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# Effects of extraction processes on the physicochemical characteristics and phenolic compound content of olive mill wastewater: a comparative study

Layla Moustafa Fleyfel, Joseph Matta, Nicole Fakhoury Sayegh, Audrey Allavena, Nathalie Karpel Vel Leitner, Marie Deborde, Nasma Hamdi El Najjar

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## ► To cite this version:

Layla Moustafa Fleyfel, Joseph Matta, Nicole Fakhoury Sayegh, Audrey Allavena, Nathalie Karpel Vel Leitner, et al.. Effects of extraction processes on the physicochemical characteristics and phenolic compound content of olive mill wastewater: a comparative study. Euro-Mediterranean Journal for Environmental Integration, 2024, 10 (3), pp.1961-1975. 10.1007/s41207-024-00672-3. hal-04790853

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Effects of extraction processes on the physicochemical characteristics
and phenolic compound content of olive mill wastewater: A comparative
study

Layla Moustafa Fleyfelª, Joseph Mattaª,>d, Nicole Fakhoury Sayeghª, Audrey Allavenaᵇ, Nathalie Karpel Vel Leitnerᵇ, Marie Debordeᵇ,c, Nasma Hamdi El Najjarª.

a, Saint Joseph University of Beirut, Faculty of Pharmacy, Department of Nutrition, Medical Sciences Campus, Damascus Road, P.O.B. 11-5076, Riad Solh Beirut, 1107 2180, Lebanon.

b, University of Poitiers, Institut de Chimie des Milieux et des Matériaux de Poitiers (IC2MP UMR 7285), Equipe Eaux Biomarqueurs Contaminants Organiques Milieux (E.BICOM), 1 rue Marcel Doré, Bâtiment B1, TSA 41105, 86073 Poitiers Cedex 9, France.

c, University of Poitiers, Faculty of Medicine and Pharmacy, 6 rue de la Milétrie, Bâtiment D1, TSA 51115, 86073 Poitiers Cedex 9, France.

d, Industrial Research Institute, Lebanese University Campus, Hadeth Baadba, Lebanon.

Corresponding author:

Saint Joseph University of Beirut, Beirut, Lebanon.

Phone: +961 71202804

Email: [layla.fleyfel@net.usj.edu.lb](mailto:layla.fleyfel@net.usj.edu.lb)
**Abstract**

The olive oil industry generates two types of olive liquid waste, i.e. olive mill wastewater (OMWW) and olive washing wastewater (OWW), which are considered a major environmental issue, particularly in Mediterranean areas. This study was designed to assess the influence of trituration systems on OMWW physicochemical characteristics, and of the olive pressing seasons on OMWW and OWW. The results showed that olive trituration systems had a significant impact on OMWW quality, with the traditional system producing the most polluting effluent in terms of chemical oxygen demand, total suspended solid, total solids, ash and metal levels. The phenolic compound study revealed high tyrosol concentrations in all OMWW (384 ±71 mg/L, 370 ±70 mg/L, and 286 ±54 mg/L for traditional, semi-traditional and modern three-phase systems, respectively), while no gallic acid was noted within our detection limit (< 0.4 mg/L). The OWW results revealed that its pollution load was lower than that of OMWW, yet it still exceeded the limits authorized by wastewater discharge legislation for total suspended solid, total solid, oil and grease, and chemical oxygen demand. Finally, our findings showed that the olive pressing season factor did not induce significant variations in most of the OMWW and OWW physicochemical components. Regardless the olive mill wastewater origin, this study revealed the need for pretreatment steps before application of a wastewater purification system to avoid hindering the system performance.

**Keywords:** olive mill wastewater, modern three-phase, traditional, semi-traditional, physicochemical characteristics, phenolic compounds.

**List of Abbreviations:**

AA : Atomic absorption

BDH: The British Drug Houses

CNRS-L: National Council for Scientific Research of the Lebanese Republic

COD: Chemical oxygen demand

EC: Electrical conductivity

EPA: Environmental Protection Agency

ERDF: European Regional Development Fund
HPLC: High performance liquid chromatography

IBM: International Business Machines Corporation

IOC: International Olive oil Council

ISO: International Organization for Standardization

LD: Limit of detection

LQ: Limit of quantification

NF: Norme Française

OCDE: Organisation for Economic Co-operation and Development

OMWW: Olive mill wastewater

OPS: Olive Pressing Season

OWW: Olive washing wastewater

PC: Phenolic compound

PCA: Principal components analysis

PDA: Photodiode array

pH: Power of hydrogen

PHC : Partenariats Hubert Curien

SPSS: Statistical Package for Social Sciences

TDS: Total dissolved solids

TKN: Total Kjeldahl nitrogen

TS: Total solid

TSS: Total suspended solids

UN: United Nations

UV : Ultraviolet

w/w: Weight/weight
# 1. Introduction

Access to water and sanitation is considered a basic human right. Water use has been increasing worldwide by 1% per year since the 1980s and is expected to increase at the same rate between now and 2050 (UN World Water Development Report, 2019). Moreover, in the coming years, water-related challenges will be exacerbated by climate change, population and urban growth, as well as by rapid global economic development which will lead to a surge in water consumption. Water is therefore a crucial factor in recent milestone agreements such as the UN 2030 Agenda for Sustainable Development. Moreover, it has been reported that agroindustrial production is the largest using sector and a major polluter of water (OCDE).

Olive oil production has a long history as a vital agroindustrial activity, especially in the Mediterranean Basin. The global olive oil production volume reached 3,010,000 t for the 2021/2022 crop year. Spain is the leading olive oil producer in the world, e.g. accounting for 42% of the global annual olive oil production volume for the 2021/2022 crop year (International Olive Oil Council, 2022). In Lebanon, around 22% of the country's agricultural land is dedicated to olive tree cultivation (In Olio Veritas), with 26,000 t of olive oil being produced for the 2020/2021 crop year, which was 85.7% more than that of the 2019/2020 crop year (IOC, 2020).

It was reported that the amount of water used in oil production is about four times that of the oil produced (Michael et al., 2014). Olive oil-producing countries are therefore hampered by serious environmental issues related to the huge volumes of heavily polluted olive mill wastewater (OMWW) produced. Moreover, the composition of this wastewater is not uniform due to a range of different factors, such as the trituration method applied, which has a major impact (Fleyfel et al., 2022). There are three main olive trituration processes: the traditional method, modern methods using a two-phase or three-phase system, and the semi-traditional method (El Moudden et al., 2020; Zahi et al., 2022). Olives are crushed with stones and pressing machines are used for oil extraction in the traditional method. Otherwise, a hammer crusher and a centrifuge system are used in modern olive trituration processes. The semi-traditional system is based on a combination of traditional and modern methods and machines.

The physicochemical OMWW quality parameters often exceed the limits set in basic water legislation for safe waste disposal. OMWW is characterized by its high pollution load due to
its substantial concentration of relatively non-biodegradable organic compounds such as phenolic compounds (PCs) and long-chain fatty acids (Amaral-Silva et al., 2017). The chemical oxygen demand (COD) of OMWW can reach up to 200 g/L which is much higher than the COD of municipal wastewater, i.e. around 0.3 g/L (Phanwilai et al., 2020). In Lebanon, this value should not legally exceed 0.5 g/L according to the Lebanese decision n°8.1-2001, which sets maximum municipal wastewater pollution levels. Regarding the total phenolic content, it has been found that 2% of the PCs remain in the oil fraction, approximately 45% in the solid waste fraction, and 53% in the OMWW (Rodis et al., 2002). This pattern is correlated with the polarity and solubility of the phenols between phases, as well as the temperature, composition and relative amounts of resulting phases, while the processes involved after crushing and malaxation especially influence this distribution (Jerman Klen and Mozetič Vodopivec, 2012; Rodis et al., 2002). Aggoun et al. (2016) found that the ash content was more dependent on the extraction process than the PC concentration, which was more affected by the olive varieties. However, Jerman Klen and Mozetič Vodopivec (2012) showed that the olive fruit phenol profile was both transferred and transformed during processing. In addition, it was clearly shown that there were no qualitative differences in these phenol profiles when comparing different processing types, whereas significant differences were observed at the quantitative level. The highest rate of phenol transfer to oil was generated during processing in a modern two-phase system (1.5%), followed by a traditional olive press (1.2%) and a modern three-phase centrifuge (0.5%), where most of the phenols were flushed away with the wastewater produced (>30%).

The present study was carried out to analyse the physicochemical parameters of olive mill wastewater resulting from traditional, modern three-phase and semi-traditional systems over two pressing seasons in Lebanon. In addition, the physicochemical characteristics were analyzed in the olive washing wastewater (OWW). Finally, a statistical analysis was carried out to compare and identify the effects of the oil extraction method and crop year on the quality of the OMWW and OWW produced. In addition to the overall parameters commonly monitored in OMWW (pH, COD, etc.), a more specific analysis of 5 PCs (tyrosol, vanillyl alcohol, vanillic acid, p-coumaric acid, and gallic acid) was performed. Previous studies on this topic have generally only focused on a few criteria, but never on a complete set of pollution parameters, as was the case here. This is the first in-depth study that has focused on the characteristics of OMWW and OWW generated by Lebanese olive trituration mills.
## 2. Materials and methods

### 2.1. Site description and sample collection

OMWW were collected from three different trituration systems: traditional, modern three-phase system, and a semi-traditional process involving a hammer crusher and olive pressing machine. OMWW were sampled from Said Saifan olive mills in North Lebanon, during two olive pressing seasons; olive pressing season 1 (OPS1) and olive pressing season 2 (OPS2). For each pressing season, 8 to 10 samples per trituration unit were collected over a 1 month period. Each sample was collected at the outlet of the liquid-liquid separation stage and bottled in plastic containers that had been prerinased several times with OMWW. The bottles were filled to the brim, without leaving any air bubbles, and stored at 4°C until analysis.

OWW were collected from a modern three-phase system in which olives were mechanically washed before processing. 10 samples were collected over a 1-month period in each olive pressing season (OPS1 and OPS2). The samples were poured in plastic bottles which were stored at 4°C as soon as possible. Note that prior to the semi-traditional processing, the olives were cleaned via an air-blowing process instead of being washed in water, thus no OWW had resulted from this type of process.

### 2.2. Chemical compounds and solutions

All chemicals used during this study were analytical grade and used without further purification.

For the physicochemical analysis, potassium dichromate, double salt K₂SO₄ and CuSO₄ were purchased from Sigma-Aldrich. calcium hydroxide (lime water) (95%), potassium sodium tartrate, ammonium hydroxide and blue methylene were acquired from BDH Chemicals. Aluminum sulfate (90%) was obtained from Lebanon Chemicals. 4-aminoanthpyrine and red methylene were from VWR International. Ferrous ammonium sulphate and ammonium chloride were purchased from Merck. Zinc was obtained from Chem Lab. Potassium hexacyanoferrate (III) (98%) was purchased from Alfa Aesar. Hydrochloride acid, phosphoric acid, sulfuric acid, and n-hexane were purchased from VWR/Fluka-Sigma Aldrich, Merck, VWR International and Sigma-Aldrich, respectively. Ethanol, sodium hydroxide and boric acid were purchased from Fischer Scientific.
For phenolic compound analysis, tyrosol (98%) and gallic acid (98%) were obtained from Acros Organics. Vanillyl alcohol (98%) was acquired from Alfa Aesar. P-coumaric acid (98%), vanillic acid (97%), and ethyl acetate (≥ 99.7%) were purchased from Sigma-Aldrich. Methanol (99.9%) was obtained from Carlo Erba reagents and formic acid (80%) was purchased from Labosi.

Standard solutions used for HPLC analysis were prepared in acidified purified water (18.2 MΩ.cm; Millipore) with HCl (pH 2-3). All other solutions were prepared with double-distilled water.

**2.3. Physicochemical analysis**

Physicochemical analyses were undertaken at the Industrial Research Institute.

Samples were homogenized before the analyses, with all measurements performed in triplicate. The density was assessed according to the APHA 2520 C standard. pH was measured with an IQ 150 pH meter. Electrical conductivity (EC) was determined with a Sprite Industries, 6,000 Conductivity Meter (ISO 7888).

Total suspended solids (TSS) in OMWW were analyzed according to the NF T9-105-2 standard. Centrifugation was thus applied and residue deposits on the bottom of the centrifuge pot were rinsed and placed on a porcelain capsule for drying at 105°C overnight until total evaporation of the distilled water. The weight difference was calculated between dishes before and after evaporation. However, in the case of OWW, EPA 160.2 was instead applied due to the lower suspended solid concentrations. Filtration over a 2 µm porosity filter was thus used instead of centrifugation.

The total solid (TS) content was determined based on the weight difference of the liquid samples before and after drying at 105°C for 12 h according to the EPA 160.3 standard. The ash content was continuously measured after 12 h incineration (at 500°C) of the OLW samples that had been predried at 105°C overnight. Total dissolved solids (TDS) encompassed the organic, inorganic, cations and anions that could pass through a 2 µm and smaller pore size filter. For analysis, precentrifugation at 10,000 rpm for 5 min was conducted, filtration was applied and then the weight difference of the filtrate before and after overnight drying at 105 °C was assessed (EPA 160.1).

The oil and grease content was determined by the hexane extraction method. In a separating funnel, 100 mL of OMWW was mixed with 100 mL of hexane for 1 min, the
organic phase was collected after 15 min of decantation and the aqueous phase was subjected
to further extraction with 50 mL of hexane. The weight difference of the liquid samples was
calculated before and after drying at 105°C for 1 h (EPA 413.1). The organic nitrogen and
ammoniacal nitrogen contents were determined based on the two-stage total Kjeldahl
nitrogen method (ISO 5663-1984 (E)), including a digestion stage and a distillation stage.
Organic nitrogen in the form of azide, azine, azo, hydrazone, nitrite, nitro, nitroso, oxime and
semi-carbazone was not quantitatively determined.

The chemical oxygen demand (COD) was monitored based on the ISO 15705:2010 method. An appropriate volume of OMWW sample was mixed with potassium dichromate and sulfuric acid solution. The mixture was then poured into a specific flask and incubated for 110 min at 148°C. The COD concentration was determined based on the titration method with ferrous ammonium sulfate (0.12 M).

Primary analysis of minerals (Pb, Fe, Al) was performed on specific OPS2 samples.
The atomic absorption technique with an AA-7,000 atomic absorption spectrophotometer was
used. OMWW was transformed into ash which was then dissolved in hot nitric acid (6.5%).
The mixture thus underwent filtration through a 0.45 µm pore size membrane filter and then
analyzed with the atomic absorption equipment. Color analysis was also performed on the
OPS2 samples. Filtration through a 0.45 µm pore size membrane filter was performed before
optical analysis using a spectrophotometer at 436 nm wavelength (ISO 7887:1994).

**2.4. Phenolic compound analysis**

Phenolic compound (PC) analyses were conducted at the University of Poitiers
(France).

Phenolic compound concentrations (tyrosol, vanillyl alcohol, vanillic acid, p-coumaric acid and gallic acid) were determined by high-performance liquid chromatography (HPLC) coupled with a UV detector. All analyses were done after sample collection.

***Phenolic compound extraction method***

A liquid/liquid extraction method was conducted before HPLC analysis due to the
high organic compound content of OMWW.

Phenolic compound extraction was performed as follows: 20 mL of OMWW sample was
centrifuged at 5,000 rpm for 10 min. 10 mL of centrifuged sample was acidified with
hydrochloric acid (37%) until reaching pH 2-3. For oil and grease elimination, 5 mL of n-
hexane (98.5%) was used to wash 5 mL of acidified sample. The mixture was agitated for 10 min prior to liquid-liquid extraction. After 2 h, the phases were separated and the hexane phase was discarded. Ethyl acetate was used as phenolic compound extraction solvent. The delipidated sample was thus gently mixed with 5 mL of ethyl acetate in a dispensing funnel for 10 min based on the liquid-liquid extraction method. The extraction process was carried out in triplicate to enhance the phenolic compound extraction. The organic phases were pooled for solvent evaporation using a distillation rotary evaporator at room temperature.

The dry residue was solubilized with a 5 mL mixture composed of the initial mobile phase used for HPLC analysis (10% methanol and 90% purified water acidified with 0.1% formic acid).

Under these conditions, 10 recovery tests of the five phenolic compounds were carried out using different water matrices (purified water, mineral water, river water and wastewater). No significant variations in the extract yield were observed regardless of the studied matrix. Similar results were then suspected in OMWW. Accordingly, the results presented in Table S1 (Supporting information) were considered during this study in OMWW.

## Phenolic compound quantification

An Alliance HPLC system equipped with an X-Bridge phenyl column (5 µm, 4.6 x 150 mm, Waters) and a photodiode array (PDA 2,998 Waters) detector were used for PC analysis. The mobile phase was composed of a mixture of two solvents (solvent A composed of 0.1% formic acid in purified water and solvent B composed of 0.1% formic acid in methanol) and the following linear gradient elution was carried out at a 1 mL/min flow rate: 10% B under the initial conditions, an increase to 100% B for 17 min, maintenance of 100% B for 3 min, then a gradual return to initial conditions for 5 min. For all samples, the injection volume was 100 µL with a 210 to 320 nm wavelength. All the samples were analyzed by HPLC-UV either directly or after dilution (1/10 or ½) in the mobile phase if necessary. PC were quantified by external calibration using standards prepared in acidified purified water at 1, 2, 5, 10, 20, 50, 100, 200, and 500 µM. Triplicate measurements were conducted for each point in the range. Under these conditions, HPLC quantification limits of 0.5-1.1 µM were obtained according to the chemometric method proposed by Harris (2004).

The retention time, specific wavelength detection and the HPLC quantification and detection limits obtained for each studied PC are described in Table 1.
**2.5. Statistical analysis**

The results were expressed as means and standard deviations for continuous variables. Log10 and Square root with non-normal distributed continuous variables were used. Statistical analyses were performed using the Wilcoxon signed-rank test to compare continuous variables between PS1 and PS2. The Kruskal-Wallis test was also performed to compare continuous variables between the traditional, semi-traditional and modern three-phase systems, followed by a pairwise comparison test. The significance level was set at *p*<0.05. Statistical analysis was performed using SPSS 23.0 software for Windows (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY, USA, IBM Corp.).

Principal components analysis (PCA) was performed using XLSTAT version 2021 –
Life Sciences. The data (for each process and season, parameter values for each of the 8 to 10
samples) were mean-centred and normalized by the standard deviation prior to PCA.

**3. Results and Discussion**

**3.1. Physicochemical characteristics of OMWW**

The average and standard deviation of the physicochemical characteristics obtained for the 8 to 10 OMWW samples resulting from each trituration system and each pressing season are presented in Figure 1 and Table 1.

The results showed that the majority of the OMWW physicochemical parameters largely exceeded the wastewater discharge limits for treatment plants authorized under national and international legislation (Lebanese decision 8,1-2001 and the Turkish Standard outlined in the Water Pollution Regulations of Turkey; see Table 1).

Statistical analyses were performed to study the effects of trituration types on the OMWW quality based on a Kruskal-Wallis test, and the findings are presented in Table S2 (Supporting information). Moreover, a Wilcoxon test was used to analyze significant differences between OMWW samples over the pressing seasons, as shown in Table S3 (Supporting information). Under these conditions, a significant variation was detected when the p-value was < 0.05.

***pH and density***
In this study, the OMWW density ranged from 0.99 ± 0.02 g/mL to 1.00 ± 0.03 g/mL, and the pH values ranged from 4.72 ± 0.08 to 4.85 ± 0.09. This acidic pH could be attributed to the presence of phenolic acids such as vanillic, caffeic, gallic acid, and 3,4-dihydroxyphecetic acid in the OMWW (Aggoun et al., 2016), while these pH values were below the legal limits for wastewater discharges and could affect the performance of biological and physicochemical treatments applied in wastewater treatment plants. From a statistical standpoint, no significant differences in the density and pH measurements between the 3 trituration systems and the season were noted. Yet a significant difference in the OMWW pH resulting from traditional and semi-traditional systems in PS1 was observed.

## Electrical conductivity

OMWW EC values were between 8.94 ± 0.17 and 12.42 ± 0.23 mS/cm. These high values indicate the need for a treatment step essentially when the effluent is intended for irrigation. This study revealed no significant differences in EC between the 2 pressing seasons. However, this parameter seemed to be affected by the trituration system. Indeed, for both studied seasons, the highest values were obtained for the semi-traditional system, followed by the traditional system and the modern system (Table 1). This may have been due to the absence of an olive washing stage and the use of a blowing machine before crushing in the semi-traditional system. In the literature, a similar variation pattern been reported for traditional (9.15 mS/cm) and modern (6.28 mS/cm) olive trituration systems (Haddad et al., 2015).

## Color

A dark brown color, with absorbance ranging from 404.4 ± 0.57 to 408.7 ± 1.41 at 436 nm, but without any real difference between the OMWW was noted in this study. Similarly, absorbance of 575 at 436 nm was previously reported for OMWW (Kirmaci et al., 2018). These values were very high in comparison to those of other wastewaters, e.g. wastewater resulting from denim production was reported to have an absorbance of 57.2 at 436 nm (Kahraman Ö and Şimşek İ, 2020).

## Total suspended solids, total dissolved solids and total solids

No statistical between-season differences were observed for TSS, TDS and TS, with the exception of the semi-traditional system for which the TSS values were slightly lower in
OPS2. In contrast, significant differences between trituration systems were noted for these parameters.

The TSS concentrations ranged from 2.06 ± 0.29 to 9.72 ± 0.80 g/100 mL and the highest values were obtained for traditional systems, followed by semi-traditional and then modern systems (Table 1 and Figure 1). These results are in agreement with a previous study, which reported a similar variation pattern, with TSS values ranging from 0.002 to 0.2 g/100 mL obtained for the modern system and from 0.4 to 7 g/100 mL for the traditional system (Achak et al., 2019). This difference could be explained by the better separation between solids and OMWW with the modern system. Moreover, it has been reported that pressing mats are used in the traditional system, which could be a source of TS contamination, especially when the mats are not sufficiently washed between trituration runs (Khdair et al., 2015). The centrifugal force with a large amount of water applied in the modern system seems better for maximum liquid, oil and solid phase separation (Torres and Maestri, 2006).

Similarly, the statistical study revealed a TDS variation that was dependent on the trituration system applied. Lower TDS concentrations were thus obtained with the traditional system compared to the other systems. This was in agreement with a recent study in which a low TDS concentration (2.21 g/100 mL) was obtained for OMWW derived from the traditional system (El Ghadraoui et al., 2021). Moreover, Zharra et al. (2017) described a higher TDS concentration (8.71 g/100 mL) for the modern system. In this latter case, the high volumes of water used in the modern system could explain why it generated the highest TDS concentration (Khdair et al., 2019).

Finally, the TS fraction encompassed the TSS and TDS. According to the Kruskal-Wallis test, the results showed that the trituration system had a significant impact on the TS concentration. A profile similar to that of TSS was observed. For both pressing seasons, this parameter was significantly higher in OMWW derived from the traditional system than in other OMWW. This pattern was in accordance with the findings of Haddad et al. (2015), which showed that the traditional system generated the highest OMWW TS concentration (12.32 g/100 mL) while the modern system generated the lowest OMWW TS concentration (4.59 g/100 mL).

## Oil and grease
OMWW are effluents with a high oil and grease concentration, which could impact aquatic ecosystems and organisms by preventing solar ray penetration, which in turn affects the water temperature (Environment, Essays and Paragraphs, 2016).

In this study, no real seasonal differences were observed for this parameter, except with regard to the modern system. However, the statistical findings revealed a variation dependent on the trituration process. The highest concentrations were found in OMWW derived from the modern system, followed by the traditional system and the semi-traditional system (Table 1 and Figure 1). Similarly, in the literature, higher oil and grease concentrations (0.3 to 1.08 g/100 mL) were generally reported for OMWW from the modern three-phase system (Khemakhem Sellami et al., 2016; Khdair et al., 2019), whereas lower levels (0.12 and 0.001 g/100 mL) were reported for the semi-traditional system (Ben Sassi et al., 2006; Elabdouni et al., 2020). Finally, intermediate oil contents (0.001 to 0.63 g/100 mL) have commonly been noted for the traditional system (Aktas et al., 2001; Ben Sassi et al., 2006). OMWW from the semi-traditional system thus seems to have the lowest oil and grease concentration, which is interesting for olive oil producers and seems to indicate that the mechanical olive crusher and the press machine are optimal for generating maximum oil yields. According to the literature, oil and grease concentrations in OMWW can be related to two factors (i.e. the dough temperature during crushing and the mixing time), which must be controlled during trituration to avoid oil emulsion and a reduction in the oil extraction efficiency (Medouni-haroune et al., 2018; Petrakis, 2006).

## Total Kjeldahl nitrogen

In this study, a slight season-dependent variation in TKN values was noted in OMWW derived from traditional and modern systems (Table 1 and Figure 1). TKN was also affected by the trituration system. The highest values were recorded in OMWW from the modern system, in accordance with the findings of Issa et al. (2023), Belaqziz et al. (2016), and Haddad et al. (2015).

This could have been due to the larger volumes of water added in the modern system than in the traditional system. More dissolved nitrogen would thus be obtained in OMWW from modern systems. Similarly, this pattern has also been observed with water-soluble salts, which were essentially concentrated in OMWW due to the high water content (Dermeche et al., 2013).

## Chemical oxygen demand
There were significant differences in COD according to the processing type. The results demonstrated that traditional olive trituration generated a more polluted effluent than modern and semi-traditional systems. This was in agreement with previously reported concentrations of 213.44 g O₂/L, 96.07-105 g O₂/L, and 113 g O₂/L in OMWW resulting from a traditional system (Elabdouni et al., 2020), a modern system (Majbar et al., 2018; Issa et al., 2023), and a semi-traditional system (El-Abbassi et al., 2012), respectively. For this parameter, despite variations according to the type of trituration system, the results showed the significant pollution characteristic of OMWW, which highly exceeded the limits authorized by wastewater discharge legislation (0.5 g/L). These values could be compared with other agroindustrial wastewaters derived, for instance, from molasses production with a COD of around 134 g/L (Mahimairaja et al., 2004) or from the dairy industry with a COD of around 15 g/L. The high COD concentration in OMWW could be attributed to the organic fraction. It has been mentioned that OMWW is composed of 83-94% water (w/w), 4-16% organic matter (w/w), and 0.4-2.5% mineral salts (w/w) (Souilem et al., 2017). Note that the organic fraction includes an insoluble part, e.g. olive pulp, suspended matter and colloidal particles, whereas the soluble fraction contains polyphenols, proteins, sugar, vitamins, lipids, organic acids, pectins, tannins and traces of pesticides (Esteves et al., 2019; Leontopoulos et al., 2020). Crucial concentrations of different organic matter fractions have been previously reported, including the total phenolic content (1,036.13 ± 652.54 g/Kg OMWW) (Issa et al., 2023), fat (16.73 g/L) (Sar et al., 2020), glucose and other sugars (10.47 ± 0.46 g/L) (Sar et al., 2020), protein (8.26 ± 0.03 g/L) (Sar et al., 2020), total solids (81.6 g/L (El Moudden et al., 2020); 270.7 ± 22.34 g/L ((El Hadrami et al., 2004)). These compounds could therefore be responsible for the high COD concentration noted in this study.

## Ash

The ash content reflects the mineral concentration in the sample. In this study, the ash content ranged from 0.85 ± 0.02 to 1.71 ± 0.08 g/100 mL, and significant differences were observed according to the trituration system. Hence, the traditional system had the highest ash content, followed by the semi-traditional system then the modern system. These results were in accordance with those of Aggoun et al. (2016), who showed that the ash content of OMWW derived from a traditional system was almost threefold higher than that produced by a modern system. These results were also in agreement with those of El-Abbassi et al. (2012), who reported significant differences according to the olive variety and processing technique.
## Metals

Among the studied metals, aluminium was found to be the most abundant, followed by lead and iron. In contrast to the other parameters, none of the studied mineral contents exceeded the legal limits for discharged wastewater outlined in the Lebanese decision n°8.1-2001. Iron concentrations found in this study (6.4 to 25.2 µg/L) were lower than those reported in other studies (9 to 23 mg/L) (Zahi et al. 2022; Danellakis et al., 2011; Mekki et al., 2006). However, Pb concentrations found in this study (57.8 to 119.6 µg/L were higher than those reported in the literature (6.7 to 10 µg/L) (Zahi et al. 2022). According to the Kruskal-Wallis test, there were no significant differences between trituration processes for aluminium. However, higher Pb and Fe concentrations were found in OMWW derived from the traditional system, followed by the semi-traditional system and the modern system. A similar pattern has been previously reported, where Fe and Pb were the most concentrated in a traditional system and the least concentrated in a modern three-phase system (Ben Sassi et al., 2006).

In conclusion, these results showed the very high pollution load in OMWW, with most of the physicochemical parameters highly exceeding the limits authorized by wastewater discharge legislations (bold values in Table 1). The influence of the crushing system used was clearly noted for many of the parameters. Hence, TS, TSS, ash, and COD were found to be significantly higher in traditional systems, while oil contents and TKN were found to be higher in modern systems and EC values were higher in semi-traditional systems. In this study, no significant differences between the physicochemical characteristics in the majority of the samples were observed between seasonal campaigns. Yet it has been demonstrated that the olive type, maturity, harvesting, storage conditions and trituration type are key factors that influence the characteristics of the resulting OMWW (Fleyfel et al., 2022). For instance, fertilizer has a cumulative effect and therefore could affect the end-product mineral concentration (Lechhab et al., 2022; Ntougias et al., 2013). Moreover, the harvesting method, i.e. manual or mechanical, should be taken into account since it can influence the picked fruit quality (Lechhab et al., 2022). Finally, the harvest-to-processing latency time and the olive storage conditions may influence the olive oil and OMWW quality. Several factors may therefore interfere to change the end-product quality, e.g. the temperature
and activation of enzymes such as fungal lipase, peroxidase and polyphenoloxidase, which
influence the free acidity and phenol levels (Jabeur et al., 2015).

**3.2. Phenolic compound concentration in OMWW**

HPLC analyses were carried out after PC extraction from different types of OMWW.
Figure 2 represents the chromatograms obtained for the analysis of OMWW extracts at 1/10
dilution. They clearly show that all PC, except gallic acid, were detected in all types of
OMWW.

Table 2 and Figure 3 present the PC concentrations in various types of OMWW when different dilution rates were used for the analysis. Similar concentrations were obtained for the different PC regardless of the dilution rate. Only the concentrations obtained at 1/10 dilution were thus taken into account in Figure 3.

These results showed that tyrosol was the main PC detected in OMWW (249-384 mg/L) followed by vanillyl alcohol (44-67 mg/L), vanillic acid (20-45 mg/L), p-coumaric acid (18-43 mg/L), and finally gallic acid (not detected). Other peaks were also noted, including a particularly intense peak recorded at 4.8 min, which did not correspond to the studied molecules. A standard solution was injected so as to be able to correlate this compound with hydroxytyrosol, which generally represents the most concentrated PC in OMWW according to previous studies (Ladhari et al., 2021; Allouche et al., 2004). It has also been reported that the main phenolic compounds in OMWW are hydroxytyrosol, tyrosol and sometimes oleuropein (Allouche et al., 2004; Visioli et al., 2001). Vanillic acid and p-coumaric acid have also frequently been reported in OMWW (Leouifoudi et al., 2014). Finally, similar tyrosol concentrations were previously obtained, with values ranging from 208 to 345 mg/L (Allouche et al., 2004; Aktas et al., 2001), whereas higher concentrations of p-coumaric acid (169 mg/L) and vanillic acid (174 mg/L) have been reported (Allouche et al., 2004; Aktas et al., 2001). Yet different factors can interfere with the PC quantification, such as the olive type and maturity, or the environmental conditions (weather and geographical area) (Leouifoudi et al., 2014). Moreover, it has been pointed out that some phenolic compounds may be affected by the cultivation site. For instance, p-coumaric acid was only detected in flatland areas whereas tyrosol and vanillic acids were found in samples from mountainous areas (Leouifoudi et al., 2014).

Few differences were found between traditional and semi-traditional OMWW in this
study. However, slightly lower PC contents were noted in OMWW from the modern three-
phase system (except for vanillyl alcohol). Differences have often been observed between
processes in other studies (El-Abbassi et al., 2012). It was reported that several factors could
affect the PC pattern. For instance, the quantity and quality of the added water, as well as the
treatment temperature, could play a major role in the PC distribution (El-Abbassi et al., 2012;
Obied et al., 2005; Rodis et al. al., 2002; Caponio and Catalano, 2001). Therefore, contrary to
our results, a modern system was reported to retain a greater amount of PC than a traditional
system. This pattern could be explained by the greater volume of water added in the modern
system compared to the traditional system, and the better separation of olive pulp could thus
be responsible for the greater quantity of PC released (Di Giovacchino et al., 2002; Rodis et
al., 2002).

However, it has been pointed out that in modern three-phase and semi-traditional systems,
the temperature reached higher values than those reached in the traditional system, which led
to phenolic compound oxidation. This could explain the PC patterns observed in this study.

**3.3. Physicochemical characteristics of olive washing wastewater**

Olive washing wastewater (OWW) was also analyzed and compared to the limits
authorized by wastewater discharge legislation. Since this latter decision does not cover all of
the studied physicochemical characteristics, the water pollution regulations of Turkey were
also taken into consideration (Table 3).

The results showed that this type of wastewater had a neutral pH (7.56 ± 0.03 to 8.68 ± 0.03), which differed from that of OMWW. This variation was essentially related to the absence of acidic compounds, since these compounds are bound within the olive fruit (Torrecilla, 2010; Souilem et al., 2017). Despite the fact that values for all other parameters were much lower than those in OMWW, the levels in this effluent exceeded the limits authorized for disposal in wastewater treatment plants and systems. The parameters that exceeded the legal limits (bold values in Table 3) by around 20-fold was COD, as well as oil and grease, and TS to lesser extent (by 3- to 5-fold).

Lower pH (5.9-6.4) and COD (2-5 g/L) values have been previously reported (Maza-Márquez et al. 2013; Nieto et al. 2009). Conversely, higher EC, TSS, and TS concentrations were reported (2.3-2.7 mS/cm, 3-3.3 g/100 mL, and 4.8 g/100 mL, respectively) (Maza- Márquez et al., 2013). It has been stated that dust and soil debris represent the main suspended particles in OWW (Maza-Márquez et al. 2013).
Here it was found that the TDS values were equal to (0.08 and 0.09 ± 0.01 g/100 mL), lower than the concentrations obtained by Maza- Márquez et al. (2013) (1.8-1.5 g/100 mL).

Finally, the TKN content was 0.002 g/100 mL (20 mg/L), which was lower than the content reported previously, i.e. 0.006 g/100 mL (60 mg/L) (Maza-Márquez et al. 2013), but without exceeding the concentration limit. However, even though this type of water is less polluted than OMWW, pre-treatment steps would be essential before disposal. This has been emphasized by Nieto et al. (2009), where OWW was used in irrigation after simple oxidation treatment.

Similarly to OMWW, a Wilcoxon test was applied to compare the differences between OWW resulting from two pressing seasons (Table 3). A statistical difference was noted for TS, TSS, ash, and COD (p= 0.005, 0.004, 0.028 and 0.005, respectively). This variation is known to be essentially affected by the olive harvesting method and storage conditions used (Lechhab et al., 2022).

**3.4. Multivariate analysis**

Finally, PCA was performed to further determine the overall correlation between the physicochemical parameters and the samples. The biplot including both samples (i.e. OMWW and OWW for both seasonal campaigns) and physicochemical parameters (i.e. pH, density, TKN, oil and grease, TDS, TS, TSS, EC, COD and ash) is presented in Figure 4.

Concerning the samples, a separation of OWW and OMWW along the F1 axis was observed, which was consistent with previous descriptions of these parameters (Table 1 and 3) and the lower values observed in OWW. A second separation along the F2 axis clearly discriminated the 3 trituration systems for OMWW. This separation could be explained by changes in the OMWW composition depending on the trituration system (Table 1). For each crushing system, the samples were grouped together, which confirmed that there was no clear distinction between the two seasons. Only a very slight separation of samples from the 2 seasons was noted for the modern system. Hence, according to these results, the F1 axis was representative of the type of olive liquid waste (OMWW vs OWW) whereas the F2 axis illustrated the difference in OMWW.

Based on the physicochemical parameters, this figure shows that OMWW derived from the modern system was closely linked to the TKN parameter. OMWW from the traditional system was mainly distinguished by the ash and TSS parameters, and to a lesser extent by the
COD and TS parameters. Lastly, intermediate values were noted for OMWW from semi-traditional systems. These observations were in agreement with results presented in Table 1 which show higher TKN values in the modern system, alongside significantly higher values for ash, TSS, COD and TS in the traditional system. In this figure, the central position of the density reflects the fact that the samples cannot be differentiated on the basis of this parameter.

PCA is an essential tool to facilitate the interpretation of results when large datasets have to be taken into account.

## 4. Conclusion

The results obtained in this study indicated that the OMWW quality was highly influenced by the type of trituration applied. The traditional system produced the most polluted OMWW in terms of TSS, TS, ash, COD and metal contents. The modern system generated a liquid effluent with a high TKN, and oil and grease content. Otherwise, in the semi-traditional system, the EC was essentially concentrated in the OMWW produced. PC analysis showed that tyrosol was the most abundant compound regardless of the type of trituration system used. Conversely, the sampling campaign did not significantly impact the OMWW and OWW quality. The overall results highlight the need to clearly pinpoint the OMWW origin to be able to determine the optimal treatment to adopt.

## 5. Acknowledgments

This research was supported by grants from the Hubert Curien CEDRE Partnership (PHC CEDRE) and the National Council for Scientific Research of the Lebanese Republic (CNRS-L) and we gratefully acknowledge their financial support. The authors thank the European Regional Development Fund (ERDF) and the *Région Nouvelle Aquitaine* (France) for their financial support. We gratefully thank Mr. David Manley for his valuable work in correcting the English.

On behalf of all authors, the corresponding author states that there is no conflict of interest.

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