5. Conclusion OMW amendment without pre-treatment seems to affect the structure and the composition of the soil. This work showed the presence of phenolic compounds at different depths of all the OMW amended soils 4 months after OMW spreading. A new specific phenolic fraction (F) not detected in control soil nor originated from OMW was synthesized and persisted in the soil 1 year after OMW application. This fraction displayed a certain ecotoxicity. The practice of OMW spreading in agricultural soil has until now been subject to a great controversy in the scientific communities. Our survey showed that the phytotoxic phenolic monomers tend to disappear from the upper layer of soil after some time due to degradation
This research was funded by E.C contract ‘‘ICA3CT2002-10034’’ and contracts programmes (MRSTDC, Tunisia). The authors acknowledge the help of Dr. Bechir Ben Rouina from ‘‘Institut de l’Olivier de Sfax, Tunisie’’. References Aggelis, G., Iconomou, D., Christou, M., Bokas, D., Kotzailias, S., Christou, G., Tsagou, V., Papanikolaou, S., 2003. Phenolic removal in a model olive oil mill wastewater using Pleurotus ostreatus in bioreactor cultures and biological evaluation of the process. Water Research 37, 3897–3904. Allouche, N., Fki, I., Sayadi, S., 2004. Toward a high yield recovery of antioxidants and purified hydroxytyrosol from olive mill wastewaters. Journal of Agricultural Food Chemistry 52, 267–273. Angus, R.A., 1983. Phenol tolerance in populations of Mosquito fish from polluted and non polluted water. Transactions of the American Fishery Society 112, 794–799. Bellido, E., 1989. Contaminacio´n por alpechin: Un nuevo tratamiento para disminuir su impacto en diferentes ecosistemas. PhD Thesis, University of Cordoba. Ben Rouina, B., 1994. Re´percussions agronomiques de l’e´pandage des margines comme fertilisant. International Conference on Land and Water Resources Management in the Mediterranean Region II, 583–594. Ben Rouina, B., Taamallah, H., Ammar, E., 1999. Vegetation water used as a fertilizer on young olive plants. Acta Horticulturae 474 (1), 353–355. Ben Sassi, A., Boularbah, A., Jaouad, A., Walker, G., Boussaid, A., 2005. A comparison of Olive oil Mill Wastewaters (OMW) from three different processes in Morocco. Process Biochemistry. in press. Box, J.D., 1983. Investigation of the Folin-Ciocalteau phenol reagent for the determination of polyphenolic substances in natural waters. Water Research 17, 511–522. Capasso, R., Cristinzio, G., Evidente, A., Scognamiglio, F., 1992. Isolation, spectroscopy and selective phytotoxic effects of polyphenols from vegetable waste waters. Phytochemistry 31, 4125–4128. Casa, R., D’Annibale, A., Pieruccetti, F., Stazi, S.R., 2003. Reduction of the phenolic components in olive-mill wastewater by enzymatic treatment and its impact on durum wheat (Triticum durum Desf.) germinability. Chemosphere 50, 959–966. Cox, L., Celis, R., Hermosin, M.C., Beker, A., Cornejo, J., 1998. Porosity and herbicide leaching in soils amended with olive-mill wastewater. Agricultural Ecosystem Environment 65 (2), 151–161. D’acqui Luigi, P., Sparvoli, E., Agnelli, A., Santi Carolina, A., 2002. Olive oil mills waste waters and clays minerals interactions: organics transformation and clay particles aggregation, 17th World Congress of Soil Science (WCSS), Thailand, August, 14–21. Symposium No 47, Paper No 1578, p. 1110–1130.
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Paredes, C., Cegarra, J., Roig, A., Sanchez-Monedero, M.A., Bernal, M.P., 1999. Characterization of olive mill wastewater (alpechin) and its sludge for agricultural purposes. Bioresource Technology 67, 111–115. Paredes, C., Roig, A., Bernal, M.P., Sanchez-Monedero, M.A., Cegarra, J., 2000. Evolution of organic matter and nitrogen during cocomposting of olive mill wastewater with solid organic wastes. Biology of Fertile Soil. 32 (3), 222–227. Ramos-Cormenzana, A., Juarez-Jime´nez, B., Garcia-Pateja, M.P., 1996. Antimicrobial activity of olive mill waste-waters (Alpechin) and biotransformed olive oil mill wastewater. Internatioanal Journal of Biodeterioration and biodegradation. 38 (3–4), 283–290. Ranalli, A., 1989. Il problema dei reflui di frantoio: Aspetti tenici e normative. L’informatore agrario 16, 29–52. Rinaldi, M., Rana, G., Introna, M., 2003. Olive-mill wastewater spreading in southern Italy: effects on a durum wheat crop. Field Crops Research 84 (3), 319–326. Rodriguez, M.M., Perez, J., Ramos-Cormenzana, A., Martinez, J., 1988. Effect of extracts obtained from olive oil mill waste waters on Bacillus megaterium ATCC 33085. Journal of Applied Bacteriology 64, 219–226. Saiz-Jimenez, C., De Leeuw, J.W., Gomez-Alarcon, G., 1987. Sludge from the waste water of the olive processing industry: a potential soil fertilizer? Science of Total Environment 62, 445–452. Sierra, J., Marti, E., Montserrat, G., Cruanas, R., Garau, M.A., 2001. Characterization and evolution of a soil affected by olive oil mill wastewater disposal. Science of Total Environment 279, 207–214. Solinas, M., Di Giovacchino, L., Cucurachi, A., 1975. Polifenoli delle olive e delfolio di oliva. Nota 1: variazioni che subiscono alcuni polifenoli delle olive col procedere della maturazione. Annali Istituto Sperimentale per l’Elaiotecnica 105, 3–24. Spandre, R., Dellomonaco, G., 1996. Polyphenols pollution by olive mill waste waters, Tuscany-Italy. Journal of Environomental Hydrology 4, 1–13. Tomati, U., Galli, E., 1992. The Fertilizing Value of Waste Waters from the Olive Processing Industry. In: Kubat, J. (Ed.), Humus, its Structure and Role in Agriculture and Environment, vol. 25. Elsevier Science, Amsterdam, pp. 117–126. Vazquez, A., Maestro, R., Graciani, E., 1974. Componentes Fenolicos de la aceituna : Polifenoles del alpechin. Grasas y Aceites 25, 341–345. Yesilada, E., O¨zmen, M., Yesilada, O¨., 1999. Studies on the toxic and genotoxic effect of olive oil mill wastewater. Fresenius Environment Bulletin 8, 732–739. Zanjari, B., Nejmeddine, A., 2001. Impact of spreading olive mill waste waters on soil characteristics: Laboratory experiments. Agronomie 21, 749–755. Zucconi, F., Forte, M., Monac, A., Beritodi, M., 1981. Biological evaluation of compost maturity. Biocycle 22, 27–29.
Journal of Environmental Management 84 (2007) 134–140 www.elsevier.com/locate/jenvman
Polyphenols dynamics and phytotoxicity in a soil amended by olive mill wastewaters Ali Mekki, Abdelhafidh Dhouib, Sami Sayadi Laboratoire des Bioproce´de´s, 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 1.2 m 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 is caused by industrial, agricultural and urban wastewaters which cannot be sent to ordinary systems of wastewater treatment. These include olive mill wastewaters (OMW) (Spandre and Dellomonaco, 1996), which have the following chemical properties: an enormous content of organic matter (COD between 60 and 185 g l1; BOD5 between 14 and 75 g l1), a low pH, and high polyphenols, potassium and phosphorus contents (Tomati and Galli, 1992; Rinaldi et al., 2003). These wastes constitute a pollution constraint for 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 the producers, millers, and for the whole community due to its potential pollution risk (Vazquez et al., 1974; Martinez et al., 1986; Rinaldi et al., 2003). Although the observed toxicity of OMW has been generally related to the concentration of phenols, nonphenolic-related toxicity was also reported by Capasso et al. (1992).
Corresponding author. Tel./fax: +216 7444 0452.
E-mail address: [email protected] (S. Sayadi). 0301-4797/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvman.2006.05.015
To solve the problems associated with OMW, different elimination methods based on physicochemical and biological 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, the most frequently used methods nowadays are the direct application to agricultural soils and storage in evaporation ponds (Fiestas and Borja, 1992; Martinez Nieto and Garrido, 1994). Agriculturally, these wastewaters can be used as soil biofertilizers (Solinas et al., 1975; Ranalli, 1989; Lo´pez et al., 1993). Moreover, positive effects on chemical fertility have been generally reported, but less attention has been paid to the effect on the underground water resource quality. D’acqui Luigi et al. (2002) reported that OMW can constitute a serious organic pollution risk for the water table and the deep underground waters. According to Levi-Menzi et al. (1992), the high COD value (150–200 g l1) and the presence of phytotoxic and antibacterial polyphenols (1–10 kg t1) in this waste can be a serious pollution risk for superficial and underground waters. Furthermore, the presence of phenolic compounds in 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 the crustacean Daphnia magna are severely intoxicated on
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exposure to phenol derivatives at concentrations of 40 mg l1 for only 15 min. OMW proved to have an almost immediate toxic action on gold fish Carassius auratus at a concentration of 10% (v/v) (Bellido, 1989), on fish and rayfinned fish Cyprinus carpio and Chondrostoma polylepis at concentrations 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 incongruens (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 phytotoxic effects in plants when OMW is used directly as an organic fertiliser. The toxicity of OMW and its phenolic content against the seeds of Lepidium sativum was well detailed in the study of Aggelis et al. (2003). However, works concerning the evolution at the real scale of OMW phenolic compounds on the soil and their consequences on the groundwater are rare (Spandre and Dellomonaco, 1996). The aim of this work was to characterize a soil which was amended with raw OMW and to demonstrate that in spite of the considerable microbiological activity of the soil to degrade and to attenuate the toxic effect of the OMW phenolic compounds, these chemicals persist or change and keep their toxicity even long after the spreading period. They infiltrate in soil and can pollute the groundwater. 2. Materials and methods 2.1. OMW characterization The fresh OMW was taken from a three-phase discontinuous extraction factory located in Chaaˆl-Sfax southern Tunisia. This OMW was first recovered in a storage basin for 1 week then characterized before application. pH and electrical conductivity (EC) were determined according to Sierra et al. (2001) standard method. Organic matter (OM) was determined by combustion of the samples in a furnace at 550 1C for 4 h. Total organic carbon was determined by dry combustion (TOC Analyser multi N/C 1000). Total nitrogen was determined by Kjeldahl (1883) method. COD was determined according to Knechtel (1978) standard method. BOD5 was determined by the manometric method with a respirometer (BSB-Controller Model 620 T WTW) and phenolic compounds (orthodiphenols) were quantified by means of the Folin–Ciocalteu colorimetric method (Box, 1983) using caffeic acid as standard. The absorbance was determined at l ¼ 765 nm. Phosphorus, iron, magnesium, potassium, sodium and chloride were determined by atomic absorption. 2.2. Study area and soil sampling The study area consisted of a field of olive trees located in Chaˆal at 60 km to the South-West of Sfax, Tunisia,
135
North latitude 341 30 , East longitude 101 200 . The mean annual rainfall is 200 mm (Ben Rouina et al., 1999). Annually, since 1994 three experimental plots P1, P2 and P3 had been respectively amended with 50, 100, and 200 m3 ha1 of unprocessed OMW. This amendment was realised in February in one application (Ben Rouina, 1994). The plot PC was not amended and served as control. Soil samples (Sc, S50, S100 and S200) were collected monthly from different parts and at variable depths (0–120 cm) of each plot, using a soil auger. All soil samples, taken from each plot were mixed and the water content was immediately determined. Then they were air dried and sieved with a mesh size of 450 mm and stored at 4 1C prior to use. 2.3. Soil analysis pH, electrical conductivity, organic matter and phenolic compounds content in all soil samples were determined. 2.4. Soil phenolic compounds determination For soil phenolic compounds, only water soluble substances were determined. A 1/25 (w/v) soil/aqueous mixture was shaken for 12 h in a mechanical shaker. The supernatant was extracted 3 times with ethyl acetate. The collected organic fraction was dried and evaporated under vacuum. The residue was extracted twice with dichloromethane in order to remove the non-phenolic fraction (lipids, aliphatic and sugars). The liquid phase was discarded while the washed residue was analysed by the Size exclusion-HPLC technique (Allouche et al., 2004). Using this same extraction and characterisation method, a residual phenolic fraction (F) was extracted from the upper layer (0–10 cm) of P3 plot in January 2001, one year after the last application of OMW (February 2000). This year was the last year of the 7 years of OMW application. The molecular-mass (MM) of the soil extract or OMW phenolic compounds was estimated by the comparison of their retention time to the retention time of standard compounds having a known molecular-mass and injected in the same conditions. 2.5. Phytotoxicity tests Phytotoxicity of OMW phenolic compounds was assessed by the determination of the germination index (GI) of a very sensitive plant belonging to the family of Cruciferae Brassicaceae (Brassica cernua) according to Zucconi et al. (1981) standard method. MS medium described by Murashige and Skoog (1962) was used for control germination index. For germination index of (F) fraction and OMW, the water in MS is replaced by the 1/25 (w/v) soil/aqueous mixture extraction solution of (F) or OMW diluted in distilled water 4, 25 and 50 times.
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2.6. Statistical analysis For soil analysis, three replications were used for each parameter. For the germination test, three replications were used in each issue. Data were analysed using the ANOVA procedure. Variance and standard deviation were determined using Genstat 5 (second edition for windows). 3. Results
was very low and it varied between 0.8% and 1.15% w/w. The water-holding capacity, the salinity and the content of total organic carbon, humus, total nitrogen, phosphate and potassium increased when the spread amounts of the treated or untreated OMW increased. Soil irrigated with raw olive mill wastewater showed also a significant inhibition of some enzymatic activities such as ureases, ammonium oxydases and nitrate reductases while some other enzymatic activities such as xylanases and cellulases were slightly stimulated (data not shown).
3.1. OMW characterization 3.3. Analytical results of soil profiles The physicochemical characteristics of crude OMW are summarized in Table 1 and correspond to the mean values of 3 analysis. The high pollutant load of OMW (72 g l1 of COD) and its elevated salinity (6.75 g l1) could be observed. The OMW C/N ratio was unfavourable for the biodegradation and humification processes. OMW contains an enormous supply of organic matter very rich in phenolic compounds (9.2 g l1) which are toxic. 3.2. Soil characterization The soil of the study area was described in a previous study (Mekki et al., 2005). It had an important content of active calcareous (0.6% w/w) at the surface, and was composed of sand (89.82% w/w), clay (7.44% w/w) and silt (2.74% w/w). It had an alkaline pH and a weak electrical conductivity. The soil was very poor in nitrogen (0.5 g kg1 dry soil), and in organic matter (0.16% w/w). The levels of potassium and phosphorus were 0.014% w/w and 0.002% w/w, respectively. As it was discussed in that previous study (Mekki et al., 2006), the soil water content
For soil profiles, pH, electrical conductivity, organic matter and polyphenols contents were analysed 4 months after OMW spreading. Taking into consideration the deviation of the pH determination, it can be said that the pH does not vary as a function of depth. However, a very weak decrease was observed when OMW spreading quantity increased. The good buffering capacity of the soil related to its calcareous content neutralized the relatively acidic pH 5.12 of OMW (Table 2). This table shows that electrical conductivity increased proportionally with the increase of the quantity of unprocessed OMW and decreased with depth in all analysed soils. The electrical conductivity increased in all soil depths compared to the control soil which suggests the leaching of OMW from the surface to the soil deeper layers. The analysis of the organic matter content of the soil showed an increase in all soil depths compared to the control soil. This organic matter increase is proportional to the OMW spread quantities (Table 2). 3.4. Phenolic compounds dynamics
Table 1 Physico-chemical characteristics of unprocessed olive mill wastewater used in ferti-irrigation (values after7represent standard deviation) Characteristics
Data
pH (25 1C) Electrical conductivity (25 1C) (dS m1) Chemical oxygen demand (g l1) Biochemical oxygen demand (g l1) COD/BOD5 Salinity (g l1) Water content (g l1) Total solids (g l1) Mineral matter (g l1) Volatile solid (g l1) Total organic carbon (g l1) Total nitrogen Kjeldahl (g l1) Carbon/nitrogen P (mg l1) Na (g l1) Cl (g l1) K (g l1) Ca (g l1) Fe (mg l1) Mg (mg l1) Ortho-diphenols (g l1)
5.170.2 8.970.1 7272.8 1370.9 5.5370.72 6.7570.66 948717.2 5272.98 870.43 4471.85 25.5271.18 0.670.06 4378 3673.6 0.9470.09 1.670.15 8.870.8 1.270.11 3272.9 187718.8 9.271.8
This part of the study proposed to investigate the dynamics of the phenolic compounds in a very porous and permeable sandy soil irrigated by increasing doses of unprocessed OMW 50, 100 and 200 m3 ha1 yr1 and to trace the change of the phenolic compounds content as a function of time and depth. Results showed that the application of increasing OMW doses increased the total phenolic compounds content in all soil layers (Table 2). Besides, Table 2 shows that the majority of phenolic compounds are kept in the soil upper layers. The phenolic concentration decreased rapidly from 0 to 25 cm then continued to decrease weakly with depth but remained even detectable at 120 cm. The control soil showed very weak detectable phenolic substances. Comparison of phenolic compound spectra shows that especially the high molecular-mass compounds decreased with depth while the low molecular-mass polyphenols remained more abundant. According to the HPLC principle, polymers were eluted with low retention time, while monomers needed higher retention time. Fig. 1 shows that phenolic compounds with low retention time (22.8–25.8 min) 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
. pH . .
SC S50 S100 S200
8.170.2 8.170.2 870.2 7.970.2
8.270.2 8.170.2 870.2 7.770.2
8.370.2 8.370.2 8.170.2 7.870.2
8.2670.2 8.270.2 8.170.2 870.2
Electrical conductivity (mS cm1) .
SC S50 S100 S200
19870.1 24870.1 37570.1 74370.1
15470.1 20570.1 31570.1 69570.1
11270.1 18370.1 28470.1 54870.1
8570.1 12670.1 19670.1 45270.1
Organic matter (%, w/w) .
SC S50 S100 S200
0.1670.003 0.3470.007 0.6270.012 1.0770.02
0.1470.002 0.270.004 0.5470.01 0.9570.019
0.0870.002 0.1470.002 0.1270.002 0.3270.006
0.0270.0004 0.0970.0018 0.0870.0016 0.1170.002
Phenolic compounds (g kg1) .
SC S50 S100 S200
0.02070.0004 0.06070.0012 0.17870.003 0.31370.006
0.00570.0001 0.01570.0003 0.04570.0009 0.05770.001
0.00470.0001 0.01270.0002 0.03470.0007 0.04970.001
0.00370.0001 0.00870.0002 0.02370.0005 0.04270.0008
layers (0–25 cm), while higher retention time phenolic compounds (28.8–31.9 min) infiltrated in deeper layers of the soil (50–120 cm). The HPLC analysis realised on phenolic compounds of the surface layers (0–10 cm) of P3 plot treated with 200 m3 ha1 y1 (S200), 1 year after OMW application showed that only a residual phenolic fraction (F) persisted. This fraction which had a molecular-mass between 17 and 25 kDa (Fig. 2) neither originated from the soil nor from the OMW. 3.5. Phenolic fraction (F) phytotoxicity The residual phenolic fraction extracted from the soil treated annually with 200 m3 ha1 yr1 of OMW (S200) proved to be phytotoxic to the plant B. cernua. Fig. 3 illustrates the germination index of B. cernua seeds in control medium (CM), phenolic fraction (F), OMW diluted 4 times (OMW/4), 25 times (OMW/25) and 50 times (OMW/50). In the CM, the germination index reached 100% after 24 h of incubation. In the medium containing the soil phenolic residual fraction (F), the seed germination index reached 100% after 72 h of incubation. In the medium containing 4, 25 and 50 times diluted fresh OMW, the germination index showed a delay or even a total absence of the B. cernua germination. 4. Discussion The unprocessed OMW pH seemed to be neutralized by soil alkalinity. This can be explained by the fact that the OMW acidity was neutralized by carbonates present in soil horizons thus generating soluble calcium bicarbonate as was shown in earlier works (Sierra et al., 2001). The increase of the soil salinity involved the alteration of the cation exchange capacity (CEC) and could affect the soil
fertility. The salinity decrease at the lower horizons could be explained by the phenomena of adsorption and evapotranspiration which concentrate ionic species (especially Na, Cl and K) in the upper horizons (Zanjari and Nejmeddine, 2001). According to the sandy composition and the porous property of the studied soil, we observed an increase of the OM in all layers of soil with the increase of the dose of OMW used in the irrigation. This suggests an important leaching of OMW from the surface to the soil deeper layers and therefore of phenolic compounds. The increase of the OM content was not accompanied with an increase of the respiration potential. On the contrary, it was found that soil respiration decreases when the OMW amendment becomes higher than 50 m3 ha1 yr1 (Mekki et al., 2005). This finding seems to differ with the previous findings by Cox et al. (1998) who reported that the mineralization of the OMW organic matter in the soil upper layers could be explained by the proportion of biodegradable OM in OMW. The beneficial effect of waste on soil organic matter depends rather on the quality of waste, the type of soil and the environment. The study of the dynamics of the polyphenols showed that phenolic compounds of low molecular-mass migrated more in depth than those of high molecular-mass. The detection of monomers in depths can have two explanations. First, the polyphenols can be adsorbed by the soil and monomers can be mobilized and transported by infiltrating rain water. Second, the monomers are easier to degrade by the aerobic microorganisms at the soil surface. This is in line with previous findings of a correlation between places of olive oil mill waste spreading and wells with high phenolic concentrations (Spandre and Dellomonaco, 1996). The residual phenolic fraction (F) extracted 1 year after OMW application in the soil annually treated with
ARTICLE IN PRESS 22.96 25.78
OMW/25
S100
22.62 25.69
S50
22.74 24.19 29.63
0m S200
S50
27.335 29.62 31.072
0.5 m S100
27.365 29.32 30.7
S100
27.613 29.18 31.243
0.215
42.987
S50
28.172 30.922
0.25 m S200
23.137 25.693 33.543 34.832
S200
27.238 29.41 30.932
S100
S200
28.818 31.917
22.86 25.76
22.803 23.61
S50
25.6 26.3
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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).
200 m3 ha1 yr1 of OMW was not detected in control soil. Therefore, it was neither originated from the soil nor was it found in the OMW. Hence, it might have been newly synthesized during the incubation time of OMW. Indeed, Saiz-Jimenez et al. (1987) observed the simultaneous degradation of high molecular-mass fractions to produce fractions of a smaller molecular-mass and the synthesis of compounds which were more resistant to degradation. Feria (2000) reported that polyphenols residual levels can be important even 6 years after OMW spreading. The phytotoxicity effect of the OMW phenolic compounds on vegetative production of olive trees was evaluated by Ben Rouina (1994) and Ben Rouina et al. (1999). The authors showed no apparent effects on the physiology and growth of olive trees after 5 successive years of irrigation with OMW whereas better olive production yields were reported. However, olive trees can not be used for monitoring phytotoxicity since they are
very resistant to stresses such as drought, salinity, etc. In our study, the model sensitive plant B. cernua was used and demonstrated that spreading OMW, even in one time a year, may modify the soil structure and composition which could affect olive trees after long-term application. The phenolic fraction (F) extracted from soil 1 year after OMW application is not very phytotoxic and it only increased the time required to obtain 100% of germination of Brassica cernua seeds. Until now, nothing has proved that long-term accumulation of this fraction in soil makes it much more phytotoxic. Moreover, this fraction could serve as precursor for the formation of humic acids in soil. More studies should be investigated to understand the role and evolution of this fraction in the soil. On the other hand, the successive load of acidic and salt effluent would necessarily lead to the acidification and salinisation of soil which, in the long term, could impair its cation exchange capacity and affect its fertility.
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or polymerization or to their important leaching to the soil deeper layers that may provoke the contamination of the ground water in the long term. Nevertheless, the high molecular weight polyphenol (fraction F) formed in the soil is moderately phytotoxic. Therefore, if a biological pretreatment of OMW is conceived for reducing its toxicity prior to its soil spreading, this would allow an ecological use of this waste in agriculture. However, this practice should take into account the cumulative effect of soil salinization, which would transform little by little the soil into an unproductive one. Acknowledgements
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 200 m3 ha1 yr1 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).
CM
120
OMW/4
OMW/25
OMW/50
F
100 GI (%)
80 60 40 20 0 0.5
1
1.5
2 Time (day)
2.5
3
3.5
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).
5. Conclusion OMW amendment without pre-treatment seems to affect the structure and the composition of the soil. This work showed the presence of phenolic compounds at different depths of all the OMW amended soils 4 months after OMW spreading. A new specific phenolic fraction (F) not detected in control soil nor originated from OMW was synthesized and persisted in the soil 1 year after OMW application. This fraction displayed a certain ecotoxicity. The practice of OMW spreading in agricultural soil has until now been subject to a great controversy in the scientific communities. Our survey showed that the phytotoxic phenolic monomers tend to disappear from the upper layer of soil after some time due to degradation
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