Profiling and evaluation of phenolic compounds in olive mill wastewater in Jordan

Olive oil is a key component of the Mediterranean diet due to its many uses, health benefits, and long shelf life. The International Olive Council estimated worldwide production at 2,444 tons in 2015 (Kiritsakis et al. 2017). Approximately 10 million hectares of land worldwide are home to a staggering 900 million olive trees, with the Mediterranean Basin containing 98% of them (Zghari et al. 2017). The olive oil production system generates olive oil, solid residue, and liquid phase. The liquid phase is called the olive mill wastewater (OMW).

Global OMW production exceeds 30 million cubic meters annually (De Marco et al. 2007).

Jordan ranks 8th in the world in the production of olive and olive oil. The olive milling industry is a major contributor to the Jordanian economy, with olive oil generating approximately 100 million Jordanian dinars in annual revenue (Abdel & Hiary 2015). In Jordan, there are 25 million olive trees that produce approximately 220,000 tons of olives, 35,000 tons of oil, 60,000 tons of olive pomace, and about 200,000 m³ of OMW annually. These products come from around 130 olive mills scattered throughout the Kingdom of Jordan (Khdair et al. 2019).

Olive oil can be obtained through two main processes, a two-phase process and a three-phase process (Frascari et al. 2016). The three-phase process typically generates more OMW, producing 1,200 L/ton compared to 200 L/ton for the two-phase process (Khdair & Abu-Rumman 2020).

OMW consists mainly of vegetation water (from the olive fruit), washing water, and water added during malaxation and during pressing (Khdair et al. 2019).

OMW is acidic, and varies in color from dark red to black, with a mixture of water (83–96%), minerals (2%), and organic matter (4–16%) (Zghari et al. 2017). The organic content of OMW includes sugars, tannins, nitrogen-containing compounds, pectins, lipids, and phenolic compounds (PCs) (Belaid et al. 2013). The chemical composition of OMW is subject to various factors, such as weather conditions, olive variety, fruit ripeness, and the method used for olive oil extraction (Khdair et al. 2019).

PCs are a significant group of naturally occurring compounds in plants. The health benefits of PCs and their use in the food, cosmetics, and pharmaceutical industries can justify the necessary work required to extract these compounds (Jerman Klen & Mozetič Vodopivec 2011). The beneficial health properties of PCs can be attributed to their antioxidant, anti-inflammatory, antimicrobial, and anticarcinogenic activities (Chanioti & Tzia 2018).

The PCs in olive fruits and their pulp are more soluble in water than in oil. Therefore, PC concentrations in the aqueous phase tend to be higher than in the oil phase, with concentrations in the OMW ranging from 0.5 to 25 g/L (Kallel et al. 2009). Thus, recently, OMW has been widely viewed as a natural source of PCs.

On the other hand, the presence of PCs in wastewater is one of the biggest environmental challenges facing the olive milling industry. The toxic effect of PCs on plants, microorganisms, and aquatic life limits the possibility of the direct reuse of OMW in irrigation. Therefore, OMW is usually dumped in landfills or waterways. This, in turn, generates another problem due to the unpleasant odors that arise from the interactions of PCs with other materials (Solomakou & Goula 2021). Moreover, there is an additional cost associated with transporting the OMW away from agricultural land for dumping.

The development of analytical methods for determining PCs in OMW serves a dual purpose, addressing environmental concerns and regulatory compliance, as well as ensuring efficient wastewater treatment and the overall sustainability of olive oil production processes. In addition, it allows for tapping into the economic potential of these compounds.

Several studies focused on developing new extraction methods for PCs in order to utilize them as natural antioxidants in OMW (Venturi et al. 2017). Some researchers employed liquid–liquid extraction, with ethyl acetate as the major solvent, while other articles reported the use of solid-phase extraction (SPE) (Jerman Klen & Mozetič Vodopivec 2011).

For the quantitation of PCs, chromatographic methods were mainly applied; for example, Zghari et al. (2017) applied gas chromatography with a mass spectrometry detector (GC–MS) for the determination of tyrosol, catechol benzoic acid, and p-hydroxyl coumaric acid in OMW, and Azaizeh et al. (2012) used high-performance liquid chromatography with an ultraviolet detector (HPLC-UV) for the determination of hydroxytyrosol (HTy), tyrosol, vanillic acid, ferulic acid, and p-coumaric acid in OMW.

However, up to our literature survey, no HPLC coupled with a diode array detector (HPLC-DAD) method is available for the simultaneous determination of HTy, protocatechuic acid, tyrosol, homovanillyl alcohol, 4-hydroxy benzoic acid, caffeic acid, and vanillic acid in OMW.

The main objective of this study was to provide a comprehensive understanding of the quantitative and qualitative aspects of phenolic content in OMW in Jordan. This goal was achieved by (1) developing a cost-effective and efficient technique for extracting PCs from OMW, (2) fine-tuning an HPLC-DAD method for precisely quantifying seven PCs in OMW, (3) profiling the PCs present in OMW using GC–MS, (4) quantifying the total phenolic content of OMW, (5) evaluating the antioxidant activity of OMW, and (6) analyzing the Fourier transform infrared (FTIR) spectra of OMW samples.

4-hydroxy benzoic acid (HBA) (99%), homovanillyl alcohol [4-hydroxy-3-methoxyphenethyl alcohol] (HVA) (99%), tyrosol [2-(4-hydroxyphenyl) ethanol] (Tyr) (98%), caffeic acid (CA) (98%), vanillic acid (VA) (97%), protocatechuic acid (PCA) (99.71%), hydroxytyrosol [3-hydroxytyrosol] (HTy) (98%), homogentisic acid (HGA) (98%) were used as internal standard (IS) for HPLC-DAD. These compounds along with N,O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) and 1 mg/L of α-naftol as IS for GC–MS were procured from Sigma Aldrich. Folin-Ciocalteu reagent (2 M) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (98%) were bought from Sigma Aldrich (Germany). All the solvents employed were of HPLC or HPLC/GC grade.

The optimized chromatographic analysis was performed utilizing a Shimadzu HPLC system (LC-20A) featuring a DAD (SPD-M20A). The HPLC-DAD method involves the separation of PCs on a C8 column (4.6 × 150 mm, 5 μm) that was maintained at a constant temperature of 40 °C. The mobile phase was comprised of solvent A, which was 0.5% (v/v) acetic acid deionized water, and solvent B, which was methanol. The system was run with the following gradient elution program 0–10 min, 5% B; 10–12 min, 30% B; 12–17 min, 33% B; 17–18 min, 38% B; 18–20 min, 50%; 20–23 min, 70%; 23–27 min, 95%; 27–35 min, 5%. The flow rate was 0.7 mL/min, the injection volume was 10.0 μL, and the data were collected at 279 nm.

A SPE C18 cartridge was conditioned with 5 mL of n-hexane. Then, a mixture of 5 mL of the OMW sample and 5 mL of n-hexane was uploaded on the cartridge and eluted under vacuum. After that, PCs were eluted by 10 mL of methanol. Finally, 10 μL of this eluent was injected into the HPLC-DAD.

A blank solution of OMW was prepared by extracting and cleaning the OMW sample 10 times. Then, a series of standard mixture solutions (5, 20, 40, 50, 100, 150, and 200 mg/L) containing all the investigated PCs (HTy, PCA, Tyr, HVA, HBA, CA, and VA) and the IS (100 mg/L) were prepared. The peak area of each compound relative to the peak of IS was calculated and plotted against the concentration to construct the calibration curves.

To account for the specificity of the method, three injections were performed: (1) blank solution, (2) blank spiked with IS, and (3) the standard mixture of 20 mg/L of PC.

The blank OMW was spiked with IS and injected six times into HPLC-DAD to find the limit of detection and limit of quantification (LOD and LOQ).

The precision of the instrument was evaluated through the injection of standard mixtures of PCs six times at three levels (50, 80, and 100 mg/L).

Three solutions were prepared by fortifying 10.0 mL of a blank OMW sample with a standard mixture of PCs, resulting in concentrations of 50, 100, and 150 mg/L. Subsequently, these solutions underwent the extraction, purification, and analysis steps, as outlined in Figure 2. The recovery tests were conducted in triplicate.

The robustness of the method was evaluated by running the analysis of a standard mixture of PCs of 100 mg/L at different conditions of flow rate (0.6, 0.7, 0.8, 0.9, and 1 mL/min), column oven temperature (30, 35, 40, and 45 °C), and detector wavelength (279, 280, and 281 nm).

Every OMW sample was meticulously prepared following the steps outlined in Figure 2, and it was subsequently injected into the HPLC-DAD column three times (n = 3). The relative peak area for each compound present in the chromatogram was determined, and the concentration of each compound was then computed by employing the previously established calibration curves.

For GC–MS analysis, the ethyl acetate extract was subjected to derivatization to enhance the volatility and thermal stability of PCs. BSTFA is mostly used as a derivatizing agent due to its high reactivity. Derivatization of PCs was performed according to the procedure described by Zafra et al. (2006). A mixture of 20 : 5 : 25 (v/v/v) BSTFA–pyridine–ethyl acetate (containing PCs) was prepared. Then, the mixture was shaken well for 2 min to derivatize the PCs. After that, 50 μL of the last solution was mixed with 50 μL of 1 mg/L of α-naftol as IS. Finally, a volume of 1 μL of the prepared solution was injected into the GC–MS instrument. To obtain a methanol extract, the same method was used but with methanol as the solvent instead of ethyl acetate.

The GC analysis was performed using a gas chromatograph fitted with an autosampler injector (Shimadzu QP2010 Ultra, Japan). A capillary column HP-5-fused silica column (30 m × 0.25 mm, film thickness 0.25 μm, (5%)-biphenyl–(95%)-dimethylsiloxane copolymer) was used. A silanized injector liner split/splitless (2 mm ID) was employed. The detection was carried out with a mass-selective single quadrupole detector. The injector temperature was 250 °C. The oven temperature was held at 80 °C for 3 min and then increased to 240 °C at a heating rate of 13 °C min⁻¹, and the temperature was held for 20 min. The total run time was 38.5 min. The detector temperature was 280 °C. The carrier gas used was helium (purity 99.999%) at a flow rate of 1.0 mL min⁻¹. The samples were injected in the splitless mode, and the splitter was opened after 7 min (delay time). The sample volume in the direct injection mode was 2 μL. The ion energy used for the electron impact ionization mode was 70 eV. The mass range scanned was 150–550 m/z.

The total phenolic content (TPC) in each extract was evaluated spectrophotometrically using the Folin-Ciocalteu method described by Waterhouse (2002). Firstly, a series of gallic acid standard solutions were prepared (50, 100, 250, 500, 250, 500, and 750 mg/L). Then, 0.5 mL of each standard/sample was treated with Folin-Ciocalteu reagent. After that, 7.5 mL of a 20% sodium carbonate solution was added to the mixture, the volume was completed to 50 mL with deionized water, and the reaction was incubated for 2 h. In conclusion, the absorbance of the prepared solutions was measured at 765 nm.

The DPPH radical scavenging assay described by Chanioti & Tzia (2018) was used to assess the antioxidant activity of OMW samples. First, 0.1 mL of the sample extract, which was constituted in methanol, was added to 3.9 mL of a 25 mg/L DPPH solution. Then, the solution was incubated in the dark at room temperature for 20 min. After that, the absorbance was measured at 515 nm. A blank solution was prepared by mixing 0.1 mL of methanol with 3.9 mL of 25 mg/L DPPH solution.

FTIR spectra were acquired utilizing a Bruker Vector 22 spectrometer from Germany, equipped with an integrated Michelson interferometer, and operated with Opus 5.5 software. The spectra were obtained by applying crude samples onto an attenuated reflection cell that featured a diamond crystal.

The experimental outcomes were replicated three times, and the results were expressed as means ± standard deviation. An analysis of variance test was conducted to estimate significant differences among samples. Differences among samples were considered to be statistically significant at p < 0.05.

The identification of PCs in the OMW extracts was performed by HPLC-DAD by comparing the relative retention times with those of standard solutions. In the optimization of the HPLC method, the most suitable was selected for the following parameters: stationary phase, mobile phase, flow rate, wavelength, and column oven temperature. The criteria were to achieve the highest response and the best resolution at a suitable run time.

The second important step in the HPLC method development is the choice of a suitable mobile phase. Methanol and acetonitrile were tested as organic solvents in the HPLC system. Methanol gave better resolution for the seven PCs, as shown in Figure 3(b). The pH value for the aqueous mobile phase plays an important role in the separation of the target compounds. The most suitable pH was 3.1, which gave the best resolution. Several factors can affect the elution of the examined target compounds. Firstly, the polarity of the individual PCs is reflected by the log p-value, as indicated in Table 1 where the lower log p-value refers to a less polar compound (Agach et al. 2014). The second factor is the molecular weight since PCs with low molecular weight are more polar and strongly interact with the relatively polar C8 stationary phase. Thirdly, the pKa values of each compound (Table 1) are important because when the pH value of the mobile phase is within the range of pKa ±1, the compound will be in its unionized form. Increasing the pH above the range leads to the ionization of the compound. The combined effect of these factors, including the type of stationary phase, causes the compounds to elute in the following order: HTy, PCA, Tyr, HVA, HBA, CA, and VA.

Different flow rates (0.6, 0.7, 0.8, 0.9, and 1 mL/min) were examined, and the best resolution was achieved at a flow rate of 0.6 mL/min. However, flow rate (0.7 mL/min) gave a slightly lower resolution at a shorter run time; hence, it was selected.

In the optimization of wavelength values 279, 280, and 281 nm wavelengths for detector reading were tested, and the highest average response for the seven target compounds was at 279 nm.

The chromatograms of the target PCs were obtained at four different oven temperatures, 30, 35, 40, and 45 °C, as shown in Figure 3(c). It can be noted that the best resolution was observed at a temperature of 40 °C. Meanwhile, at 45 °C, some peaks of the target compounds were absent, since it seems that HBA co-eluted with HVA at this temperature.

Therefore, the optimized conditions include using C8 as a stationary phase, methanol in the mobile phase, a flow rate of 0.7 mL/min, temperature adjusted to 40.0 °C, and a 279 nm detector wavelength. To the best of our knowledge, this is the first method involving the separation of the seven PCs: HTy, PCA, Tyr, HVA, HBA, CA, and VA and their determination using HPLC-DAD. Figure 3(d) represents a typical chromatogram of a standard mixture of the seven PCs under optimal conditions.

System suitability is a test that is based on the concept that the equipment, electronics, analytical operations, and samples to be analyzed constitute an integral system that can be evaluated (Branch 2005). System suitability parameters were evaluated for the newly developed HPLC-DAD method according to the Food and Drug Administration (FDA) guidelines (FDA & CDER 2018).

The evaluated system suitability parameters were the number of theoretical plates/efficiency (N), capacity factor (k), resolution (R_s), tailing factor or asymmetry factor (A_s), and separation factor (α).

Table 1 shows the calculated system suitability parameters and the acceptance criteria for each one. It can be observed that all system suitability parameters for all target compounds met the accepted criteria (FDA & CDER 2018). However, the resolution of HBA and VA was less than the required criteria.

The optimization of the extraction procedure involves selecting the extraction method, extraction solvent, solvent ratio, and adding sodium hydroxide. The criteria were to achieve the highest recovery, analytical signal, and the simplest procedure. The chosen extraction procedures were evaluated and applied for the extraction of the analytes using several replicates of the same sample.

Figure 4(b) shows that adding NaOH before the n-hexane step gives a higher response compared to the response with no NaOH added. This can be attributed to the fact that sodium hydroxide ionizes the PCs, thus increasing their solubility in the aqueous layer. Therefore, the addition of NaOH enables the hexane layer to be discarded without worrying about the presence of residual PCs in it. It should be clarified also that n-hexane was added in order to remove most of the lipids and nonpolar compounds from the sample.

Acidification is expected to increase the solubility of PCs in organic solvents (de Marco et al. 2007). Therefore, the pH was adjusted to 2 after the removal of the n-hexane layer to ensure that all PCs were in their unionized form.

Ethyl acetate is the most used solvent to extract PCs from aqueous matrices such as OMW. Ethyl acetate was more effective at extracting PCs than other solvents, such as methyl ethyl ketone and diethyl ether (Zghari et al. 2017). Therefore, it was selected in our study as the extraction solvent.

Two ratios of solvent to the sample were tested: 1:1 and 1:2. As shown in Figure 4(c), the 1:1 ratio gave the best recovery for the seven compounds.

The number of replicates for the extraction step with ethyl acetate was adjusted. Figure 4(d) shows chromatograms of PCs after extraction 2 and 4 times, and as can be noticed, the highest recovery was achieved. This may be attributed to the loss of some PCs upon employing more extraction steps.

Considering all the tested optimizations, the optimal extraction conditions for the PCs from OMW include LLE with the preliminary treatment of the sample using NaOH and hexane. This is followed by extraction with ethyl acetate in a ratio of 1:1 with the sample, and the pH is adjusted to 2. These conditions, along with a full description of the extraction process, are shown in Figure 2.

The developed HPLC-DAD method was validated in terms of linearity, precision, accuracy, recovery, robustness, the limit of detection, and limit of quantitation. The results of the validation process are discussed in the following sections.

Table 2 shows the slope, intercept, and the correlation of determination R² for the seven target PCs. R² values ranged between 0.95 and 0.99 for all compounds. This indicates a good linear relationship between the area ratio and the concentration.

Table 2 represents the LOD and LOQ values of each PC with their retention times. For example, the LOD value of HVA was 0.042 mg/L, which is lower than the value obtained by Shmeis et al. (2021) (1.57 mg/L) using HPLC-UV.

The instrument precision was evaluated by injecting a standard mixture of PCs six times at three concentration levels and calculating the RSD. The RSD values for all the target compounds ranged from 0.24 to 1.07%. This indicated good instrument precision where RSD values meet the accepted criteria (15%) for trace analyses related to the HPLC-DAD instrument (Gustavo González & Ángeles Herrador 2007).

The recovery outcomes are detailed in Table 2. It is notable that all recovery percentages fell within the range of 81.6–97.6%, which aligns with the acceptable bounds for trace analyses (70–120%) as suggested by Gustavo González & Ángeles Herrador (2007). This range surpasses the limits described by Rahmanian et al. (2014).

The results suggest that the method demonstrated robustness in the face of variations in both mobile phase flow rate and wavelength. This is evident from the fact that the relative standard deviation (RSD%) values remained within the acceptable range (Gustavo González & Ángeles Herrador 2007). In contrast, the method exhibited susceptibility to changes in the column temperature, as evidenced by a relatively high RSD% value. This highlights the importance of exercising caution when adjusting the column temperature during analysis.

The significance of this method lies in its applicability to any study focused on the removal of the seven PCs from OMW. Moreover, the method has the potential to evaluate the economic feasibility of extracting these PCs from OMW by leveraging information about their initial concentrations.

The olive cultivar types could also contribute to variations in the levels of PCs. However, it is important to note that the specific olive types collected from each region are unknown, and there is a possibility that the samples consist of a mixture of different olive varieties (Khdair et al. 2019).

Additionally, it was noted that HTy emerged as the most prevalent compound, with an average concentration of 99.5 mg/L in both Amman and Irbid samples, followed by tyrosol with an average value of 65.3 mg/L. This result is consistent with findings reported by Di Mauro et al. (2017), where HTy was identified as the most abundant compound. However, it is worth noting that the reported concentration in their study (545 mg/L) is higher than our findings. Meanwhile, in the Karak sample, the PCA was identified as the dominant compound.

Gallic acid was employed as a representative PC, and the TPC was quantified and expressed in mg/L of gallic acid equivalent. The TPC was determined utilizing the linear equation: y = 0.0018x − 0.0774, where y represents the absorbance measured at 760 nm, and x corresponds to the total phenolic content derived from the calibration curve, exhibiting a coefficient of determination (R²) of 0.997.

The results of TPC in each extract are tabulated in Table 3. A significant difference (p < 0.05) was observed in TPC amounts among the different samples. Among the examined OMW, Irbid OMW was found to be the highest TPC with an average value of 1,839 mg/L, followed by Amman with 1,239.1 mg/L, Karak with 748.1 mg/L, and least TPC in Al-Salt with an average value of 485.2 mg/L. However, these results were higher than the Σ7 PCs, as the tested samples are expected to contain PCs other than the target seven PCs such as Gentisic acid, 4-Coumaric acid, and Acetovanillone (Shmeis et al. 2021). Also, the richness of the samples in PCs has been proven by the results of GC − MS analysis and is indicated in Figure 8 and Table S1.

Comparisons with TPC in OMW from other countries revealed significant variations in results. In Greece, Sygouni et al. (2019) reported a maximum TPC value of 1,020 mg/L. Additionally, Baazaoui et al. (2023) obtained a TPC value of 8,300 mg/L, which is considerably higher than the findings in this study. This variability can be ascribed to distinctions in weather conditions, olive cultivar varieties, fruit ripeness, and the methodology employed for olive oil extraction (Khdair et al. 2019).

The computed percentages of scavenging activities are detailed in Table 3. The Al-Salt OMW sample exhibited the highest scavenging activity percentage. It is worth noting that although a portion of the % scavenging activity can be attributed to the presence of PCs (as reflected by the TPC values), the Al-Salt sample does not possess the highest TPC. This could be attributed to substances with antioxidant properties other than PC, such as β-carotene (Murillo-Cruz et al. 2021). The percentage of scavenging activity ranged from 93.8 to 95.8% in the samples. These results reveal relatively high levels of scavenging activity in comparison to findings in Germany and Greece, where the range was 56–66 and 34.2–59.8%, respectively (Lafka et al. 2011; Azaizeh et al. 2012).

Although the work presented in this study shares a common purpose with other reports, it distinguishes itself through several novel aspects. Firstly, it signifies the inaugural analysis of PCs in Jordan, emphasizing a unique exploration of this crucial aspect. The methodology employed demonstrates innovation by substituting acetonitrile with the more abundant methanol and incorporating NaOH in the extraction process. A distinctive feature of this study lies in the simultaneous application of liquid and gaseous chromatography, offering a comprehensive analytical approach. Notably, our results surpass previous studies, attesting the efficacy of our methodology. Furthermore, our research breaks new ground by providing, for the first time, data on total phenolic contents and antioxidant activity in treated wastewater from Jordan. Lastly, the study addresses a gap in the existing literature by conducting an HPLC-DAD analysis in OMW, contributing uniquely to this specific field of research.

The study's outcomes demonstrate the abundance of PCs in OMW, positioning it as a sustainable source with significant health and nutritional benefits. Consequently, future research should prioritize an economic feasibility study to explore the potential of upscaling the presented extraction method for generating commercial quantities of PCs. Given the established value of HTy, the primary phenolic component in our OMW samples (valued at $1,000–2,000 per gram for scientific/experimental purposes), we anticipate the feasibility of scaling up the extraction process. This transformation of what was once an environmental concern into a financially viable resource exemplifies the achievement of sustainability.

Moreover, the knowledge gained about PC concentrations in OMW holds paramount importance for developing future methods to treat OMW, enabling its safe reuse in irrigation or proper disposal. Subsequent studies could further expand on this research by exploring additional PCs through preparative HPLC and incorporating a larger sample size of OMW for a more comprehensive understanding.

In this study, we developed a novel, efficient, and cost-effective method for extracting and quantifying seven key PCs present in OMW. The method was modified to establish optimal conditions for extraction, separation, and analysis. Through a comprehensive validation process and adherence to international guidelines for method analysis, efficacy and success were demonstrated by the method. The developed method was then employed to assess PC levels in OMW in Jordan. Significant levels of PCs were observed, with HTy identified as the most abundant compound. All target compounds were found in all the samples, albeit at varying concentrations and distributions that could be attributed to differences among the samples in soil quality, climate, surrounding environment, type of olive fruit, and fruit ripeness. More careful sampling should be considered in the future to account for these variations. The total phenolic content and antioxidant activity were also evaluated and revealed consistently high values across all samples, reinforcing the substantial value of OMW. The economic potential of OMW as a source of PCs, boasting both health and economic benefits, is highlighted by this study. Additionally, a database for selecting the proper treatment method for OMW is provided.

The authors would like to acknowledge the financial support of Al Balqa Applied University.

This work received funds from Al Balqa Applied University. The fund code is DSR-2020#320.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.