Olive oil mill wastewaters before and after treatment: a critical review from the ecotoxicological...

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Ecotoxicology ISSN 0963-9292Volume 21Number 2 Ecotoxicology (2012) 21:615-629DOI 10.1007/s10646-011-0806-y

Olive oil mill wastewaters before andafter treatment: a critical review from theecotoxicological point of view

Celine I. L. Justino, Ruth Pereira, AnaC. Freitas, Teresa A. P. Rocha-Santos,Teresa S. L. Panteleitchouk & ArmandoC. Duarte

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Olive oil mill wastewaters before and after treatment: a criticalreview from the ecotoxicological point of view

Celine I. L. Justino • Ruth Pereira •

Ana C. Freitas • Teresa A. P. Rocha-Santos •

Teresa S. L. Panteleitchouk • Armando C. Duarte

Accepted: 27 September 2011 / Published online: 1 November 2011

� Springer Science+Business Media, LLC 2011

Abstract The olive oil mill wastewater (OMW) is a

problematic and polluting effluent which may degrade the

soil and water quality, with critical negative impacts on

ecosystems functions and services provided. The main

purpose of this review paper is presenting the state of the

art of OMW treatments focusing on their efficiency to

reduce OMW toxicity, and emphasizing the role of eco-

toxicological tests on the evaluation of such efficiency

before the up-scale of treatment methodologies being

considered. In the majority of research works, the reduction

of OMW toxicity is related to the degradation of phenolic

compounds (considered as the main responsible for the

toxic effects of OMW on seed germination, on bacteria,

and on different species of soil and aquatic invertebrates)

or the decrease of chemical oxygen demand content, which

is not scientifically sound. Batteries of ecotoxicological

tests are not applied before and after OMW treatments as

they should be, thus leading to knowledge gaps in terms of

accurate and real assessment of OMW toxicity. Although

the toxicity of OMW is usually high, the evaluation of

effects on sub-lethal endpoints, on individual and multi-

species test systems, are currently lacking, and the real

impacts yielded by its dilution, in freshwater trophic chains

of receiving systems can not be assessed. As far as the

terrestrial compartment is considered, ecotoxicological

data available include tests only with plants and the eval-

uation of soil microbial parameters, reflecting concerns

with the impacts on crops when using OMW for irrigation

purposes. The evaluation of its ecotoxicity to other edaphic

species were not performed giving rise to a completely lack

of knowledge about the consequences of such practice on

other soil functions. OMW production is a great environ-

mental problem in Mediterranean countries; hence, engi-

neers, chemists and ecotoxicologists should face this

problem together to find an ecologically friend solution.

Keywords Biological and chemical treatments �Ecotoxicological evaluation � Olive oil mill wastewater

(OMW) � Soil and freshwater receiving systems

Introduction

According to recent statistics of the International Olive Oil

Council (November 2010), the major world producers of

olive oil are in the EU (74.3%), accounting for 46.2% in

Spain, 15.2% in Italy, 10.6% in Greece, and 1.9% in

Portugal. Outside the EU, yet in the Mediterranean area,

the relevant olive oil producers are: Morocco (5.9%), Syria

and Tunisia (each 5.0%), and Turkey (4.9%). The majority

of the world consumption of olive oil is also in the Med-

iterranean area: Italy (23.5%), Spain (19.1%) and Greece

(7.8%). The USA, with 9.0% of the total world olive oil

consumption, plays also an important role in olive oil

business. It is generally accepted that olive oil consumption

brings benefits to human health such as, reduction of

risk factors of coronary heart disease, prevention of sev-

eral types of cancers, and modifications of immune and

C. I. L. Justino (&) � A. C. Freitas � T. A. P. Rocha-Santos �A. C. Duarte

Department of Chemistry and CESAM, University of Aveiro,

3810-193 Aveiro, Portugal

e-mail: [email protected]

R. Pereira

Department of Biology and CESAM, University of Aveiro,

3810-193 Aveiro, Portugal

A. C. Freitas � T. A. P. Rocha-Santos � T. S. L. Panteleitchouk

ISEIT/Viseu, Instituto Piaget, Estrada do Alto do Gaio,

Galifonge, 3515-776 Lordosa, Viseu, Portugal

123

Ecotoxicology (2012) 21:615–629

DOI 10.1007/s10646-011-0806-y

Author's personal copy

inflammatory responses (Tuck and Hayball 2002; Visioli

and Galli 2002; Zafra et al. 2006; Bendini et al. 2007).

According to Tuck and Hayball (2002) the occurrence of

such health problems are considerably lower in Italy,

Spain, and Greece which may be related to the consump-

tion of olive oil as the main source of fat in Mediterranean

diet.

Despite the health and economical benefits of the pro-

duction and consumption of OMW, producing countries

have to deal with two serious environmental issues: the

high water consumption, and the extremely high toxicity of

resulting effluents. The traditional process of olive oil

extraction by batches has been discontinued for some time,

and nowadays the extraction of olive oil can be obtained by

one of two different continuous processes: the two-phase or

the three-phase systems. The two-phase and the three-

phase extraction processes generate two main wastes: a

brownish-black liquid effluent called olive oil mill waste-

water (OMW), and a solid waste, generally called pomace

or husk (Obied et al. 2005; Asses et al. 2009).

OMWs have been spread, without any valorisation

treatment, into soil or nearby streams and rivers, for many

years, being very harmful to soil microflora, plants and

freshwater species (Aggelis et al. 2003; Roig et al. 2006).

Yearly, large volumes of OMW are produced due to the

extraction of olive oil. For example, in the Mediterranean

area, an annual production of OMWs between 7 9 106 and

3 9 107 m3 of OMWs has been recorded (Kavvadias et al.

2010). The management and treatment of OMW have been

considered as the main goal of the majority of research

studies in this field. In order to minimize these environ-

mental impacts, olive mills have been obliged to treat or

even reduce substantially their wastes. However, the

complex physico-chemical composition of OMW repre-

sents a technical difficulty to achieve efficient treatments,

since its recalcitrant compounds-rich composition is highly

variable, depending on many factors such as the type of

olive oil extraction process, the local and seasonal nature of

oil production, the climatic conditions and cultivation

methods (Roig et al. 2006; McNamara et al. 2008).

Some physico-chemical (e.g., coagulation and electro-

chemical oxidation) and biological treatments (with aero-

bic and anaerobic microorganisms) have been applied to

OMW. Such processes have proved to be highly effective

for the reduction of OMW colour, chemical oxygen

demand (COD) and phenolic content. Several studies

(Sayadi et al. 2000; Casa et al. 2003; Fiorentino et al. 2003;

Isidori et al. 2005; Celano et al. 2008; Mekki et al. 2008;

Kallel et al. 2009) emphasized the roles of phenolic and

polyphenolic compounds (molecular mass up to 60 kDa) in

the toxicity of OMWs towards microorganisms and plants.

However, and despite the degradation of most of these

compounds (Kallel et al. 2009), with some of the treatment

methodologies proposed, there are few studies (e.g.,

Aggelis et al. 2003; Andreozzi et al. 2008; Justino et al.

2009) reporting the application of ecotoxicological tests on

OMW before and after such treatments to obtain a real

evaluation of its effectiveness in terms of toxicity reduc-

tion. Then, the objective of this review is to compile and

make a critical review of the relevant information to

demonstrate that a treatment methodology can not be

chosen based only on its efficiency to remove COD and

colour, and that is important to focus our attention in this

research topic, which represents a great environmental

problem for several Mediterranean countries.

Physico-chemical composition and valorisation

of olive oil mill wastewaters

According to Asses et al. (2009) the average composition

of OMW is 83–96% of water, 3.5–15% of organic matter

and 0.5–2% of mineral salts. In addition, the typical colour

of OMW has been related to the presence of polymeric

phenolic compounds (corresponding to 10% of the organic

matter) that display a lignin-like structure constituting the

most recalcitrant fraction of this effluent (Tsioulpas et al.

2002). Table 1 presents values for the main parameters

assessed in the characterization of different OMWs

according to their origin and olive oil extraction processes.

It can be observed the presence of large amounts of phenols

and suspended solids, as well as acidity which contribute

for characterizing OMWs as a recalcitrant effluent.

Alburquerque et al. (2004) and Crognale et al. (2006) also

recorded a high organic content constituted by long-chain

fatty acids, lipids, simple and complex sugars and proteins,

and high content of inorganic salts, mainly potassium, but

also calcium, sodium, magnesium, and ferrous salts. The

amount of phenols in OMWs depends on the olive fruit

cultivar and maturity, climatic conditions, storage time as

well as the extraction process (Allouche et al. 2004; Roig

et al. 2006; Kallel et al. 2009). According to Rodis et al.

(2002) only 2% of the total phenolic content of the olive

fruit remains in the oil phase, while the greatest percentage

is lost in the OMW (approximately 53%) which is in

agreement with studies of McNamara et al. (2008) where

they found that phenols present in olive stone and pulp tend

to be more soluble in the water phase than in oil.

The quantification of total phenols in OMW is usually

made by Folin-Ciocalteu procedure (Bouaziz et al. 2005).

However the separation of individual phenolic compounds

by either gas or liquid chromatography has allowed

understanding their proportions and their role in the com-

position of OMW (Justino et al. 2010). According to

Mantzavinos and Kalogerakis (2005) the phenolic com-

pounds commonly found in OMWs can be divided into

616 C. I. L. Justino et al.

123


three major families, as shown in Fig. 1: the cinnamic acid

derivatives, the benzoic acid derivatives, and tyrosol

derivatives. The concentration of such phenolic compounds

varies in a wide range of values as verified by Asses et al.

(2009): 3,451 mg ml-1 for hydroxytyrosol, 439 mg ml-1

for tyrosol, 191 mg ml-1 for vanillic acid, 67 mg ml-1 for

2-(4-hydroxyphenyl) acetic acid, 3 mg ml-1 for caffeic

acid, 2 mg ml-1 for ferulic acid, and 1 mg ml-1 for

p-coumaric acid. Lesage-Meessen et al. (2001) showed that

the phenolic profiles (caffeic acid, hydroxytyrosol, ferulic

acid, tyrosol, p-coumaric acid, vanillic acid) identified by

HPLC coupled to a UV detector (280 nm) were similar for

OMW from both oil extraction processes (two-phase or

three-phase mills), but the contents of individual phenols

with the exception of vanillic acid, were higher for the two-

phase system which was also confirmed by Alburquerque

et al. (2004). However, and since published literature show

that OMW characterization has not been based on a stan-

dard evaluation concerning chemical parameters and ana-

lytical procedures used, this leads to some difficulties in

comparing the chemical composition of OMWs from

different regional origins and production processes and to

relate their composition with their toxicity. The absence of

such standardisation will also impair the establishment of

legal limits for discharges of OMWs in receiving systems.

Despite their beneficial effects, mainly as antioxidant

agents (Tuck and Hayball 2002; Bendini et al. 2007;

Asses et al. 2009), phenolic compounds have been pointed

as responsible for the high toxicity of OMWs. Such

negative effects are mainly due to: (1) the ability of some

of these phenolic compounds to undergo auto-oxidation

with the subsequent generation of semiquinone and reac-

tive oxygen species (e.g., superoxide and hydrogen per-

oxide); (2) their role as uncoupling agents breaking the

linkage between the respiratory chain and the mythoc-

ondrial phosphorylation system; and (3) unspecific polar

narcosis (Aptula et al. 2002; Ren and Frymier 2002).

Furthermore, and despite the scarce knowledge about the

toxicity of phenolic compound mixtures, like those pres-

ent in OMW, Chen et al. (2010) have shown that syner-

gisms between some phenolic compounds, could be also

responsible by their high toxicity.

Table 1 General physical and chemical characterization of OMW from different regional origins and olive oil extraction processes

OMW origin Parameters References

Extraction

process

Local of

extraction

pH BOD5c (g l-1) CODd (g l-1) TSSe (g l-1) TPf (g l-1)

DTPa Morocco 5.2 – 124 92.4 8.2 Fadil et al. (2003)

Morocco 4.1 3.4 255 25.0 6.3 El Hadrami et al. (2004)

Greece 5.3 – 61.1 36.7 3.5 Ginos et al. (2006)

3-Phase system Greece 5.4 47.8 105 46.1 0.01 Fountoulakis et al. (2002)

Greece 5.3 50.0 140 95.0 3.4 Tsioulpas et al. (2002)

Italy 5.3 – 38.0 13.0 3.9 Celano et al. (2008)

Tunisia 4.8–5.2 – 9.3–9.7 14.4–15.3 – Asses et al. (2009)

Greece 5.1 – 123 32.7 6.2 Blika et al. (2009)

Portugal 3.9–4.1 – 21.8–45.0 – 0.1–0.4 Justino et al. (2009)

Spain 5.0 – 90.0 – 0.7 Martinez-Garcia et al. (2007)

N.I.b Tunisia 4.9–5.4 – 120–160 15.0–25.3 – Assas et al. (2002)

Morocco 4.2 – 50.0 4.00 12 Kissi et al. (2001)

Morocco 4.8–5.2 – 77–87 – 2.9–3.5 Aissam et al. (2007)

Morocco 4.8 – 135 – 1.0 El Hajjouji et al. (2007)

Tunisia 5.2 12.5 36.9 4.50 – Khoufi et al. (2007)

Tunisia 4.6–5.0 15.6–18.4 54.6–62.5 38.6–43.8 – Mekki et al. (2008)

Turkey 4.5–5.2 50–100 80.0–200 20.0–120 2–15 Mert et al. (2010)

a DTP Discontinuous traditional processb N.I. Not identifiedc BOD5 Biological oxygen demandd COD Chemical oxygen demande TSS Total suspended solidsf TP Total phenols

Olive oil mill wastewaters before and after treatment 617

123


Olive oil mill wastewaters ecotoxicity to aquatic and soil

organisms

There is a wide variety of standard ecotoxicological tests

with soil and aquatic species which can be used for eval-

uating the toxicity of wastewaters. In fact, different groups

of organisms such as bacteria, crustacean and plants have

been used to assess the toxicity of OMW. Table 2 lists

some of these tests commonly applied to OMW as well as

ecotoxicological data for the endpoints evaluated. Never-

theless, acute toxicity tests with crustaceans (e.g., Daphnia

magna and Brachionus) and with the bacteria Vibrio

fischeri have been mainly used, since they are considered

as efficient ecotoxicological tests for providing a rapid

assessment of OMW toxicity. In fact, these assays have a

short duration and are quite simple to perform, even

without expertise in ecotoxicology. Additionally, some of

them are available on kits which also make the mainte-

nance of laboratorial cultures of test organisms not neces-

sary. This has probably contributed for the lack of current

applications of a battery of ecotoxicological assays with

species from different trophic levels and with different

Cinnamic acid and derivatives

(E)-3-phenylprop-2-enoic acid (148.17)

C9H8O2

3-(4-hydroxyphenyl)prop-2-

enoic acid (164.16)

p-Coumaric acid

C9H8O3

3-(3,4-dihydroxyphenyl)prop-

2-enoic acid (180.16)

Caffeic acid

C9H8O4

(E)-3-(4-hydroxy-3-methoxyphenyl)prop-

2-enoic acid (194.18)

Ferulic acid

C10H10O4

Benzoic acid and derivatives

Benzoic acid (122.12)C7H6O2

4-hydroxy-3-methoxybenzoic acid

(168.15)

Vanillic acid

C8H8O4

3,4,5-trihydroxybenzoic acid

(170.12)

Gallic acid

C7H6O5

3,4-dimethoxybenzoic acid

(182.17)

Veratric acid

C9H10O4

4-hydroxy-3,5-dimethoxybenzoic

acid (198.17)

Syringic acid

C9H10O5

3,4-dihydroxybenzoic acid

(154.12)

Protocatechuic acid

C7H6O4

4-hydroxybenzoic acid (138.12)

Hydroxybenzoic acid

C7H6O3

Tyrosol and derivatives

4-(2-hydroxyethyl)phenol (138.16)

C8H10O2

4-(2-hydroxyethyl)-1,2-benzenediol (154.16)

Hydroxytyrosol

C8H10O3

2-(4-hydroxyphenyl)acetic acid (152.15)

4-Hydroxyphenylacetic acid

C8H8O3

Fig. 1 Most abundant phenolic

compounds detected in OMW:

IUPAC name (molecular

weight), chemical formula and

structure


123


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123


sensitivities, and including the evaluation of sub-lethal

endpoints, leading to a better understanding of OMW

impacts on environment. Despite being concerned with the

ecotoxicological evaluation of OMWs, but following in an

opposite direction, Paixao et al. (1999) conduct an acute

ecotoxicological evaluation with three different non-

related species D. magna, Thamnocephalus platyurus and

V. fischeri, from marine and freshwater systems, aimed at

evaluating their sensitivity to the OMW effluent, and to

select one for current application in the ecotoxicological

evaluation of this effluent. V. fischeri test was found to be

highly sensitive (but without correlation with OMWs

physical and chemical parameters), useful for a daily rou-

tine evaluation of the toxicity of OMWs, but enable to

discriminate different OMW samples and to predict the

risks to aquatic ecosystems. In the same direction of the

previous study, Mekki et al. (2008) also verified the high

toxicity of raw OMW (COD = 58.5 g l-1; phenolic con-

tent = 9.1 g l-1) which has completely inhibited the

luminescence of V. fischeri being such inhibitory effect still

high (96%) even after a great dilution (24 times) of the

OMW. Andreozzi et al. (2008) have chosen the green

freshwater algae Pseudokirchneriella subcapitata to assess

the toxicity of a centrifuged OMW, verifying that even

after a dilution of 1:640 (v/v), an inhibition of about 50% in

growth occurred. All the studies reported are enable to

relate the data gathered with their ecological consequences,

although direct and serious indirect effects would be

expected, as those provoked by a depression of the phy-

toplankton community in the freshwater ecosystems.

As far as the soil compartment is considered, Mekki

et al. (2006) and Saadi et al. (2007) have demonstrated that

the irrigation of agricultural lands with a moderate quantity

of OMWs can provide a fertilizing effect. Then, the use of

OMW as a liquid fertiliser, in controlled doses, also pro-

vides an inexpensive option for the valorisation for OMWs

(Yaakoubi et al. 2010), and has been applied in many

Mediterranean countries. However, data available are

mainly focused on the evaluation of the toxicity of OMW

to plants and soil microflora, since main concerns are

related with predicting and preventing disturbances in crop

production, and no data exist about the impact on other

edaphic species (e.g., invertebrates), allowing an evalua-

tion of the long-term risks of such practices on soil

ecosystems.

Concerning the microbial community, several studies

have already addressed the impacts in its structure and

function derived from the use of OMW as irrigation water

(Kotsou et al. 2004; Saadi et al. 2007; Karpouzas et al.

2010). Mekki et al. (2006) reported a strong increase in the

carbon content of soil (17 mg g-1) in soil amended with

200 m3 h-1 OMW, in parallel with a low specific soil

respiration. Such parameter expressed by C–CO2/Ctot has

decreased from 1.7 in the control soil to 0.5 in soil

amended with 100 m3 ha-1 of OMW. This decrease

has occurred in parallel with a decrease in CFU number

for nitrifiers and an increase for fungi, actinomycetes

and spore-forming bacteria. In soils amended with

200 m3 ha-1 of OMW a general decrease in CFU numbers

was recorded, suggesting a strong impact in the soil

microbial community. Despite using a culture dependent-

method, which do not reveal the overall soil biodiversity,

and without a clear indication for how long soil plots were

irrigated with OMW, this study was able to demonstrate the

impact on the major groups of soil microorganisms. These

observations were coincident with those from Karpouzas

et al. (2010) which used a PCR-DGGE method to follow

changes in profiles of actinobacteria and ammonia oxidiz-

ing bacteria of soils regularly irrigated with OMW highly

diluted (2 and 4% v/v) for 49 days. In parallel with a sig-

nificant reduction in N–NO3, and an increase in soluble

phenolic compounds, significant soil and dose dependent

changes were observed in ammonia-oxidizing bacteria

profiles, with shifts in dominant groups of Nitrosospira

genus. Nevertheless, evidences of changes in soil functions

caused by observed changes in the soil microbial com-

munity structure were not provided by both studies. Such

changes were shown by Piotrowska et al. (2011) occurring

in the former days after amendment with OMW has started,

even though the initial status is re-established after a while.

Hence, the addition of OMW to soil has caused contra-

dictory effects due, on one hand, to the addition of an

available source of C, and on other hand to the addition of

inhibitory compounds such as phenols. Hence, a decrease

in the qCO2 quotient, despite an increase in soil microbial

biomass C and N, reflects an impact not in the soil

microbial community biomass but in its ability to miner-

alize organic matter which was observed in parallel with

the inhibition of the activity of urease and glucosidase

enzymes mainly during the former 14 days of soil

amendment with 80 m3 ha-1 of OMW. Despite the evi-

dences of changes in soil microbial community structure

and functions, the differences in experimental designs in

terms of temporal evaluations, volumes and dilutions of

OMW used for irrigation, test soils and treatment extension

does not allow to establish any correlations, and sometimes

contradictory results may arise in different studies.

The phytotoxic effects of OMW seemed to be less

contradictory since several authors have shown that the

high loads of phenolic compounds both in raw and diluted

OMWs may cause toxic effects at different growth stages

and on seed germination of several crops, such as maize,

radish, cucumber, lettuce, wheat, chickpea, tomato durum

wheat, and English cress (Casa et al. 2003; El Hadrami

et al. 2004; Ben Sassi et al. 2006; Andreozzi et al. 2008).

The phytotoxicity of OMW effluent is high even after a


123


dilution of 1:8 (v/v) which can cause an inhibition of 55, 60

and 98% in the germination index of radish, cucumber and

lettuce species (Andreozzi et al. 2008). This conclusion is

further enhanced by the fact that seed germination in crop

species has been pointed out as the less sensitive parameter

to different types of soil contamination, due to the pro-

tection given to the embryos by the seed coat. However,

information is lacking about the effects of OMW on other

endpoints in crop standard species (growth above soil, or

growth of roots). Additionally, no information is available

about the toxicity of soils irrigated with OMW, on edaphic

invertebrate species, like those used in standard ecotoxi-

cological assays, or in the overall biodiversity of the

edaphic community of these soils.

Olive oil mill wastewaters cytotoxicity and genotoxicity

Data regarding the evaluation of the effects of OMW on

sub-lethal endpoints is scarce, being available mainly at

sub-individual levels resulting from the evaluation of

molecular biomarkers (Karaouzas et al. 2010). Hence, the

very recent study from Danellakis et al. (2011) is the only

one reporting the application of a battery of biomarkers to

assess the cytotoxic effects caused by OMW in an aquatic

invertebrate species (Mytillus galloprovinciallis). Despite

the lower concentrations of the effluent (0.1 and 0.01% v/v)

tested, intentionally selected to represent the environmental

concentrations to which this coastal species could be

exposed in the field, a significant increase in the loss of

lysossomal membrane integrity on haemocytes was recor-

ded, for organisms exposed to both concentrations for

5 days, in parallel with a significant inhibition on the

activity of the acetilcholinesterase (AChE), an enzyme on

body tissues, a significant increase in the level of mal-

ondialdehyde (MDA), a by-product of lipid peroxidation,

and in the level of metallothionein (MT) both in haemo-

lymph and gills of the organisms. Phenols have been sug-

gested (Danellakis et al. 2011) as the main responsible by

such effects, followed by metals also found in high con-

centrations in the OMW tested.

The genotoxicity, i.e., the toxicity of any substance to

cell’s genetic material which provokes a loss of cell

integrity, was also detected after exposure to OMW. Vicia

faba micronucleus test has been used for detecting chro-

mosomal aberrations, like chromosomal breakage and

aneuploidy in organism cells. El Hajjouji et al. (2007) were

the first applying the detection of micronuclei in V. faba

root tips, showing that raw OMW was toxic at a concen-

tration of 10%, and higher concentrations were responsible

for root tips blackening and inhibition of mitosis. El

Hajjouji et al. (2007) also tested the genotoxicity of the six

major phenols of an OMW, 10% diluted (the highest

concentration allowing the identification of micronucleus),

in order to perceive which were the compounds responsible

by the genotoxicity of the effluent. Although hydroxy-

phenyl acetic acid and veratric acid were identified in

undiluted OMW at high concentrations, toxicity results

showed that only gallic acid and oleuropein induce a sig-

nificant increase in micronucleus frequency in V. faba.

Therefore, the authors have associated such phenols to the

genotoxicity of OMW.

The inhibition of DNA synthesis is another mechanism

which it is affected by OMW toxicity, leading to inter-

ruption of protein synthesis on root tips of Triticum

aestivum (Aybeke et al. 2008), which is probably related to

polyphenols content.

Once again, the study by Danellakis et al. (2011) is the

only one reporting genotoxic effects of OMW in an aquatic

animal species, describing significant increased levels in

nuclear abnormalities and in DNA damage assessed by the

micronucleus and comet assay respectively, in mussels

exposed to OMW extremely diluted (0.1 and 0.01% v/v).

From the above considerations, it can be concluded that

the OMW toxicity induces mutagenic, genotoxic and

cytotoxic effects at very low concentrations, reinforcing the

importance of integrating the evaluation of different

molecular biomarkers in standard ecotoxicological assays,

with aquatic and terrestrial invertebrate and vertebrate

species (e.g., fish), as they can reduce the likely underes-

timation of risks posed by these wastewaters and can

provide early-warning information about their impacts,

even at lower distances from their discharging points.

Furthermore, no data exists for individual sub-lethal end-

points (e.g., reproduction, growth), which is crucial to

assess risks to ecosystems, through direct and indirect

effects on trophic chains.

Ecotoxicological assays in the evaluation

of the efficiency of olive oil mill wastewaters treatment

processes

The successive optimizations of olive oil extraction pro-

cesses together with the global climatic changes in culti-

vars have impacted the olive oil composition along the

last decades, with consequent changes in the physico-

chemical characteristics of OMW (McNamara et al. 2008).

Successful degradation of organic compounds, reduction of

COD and/or decolourization of OMW have been obtained

by physico-chemical methods (e.g., ozonation, coagulation,

ultrafiltration, electrochemical oxidation) and biological

processes (aerobic or anaerobic biological treatments) as

reviewed by Mantzavinos and Kalogerakis (2005), Roig

et al. (2006), and McNamara et al. (2008). However, in

the great majority of the studies there are no records of


123


ecotoxicological tests for the evaluation of toxicity before

and after OMW treatment.

Those few works which have evaluated the effectiveness

of OMW treatments in terms of toxicity reduction to

environmental organisms, mostly aquatic organisms such

as D. magna (Aggelis et al. 2003), despite considerable

decreases in total or individual phenols, have recorded an

increased or an unchangeable toxicity after treatment

(Chatzisymeon et al. 2009a). Reasons pointed out for

explaining such results included: (1) the oxidation of

phenolic compounds with formation of even more toxic

by-products [e.g., formation of phenoxy radicals in treat-

ments involving laccases and peroxidases (Aggelis et al.

2003)]; and (2) the inhibition of the degradative enzyme

system of ligninolytic fungi, when biological treatment

with ligninolytic fungi was applied, due to the high content

of phenolic compounds present in OMW (Sayadi et al.

2000). In fact, and according to Aissam et al. (2007), the

toxicity of OMW for microorganisms and plants depends

on the phenolic polymerisation degree, and especially on

the monomeric phenols (Fiorentino et al. 2003; Mekki et al.

2008). Tables 3 and 4 summarize some of the works

published on the evaluation of the toxicity of OMW after

biological and physico-chemical treatments, respectively,

with most representative conclusions presented in follow-

ing sub-sections.

Biological processes

Biological processes are the most environmentally well-

suited and less expensive wastewater treatments. However,

the presence of some groups of inhibitory or toxic com-

pounds, such as polyphenols and lipids, make the OMW

not amenable for direct biological treatment. Our previous

work (Justino et al. 2009) was the first comparing the

efficiency of biological treatments when applied to OMWs

collected at two different periods: one immediately after

olive oil production, and the other 1 year after olive

oil production. Fungi strains (Trametes versicolor, Phan-

erochaete chrysosporium and Pleurotus sajor caju) were

applied for performing the degradation of organic com-

pounds in diluted OMW. As shown in Table 3, at the same

dilution (25% v/v), the reduction in total phenolic content

was lower in the 1 year’s old OMW (69 ± 17%) than in

the OMW collected immediately after the olive oil pro-

duction (82 ± 6%). Such results could be based on the

tendency of OMW phenols to auto-oxidise, during storage

on open tanks, with subsequent polymerization into high

molecular weight phenolic compounds which are difficult

to degrade in biological wastewater treatment systems

(Tsagaraki et al. 2007). The storage and evaporation in

open tanks is a strategy followed by many producers to

reduce the volume and to valorise OMW, through settle-

ment of suspended solids. In terms of toxicity, Justino

et al. (2009) observed that fungi were able to promote

a remarkable reduction (91%) on acute toxicity to

D. longispina especially in diluted OMW (50% v/v) with

P. sajor caju.

In turn, Aggelis et al. (2003) showed the ability of

selected P. ostreatus strains to remove phenolic com-

pounds (86%) from a diluted (50% v/v) and sterilized

OMW, used as a model for phenolic wastewater. The

toxicity of the studied wastewater on seeds of Lepidium

sativum decreased significantly after biological treatment.

Therefore, since different species have different sensitivi-

ties, ecotoxicological tests before and after the OMW

treatments should be applied using a wide variety of spe-

cies to obtain a real assessment of mitigation in terms of

the OMW impacts on ecosystems. On the other hand,

sterilized OMW should be considered only as a model for

phenolic wastewater since sterilization of OMW may cause

unpredictable physico-chemical modifications on several

compounds such as oxidation of phenolics and quinoid

compounds followed by precipitation. Therefore, any

methodology including sterilization of OMW is not suit-

able for application in wastewater treatment because it does

not represent the actual operating industrial conditions.

From the studies reported above, it could be highlighted

that despite concerns in making an ecotoxicological eval-

uation of treated effluents the few data provided is not

sufficient to make inferences about the efficiency of such

treatment in mitigating the hazardousness of OMW to

aquatic and terrestrial ecosystems. Additionally to the

possible reasons already discussed, the low volumes of

treated effluent obtained could be another justification,

since until now treatment experiments have been carried

out in laboratorial conditions. Hence the up-scale of these

experiments should be taken in consideration in future

studies.

Using a vertebrate model species, Peixoto et al. (2008)

evaluated the OMW toxicity on the mitochondrial bioen-

ergetics of rat liver cell before and after the OMW treat-

ment with the yeast species C. oleophila. They remarked

that the mitochondrial respiration was not affected when

cells were exposed to the biologically treated effluent,

while the raw OMW has caused a decrease on mitochon-

drial membrane potential, giving an idea about the effec-

tiveness of such OMW treatments in reducing toxicity.

Physico-chemical processes

Concerning the use of physico-chemical processes for

OMW treatment, advanced oxidation processes (AOPs) are

the most commonly applied (Mantzavinos and Kalogerakis


123


Ta

ble

3P

erce

nta

ges

of

CO

D,

ph

eno

lco

nte

nt

and

tox

icit

yre

du

ctio

ns

inO

MW

saf

ter

dif

fere

nt

bio

log

ical

trea

tmen

ts,

carr

ied

ou

tu

nd

erd

iffe

ren

tco

nd

itio

ns

Ty

pe

of

OM

WT

reat

men

tp

roce

ss

(wo

rkin

gex

per

imen

tal)

Bio

log

ical

trea

tmen

t

agen

t

CO

D

red

uct

ion

(%)

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eno

ls

red

uct

ion

(%)

To

xic

ity

red

uct

ion

ob

serv

ed

Ref

eren

ces

Dil

ute

dat

20

%(v

/v)

Fu

ng

iP

.o

stre

atu

sN

.D.c

88

d6

0%

gM

arti

ran

iet

al.

(19

96

)

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dil

ute

dF

un

gi

A.

terr

eus

74

94

d8

7%

hB

orj

aet

al.

(19

98

)

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can

did

um

63

66

d5

9%

h

Bac

teri

aA

.ch

roo

cocc

um

75

90

d7

9%

h

Un

dil

ute

dF

un

gi

P.

ost

rea

tus

52

62

d89

gK

issi

etal

.(2

00

1)

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chry

sosp

ori

um

77

82

d2

09

g

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dil

ute

dF

un

gi

P.

fla

vid

oa

lba

N.D

.5

2e

70

%h

Bla

nq

uez

etal

.(2

00

2)

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dil

ute

dY

east

s(7

day

s;2

8�C

)G

eotr

ich

um

sp.

11

±3

7±

6f

N.D

.F

adil

etal

.(2

00

3)

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ute

dat

50

%(v

/v)

32

±6

34

±6

fN

.D.

Dil

ute

dat

75

%(v

/v)

48

±4

33

±6

fN

.D.

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dil

ute

dY

east

s(2

day

s;3

0�C

)C

an

did

atr

op

ica

lis

18

±7

17

±3

fN

.D.

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dat

50

%(v

/v)

54

±6

58

±3

fN

.D.

Dil

ute

dat

75

%(v

/v)

47

±7

53

±1

fN

.D.

Un

dil

ute

dF

un

gi

(7d

ays;

28�C

)A

sper

gil

lus

sp.

12

±3

11

±1

fN

.D.

Dil

ute

dat

50

%(v

/v)

32

±6

48

±4

fN

.D.

Dil

ute

dat

75

%(v

/v)

40

±7

44

±6

fN

.D.

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dil

ute

dF

un

gi

P.

chry

sosp

ori

um

20

–5

0N

.D.

26

%h

Dh

ou

ibet

al.

(20

06

)

Fu

ng

i?

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aero

bic

dig

esti

on

P.

chry

sosp

ori

um

?B

acte

ria

65

N.D

.6

2%

h

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dil

ute

dF

un

gi

?A

nae

rob

icd

iges

tio

nP

.ch

ryso

spo

riu

m?

Bac

teri

a8

18

3d

38

%h

Mek

ki

etal

.(2

00

6)

Dil

ute

dat

25

%(v

/v)

Fu

ng

i(1

4d

ays;

25

�C)

P.

sajo

rca

ju8

9N

.D.

4.6

9i

Fer

reir

aet

al.

(20

08)

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ost

rea

tus

32

N.D

.3

.39

i

Ste

rili

zed

and

dil

ute

dat

20

%(v

/v)

Fu

ng

i(7

day

s;2

5�C

)A

sper

gil

lus

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vatu

s1

46

4d

N.D

.A

fify

etal

.(2

00

9)

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cor

stri

ctu

sha

gem

12

59

dN

.D.

Pen

icil

liu

mn

igri

can

s1

65

4d

N.D

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icil

liu

mci

lrea

viri

de

22

61

dN

.D.

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erg

illu

sw

enti

i6

86

7d

N.D

.

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icil

liu

mch

erm

esin

um

30

54

dN

.D.

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erg

illu

sn

iger

25

71

dN

.D.

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ost

rea

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75

dN

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chry

sosp

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um

77

0d

N.D

.


123


Ta

ble

3co

nti

nu

ed

Ty

pe

of

OM

WT

reat

men

tp

roce

ss

(wo

rkin

gex

per

imen

tal)

Bio

log

ical

trea

tmen

t

agen

t

CO

D

red

uct

ion

(%)

Ph

eno

ls

red

uct

ion

(%)

To

xic

ity

red

uct

ion

ob

serv

ed

Ref

eren

ces

Un

dil

ute

dO

MW

-Oa

Fu

ng

i(2

0d

ays;

25

–3

0�C

;af

ter

4d

ays

of

ph

oto

-Fen

ton

trea

tmen

t)

P.

chry

sosp

ori

um

67

1d

No

nei

Just

ino

etal

.(2

00

9)

T.

vers

ico

lor

57

4d

No

nei

P.

sajo

rca

ju1

47

0d

No

nei

Un

dil

ute

dO

MW

-Nb

P.

chry

sosp

ori

um

No

ne

62

dN

on

ei

T.

vers

ico

lor

No

ne

61

dN

on

ei

P.

sajo

rca

juN

on

e4

8d

No

nei

Dil

ute

dO

MW

-Oat

25

%

(v/v

)

Fu

ng

i(2

1d

ays;

25

–3

0�C

)P

.ch

ryso

spo

riu

m5

78

0d

39

i

T.

vers

ico

lor

62

87

dN

on

ei

P.

sajo

rca

ju5

18

1d

39

i

Dil

ute

dO

MW

-Nat

50

%

(v/v

)

Fu

ng

i(2

1d

ays;

25

–3

0�C

)P

.ch

ryso

spo

riu

m6

99

0d

1.7

9i

T.

vers

ico

lor

72

94

d2

.19

i

P.

sajo

rca

ju7

29

1d

5.2

9i

aO

MW

-OO

MW

coll

ecte

d1

yea

raf

ter

oli

ve

oil

pro

du

ctio

nb

OM

W-N

OM

Wco

llec

ted

imm

edia

tely

afte

rth

eo

liv

eo

ilp

rod

uct

ion

cN

.D.

No

td

eter

min

edd

To

tal

ph

eno

lse

Aro

mat

icco

mp

ou

nd

sf

Po

lyp

hen

ols

gD

eter

min

edw

ith

Ba

cill

us

cere

us

hD

eter

min

edw

ith

V.

fisc

her

ite

sti

Det

erm

ined

wit

hD

.lo

ng

isp

ina

test


123


2005; Canizares et al. 2007; Stasinakis 2008; Justino et al.

2009; Mert et al. 2010). These methods employ strong

oxidant agents (e.g., ozone, hydrogen peroxide together

with UV radiation, and Fenton reagent) with the formation

of very reactive oxidizing free radicals (OH•), especially

hydroxyl radicals which contribute to the partial or total

removal of the OMW organic content.

Among AOPs, Fenton process is the most cost effective

and easy to apply when a high reduction of COD is

required (Lee and Shoda 2008) and it can be easily scaled

up in storage ponds, under solar radiation (Andreozzi et al.

2008). However it is expected that this type of treatment

may lead to an increase of effluent toxicity, as shown in

Table 4, due to the formation of free radicals. For example,

Table 4 Percentages of COD, phenol content and toxicity reductions in OMWs after different physic-chemical treatments, carried out under

different conditions

Type of OMW Treatment process (working experimental) COD

reduction

(%)

Phenols

reduction

(%)

Toxicity

reduction

observed

References

Undiluted FeCl3 (5 h) N.D.c 59d 82%g Fiorentino et al. (2004)

FeCl3 ? Activated sludge (4 days) 63 60d Up to 30%g

Undiluted Lime precipitation (5 h; 40 kg

of Ca(OH)2/m3 OMW)

39 71d N.D. El-Shafey et al. (2005)

Lime precipitation ? Filtration

using activated carbon

80 99d N.D.

Undiluted Chemical (pH 2; H2SO4) 38 23d N.D. Kestioglu et al. (2005)

Chemical (pH 8; H2SO4 ? FeCl3�6H2O) 95 90d N.D.

Undiluted Electrochemical oxidation with Ti–Pt

anode (2 h; 25–28�C)

40 N.D. N.D. Belaid et al. (2006)

Diluted at 50% (v/v) 65 98d N.D.

Diluted at 25% (v/v) Lime ? cationic poly-electrolytes

(200–300 mg/l)

10–40 30–80d Noneh Ginos et al. (2006)

Undiluted Electro-coagulation (Ti–Ta–Pt–Ir

anode; 8 h; 3% NaCl)

71 N.D. 2.79i Giannis et al. (2007)

8.89j

Undiluted Electro-coagulation 34 53e 34%j Khoufi et al. (2007)

Undiluted Electro-coagulation ? anaerobic digestion 91 92f 35%j Mekki et al. (2008)

Undiluted and filtered Electrochemical oxidation

(boron-doped diamond electrodes)

19 36d Nonej Chatzisymeon et al. (2009a)

Undiluted Ozonation (20�C; pH 5; 40 min) 48 80d N.D. Chedeville et al. (2009)

Undiluted OMW-Oa Photo-Fenton (4 days) 53 81d 1.49k Justino et al. (2009)

Undiluted OMW-Nb 76 92d Nonek

Undiluted Wet hydrogen peroxide catalytic oxidation

(Al–Fe pillared clay ? H2O2 ? UV radiation)

25 41d N.D. Azabou et al. (2010)

Wet hydrogen peroxide catalytic

oxidation (Al–Fe pillared clay ? H2O2)

70j

Undiluted Chemical (pH 2; 4 h; H2SO4) 46 37d 2.39l Mert et al. (2010)

Chemical (pH 10; 4 h; H2SO4 ? FeSO4�7H2O) 90 91d 1.69l

Chemical (pH 9; 4 h; H2SO4 ? FeCl3�6H2O) 93 95d N.D.

a OMW-O OMW collected 1 year after olive oil productionb OMW-N OMW collected immediately after the olive oil productionc N.D. Not determinedd Total phenolse Orto-diphenolsf Phenolic monomersg Determined with Brachionus calyciflorush Determined with seeds germination testi Determined with D. magnaj Determined with V. fischeri testk Determined with D. longispina testl Determined with activated sludge respiration inhibition


123


and although Justino et al. (2009) observed reductions of

COD and total phenolic contents for all OMW samples

after photo-Fenton oxidation, they were not followed by a

decrease in toxicity for D. longispina.

Electrochemical methods using oxidation over many

traditional anodes such as Ti–RuO2 (Panizza and Cerisola

2006; Un et al. 2008), Ti–IrO2 (Chatzisymeon et al.

2009b), Ti–Ta–Pt–Ir (Gotsi et al. 2005; Giannis et al.

2007), Fe and Al electrodes (Adhoum and Monser 2004;

Inan et al. 2004; Un et al. 2006; Khoufi et al. 2007) have

also been considered for the removal of organic com-

pounds from OMW. In some cases, the chemical oxidation

step has led to the formation of intermediates that can be

even more recalcitrant or more toxic than the originally

ones present in the effluent (Gotsi et al. 2005).

Ozone is also used as a powerful oxidizing reagent, and

despite its sparing solubility in water (Mantzavinos and

Kalogerakis 2005), some recent studies have reinforced

ozonation as a suitable OMW treatment process. However,

the ozonation efficiency in terms of toxicity reduction has

not been proved yet. In fact, as Andreozzi et al. (2008)

observed, despite ozonation for 120 min has caused a

reduction of 31.5% in COD levels and of 63.6% in poly-

phenols content of a centrifuged OMW, such was not fol-

lowed by a reduction of its phytotoxicity. Only after an

ozonation period of 2 h it was possible to observe a

reduction of about 50 and 20% in the inhibitory effect on

the germination index of three crop species [radish

(Raphanus sativus), cucumber (Cucumis sativus), and let-

tuce (Lactuca sativa)] and on the growth of the green algae

Pseudokirchneriella subcapitata, respectively. However,

the efficiency of this treatment still is questionable, since

dilutions of 1:8 and 1:80 (v/v) are required for adeuqate

toxicity reduction. In their study Andreozzi et al. (2008)

were also able to relate the phytotoxicity of the effluent

treated by ozonation with low molecular weight polyphe-

nols, since prior to the ecotoxicological evaluation, bovine

laccase was added to the treated effluent for removal of

oxygen free radicals. These results were in agreement with

Fiorentino et al. (2003) who concluded that several indi-

vidual phenolic compounds had a strong toxic potential on

different trophic levels in the aquatic system, evaluat-

ing the OMW toxicity on four organisms: the algae

P. subcapitata, the rotifer Brachionus calyciflorus, and two

crustaceans, the cladoceran D. magna and the anostracan

T. platyurus. Karageorgos et al. (2006) also observed that

in most of their experiments the toxicity values decrease

with the decrease of organic content, but an increase of

residual phytoxicity was observed possibly due to the

production of short-chain fatty acids (C8–C9) during the

oxidative process of ozonation.

From the above considerations, it can be concluded that

toxicity must be evaluated by various toxicity tests and

such evaluation is required before and after the OMW

treatment, or if possible during the treatment process, in

order to conclude about the effectiveness of the OMW

treatment in terms of simultaneous effects on organic

content and on toxicity.

Final remarks are related to the application of combined

physico-chemical and biological treatments as first stages

on treatment process, enabling an additional decrease of

organic load and OMW toxicity, as shown in Tables 3 and

4. For example, Justino et al. (2009) tested the efficiency of

two treatments, involving fungi and photo-Fenton oxida-

tion sequentially applied to OMW, in terms of organic

compounds degradation and toxicity mitigation. Although

the photo-Fenton oxidation applied after a biological

treatment seemed to be an interesting option in terms of

reduction of COD (53–76%), total phenolic content

(81–92%), and organic content (100%), the toxicity of the

effluent to D. longispina (1.49) remained very high. The

alternative sequence of treatments, namely photo-Fenton

oxidation followed by biological treatment showed great

potential, with additional reductions in COD (5–14%), and

total phenolic content (70–74%), but without toxicity

reduction.

Conclusions and future directions

The toxicity is an extremely important parameter regarding

the characterization of OMW, and it should be taken into

account before discharging the OMW into freshwater

streams or into other environmental compartments. OMW

has a variable composition with a high content in compo-

nents of very low biodegradability (e.g., long-chain fatty

acids, lipids and simple and complex sugars), and then the

magnitude of its toxicity is also affected. On the other

hand, as the chemical composition of OMW varies with

several factors such as the type of olive oil extraction

process, the local and seasonal nature of oil production, the

climatic conditions and cultivation methods as well as the

type of olive, the determination of toxicity should also

consider this variability for different wastewater origins.

From the ecotoxicological point of view, both raw and

treated OMW should be evaluated through a battery of

freshwater species and edaphic species, from different

trophic levels and with different sensitivities to allow a

sound scientific evaluation of the risks posed to these

ecosystems. According to this review, it is clearly shown

that OMW are highly toxic not only to microorganisms, but

also to invertebrates with intermediary position on trophic

chains (e.g., D. magna, D. longispina, B. calyciflorus) as

well as to primary producers (plants and algae) affecting

the sustainability of receiving systems. Similar and sig-

nificant toxicant impacts were reported for crop plants and


123


soil microbial community. However, the evaluation of soil

toxicity on edaphic invertebrate species after the soil irri-

gation with OMW is lacking and it should be encouraged.

On the other hand, and currently, data for individual sub-

lethal endpoints on species from both compartments do not

exist, contributing as an important knowledge gap on the

assessment of risks of OMW to ecosystems. In the future,

ecotoxicological data obtained for OMWs using different

species should be used and integrated to derive legal limits

for regulating discharges of both treated and untreated

OMW into aquatic and soil systems.

A plethora of wastewater treatment processes have been

tested with success mainly for reduction of phenolic

compounds, which are the most difficult to remove from

OMW. However, in the majority of the studies no eco-

toxicological evaluation is reported, and the success of

OMW treatment is only based upon the reduction of colour,

COD, phenol content, etc. In some works, although the

phenolic content was reduced, the toxicity remained con-

stant or slightly increased, resulting on a wastewater with a

less organic content but more toxic than before treatment.

Acknowledgments This work was developed under the scope of the

FCT (Fundacao para a Ciencia e a Tecnologia, Portugal) research

grants (SFRH/BD/60429/2009 and SFRH/BPD/65410/2009) funded

by QREN-POPH co-financed by the European Social Fund and

Portuguese National Funds from MCTES, through FSE and POPH

funds (Programa Ciencia 2007) and through the bilateral cooperation

FCT/CNRST.

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