Assessment of toxicity of the untreated and treated olive mill wastewaters and soil irrigated by using microbiotests

ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 69 (2008) 488–495 www.elsevier.com/locate/ecoenv

Assessment of toxicity of the untreated and treated olive mill wastewaters and soil irrigated by using microbiotests$ Ali Mekki, Abdelhafidh Dhouib, Firas Feki, Sami Sayadi Laboratoire des bioproce´de´s, Centre de Biotechnologie de Sfax, BP: ) K* , 3038 Sfax, Tunisie Received 19 March 2006; received in revised form 3 April 2007; accepted 9 April 2007 Available online 22 May 2007

Abstract Hazard assessments based on two measures of toxicity were conducted for the untreated olive mill wastewaters (U), untreated olive mill wastewaters organic extract (UOE), treated olive mill wastewaters (T), treated olive mill wastewaters organic extract (TOE) and extracts of soils ferti-irrigated with untreated (SU) and with treated olive mill wastewaters (ST). The measures of toxicity were achieved by the determination of the bioluminescence inhibition percent (IB%) of Vibrio fischeri and by the growth inhibition (GI) of Bacillus megaterium, Pseudomonas fluorescens and Escherichia coli. A bioluminescence inhibition of V. fischeri of 100%, 100%, 65%, 47%, 46% and 30% were obtained with U, UOE, T, TOE, SU and ST respectively. Indeed, even diluted 24 times, a significant bioluminescence inhibition of 96% was obtained by U. However, only 30% bioluminescence inhibition was obtained by 24 times diluted T. Whereas, 24 times diluted, SU and ST did not show a bioluminescence inhibition (3% and 1%, respectively). The GI of B. megaterium, P. fluorescens and E. coli were, respectively, 93%, 72% and 100% by U; 100%, 80% and 100% by UOE; 70%, 60% and 89% by T; 63%, 54% and 68% by TOE; 39%, 27% and 43% by SU and 23%, 0% and 34% by ST. The incubation of U or T in the soil during four months reduced their toxicity by 54% and 35%, respectively. As it was expected, the most resistant bacterium to OMW toxicity is P. fluorescens then B. megaterium and E. coli. V. fischeri remained the most sensitive strain to the toxicity of this sewage what proves again its utilisation as standard of measure of the toxicity. r 2007 Elsevier Inc. All rights reserved. Keywords: Olive mill wastewaters; Toxicity; Bioluminescence inhibition; Growth inhibition; Soil

1. Introduction The annual production of olive mill wastewaters (OMW) in Mediterranean region reached 30 million cubic meters (D’Annibale et al., 2004). The polluting properties of the Abbreviations: COD, chemical oxygen demand; BOD5, biochemical oxygen demand; EC, electrical conductivity; IB%, bioluminescence inhibition percent of Vibrio fischeri; GI, growth inhibition; OMW, olive mill wastewaters; OM, organic matter; U, untreated olive mill wastewaters; UOE, untreated olive mill wastewaters organic extract; SU, extract of soil ferti-irrigated with untreated olive mill wastewaters; ST, extract of soil ferti-irrigated with treated olive mill wastewaters; T, treated olive mill wastewaters; TOC, total organic carbon; TOE, treated olive mill wastewaters organic extract $ This research was supported by the ‘‘Ministry of Higher Education, Scientific Research and Technology of Tunisia’’ and the E.C program ‘‘Medusa water’’ Contract ICA-CT-1999-00010. Corresponding author. Fax: +216 74 440 452. E-mail address: abdelhfi[email protected] (A. Dhouib). 0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2007.04.008

OMW are essentially owed to their high organic contents (Feria, 2000), and their toxicities especially to their phenolic molecules of different molecular mass (Capasso et al., 1995; Sayadi and Ellouz, 1995; Sayadi et al., 2000; DellaGreca et al., 2001; Tsioulpas et al., 2002; Fiorentino et al., 2003). OMW physicochemical composition and characteristics are well documented (Capasso et al., 1992; Ben Sassi et al., 2006; Dhouib et al., 2006a). OMW contain relevant amounts of mineral salts and organic nutrients of potential interest for the ferti-irrigation. However, the uncontrolled disposal of OMW may cause serious environmental pollution with unforeseeable effects on the soilplant system and even transfers harmful compounds into other media, such as ground waters and surface waters (Azbar et al., 2004). In many studies, authors warmed that OMW disposal in the nature causes serious environmental problems due to its antibacterial effects and its phytotoxicity (Sierra et al., 2001; Rana et al., 2003; Cereti et al.,

ARTICLE IN PRESS A. Mekki et al. / Ecotoxicology and Environmental Safety 69 (2008) 488–495

2004; Mekki et al., 2006a; Piotrowska et al., 2006). This practice represents now a controversy discussion and a debate of actuality between those that are for and those that are against this strategy. Therefore, research has not yet reached a consensus about the effects of this by-product on the soil. Recently, Mekki et al. (2006b), Komilis et al. (2005), Iconomou et al. (2002) and Aliotta et al. (2002) reported the phytotoxicity of polyphenols from OMW on seed germination and plant growth. Yesilada and Sam (1998) reported their toxic effects on the soil bacterium Pseudomonas aeruginosa. Fiorentino et al. (2003) reported the toxic potential of this matrix on the typical organisms of the freshwater food chain. Toxicity evaluation is an important parameter in waste characterisation (Wang et al., 2003; Novotny´ et al., 2006). Many types of bioassays using representatives from microorganisms, plants, invertebrates, and fish are available (Aggelis et al., 2003; Tsui and Chu, 2003; Ricco et al., 2004; Zurita et al., 2005; Novotny´ et al., 2006). In recent years, use of bioluminescence bacterial tests has become particularly popular because they are rapid, reproducible, simple to use, and unambiguous and they cause no ethical problems (Ribo, 1997). Bioluminescence bioassays have been frequently employed to measure the toxicity of wastewaters, sludges, and solid wastes (Pe´rez et al., 2001; Wang et al., 2002; Lapa et al., 2002; Schultz et al., 2002; Farre´ and Barcelo´, 2003; Hernando et al., 2006). However, only a few attempts to use bacterial growth inhibition (GI) bioassays in conjunction with ecotoxicity tests have been made (Lambolez et al., 1994; Ferna´ndez-Sempere et al., 1997; Schultz et al., 2002; Isidori et al., 2005). In previous works, we reported the negative impact of the application of OMW on the soil physico-chemical and microbiological characteristics (Mekki et al., 2006a,b, 2007). In this work we investigated to compare antibacterial effects of untreated and treated OMWs, organic extracts of OMWs and soil ferti-irrigated, on the model biotest of V. fischeri bioluminescence and on GI of some representing soil and aquatic bacteria as Bacillus megaterium, P. fluorescens and E. coli. Among main goals of this work is to know: (i) how much the treatment method application (untreated and treated OMW) or the incubation in soil can remove its toxicity, (ii) the estimation of the sensitivity limits of B. megaterium, P. fluorescens and E. coli bacterial species for OMW phenolic compounds, and (iii) the evaluation of the potential of microbiotest for the estimation of the toxicity and the importance of the additional information for risk assessment. 2. Materials and methods 2.1. Materials 2.1.1. OMW origin The fresh OMW was taken from a continuous extraction system factory located in Sfax, Tunisia. The treated OMW, was obtained with an

489

Table 1 Physico-chemical characteristics of untreated OMW (U) and treated OMW (T) Characteristics

U

T

pH (25 1C) Electrical conductivity (25 1C) (dS m1) Chemical oxygen demand (g l1) Biochemical oxygen demand (g l1) COD/BOD5 Reducing sugars (g l1) Glucose (g l1) 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 (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)

4.870.2 8.870.1 58.573.9 1771.4 3.470.5 13 6 6.670.7 958719 41.772.1 7.570.4 3471.7 19.770.9 0.5370.05 37.275 3773.7 0.870.1 1.570.2 7.670.7 0.970.1 23.472.3 18719 9.170.9

7.270.2 10.270.1 5.270.4 2.370.13 2.370.5 Not detected Not detected 8.770.9 982719 17.970.9 12.270.6 5.770.3 3.370.2 0.3270.03 10.371.9 1471.3 170.1 1.270.1 5.370.5 4.470.4 38.373.8 281728 0.770.07

integrated process based on electro-coagulation pre-treatment followed by a decantation step then anaerobic digestion (Khoufi et al., 2006). The characteristics of the treated and untreated OMW are given in Table 1.

2.1.2. Soil samples Soil samples were collected 4 months after the OMW spreading, from two different plots ferti-irrigated with 100 m3 ha1 y1 and 400 m3 ha1 y1 of untreated and treated OMW, respectively. Samples were taken from different parts of each plot from 0–10 cm deep, using a soil auger. All soil samples, taken from each plot were then mixed, air-dried, sieved with a mesh size of 450 mm and stored at 4 1C prior to use.

2.1.3. Bacteria P. fluorescens DSM 50090, B. megaterium DSM 90 and E. coli DSM 498 were purchased from German collection of microorganisms and cell cultures DSMZ, Germany.

2.2. Methods 2.2.1. Physicochemical analysis OMW 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). Chemical oxygen demand (COD) was determined according to Knechtel (1978) standard method. Biochemical oxygen demand (BOD5) was determined by the manometric method with a respirometer (WTW BSB-Controller Model 620 T, Weilheim, Germany). Phosphorus, iron, magnesium, potassium, calcium, sodium and chloride were determined by atomic absorption.

ARTICLE IN PRESS A. Mekki et al. / Ecotoxicology and Environmental Safety 69 (2008) 488–495

490

solutions were used with each batch of bacteria to verify bacteria and reagent quality.

Ortho-diphenols were quantified by means of Folin–Ciocalteu colorimetric method (Box, 1983) using caffeic acid as standard. The absorbance was determined at l ¼ 765 nm.

2.2.4.2. GI test. P. fluorescens, B. megaterium and E. coli were cultivated on nutrient broth (NB) as control. P. fluorescens and B. megaterium were incubated at 30 1C and E. coli at 37 1C. Each one of U, T, UOE, TOE, SU and ST was mixed in 10%, 20%, 40% and 50% proportions with NB and inoculated with each bacterial strain. The bacterial growth was assessed by mixed liquor volatile suspended solids (MLVSS) determination and by cell-numeration every 2 h during 10 h culture. The GI was determined as described by Capasso et al. (1995):

2.2.2. Organic extracts of untreated (UOE) and treated OMW (TOE) Untreated and treated OMW were extracted 3 times with ethyl acetate ð12 : v=vÞ. The collected organic fractions were dried and evaporated under vacuum. The residues were extracted twice with dichloromethane in order to remove the non-phenolic fraction (lipids, aliphatic and sugars). The liquid phase was discarded while the washed residues (UOE and TOE) were recuperated in the same volume of water and were analysed by size exclusion-HPLC technique (Allouche et al., 2004).

GIð%Þ ¼ 100  ð100  Ns=NcÞ where, GI (%) is the percentage of GI, Ns the CFU ml1 or MLVSS (g ml1) in the sample, Nc the CFU ml1 or MLVSS (g ml1) in the control.

2.2.3. Soil extract preparation For soil phenolic compounds, only water soluble substances were determined. A 1/2.5 (w/v) soil/aqueous mixture was shaken for 12 h in a mechanical shaker (Hund and Traunspurger, 1994). The supernatant was extracted 3 times with ethyl acetate. The collected organic fractions (SU and ST) were recuperated in the same volume of water and were analysed by size exclusion-HPLC technique (Allouche et al., 2004).

3. Results 3.1. Treated and untreated OMW characterisation Untreated OMW is an acidic effluent with a high pollutant load (COD ¼ 58.5 g l1) and a high phenolic content (9.1 g l1). Treated OMW is a slightly alkaline effluent, rich in inorganic load such as potassium, calcium, magnesium and iron (Table 1). OMW treatment decreased the COD from 58.5 g l1 to 5.2 g l1 and orto-diphenols concentration from 9.1 g l1 to 0.7 g l1. The major phenolic monomers present in the UOE were hydroxytyrosol and tyrosol (Fig. 1 A). TOE, SU and ST extracts did not show detectable monomeric phenols (Fig. 1B, 1C and 1D).

2.2.4. Toxicity testing 2.2.4.1. Acute toxicity LUMIstox bioassay. A LUMIStox 300 luminometer, a LUMISterm incubator, and the non-pathogenic bacteria V. fischeri LCK 480 (liquid dried), all obtained from Dr. Lange GmbH, Du¨sseldorf, Germany, were used for toxicity measurements. The test consisted in the inhibition of the bioluminescence of V. fischeri according to ISO 11348-2, (1998). Since the luminescent bacterium requires marine conditions. Solid sodium chloride crystals were added to the OMW and soil extract samples to obtain a final concentration of 2% w/v. The pH was adjusted to 7.070.2. Dilutions of OMW and soil extracts were carried out with 2% sodium chloride solution according to Dr. Lange LUMIStox operating manual. Percentage inhibition of the bioluminescence was achieved by mixing 0.5 ml of OMW or soil extract and 0.5 ml luminescent bacterial suspension. After a 15 min exposure at 15 1C, the decrease in light emission was measured. The toxicity of the OMW and soil extract was expressed as the percent of the inhibition of bioluminescence (%IB) relative to a non-contaminated reference. Blank (Milli-Q water containing 2% NaCl) and positive control (K2Cr2O7 4.0 mg l1 and NaCl 7.5%)

3.2. Treated and untreated OMW antibacterial activities 3.2.1. Bioluminescence inhibition Fig. 2 shows the bioluminescence inhibition of V. fischeri by U, UOE, T, TOE, SU, ST and their 8 and 24 times

0.3

Hydroxytyrosol Tyrosol Volts

Volts

0.4 0.2 0.0

0.2 0.1 0.0

0

5

10

15

20

25 30 Minutes

35

40

45

50

0.020

0

5

10

15

20

25 30 Minutes

35

40

45

50

0

5

10

15

20

25 30 Minutes

35

40

45

50

0.2 Volts

Volts

0.015 0.010

0.1

0.005 0.000

0.0 0

5

10

15

20

25 30 Minutes

35

40

45

50

Fig. 1. HPLC-spectrum profile of ethyl acetate extracts of U (A), T (B), SU (C) and ST (D).

ARTICLE IN PRESS A. Mekki et al. / Ecotoxicology and Environmental Safety 69 (2008) 488–495 120

2 Undiluted

d/8

d/24

Bacillus megatrium

C

1.8

100

U

Biomass (g l-1)

1.6

80 IB (%)

491

60 40

1.4

UOE

1.2 T

1 0.8

TOE

0.6

SU

0.4 20

ST

0.2 0

0

0 U

UOE

T TOE Substrate

SU

2

4

ST

2.5

Fig. 2. Vibrio fischeri bioluminescence inhibition (IB%) after 15 min incubation time in various substrates (U, UOE, T, TOE, SU and ST) and their 8 and 24 times dilutions.

3.2.2. Growth inhibition Growth of B. megaterium, P. fluorescens and E. coli on nutrient broth medium (C) as control and on nutrient broth containing 50% of U, UOE, T, TOE, SU and ST were shown in Fig. 3. Growth curves of the 3 strains on the control medium and the different substrata showed different slopes, more the substratum is toxic more the growth is slow and the lag phase is prolonged. As was expected, U and UOE were highly toxic and bacteria growth was strongly inhibited for the three tested bacteria especially E. coli. In a second order of toxicity are T and TOE, then SU and ST having the lowest toxicity towards these strains. However, the bacterial responses regarding various substrates were different. The GI of B. megaterium, P. fluorescens and E. coli were, respectively, 93%, 72% and 100% by U; 99.9%, 80% and 99.8% by UOE; 70%, 60% and 89% by T; 63%, 54% and 68% by TOE; 39%, 27% and 43% by SU and 23%, 0% and 34% by ST (Fig. 4). An average reduction of 35% of the toxicity could be obtained after the treatment of the OMW, whereas the toxicity of untreated and treated OMW was, respectively, reduced at least by 54 and 35%, 4 months after their incubation in soil. A better growth of P. fluorescens was even obtained on ST than on nutrient broth. This excess of

8

10

Pseudomonas fluorescens

Biomass (g l-1)

12

C U

2

UOE

1.5 T

1

TOE SU

0.5 ST

0 0

2

2

4

6 Time (h)

8

10

Escherichia coli

12

C

1.8 U

1.6 Biomass (g l-1)

dilutions. The U totally inhibited V. fischeri bioluminescence. Even diluted 24 times, the U exerted 96% bioluminescence inhibition. Similar toxicity was obtained with the UOE which is essentially composed by hydroxytyrosol and tyrosol (Fig. 1A). Even after treatment, the T contained again traces of these monomers (Fig. 1B) generating a residual toxicity. This toxicity gave IB of 65% in T and 47% in ethyl acetate extract TOE (Fig. 2). SU showed IB of 46%, whereas IB of ST was 30%. The 24 times diluted SU and ST did not show a bioluminescence inhibition (3 and 1%, respectively). The toxicity of OMW was reduced of 35% by its treatment and again 35% were eliminated by its incubation in soil. The incubation, it alone, of raw OMW in soil reduced more than 50% of its toxicity (Fig. 2).

6 Time (h)

1.4

UOE

1.2 T

1 0.8

TOE

0.6 SU

0.4 0.2

ST

0 0

2

4

6 Time (h)

8

10

12

Fig. 3. Evolution according to the time of the biomass of Bacillus megaterium, Pseudomonas fluorescens and Escherichia coli cultivated on nutrient broth C and nutrient broth containing 50% of U, UOE, T, TOE , SU and ST.

biomass obtained on ST may be explained by the part of soil nutrients supplementation and the good adaptation of Pseudomonas to the soil (Fig. 5). 4. Discussion In the present study, the acute toxicity of U, UOE, T, TOE, SU and ST, was assessed on the marine bacterium

ARTICLE IN PRESS A. Mekki et al. / Ecotoxicology and Environmental Safety 69 (2008) 488–495

492 120

2.5

Bacillus megatrium U

GI (%)

80

T

60 TOE

40

SU

20

ST

Bacillus megatrium

E.Coli

2

UOE

Biomass (gl-1)

100

Pseudomonas fluorescens

1.5 1 0.5 0 C

0 0

10

20

30

40

50

60

ST

SU U UOE T Substrate concentration (50%)

TOE

Substrate concentration (%) 100

Fig. 5. Bacillus megaterium, Pseudomonas fluorescens and Escherichia coli, 10 h growth on nutrient broth containing 50% proportion of various substrates (U, UOE, T, TOE, SU and ST) in comparison with growth control (C) on nutrient broth.

Pseudomonas fluorescens

90 U

GI (%)

80 70

UOE

60

T

50 TOE

40 30

SU

20

ST

10 0 0

10

20

30

40

50

60

Substrate concentration (%) 120

Escherichia Coli U

100 UOE

GI (%)

80 T

60

TOE

40

SU

20

ST

0 0

10

20

30

40

50

60

Substrate concentration (%)

Fig. 4. Growth inhibition percent of Bacillus megaterium, Pseudomonas fluorescens and Escherichia coli by various concentrations (10%, 20%, 40% and 50%) in nutrient browth of U, UOE, T, TOE, SU and ST.

V. fischeri and on representing soil and aquatic bacteria as B. megaterium, P. fluorescens and E. coli. Toxicity assays based on bioluminescence in V. fischeri can provide a rapid assessment of chemical toxicity (Ribo, 1997). They are widely used for routine screening of waste effluents or as part of more elaborate environmental assessments that involve several forms of bioassay and employ a range of different organisms (Kaiser and Esterby, 1991; Astley et al., 1999; Jennings et al., 2001). But there are only a few

studies that have compared toxicity data obtained using GI of soil or water representative bacteria and the behaviour of these bacteria towards these toxic substrates. Untreated OMW totally inhibited the bioluminescence of V. fischeri. This toxicity was essentially due to its high content of phenolic compounds and more precisely to phenolic monomers as hydroxytyrosol and tyrosol (Fig. 1A). Indeed, similar toxicity was obtained with the UOE which is essentially composed by hydroxytyrosol and tyrosol (Fig. 2). These findings are in line with previous findings of Dhouib et al. (2006b) who put in evidence the toxicity exercised by the main phenolic monomers of the OMW on the microbial flora implied in the treatment of this waste. Fiorentino et al. (2003) reported that the most toxic fraction to the test organisms (Pseudokirchneriella subcapitata (alga), Brachionus calyciflorus (rotifer) and the two crustaceans Daphnia magna and Thamnocephalus platyurus) was the low molecular weight (o350 Da) and especially catechol and hydroxytyrosol, the most abundant components of this fraction. Allouche et al. (2004) and Obied et al. (2005) reported that compounds found in OMW that exhibited antibacterial activity were tyrosol, hydroxytyrosol, oleuropeine, 3–4 dihydroxyphenyl acetic acid, and 4-hydroxybenzoic acid. Our results showed that the treatment of the OMW reduced considerably its phenolic content from 9.1 to 0.7 and eliminate essentially phenolic monomers which reduced the IB from 100% to 65%. Treated or untreated OMW incubation in soil contributed also in the reduction of its toxicity and therefore, the significant role played by the soil microflora. In line with this, Cox et al. (1998), Sierra et al. (2001) and Mekki et al. (2007) showed the fast degradation of these monomers by the biologic activities of soil or their infiltration in the deep layers of soil. Bioluminescence inhibition of V. fischeri is a very appreciable and very efficient ecotoxicological test to detect and to quantify the toxicity exercised by any substratum. Moreover, in order to perform a site-specific ecotoxicological monitoring, the optimisation bioassay

ARTICLE IN PRESS A. Mekki et al. / Ecotoxicology and Environmental Safety 69 (2008) 488–495

procedure may be applied to another target microorganism, specific for the polluted site. The monitoring of the growth of bacteria representing the soil or the aquatic microflora as B. megaterium, P. fluorescens and E. coli cultivated in the presence of OMW is very instructive and permits to predict their behaviour, the day where they will be in contact with this waste or submitted to its toxicity in the nature. The strains were interesting to compare their performances with the same chemicals. Although time, conditions of exposure, and species specificity were different, it would be useful to know how the response of each test strain to other one may predict chronic toxicity. The bacterial responses regarding various substrates U, UOE, T, TOE, SU or ST were different. The GI values for B. megaterium, P. fluorescens and E. coli (Fig. 4) allow visualisation of the fact that the E. coli response is the most sensitive to the toxic effect of monomers present in U and UOE. P. fluorescens showed the high resistance to OMW toxicity. A better growth was even obtained on ST that on nutrient broth (Fig. 5). This is quite normal because this bacterium is known by its powerful capacity to degrade the recalcitrant compounds and its ubiquitous distribution in soil and water environments. This bacterium has often been found during biodegradation studies of petroleum hydrocarbons contaminated samples (Richard and Vogel, 1999; Bugg et al., 2000; Evans et al. 2004). Abbondanzi et al. (2003) observed that the lower sensitivity of P. fluorescens to phenol makes difficult its use as microbial bioassay in organic polluted samples. However, B. megaterium and E. coli were more sensitive to this toxicity. This finding confirms previous findings by Perez et al. (1992) reporting that B. megaterium was the most sensitive bacteria to OMW ethyl acetate extract. On the other hand, Ramos-Cormenzana et al. (1996) noted that antibacterial activity of OMW phenolic compounds was higher on Gram positive than on Gram negative bacteria. Indeed, the relation between the toxicity of the phenolic compound of OMW and the bacterial sensitivity are in report with the bacterial capacity to convert these compounds. The E. coli response is the most sensitive to U, UOE, T, TOE, SU and ST (GI ¼ 100%, 99.8%, 89%, 68%, 43% and 34%, respectively). So this strain can be used as standard species of measure of the phenolic compounds toxicity. The method developed for culturing and monitoring the bacterium as B. megaterium or E. coli during growth curve was fairly simple and did not required excessive speciality equipment. The data presented in this study show clearly that the results obtained in four of these ecotoxicological test systems compare very favourably and that there is a significant difference between the response obtained by these four strains for different chemicals resulting from the treatment or the incubation in soil of the OMW. Thus, according to our results we can classify the four tested bacteria according to their increasing sensitivity toward the toxic compounds of the OMW as following: P. fluorescenso B. megateriumoE. colioV. fischeri. The present work has demonstrated that E. coli is as sensitive as LUMIStox culture (V. fischeri), for phenolic

493

monomers. This makes it possible to use E. coli bioassay in toxicity testing of phenolic polluted samples, thus avoiding result distortions related to salinity correction of the LUMIStox test. Moreover, E. coli has often been found during biodegradation studies of OMW contaminated samples, due to its ubiquity in soil and water. However, further work is required to evaluate the real sensitivity of E. coli to different organic compounds in order to assess its eventual resistance or degrading capability. 5. Conclusion The results of the acute toxicity and the GI tests showed that OMW toxicity was mainly due to its monomeric phenolic compounds such as hydroxytyrosol and tyrosol. U and UOE showed highly toxic effects on the four tested bacteria (V. fischeri, B. megaterium, P. fluorescens, and E. coli). OMW treatment or incubation in soil reduces significantly its toxicity by eliminating the phenolic monomers. It can be seen from both methods that LUMIStox bioassay is the most sensitive test, while GI test exhibits a very narrow dependence to the used strain of bacterium. Indeed, E. coli is very sensitive to the phenolic compounds toxicities as well as V. fischeri of the LUMIStox bioassay. To a least degree of sensitivity comes B. megaterium, but P. fluorescens seems to be resistant enough to such substratum and not suitable for similar tests. The results indicated that bioassays with the determination of GI of E. coli or B. megaterium are suitable for evaluating the toxic effects of OMW phenolic monomers. These strains can be considered a good choice for such bioassays. As regards the comparison between the two bioassays, the GI one is simple to carry out (the experimental apparatus can easily be assembled with the ordinary lab equipment), measurement time is quite short (about 10 h) and the method is specific since the toxicity effects are evaluated directly on the wastewater or soil. On the other hand, LUMIStox has the advantage of operating with a selected pure culture and with a standardised procedure. This biotest possesses also a high sensitivity to a wide range of substrata. In conclusion, both methods can be usefully applied for toxicity detection in wastewater and soil. Acknowledgment This research was funded by E.C. program ‘‘Medusa water’’ Contract ICA-CT-1999-00010 and contract programmes (MHESRT, Tunisia). Disclaimer Authors of this manuscript declare that this work complies with national and institutional guidelines for the protection of human subjects and animal welfare. In this paper, no experiments involving humans or experimental animals were conducted.

ARTICLE IN PRESS 494

A. Mekki et al. / Ecotoxicology and Environmental Safety 69 (2008) 488–495

References Abbondanzi, F., Cachada, A., Campizi, T., Guerra, R., Raccagni, M., Iacondini, A., 2003. Optimisation of a microbial bioassay for contaminated soil monitoring: bacterial inoculum standardisation and comparison with Microtox assay. Chemosphere 53, 889–897. 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 Res. 37, 3897–3904. Aliotta, G., Fiorentino, A., Oliva, A., Temussi, F., 2002. Olive oil mill wastewater: isolation of polyphenols and their phytotoxicity in vitro. Allelopathy J 9, 9–17. Allouche, N., Fki, I., Sayadi, S., 2004. Toward a high yield recovery of antioxidants and purified hydroxytyrosol from olive mill wastewaters. J. Agri. Food Chem. 52, 267–273. Astley, K.N., Meigh, H.C., Glegg, G.A., Braven, J., Depledge, M.H., 1999. Multi-variate analysis of biomarker responses in Mytilus edulis and Carcinus maenas from the Tees Estuary (UK). Mar. Pollut. Bull. 39, 145–154. Azbar, N., Bayram, A., Filibeli, A., Muezzinoglu, A., Sengul, F., Ozer, A., 2004. A review of waste management options in olive oil production. Crit. Rev. Environ. Sci. Technol. 34, 209–247. Ben Sassi, A., Boularbah, A., Jaouad, A., Walker, G., Boussaid, A., 2006. A comparison of olive oil mill wastewaters (OMW) from three different processes in Morocco. Process Biochem. 41, 74–78. Box, J.D., 1983. Investigation of the Folin-Ciocalteu phenol reagent for the determination of polyphenolic substances in natural waters. Water Res. 17, 511–522. Bugg, T., Foght, J.M., Pickard, M.A., Gray, M.R., 2000. Uptake and active efflux of polycyclic aromatic hydrocarbons by Pseudomonas fluorescens Lp6a. Appl. Environ. Microbiol. 66, 5387–5392. 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. Capasso, R., Evidente, A., Schivo, L., Orru, G., Marcialis, M.A., Cristinzio, G., 1995. Antibacterial polyphenols from olive mill wastewaters. J. Appl. Bacteriol. 79, 393–398. Cereti, C.F., Rossini, F., Federici, F., Quaratino, D., Vassilev, N., Fenice, M., 2004. Reuse of microbially treated olive mill wastewater as fertiliser for wheat (Triticum durum Desf). Bioresour. Technol. 91, 135–140. Cox, L., Celis, R., Hermosin, M.C., Beker, A., Cornejo, J., 1998. Porosity and herbicide leaching in soils amended with olive-mill wastewater. Agri. Ecosyst. Environ. 65, 151–161. D’Annibale, A., Casa, R., Pieruccetti, F., Ricci, M., Marabottini, R., 2004. Lentinula edodes removes phenols from olive-mill wastewater: impact on durum wheat (Triticum durum Desf.) germinability. Chemosphere 54, 887–894. DellaGreca, M., Monaco, P., Pinto, G., Pollio, A., Previtera, L., Temussi, F., 2001. Phytotoxicity of low molecular weight phenols from olive mill waste waters. Bull. Environ. Contam. Toxicol. 67, 352–357. Dhouib, A., Aloui, F., Hamad, N., Sayadi, S., 2006a. Pilot-plant treatment of olive mill wastewaters by Phanerochaete chrysosporium coupled to anaerobic digestion and ultrafiltration. Process Biochem. 41, 159–167. Dhouib, A., Ellouz, M., Aloui, F., Sayadi, S., 2006b. Effect of bioaugmentation of activated sludge with white-rot fungi on olive mill wastewater detoxification. Lett. Appl. Microbiol. 42, 405–411. Evans, F.F., Seldin, L., Sebastian, G.V., Kjelleberg, S., Holmstro¨m, C., Rosado, A.S., 2004. influence of petroleum contamination and biostimulation treatment on the diversity of Pseudomonas spp. in soil microcosms as evaluated by 16 s rRNA based-PCR and DGGE. Lett. Appl. Microbiol. 38, 93–98. Farre´, M., Barcelo´, D., 2003. Toxicity testing of wastewater and sewage sludge by biosensors, bioassays and chemical analysis. Trends Anal. Chem. 22, 299–310.

Feria, L.A., 2000. The generated situation by the O.M.W. in Andalusia. Actas/Proceedings-Workshop Improlive-2000-Anexo A1/Annex A1, pp. 55–63. Ferna´ndez-Sempere, J., Barrueso-Martı` nez, M.L., Font-Montesinos, R., Sabater-Lillo, M.C., 1997. Characterization of tannery wastes comparison of three leachability tests. J. Hazard. Mater. 54, 31–45. Fiorentino, A., Gentili, A., Isidori, M., Monaco, P., Nardelli, A., Parrella, A., Temussi, F., 2003. Environmental effects caused by olive mill waste waters: toxicity comparison of low-molecular-weight phenol components. J. Agri. Food Chem. 51, 1005–1009. Hernando, M.D., Malato, O., Farre´, M., Fernandez-Alba, A.R., Barcelo´, D., 2006. Application of ring study: water toxicity determinations by bioluminescence assay with V. fischeri. Talanta 69, 370–376. Hund, K., Traunspurger, W., 1994. Ecotox-evaluation strategy for soil bioremediation exemplified for a PAH-contaminated site. Chemosphere 29, 371–390. Iconomou, D., Eˆarayanni-Christou, I´., Zanganas, P., Theocharopoulos, S., 2002. Bioremediated olive oil mill waste-waters and their use on the growth of lettuce and tomate plants. Proceedings of the sixth International Conference, on the Protection and Restoration on the Environment VI., Skiathos Island, Greece, 1–5, July 2002, pp. 651–658. Isidori, M., Lavorgna, M., Nardelli, A., Parrella, A., 2005. Model study on the effects of 15 phenolic olive mill wastewater constituents on seed germination and V. fischeri metabolism. J. Agri. Food Chem. 53, 8414–8417. ISO 11348-2, 1998. Water Quality Determination of the Inhibitory Effect of Water Samples on the Light Emission of Vibrio fischeri (Luminescent Bacteria Test). Part 2: Method Using Liquid-Dried Bacteria. DIN Deutsches Institut fu¨r Normung Burggrafenstrasse 6 DE-10787 Berlin. Jennings, V.L.K., Rayner-Brandes, M.H., Bird, D.J., 2001. Assessing chemical toxicity with the bioluminescent Photobacterium (Vbrio fischeri): a comparison of three commercial systems. Water Res. 35, 3448–3456. Kaiser, K.L.E., Esterby, S.R., 1991. Regression and cluster-analysis of the acute toxicity of 267 chemicals to six species of biota and the octanol water partition coefficient. Sci. Total Environ. 109, 499–514. Khoufi, S., Aloui, F., Sayadi, S., 2006. Treatment of olive oil millwastewater by combined process electro-Fenton reaction and anaerobic digestion. Water Res. 40, 2007–2016. Knechtel, R.J., 1978. A more economical method for the determination of chemical oxygen demand, Water Pollut. Control. 25–29. Komilis, D.P., Karatzas, E., Halvadakis, C.P., 2005. The effect of olive mill wastewater on seed germination after various pretreatment techniques. J. Environ. Manage. 74, 339–348. Lambolez, L., Vasseur, P., Ferard, J.F., Gisbert, T., 1994. The environmental risks of industrial waste disposal: an experimental approach including acute and chronic toxicity studies. Ecotoxicol. Environ. Saf. 28, 317–328. Lapa, N., Barbosa, R., Morais, J., Mendes, B., Me´hu, J., Santos Oliveira, J.F., 2002. Ecotoxicological assessment of leachates from MSWI bottom ashes. Waste Manage. 22, 583–593. Mekki, A., Dhouib, A., Sayadi, S., 2006a. Changes in microbial and soil properties following amendment with treated and untreated olive mill wastewater. Microbiol. Res. 161, 93–101. Mekki, A., Dhouib, A., Aloui, F., Sayadi, S., 2006b. Olive wastewater as an ecological fertiliser. Agron. Sustain. Dev. 26, 61–67. Mekki, A., Dhouib, A., Sayadi, S., 2007. Polyphenols dynamics and phytotoxicity in a soil amended by olive mill wastewaters. J. Environ. Manage. 84, 134–140. Novotny´, C., Dias, N., Kapanen, A., Malachova´, K., 2006. Comparative use of bacterial, algal and protozoan tests to study toxicity of azo- and anthraquinone dyes. Chemosphere 63, 1436–1442. Obied, H.K., Allen, M.S., Bedgood, D.R., Prenzler, P.D., Robards, K., Stockmann, R., 2005. Bioactivity and analysis of biophenols recovered from olive mill waste. J. Agri. Food Chem. 53, 823–837.

ARTICLE IN PRESS A. Mekki et al. / Ecotoxicology and Environmental Safety 69 (2008) 488–495 Perez, J., Delarubia, T., Moreno, J., Martinez, J., 1992. Phenolic content and antibacterial activity of olive oil wastewaters. Environ. Toxicol. Chem. 11, 489–495. Pe´rez, S., Farre´, M., Garcı` a, M.J., Barcelo´, D., 2001. Occurrence of polycyclic aromatic hydrocarbons in sewage sludge and their contribution to its toxicity in the ToxAlerts 100 bioassay. Chemosphere 45, 705–712. Piotrowska, A., Iamarino, G., Rao, M.A., Gianfreda, L., 2006. Shortterm effects of olive mill waste water (OMW) on chemical and biochemical properties of a semiarid Mediterranean soil. Soil Biol. Biochem. 38, 600–610. Ramos-cormenzana, A., Juarez-Jimenez, B., Garcia-Pateja, M.P., 1996. Antimicrobial activity of olive mill waste waters (Alpechin) and biotransformed olive oil mill waste water. Int. Biodeterior. Biodegrad. 38, 283–290. Rana, G., Rinaldi, M., Introna, M., 2003. Volatilisation of substances after spreading olive oil waste water on the soil in a Mediterranean environment. Agri. Ecosyst. Environ. 96, 49–58. Ribo, J.M., 1997. Interlaboratory comparison of studies of the luminescent bacteria toxicity bioassay. Environ. Toxicol. Water Qual. 12, 283–294. Ricco, G., Tomei, M.C., Ramadorib, R., Laera, G., 2004. Toxicity assessment of common xenobiotic compounds on municipal activated sludge: comparison between respirometry and Microtoxs. Water Res. 38, 2103–2110. Richard, J.Y., Vogel, T.M., 1999. Characterisation of a soil bacterial consortium capable of degrading diesel fuel. Int. Biodeterior. Biodegrad. 44, 93–100. Sayadi, S., Ellouz, R., 1995. Roles of lignin peroxidase and manganese peroxidase from Phanerochaete chrysosporium in the decolorization of olive mill wastewaters. Appl. Environ. Microbiol. 61, 1098–1103.

495

Sayadi, S., Allouche, N., Jaoua, M., Aloui, F., 2000. Detrimental effects of high molecular-mass polyphenols on olive mill wastewaters biotreatments. Process Biochem. 35, 725–735. Schultz, E., Vaajasaari, K., Joutti, A., Ahtiainen, J., 2002. Toxicity of industrial wastes and waste leaching test eluates containing organic compounds. Ecotoxicol. Environ. Saf. 52, 248–255. 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. Sci. Total Environ. 279, 207–214. Tsioulpas, A., Dimou, D., Iconomou, D., Aggelis, G., 2002. Phenolic removal in olive oil mill wastewater by strains of Pleurotus spp. in respect to their phenol oxidase (laccase) activity. Bioresource Technol. 84, 251–257. Tsui, M.T.K., Chu, L.M., 2003. Aquatic toxicity of glyphosate-based formulations: comparison between different organisms and the effects of environmental factors. Chemosphere 52, 1189–1197. Wang, C.X., Yediler, A., Lienert, D., Wang, Z.J., Kettrup, A., 2002. Toxicity evaluation of reactive dyestuffs, auxiliaries and selected effluents in textile finishing industry to luminescent bacteria V. fischeri. Chemosphere 46 (2), 339–344. Wang, C., Wang, Y., Kiefer, F., Yedeler, A., Wang, Z., Kettrup, A., 2003. Ecotoxicological and chemical characterization of selected treatment process effluents of municipal sewage treatment plant. Ecotoxicol. Environ. Saf. 56, 211–217. Yesilada, O¨., Sam, M., 1998. Toxic effects of biodegraded and detoxified olive oil mill wastewater on the growth of Pseudomonas aeruginosa. Toxicol. Environ. Chem. 65, 87–94. Zurita, J.L., Repetto, G., Jos, A., del Peso, A., Salguero, M., Lo´pezArtı´ guez, M., Olano, D., Camea´n, A., 2005. Ecotoxicological evaluation of diethanolamine using a battery of microbiotests. Toxicol. In Vitro 19, 879–886.