Accepted Manuscript

Enzymatic treatment and subsequent toxicity of organic micropollutants using

oxidoreductases - A review

Béla Varga, Viola Somogyi, Mónika Meiczinger, Nóra Kováts, Endre Domokos

PII: S0959-6526(19)30524-4
DOI: https://doi.org/10.1016/j.jclepro.2019.02.135
Reference: JCLP 15865

To appear in: Journal of Cleaner Production

Received Date: 5 February 2018

Revised Date: 9 February 2019
Accepted Date: 13 February 2019

Please cite this article as: Varga Bé, Somogyi V, Meiczinger Mó, Kováts Nó, Domokos E, Enzymatic
treatment and subsequent toxicity of organic micropollutants using oxidoreductases - A review, Journal
of Cleaner Production (2019), doi: https://doi.org/10.1016/j.jclepro.2019.02.135.

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 ACCEPTED MANUSCRIPT

Enzymatic treatment and subsequent

toxicity of organic micropollutants using
oxidoreductases - a review
Béla Varga1*; Viola Somogyi1; Mónika Meiczinger2; Nóra Kováts3; Endre Domokos1

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1
Institute of Environmental Engineering, University of Pannonia, Veszprém, Hungary
2
Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprém,
Hungary
3

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Department of Limnology, University of Pannonia, Veszprém, Hungary

Abstract

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Organic micropollutants like pharmaceuticals, personal care products, pesticides and industrial
chemicals have received an ever growing attention due to their adverse ecological effects in natural

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water bodies. A promising technology to remove the micropollutants from the wastewater is
biocatalytic treatment using oxidoreductase enzymes. In order to improve removal efficiency and
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feasibility, the focus has been on developing enzyme-mediator systems and immobilization
techniques. Nonetheless, since oxidoreductase enzymes often promote coupling reactions, enzymatic
reactions do not necessarily result in simpler compounds. Thus transformation products, which might
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be in equally or even more hazardous pollutants, can be still present in the wastewater after treatment.
Therefore, the toxicity and ecological effects of the reaction products have to be investigated
rigorously to ensure that the treatment mitigates the environmental and health related impacts.
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The objective of this paper is to give an overview of research carried out with oxidoreductase
enzymes to treat different organic micropollutants and their mixtures along with real wastewater and
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to collect available information on different techniques, transformation products and toxicity. Though
in most cases the reviewed enzymatic treatment methods resulted in less toxic effluent, the application
of mediators, while facilitating higher transformation rates, increased the level of toxicity. Hence, it
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was concluded that the suitability of biocatalytic methods should be assessed from the perspective of
toxicity beside the removal efficiency in order to allow the upscaling of the technology to a feasible
and sustainable wastewater treatment solution.
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Keywords:
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Enzymatic wastewater treatment; oxidoreductase; transformation product; micropollutant; ecotoxicity;

estrogenic activity

*
Corresponding author: Béla Varga, E-mail: vargabela@almos.uni-pannon.hu

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LIST OF ABBREVIATION

2,4-DCPh - 2,4-Dichlorophenol
ABTS - 2,2’-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
APAP - Acetaminophen
AS - Acetosyringone
ATZ - Atrazine
BHA - 2-hydroxybenzoic acid, Salicylic acid

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BPA - Bisphenol A
CBZ - Carbamazepine
CBZD - 10,11-dihydro-10,11-dihydroxy-CBZ

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CBZE - 10,11-dihydro-10,11-epoxy-CBZ
CLEA - Cross-linked enzyme aggregate
CLF - Clofibric acid

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CP - Chlorophene
CPS - Chlorpyrifos
CTC - Chlortetracycline

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DBP - Dibutyl phthalate
DC - Doxycycline
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DCF - Diclofenac
DCP - Dichlorophen
DEET - N,N-diethyl-m-toluamide
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DEHP - Bis(2-ethylhexyl) phthalate

DEP - Diethyl phthalate
DMBQ - 2,6-dimethoxy-1,4-benzoquinone
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DMP - 2,6-dimethoxyphenol
E1 - Estrone
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E2 - 17β-Estradiol
E3 - Estriol
EC50 - Half maximal effective concentration
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EDC - Endocrine disrupting compound

EE2 - 17α-Ethinylestradiol
EMR - Enzymatic membrane reactor
ERE - Estrogen responsive sequence
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ERYC - Erythromycin
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GFB - Gemfibrozil
HBA - 4-hydroxybenzyl alcohol
HBT - 1-Hydroxybenzotriazole
hER - human estrogen receptor
HPLC - High-performance liquid chromatography
HPLC-MS - Liquid chromatography-mass spectrometry
HRP - Horseradish peroxidase
IBU - Ibuprofen
Im - Immobilised
IPU - Isoproturon
KCZ – Ketoconazole
KET - Ketoprofen

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L - Laccase
LCA - Life cycle assessment
LiP - Lignin peroxidase
LMS - Laccase-mediator systems
LOEC - Lowest observed effect concentration
MBR - Membrane bioreactor
MFA - Mefenamic acid
MnP- Manganese peroxidase
NOM - Natural organic matter

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NP - Nonylphenol
NPX - Naproxen
OD - Optical density

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OECD - The Organisation for Economic Co-operation
OP - Octylphenol
OTC - Oxytetracycline

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PCP - Personal care product
PPCP - Pharmaceutical and personal care products
SA - Syringaldehyde

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SBE - Soybean meal extract
SDM - Sulfadimethoxine
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SMM - Sulfamonomethoxine
SMZ - Sulfamethoxazole
STH - Sulfathiazole
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STL - Sotalol
TC - Tetracycline
TCEP - Tris(2-chloroethyl)phosphate
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TCPP - Tris(1-chloro-2-propyl) phosphate

TCS - Triclosan
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TMP - Trimethoprim
TrOCs - Trace organic contaminants
TU - Toxic Unit
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UV/Vis - Ultraviolet-visible
VA - Vanillin
VLA - Violuric acid
WW - Wastewater
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YES - Yeast estrogen screen assay

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1. Introduction
The term ‘micropollutant’ is used for compounds that are present in the range of nanograms-
micrograms per litre in the environment (Virkutyte et al., 2010) and have negative environmental
effects due to their toxicity, persistence or bioaccumulative properties (Sauvé and Desrosiers, 2014).
They are also referred to as emerging pollutants or compounds of emerging concern/interest although
these expressions are relative to time and geographical location (Daughton, 2004). Figure 1 shows a
possible way to categorise the micropollutants based on their use. The scope of this review extends to

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organic micropollutants, within that pesticides, personal care products (PCPs), pharmaceuticals and
industrial chemicals.

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Figure 1. Classification of micropollutants (source: own elaboration). Pharmaceutical categories are

based on drug.com drug classes. Blue colouring indicates the classes that are covered by this review.

Examples of pesticides would be atrazine (ATZ), isoproturon (IPU) or chlorpyrifos (CPS), the former
two being herbicides while the latter is an insecticide. PCPs cover a wide range of ingredients that are
used in cosmetics (e.g. salicylic acid ((BHA), an exfoliant), insect-repellents (e.g. N, N-Diethyl-M-
toluamide (DEET)) or sun-screens (e.g. oxybenzone). In case of pharmaceutical products, they could
be further grouped into numerous classes. In this article, only those broader categories are highlighted

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that were mentioned in BIO Intelligence Service (2013) as the most consumed medicinal products in
Europe. It is not coincidental that the most researched emerging contaminants fall into one of these
categories, too. Representatives of the highlighted classes would be amoxicillin (anti-infective),
sotalol (STL, cardiovascular agent), acetaminophen (APAP, central nervous system agent), 17β-
Estradiol (E2, hormone) and gemfibrozil (GFB, metabolic agent). The category of industrial
chemicals includes surfactants (e.g. nonylphenol (NP)), plasticisers (e.g. bisphenol A (BPA)) and dyes
(not the scope of the review), among others. Endocrine disruptors (exogenous substances that alter
functions of the endocrine system causing adverse health effects (Bergman et al., 2013)) can be found
in several of the above-mentioned categories as this classification is based on purpose and not effect.

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Emission of micropollutants may occur at manufacturing stage, after consumption and when
disposing unused products. Major sources were found to be urban (Kasprzyk-Hordern et al., 2009)
and industrial (Sánchez-Avila et al., 2012), wastewater effluents, municipal landfill leachates (Xu et

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al., 2008) and agricultural runoff (Yeom et al., 2017). A considerable fraction of pharmaceuticals is
excreted or washed off unchanged (Jjemba, 2006) and while formalized protocol for patient disposal
and destruction of pharmaceuticals in the European Union (Directive 2004/27/EC) and in several

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other countries (Kusturica et al., 2016) exist, the unused or unwanted medicinal products are most
commonly disposed of in the garbage, toilet or sink (Tong et al., 2011). Micropollutants from
agricultural practices may arise from direct use of pesticide and through fertilization and irrigation

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with manures and reclaimed water polluted with pharmaceutical products (Petrovic et al., 2016).
While both inorganic and organic harmful substances can be found in soil, air and water the latter
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became the focus of concern lately due to their ever-increasing number and variety besides a new
understanding of their long-term adverse effects (Barbosa et al., 2016). The presence of antibiotics,
endocrine disruptors, anti-inflammatory drugs, cosmetics, and pesticides has been reported in
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wastewater effluents in the USA (Hignite and Azarnoff, 1977), in Europe (Ternes, 1998) and all over
the world (Heberer, 2002). A detailed measurement campaign was carried out in Liaohe River, China
screening for 1030 organic micropollutants and more than 80 were found both in the sediment and the
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surface water (Bu et al., 2015). Other investigations found micropollutants in the rivers of Romania
(Moldovan, 2006), in River Anoia, Spain (Ginebreda et al., 2010) in River Tyne, United Kingdom
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(Roberts and Thomas, 2006) and in the water bodies of Sydney, Australia (Birch et al., 2015). Conde-
Cid et al. (2018) investigated the occurrence and concentration levels of tetracyclines and
sulfonamides in manure, soil and plant samples in Galicia (Spain). They pointed out that the corn
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samples collected on land amended with manure had the antibiotics in high concentration, indicating
accumulation and thus increased public health risks.
The investigation of adverse ecological effects of emerging pollutants in the environment have been
in focus of interest since the nineties of the last century (Kümmerer, 2004). Existing literature on both
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chronic and acute toxicity of micropollutants have proven the reality of potential harmful effects of
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these compounds on both human and aquatic life. An investigation in 2004 concluded that pollution
of steroid hormones can cause reproductive failure in fish even in ng/l concentration range (Nash et
al., 2004). A paper published in 2005 revealed that bacteria resistant to certain antibiotics were found
in the wastewater (Costanzo et al., 2005), bearing the danger of evolving antibiotic resistant pathogens
by adaptation. Fent et al. (2006) pointed out that targeted ecotoxicological studies on the long-term
effects of pharmaceuticals are required, because for some compounds (diclofenac (DCF), propranolol
and fluoxetine) the concentration values in treated wastewater samples were close to the lowest
observed effect concentration (LOEC). Research on the environmental risk assessment of
pharmaceuticals in rivers proved that pharmaceuticals were intrinsically bioactive compounds and
were therefore able to cause potential negative effects in living systems (Ginebreda et al., 2010).
Galus et al. (2013) conducted experiments with selected pharmaceuticals (APAP, venlafaxine,
carbamazepine (CBZ) and GFB) and found that recurring, low concentration exposure of

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pharmaceutical and personal care products (PPCPs), except for estrogens, had chronic impacts on
multiple organ systems in zebrafish (Danio rerio). Additionally, micropollutants have detrimental
impacts at population level (Peschke et al., 2014). Since serious ecological and human risks arise from
these emerging compounds, this type of pollution has to be managed, requiring large-scale,
interdisciplinary studies (Stamm et al., 2016).
Conventional (activated sludge) wastewater treatment technologies cannot eliminate these pollutants
completely. Carballa et al. (2004) published the removal efficiencies for fragrances (70-90%), anti-
inflammatories (40-65%), antibiotic sulfamethoxazole (SMZ) (60%) and for E2 (65%) in a
conventional activated sludge wastewater treatment plant in Spain. Joss et al. (2006) concluded that

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municipal wastewater treatment plants cannot remove pharmaceuticals effectively, since only 4 of 35
reviewed pollutant concentrations decreased by more than 90% after treatment. A life cycle
assessment (LCA) showed that the PPCPs have a major impact on freshwater ecotoxicity as well as

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on the human toxicity in assessing the environmental performance of a completely autotrophic
nitrogen removal over nitrite (CANON) pilot plant (Alfonsin et al., 2014). The removal efficiency can
be enhanced by modifying the technology and/or the conditions of the treatment (i.e. increasing the

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sludge age or adding polishing steps) (Papa et al., 2016). Nonetheless, a certain concentration of
recalcitrant compounds would remain in the effluent (Grandclément et al., 2017). In addition to this,
the sludge adsorbs large amount of pharmaceuticals (Martín et al., 2012), thus the sludge has to be

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treated as well.
Even the advanced wastewater treatment methods have challenges with micropollutants due to large
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variety of target compounds, not to mention the countless intermediates, whose concentration and
ecological effects are rarely determined (Prasse et al., 2015). One of the largest European monitoring
surveys regarding the micropollutant contamination in wastewater (conducted in 90 sites) concluded
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that 125 out of 156 investigated micropollutants could be detected in European wastewater effluents
(Loos et al., 2013).
Several solutions have been tested to treat micropollutants: advanced oxidation processes (Jiang et al.,
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2013) such as photocatalytic degradation (Eskandarian et al., 2016) or ferrate technology (Jiang,
2013), membrane filtration (Yoon et al., 2006), adsorption (Lozano-Morales et al., 2018) as well as
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biological processes (Szilveszter et al., 2016). Coupling membrane bioreactor (MBR) with advanced
oxidation by pulsed corona discharge provided a removal efficiency of more than 90% of
micropollutants, exceeding even the combination of nanofiltration with MBR (Arola et al., 2017).
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Nonetheless, cost effective removal is still an open question, as most of these solutions have high
electricity consumption (Foteinis et al., 2018) and/or are still in experimental stage. Also, the
formation of toxic by-products poses additional risks (Benner et al., 2013).
Enzymatic treatment in other areas, for instance food, textile and paper industry, have proven to be
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viable solutions (Choi et al., 2015) because enzymes catalyze reactions with high specificity
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(Hedstrom, 2010) usually under moderate conditions (Madhavan et al., 2017), and it may serve as an
alternative to treat organic micropollutants. As opposed to activated sludge systems, enzymes are less
susceptible to inhibition by substances toxic to living organism, they are highly selective and may be
used over a broad concentration range effectively, even in diluted solutions (Agarwal et al., 2016).
Compared to other chemically catalyzed processes, enzymatic systems consume less chemicals, water
and energy and produce less waste (Grandclément et al., 2017), though the efficiency of
micropollutant removal depends on the compound and the operational circumstances (Singhal and
Perez-Garcia, 2016). Comparative environmental assessments pointed out that using enzymatic
processes in industrial applications often mitigate the impact on the environment (Jegannathan and
Nielsen, 2013). For example, using enzyme-enhanced membrane filtration to treat drinking water had
lower negative environmental impact than adsorption to activated carbon (Manda et al., 2014).

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However, cost-effective solutions for waste stream remediation is yet to be investigated (Naghdi et al.,
2018).
In this review, the focus is on the enzymatic treatment of organic micropollutants (excluding dyes and
drugs of abuse) with oxidoreductase enzymes and the evaluation of these from the perspective of
toxicity.
Methodological search of scientific databases was carried out to explore the topic in detail. Answers
are sought to the following questions:
● What are the main products of the micropollutants in case of treatment with oxidoreductases
under different circumstances?

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● It is possible to achieve lower toxicity in the treated effluent or are the transformation products
more toxic than the parent compounds?
● What are the limits of the enzymatic treatment of water-borne micropollutants and how to

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overcome those?
The aim of this manuscript is to identify the areas where the enzyme catalytic processes could be
improved in order to facilitate the upscaling of the technology. If solutions to treat the micropollutants

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present in wastewater and surface water were technically and economically feasible that would allow
closing the loop in the urban water cycle.

2. Oxidoreductase enzymes
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Oxidoreductases are able to treat wastes of chemical origins, containing phenols, drugs and hormones
(Demarche et al., 2012). In this section the characteristics of three oxidoreductase enzymes (EC 1) are
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discussed: laccases, tyrosinase and peroxidases as these are the most researched enzymes to treat
organic micropollutants.
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Laccases (EC 1.10.3.2.) are multi-copper containing oxidoreductases that can be used to oxidize a
wide range of aromatic and nonaromatic compounds (Hautphenne et al., 2016) utilising oxygen as
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substrate. Copper sites have historically been divided into three classes: type 1 (T1) or blue copper,
type 2 (T2) or normal copper, and type 3 (T3) or coupled binuclear copper centres (Solomon et al.,
1996). In laccases T1 site is mononuclear, and it is the primary acceptor of electrons from reducing
substrates while T2 and T3 copper centres form a trinuclear copper cluster and are responsible for the
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binding and the reduction of oxygen (Morozova et al., 2007). The efficiency of the oxidation depends
on the redox potential difference between the T1 copper of the enzyme and the substrate, hence some
organic compound with lower ionization potential cannot be directly oxidized by laccase (Lloret et al.,
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2010). The catalytic reaction is thought to be the following: the T1 site accepts electrons from
reducing substrates which are transferred onto the T2/T3 cluster where molecular oxygen is reduced
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to water (Senthivelan et al., 2016). During this process the laccase may form oligomers, quinone-type
substances and even cause partial degradation of phenolic compounds (Naghdi et al., 2018).
Different types of laccases are distinguished depending on the source of the enzyme. Laccase from
Trametes versicolor is often used for experiments, probably because of the relatively high potential of
T1 site compared to other sources (Morozova et al., 2007). However, laccases from Pycnoporus
sanguineus CS43 were effective against the endocrine disrupting compounds (EDC) NP and triclosan
(TCS) at lab scale experiments (Ramírez-Cavazos et al., 2014). At larger scale, the production of
laccase enzyme by Pycnoporus sp. SYBC-L3 can be done effectively in air-lift reactor (Liu et al.,
2013). Also, the production of laccase enzyme by growing fungal strains on poultry litter has been
demonstrated that showed high removal efficiency of estradiol (Liu et al., 2016).

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Tyrosinase (EC 1.10.3.1) is another copper-containing enzyme that can also be used to treat
pharmaceuticals. This enzyme contains one T3 copper centre and uses oxygen as a cofactor but the
mechanism is different than of the laccases (Ben-Yosef et al., 2010). The T3 site catalyses the
hydroxylation of monophenols to form o-diphenols and the oxidation of o-diphenols to o-quinones
(Durán et al., 2002). Due to their broad specificity tyrosinases are also able to react with a wide range
of phenolic compounds (Faccio et al., 2012). The process tends to form polymers which can be
removed from the wastewater by precipitation (Fairhead and Thöny-Meyer, 2012).
Peroxidases (EC 1.11.1.X) are oxidoreductases which use hydrogen peroxide as an electron acceptor
to catalyse oxidative reactions (Medina et al., 2017). Peroxidases like horseradish peroxidase (HRP)

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from the plant horseradish (Armoracia rusticana) (Melo and Dezotti, 2013), chloroperoxidase from
Caldariomyces fumago (Li et al., 2017), lignin peroxidases (Mao et al., 2010), manganese peroxidase
(MnP) from Phanerochaete chrysosporium (Inoue et al., 2010), soybean peroxidase (Al-Ansari et al.,

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2009), black radish peroxidase (Altinkaynak et al., 2017) or turnip peroxidase (Quintanilla-Guerrero
et al., 2008) can be used to transform and/or detoxify organic pollutants (Torres et al., 2003). One of
the most frequently tested peroxidase enzymes is HRP. Horseradish is a rich source of heme-

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containing oxidoreductase enzymes, which can catalyse the oxidation of a wide range of organic and
inorganic compounds (Azevedo et al., 2003) using H2O2 or other peroxides. The most often used
isoenzyme of horseradish is isoenzyme-C (Veitch, 2004). In the catalysed reaction, first the native

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enzyme (Fe3+) is oxidized by peroxide forming water as by-product. Then the oxidised form of
peroxidase (Fe4+-R+∙) oxidises one molecule of substrate (serving as electron donor), resulting in a
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substrate radical and a red intermediate (Fe4+) which is then further reduced by a second substrate
molecule, regenerating the peroxidase and producing another free radical (Martinez, 2002).
Ultimately this process can result in different reaction products, mainly dimers, trimers and oligomers
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(Margot et al., 2015).

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3. Method of literature review

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In order to answer the questions presented in Chapter 1, scientific articles were searched for in the
following databases: ScienceDirect, Web of Science and Scopus, without time limitation. The queries
were performed last time in July 2018. In order to filter articles on sciencedirect.com for enzymatic
treatment of micropollutants, keywords were chosen considering the difference between British and
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American English. Since treatment of micropollutants in air and soil are not considered relevant to the
scope of this paper, these were excluded. In case of ScienceDirect and Scopus the advanced search
function was used. In the Web of Science database, the syntax of query was slightly modified but the
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keywords remained the same. Another search was carried out to make sure to include specific
micropollutants in the review. The list of common pollutants reported by Luo et al. (2014) was used
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for the queries. Search terms were written as “name of the micropollutant” AND “name of the
enzyme”. The keywords and numbers of search results are given in the Table 1.

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Table 1. Search results of scientific databases
Keyword form ScienceDirect Scopus Web of Science

(oxidoreductase OR laccase OR peroxidase OR tyrosinase) 1824 669 29

AND (degradation OR removal OR treatment) AND
(micropollutant OR micro-pollutant) in all fields
NOT air NOT soil in abstract, title or keywords

(Acetaminophen OR Diclofenac OR Ibuprofen OR Ketoprofen 1541 2154 1114

OR Mefenamic acid OR Naproxen OR Salicylic acid OR

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Carbamazepine OR Bezafibrate OR Clofibric acid OR
Gemfibrozil OR Erythromycin OR Sulfamethoxazole OR
Trimethoprim OR Atenolol OR Metoprolol OR Caffeine OR
Galaxolide OR Tonalide OR Triclosan OR DEET OR

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Benzophenone-3 OR Estrone OR Estradiol OR 17α-
Ethynylestradiol OR Estriol OR Nonylphenol OR Octylphenol
OR Bisphenol A OR DBP OR DEHP OR DMP OR TCEP OR
TCPP OR Atrazine OR Diuron OR Diazinon OR Clotrimazole

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OR Tebuconazole) AND (laccase OR peroxidase OR
tyrosinase)

Since there are overlaps between the three databases as well as among the records of the two queries

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within one database, a preliminary screening to remove duplicates was carried out. As a large amount
of not relevant articles were still included in the outcome, these were filtered out based on the title and
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abstract manually. Articles were considered to be irrelevant if none of the following topics were dealt
with: enzymatic catalysis, micropollutants in water bodies or toxicity of the enzymatic treatment.
Also, dyes as micropollutants were omitted since several review articles on the industrial application
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of enzymatic decolorization had already been published. This process leads to altogether 333 articles
from all three databases serving the core of this review.
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4. Results

4.1 Enzymatic treatment of micropollutants

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After reviewing the articles, information on the micropollutants, enzymes used, experimental
conditions (pH, temperature, type of solution, treatment time, including the possible presence of
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mediators) and removal efficiency were collected and presented in table form (Table S.1) as
supplement. The units of the original sources were used. When available, the reaction products and
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toxicity or estrogenic activity changes were also provided.

A sum of 91 different micropollutants were found in the articles (Figure 2, inventory of compounds is
included in Table S.2). In 14 cases, only the decrease in concentration was discussed while the
transformation products were investigated for 17 (5+12) compounds. The environmental effects, i.e.
toxicity and/or estrogenic activity were measured in case of 72 chemicals altogether, though it has to
be pointed out that the majority of pollutants were tested in mixtures (e.g., solution of 38
pharmaceuticals in Becker et al. (2016)) and not separately for each micropollutant. Table S.1
includes information on 47 articles out of which 20 discussed transformation products and 25 studied
the toxicity and/or estrogenic activity (some papers examined both).

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Figure 2. Types of investigations on the enzymatic treatment of 91 organic micropollutants discussed

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in this review based on 47 articles.
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The distribution of the organic micropollutants based on the classification of Figure 1 showed the
dominance of pharmaceuticals (60) and within that the anti-inflammatory drugs (44) that contains the
antibiotics (number of compounds within other subcategories of pharmaceuticals: 7 central nervous
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system agents, 5 hormonal products, 2 metabolic agents and 1-1 cardiovascular and psychotherapeutic
agents). It was followed by PCPs (11), industrial chemicals (9) and pesticides (8). Two compounds
could not be fitted into any of these classes, these were phytoestrogens included in the study of
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Nguyen et al. (2014a) where a mixture of 30 trace organic compounds was treated with crude enzyme
extract.
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In the following sections experiments using crude (i.e. unpurified) enzymes and mediators as well as
immobilization are discussed in detail. Since in many papers the term ‘degradation’ for the enzymatic
reaction is used even if the results did not support that, the reaction products and the change in the
toxicity or estrogenic activity of the processes in question were examined further. In those cases,
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where no information on the products was available, the expression ‘degradation’ was avoided even if
the original sources used it. A process was called degradation only if the results supported that, i.e. at
least one product of smaller molecular weight was stated. The term ‘removal’ refers to the decrease in
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concentration compared to the initial value. It has to be pointed out that neither the lower
concentration of the parent compound nor the smaller molecular mass of the product leads necessarily
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to mitigating the negative environmental effects. Hence information was collected on the toxicity or
estrogenic activity of the experiments when available and are discussed in a separate section.
The optimal conditions of treatment solutions are different because the activity of enzymes highly
depends on pH, temperature, the presence of mediators and in some cases, co-factors (Caza et al.,
1999) leading to difficulties in comparing the results. For example, the amounts of enzymes used in
the experiments are often defined as enzyme activity, but these measurements may have been carried
out under various conditions with different substrates. The most commonly used substrates are 2,2’-
azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) for laccase and 2,6-dimethoxyphenol (DMP)
for HRP, but others are also used. In case of laccase, 1 activity unit equals 1 µmol ABTS radical
formed in 1 minute - measured by the change of absorbance at 420 nm, regardless of the conditions.
For example, Becker et al. (2017) used an activity assay, which followed the oxidation of ABTS at

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room temperature and pH 5.0 in sodium-acetate buffer using 0.5 mM initial ABTS concentration.
Meanwhile Auriol et al. (2008) defined the activity unit similarly and used pH 5.0 buffer, but the
initial ABTS concentration was 50 mM, and temperature was raised to 37°C, which could cause a big
difference in enzyme activity (Azimi et al., 2016). Li et al. (2008) compared laccase activity assays
with three different substrates and indicated significant differences. Similarly, Ademakinwa and
Agboola (2016) tested reaction kinetics with phenolic and non-phenolic substrates including guaiacol
and catechol resulting in different reaction rates. Most of the enzymes have the highest activity under
acidic conditions (Margot et al., 2013) that is not present in municipal wastewater treatment. Also,
while there are protocols to determine the activity, the conditions fixed there might not collide with

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those of interest. Therefore, the activity values of enzymes stated in Table S.1 (protocols not indicated
in the table) are not directly comparable to each other and the mass of enzyme cannot be deducted
from these values. In addition to that, most of the experiments were done in synthetic solutions

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instead of real wastewater which also may lead to different results in environmentally relevant
conditions.
Figure 3 shows the removal percentages of 28 selected compounds (under varying conditions,

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altogether 42 results are included). The selection was made so that all types of treatment solutions as
well all discussed micropollutant classes would be represented. As the enzymatic treatment of 91
different organic micropollutants was examined it was not reasonable to visualise all of them. Bearing

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these in mind some general remarks may be concluded based on the collected information regarding
the micropollutant removal efficiency by oxidoreductase enzymes. It can be stated that higher than
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90% removal rate can be achieved with enzymatic treatment in case of 17 instances including
estrogenic compounds, BPA, DCF, APAP, tetracycline (TC), CPS and IPU. However, there are some
recalcitrant compounds, such as ibuprofen (IBU), a quite common non-steroidal anti-inflammatory, or
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CBZ, an anticonvulsant, in which cases the removal would not go higher than 11% and 16%,
respectively even if it was treated by laccase-mediator system.
An ultimate winner among the examined oxidoreductases cannot be chosen either (see Table S1). For
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example, for TCS with HRP around 45% removal in one hour could be achieved while MnP (94% in
0.5 h) performed better (Melo and Dezotti, 2013). Though laccase also removed the pollutant
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completely, the process took 6 hours under the same circumstances. In another experiment BPA and
NP were treated with MnP, laccase and laccase-HBT (Tsutsumi et al, 2001). The MnP resulted in
complete removal of both compounds and in this case the laccase could only oxidise 70% of BPA and
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60% of NP. When repeating the process in the presence of mediator, the removal could be improved
up to 100% and 80%, respectively. Looking at these results only, the MnP would be preferred, but one
also has to consider the additional compounds required to catalyse the process. In many cases the
laccase could tackle the pollutant and only oxygen is required for the oxidation.
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Figure 3. Removal efficiency of selected organic micropollutants by enzymatic treatment.
References: 1: Dahili et al. (2015); 2: Ba et al. (2014a); 3: Ratanapongleka and Punbut (2017); 4: Nguyen et al. (2014b); 5:
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Nguyen et al. (2014a); 6: Huang et al. (2005); 7: Tsutsumi et al. (2001); 8: Zhang and Geißen (2010); 9: Das et al. (2017);
10: Tran et al. (2013); 11: Spina et al. (2015); 12: Xia et al. (2014); 13: Auriol et al. (2007); 14: Yang et al. (2017); 15: Zeng
et al. (2017); 16: Yousefi-Ahmadipour et al. (2016); 17: Margot et al. (2013); 18: Catapane et al. (2013); 19: Becker et al.
(2016); 20: Rahmani et al. (2015); 21: Llorca et al. (2015); 22: Suda et al. (2012); 23: Inoue et al. (2010)
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4.1.1 Experiments using crude enzyme extracts.

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Many experiments were done with pure enzymes (see Table S.1), thus the feasibility of the
technology highly depends on the cost of commercial enzymes. In order to resolve this and to possibly
make the process cheaper, crude enzyme extracts may be used for the treatment (Lonappan et al.,
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2018) making the purification process as simple as possible (Ji et al., 2017). Detailed information on
the experiments with crude enzymes can be found in Table S.1.
The use of crude MnP was investigated and confirmed the potential to treat TC and oxytetracycline
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(OTC) achieving removal percentages of 72.5% and 84.3%, respectively (Wen et al., 2010). Zhang
and Geißen (2010) tested the in vitro reaction of CBZ and DCF in a mixed solution with crude lignin
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peroxidase. Complete removal of DCF was achieved after 2 hours while CBZ proved to be
recalcitrant. Garcia-Morales et al. (2015) conducted a series of experiments with crude laccase from
Pycnoporus Sanguineus CS43 fungus and reported 89-100% removal rate of endocrine disruptor
compounds (BPA, 4-NP, 17α-ethynylestradiol (EE2), TCS). Experiments on the removal of organic
micropollutants were carried out successfully using lysates from activated sludge containing a variety
of native enzymes (Krah et al., 2016), showing similar removal rates as observed in conventional
wastewater treatment plants (Joss et al., 2006).

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4.1.2 Experiments using immobilized enzymes.
Despite the high removal efficiencies that could be achieved under laboratory conditions in most
cases, there are certain disadvantages to using enzymes such as inactivation by various denaturants
(Iyer and Ananthanarayan, 2008) and inhibition (Rao et al., 2014). Another issue is the reusability of
enzymes. In order to facilitate reuse and to improve enzyme stability, different immobilization
techniques such as bonding to magnetic nanoparticles (Fortes et al., 2017), cross-linked enzyme
aggregates (CLEAs) (Hassani et al., 2013), enzyme grafted membrane (de Cazes et al., 2015) and
enzyme encapsulation (Le et al., 2016) have been developed. Results of different immobilisation

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methods are summarised in Table 2.
Experiments concluded that immobilized laccase, which was repeatedly used for 7 days, was able to
accomplish 60% removal efficiency of 2,4-dichlorophenol (2,4-DCPh) at an optimum pH of 5.5

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(Zhang et al., 2008). The same component was removed efficiently (by more than 90%) by
immobilized HRP (Dahili et al., 2015). When turnip peroxidase was immobilised both in alginate
matrix and via covalent binding to Affi-Gel 10 in the presence of polyethylene glycol to remove

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phenolic compounds, the operative stability could be also sustained for several cycles (Quintanilla-
Guerrero et al., 2008).
Chemical (Hommes et al., 2012) or physical (Zdarta et al., 2018) immobilization of the enzymes may

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also improve the storage stability so that the activity would not decline quickly and diminish in a few
days, as it would be the case with free laccase. Using magnetic core-shell alginate beads in
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wastewater resulted in improved stability in wastewater compared to free laccase (Le et al., 2016).
Moreover, it was possible to preserve the activity of laccase up to one year by storing at 4°C when
immobilized as CLEAs containing magnetic nanoparticles (Kumar and Cabana, 2016).
Since immobilization slightly changes the structure of the enzymes and may result in blocking the
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active site (Barrios-Estrada et al., 2018), it is important to test the performance of each unique system.
Immobilization of laccase in hydrogels improved the stability for storage and reusability (Yamak et
al., 2009), although the affinity of the enzyme to substrate may decrease. Controversially, Gassara et
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al. (2013) reported higher removal efficiency (86-90%) with encapsulated enzymes compared to free
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enzymes when treating BPA. Similarly, laccase immobilized on polyamide/chitosan nanofibers using
spacers were successfully used to transform BPA and 17α-ethinylestradiol more effectively than free
enzyme (Maryšková et al., 2016). A possible explanation for the higher removal rate could be that the
micropollutants adsorb to the surface of the immobilisation material, which was found to be the case
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in the experiments of Ba et al. (2014b).

Nicolucci et al. (2011) compared the removal efficiency of four bisphenol derivatives with laccase
and tyrosinase in immobilized form and achieved 90-100% reduction in 1.5 hours with both enzymes,
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though tyrosinase was found to be less effective. In some cases, immobilization may enhance the
activity of the enzyme in environmentally relevant conditions, e.g. neutral pH, (Sathishkumar et al.,
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2012) or ambient temperature (Naghdi et al., 2017). Arca-Ramos et al. (2016) achieved nearly
complete removal of BPA in slightly alkaline solution by using laccase immobilized on fumed silica
nanoparticles. Likewise, laccase immobilization by cross-linking combined with magnetic
nanoparticles improved the activity at neutral pH (Kumar and Cabana, 2016).
Immobilizing enzymes on the surface of a membrane can integrate the catalytic reaction and
separation process of the treatment. De Cazes et al. (2014) designed and optimized an immobilized
enzymatic membrane reactor (EMR) containing laccase for the treatment of TC, and reached a
removal efficiency of 56% and constant transformation rate of 0.34 mg/h for 10 days. Another EMR
was developed by immobilizing laccase on a carbon nanotube coated polymer support and it was not
only capable of achieving high removal for a mixture of pharmaceuticals, the membrane could be
rejuvenated by reversible physical adsorption of laccase (Ji et al., 2016a), as well.

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Table 2. Results of different immobilisation methods

Immobilisation Enzyme Treated Notable results Source
method micropollutants
Grafted ceramic laccase mixture of 38 mediator had to be used Becker et al.
membrane antibiotics for efficient removal (2016)
mixture of EDCs grafted ceramic
membrane had low
activity, removal was
achieved by adsorption

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on the membrane
TC 56% removal (vs. 30% de Cazes et al.
with free laccase) (2014)
increased stability

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BPA development of A high Barrios-Estrada et
load enzymatic al. (2018)
membrane
CLEA laccase, APAP broader pH profiles, Ba et al. (2014a)

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tyrosinase increased stability,
higher removal rate;
tyrosinase had higher
activity yield than

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laccase
laccase APAP, MFA, CBZ synergistic effect of Ba et al. (2014b)
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using microfiltration and
CLEA together
Barium alginate laccase APAP enhanced characteristics Ratanapongleka
beads after immobilisation, and Punbut (2017)
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activity >70% after five

cycles
Calcium-alginate turnip phenol improved stability for Quintanilla-
beads peroxidase several cycles Guerrero et al.
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Covalent bonding to phenol immobilisation without (2008)

Affi-Gel 10 activity loss, stability
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was kept for 15 cycles

Acrylic beads laccase mixture of EDCs removal was partly Becker et al.
occurring by sorption to (2017)
immobilization supports
Polyacrylonitrile laccase, Bisphenol 80% of initial activity Nicolucci et al.
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beads tyrosinase derivatives retained after 30 days of (2011)

work
laccase NP, OP use of fluidised bed Catapane et al.
reactor, (2013)
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95% of original activity

after 50 days
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Porous silica beads laccase STZ, stability at higher Rahmani et al.

SMZ temperature and range of (2015)
pH increased
Magnetic CLEA laccase mixture of 13 biocatalyst can be Kumar and
pharmaceuticals recovered within 20 s Cabana (2016)
1 year storage stability,
laccase TC, OTC, AMP, maximal activity Yang et al. (2017)
SMZ, ERYC, TMP, recovery of 46.8% was
CHL obtained
Magnetic alginate laccase TCS activity retained in real Le et al. (2016)
beads wastewater

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Magnetic laccase CPS 99% of CPS could be Das et al. (2017)
nanoparticles with removed at pH 7
chitosan coating
Fumed silica laccase BPA significantly improved Hommes et al.
nanoparticles enzyme stability (2012)

laccase BPA, DCF complete removal of Arca-Ramos et al.

BPA in pH 8.4 solution (2016)

Ethyl cellulose HRP 2,4-DCPh high removal rate, Dahili et al.

nanoparticles improved pH tolerance (2015)

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and storing stability
Polyamide 6/chitosan laccase BPA, EE2 Immobilized enzyme Maryšková et al.
nanofibers were more effective than (2016)
free.

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Chitosan carrier with laccase 2,4-DCPh, CP activity decreased with Zhang et al.
GA cross-linking immobilisation by could (2008)
be reused for 7 day

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Encapsulation in ligninolytic BPA encapsulation protected Gassara et al
polyacrylamide enzymes enzymes from (2013)
microgel inactivation by non-
competitive inhibition
Poly (lactic-co- laccase DCF improved activity on Sathishkumar et

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glycolic acid) neutral pH al. (2012)
nanofiber
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Oxygen laccase CBZ improved storage, pH Naghdi et al.
functionalized and thermal stability (2017)
nanobiochars
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4.1.3 Experiments involving mediators.

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Using enzyme mediators (diffusible electron carriers from natural or synthetic sources) can broaden
the range of substrates and to improve the removal efficiency. Mediators are low weight molecular
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compounds that are stable but reactive and facilitate the oxidizing process due to their smaller size or
expand the oxidizing capability (Munk et al., 2018). If the redox potential of the organic compound is
lower than the redox potential of the enzyme, mediators can be applied to enable the oxidation process
(Madhavi and Lele, 2009). By doing so, the laccase enzyme oxidizes the mediator compound,
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forming reactive intermediates that can further react with the target compounds (Fabbrini et al., 2002).
Molecules that are already present in the environmental matrix are considered natural mediators while
synthetic mediators were developed to mimic the effect (Johannes and Majcherczyk, 2000).
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Representatives of natural mediators are for example syringaldehyde (SA), acetosyringone (AS),
vanillin (VA), acetovanillone, methyl vanillate and p-coumaric acid. 4-hydroxybenzyl alcohol (HBA)
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(Cañas and Camarero, 2010), while ABTS and 1-hydroxybenzotriazole (HBT), violuric acid (VLA)
are of synthetic origin (Guan et al., 2018). Detailed information on the experiments with mediators are
provided in Table S.1 of the supplementary data.
In the following examples the synthetic mediators seemed to provide better results. Margot et al.
(2015) tested different laccase-mediator systems (LMS) to treat solutions containing IPU and SMZ.
None of the compounds reacted with the enzyme in the presence of natural mediators (SA, AS) during
the incubation. However, laccase with ABTS completely oxidized both pollutants in relatively short
time. HBT as mediator was tested to treat ketoconazole (KCZ) (Yousefi-Ahmadipour et al., 2016) and
IPU (Zeng et al., 2017) with laccase. In both cases, improvement of removal efficiency was reported.
Treatment of sulfonamide antibiotics (sulfamonomethoxine (SMM) and sulfadimethoxine (SDM))
with laccase mediators (VLA, HBA, SA and ABTS) was investigated regarding the efficiency and

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toxicity by Weng et al. (2013). The study showed that complete elimination of target compounds can
be achieved within 30 minutes in laccase-ABTS or within 15 minutes in laccase-VLA solutions,
however, the removal efficiency and toxicity of the solution varied with the used mediator.
Despite their efficiency, most synthetic mediators are expensive and probably have some ecological
effects, thus alternative natural mediators have been tested. Lloret et al. (2010) compared the
oxidation of several pharmaceuticals (DCF, naproxen (NPX), estrone (E1), E2, EE2 with laccase and
various mediators and reported excellent removal efficiencies. Liang et al. (2017) used laccase and
soybean meal extract (SBE) to degrade sulfadimethoxazole and proved that the soybean extract
(containing VA, apocynin, daidzein) can replace synthetic laccase mediators with comparable

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efficiency. Phenolic compounds in white-rot fungi that play a role in the biodegradation of lignin
polymer also act as mediators to oxidise non-phenolic structures (Cañas and Camarero, 2010). Other
natural organic compounds present in the matrix might act as mediators, too. Tran et al. (2013)

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reported higher removal rate in case of DEET in wastewater, which was attributed to some phenolic
compound acting as natural mediator.

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4.2 Products of enzymatic treatment

4.2.1 Analytics
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The measurements assessing the change of target compound due to enzymatic treatment in higher
concentration range can be done with relatively simple methods, for instance HPLC-UV/VIS or even
using UV/VIS spectrometry. On the other hand, diluted samples (in the range of micrograms or
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nanograms per litre) call for more sophisticated sample preparation and analytic methods (see Table
S.1). In order to identify the products, the use of liquid/gas chromatography with mass spectrometry
(HPLC-MS) is required (Fatta et al., 2007). In case of chiral compounds measurement at the
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enantiomeric level is suggested because these pollutants may be transformed stereoselectively by

bacterial activity in wastewater treatment plants (Petrie et al., 2017) and the enantiomers may have
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different toxicity in the environment (Evans and Kasprzyk-Hordern, 2014).

There is a difference between the products formed in synthetic and real wastewater since the enzymes
react with the natural organic compounds present in the solution as well (Mao et al., 2010), thus
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investigation of reaction products is important. Relatively few papers deal with enzymatic treatment
in real wastewater, probably because the measurement of transformation products under such
circumstances are challenging and require sensitive analytical methods (Radjenović et al., 2009) due
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to low concentrations in the nanogram per litre range and matrix effect (Wang and Wang, 2016).
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4.2.2 Transformation products in synthetic wastewater

Enzymatic treatment of organic micropollutants does not necessarily lead to degradation (Table 3).
Phenolic coupling products of BPA were identified as products of HRP catalysed treatment using
molecular modelling supported by liquid and gas chromatography measurements (Huang and Weber,
2005). Similarly, based on investigation of the HRP catalyzed transformation of seven
pharmaceuticals with HPLC-MS, 4 possible reaction products of DCF were proposed: DCF-dimer,
OH-dimer, dimer-iminoquinone and reduced dimer (Stadlmair et al., 2017). Ba et al. (2014a) tested
the treatment of APAP with tyrosinase and laccase and reported dimers, trimers and oligomers of
APAP, 3-hydroxyacetaminophen as intermedier, and 4-acetamido-o-benzoquinone as final product of
the treatment. HPLC-MS experiments confirmed that a possible pathway during the treatment of
antimicrobial pollutants chlorophene (CP) and dichlorophen (DCP) with laccase-catalysed system was

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through nucleophilic substitution of chlorine by a hydroxyl group and further oxidation, resulting in
coupled transformation products (Shi et al., 2016).
Though using mediators may improve the efficiency of the treatment, it changes the mechanism of the
reaction as well, thus, transformation may produce different products. Laccase proved to be
nonselective regarding the transformation of enantiomers (IBU, KET and NPX) but the addition of
mediators made the reaction enantioselective (Nguyen et al., 2017). Daâssi et al. (2016) reported that
degradation of BPA with laccase from Coriolopsis gallica showed a different pathway when it was
used with HBT as mediator. Without the mediator, the product was β-hydroxybutyric acid, but in
presence of mediator two products were identified: tartaric acid and pyrogluamic acid (Daâssi et al.,

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2016). Zeng et al. (2017) identified trimers and dimers of IPU after treating it with laccase and HBT
as mediator. The transformation product of HBT was also found in the solution after the treatment.
Margot et al. (2015) identified several transformation products of SMZ and IPU when oxidised by

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laccase-mediator systems, including those of the mediator and found that the ratio of the different
products depended on the pH. They also reported, that oxidation of SMZ in the presence of laccases
and AS produces five different products: 2,6-demethoxy-1,4-benzoquinone (DMBQ) and SMZ-

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DMBQ coupled products, among others. DMBQ was also identified as an oxidation product of
syringyl compounds by Ibrahim el al. (2013).
Ji et al. (2016b) identified metabolites of CBZ such as 10,11-dihydro-10,11-dihydroxy-CBZ (CBZD),

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10,11-dihydro-10,11-epoxy-CBZ (CBZE) and acridone when it was treated in a hybrid membrane
reactor containing immobilized laccase and TiO2 nanoparticles. Das and Singh (2017) reported that
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treatment of CPS with immobilized laccase produces 2,4-bis(1,1-dimethylethyl) phenol, 1,2-
benzenedicarboxylic acid, bis(2-methyl propyl) ester.
In case of TC removal by laccase, three reaction pathways were identified through which the TC was
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transformed (Yang et al., 2017) resulting in oxidized and anhydride forms (de Cazes et al., 2014).
Llorca et al. (2015) detected two transformation products with similar molecular mass and one smaller
using on-line turbulent flow chromatography equipped with mass spectrometry detector. OTC as a
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transformation product of TC were reported by de Cazes et al. (2014), which is also known for its
antibiotic properties. Nonetheless, transformation of OTC with MnP and laccase-HBT solution was
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already reported by others (Suda et al., 2012), which indicates that the removal of product may be
achieved with enzymatic treatment.
The poor removal efficiency of some organic micropollutants can be explained by their chemical
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structure. Yang et al. (2013) demonstrated if the compound contains relatively strong electron
drawing group like halogenated, amide or carboxylic acid groups, then the reaction is less likely to
occur. Similarly, non-phenolic organic compounds containing withdrawing electron groups are
difficult to oxidize for laccase without the assistance of a redox mediator with the exception of DCF
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and indomethacin (Tran et al., 2013).

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4.2.3. Experiments regarding treatment of real wastewater

While researches with synthetic solutions provide valuable information on the mechanisms of
enzymatic treatment, it is crucial to gather information on the reaction mechanisms, efficiency and
products when treating real wastewater. Natural organic matter (NOM) may cause inhibition, act as
natural mediator or influence the reaction in a way that different products are formed.
While oligomer production in synthetic solutions can be achieved (Nicotra et al., 2004), their coupling
to NOM in real wastewater may limit that process (Li et al., 2017) resulting in various transformation
products. NOM was found to inhibit the oxidative coupling processes of laccase catalyzed E2 removal
slightly in surface water (Xia et al., 2014). However, dimers were identified as products, suggesting

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that the reaction mechanism did not change. In case of APAP, experiments conducted in the effluent
of a wastewater treatment plant with free and cross-linked tyrosinase and laccase showed high
conversion to oligomers (Ba et al., 2014a). Mao et al. (2010) reported that E2, when treated with
lignin-peroxidase enzyme, formed coupling products via the covalent bonding at their unsubstituted
carbons in phenolic rings that may go through further coupling reactions. In case of high
concentration of NOM, the efficiency of E2 transformation was reduced because these organic
compounds also reacted with lignin-peroxidase and were prone to couple to each other. According to
Tran et al. (2013) laccase activity regarding the target component (in their case: DEET) may decrease
in real wastewater due to the presence of other, phenolic compounds or the specific degradation of

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laccase by extracellular protease. Other experiments with pure humic acid (HA), as the model
compound of NOM, and TCS (Dou, 2018) showed that HA inhibited self-coupling and
simultaneously altered the transformation pathway.

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Controversially, Zeng et al. (2017) measured faster removal of IPU in real wastewater than in citrate
buffer by laccase-HBT (in case of 0.2 mM HBT), indicating, that NOM may serve as efficient natural
mediator, too. On the other hand, the wastewater matrix did not affect the removal of estrogens by

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laccase (Auriol et al., 2007) or by HRP (Auriol et al., 2008) resulting in elimination of estrogenic
activity within an hour. For other compounds the removal of parent compounds (ACT, MFA and
CBZ) could be achieved but required considerably more time (Ba et al., 2014b).

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Changes due to the different characteristics of real versus optimised conditions may also alter the
conversion. DCF proved to be more recalcitrant in wastewater effluent (pH 7.9) showing a removal
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rate of 30% as opposed to 90% in buffer solution (Nair et al., 2013). Contrary to that, concentrations
of BPA and EE2 in the same experiment showed similar decrease in the synthetic (90%) and real
(85%) wastewater.
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It has to be pointed out that while several articles focused on reaction products in synthetic solution, it
was difficult to find information on products regarding wastewater effluent. Some researchers chose
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to use HA as a model compound and only a few used environmental samples (9 of 47 articles
processed in Table S1). In most of the latter cases (6 out of 9) wastewater effluent was used to
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examine the change in the removal of micropollutants; product identification was not the goal.
Probable reasons to this could be that the measurement methods are complex and require high level of
expertise and resources, and that the ever-changing quality of wastewater effluents complicate
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repeating experiments. Based on the results, treatment of pollutants with oxidoreductase enzymes
tended to produce dimers, quinons and oligomers instead of simpler compounds (Table 3), thus the
preferred pathway of removing these after the enzymatic treatment could be precipitation (Xia et al.,
2014) or filtration (Asif, 2017).
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Table 3. Products of the enzymatic treatment

Compound Enzyme used Solution Products of the reaction Reference

APAP Laccase wastewater Dimer, trimer, tetramer Ba et al.

(2014a)
Tyrosinase synthetic 4-acetamido-o-benzoquinone Valero et al.
(2002)
wastewater 3-hydroxyacetaminophen Ba et al.
(2014a)

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BPA Enzyme extract synthetic Dimers, trimers, tetramers Cabana et al.
from Coriolopsis (2007)
polyzona
Horseradish synthetic 13 intermediates and coupled products Huang et al.
peroxidase (2005)
Laccase synthetic β-hydroxybutyric acid Daâssi et al.
(2016)
Laccase – HBT synthetic tartaric acid Daâssi et al.
pyroglutamic acid (2016)

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Laccase from synthetic 13 aromatic and aliphatic metabolizes de Freitas et
Pleurotus ostreatus including ring opening products al. (2017)
and Pleurotus

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pulmonarius
Lignolitic enzymes synthetic Final product: 4,5-bisphenol-o-quinone Gassara et al.
(Manganase (2013)

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perodiase,
Lignin peroxidase,
Laccase)
CBZ Laccase – TiO2 synthetic metabolites: Ji et al.

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CBZD, (2016b)
CBZE,
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acridone
CP Laccase synthetic dimers, trimers Shi et al.
(2016)
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CPS Laccase synthetic 2,4-bis(1,1 dimethylethyl) phenol, Das and

1,2 benzenedicarboxylic acid, bis(2- Singh (2017)
methyl propyl) ester
DCF Chloroperoxidase synthetic 6 different hydroxydiclofenac Li et al.
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(2017)
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Horseradish synthetic DCF-dimer, OH-dimer, dimer- Stadlmair et

peroxidase iminoquinone, reduced dimer al. (2017)
DCP Laccase synthetic dimers, trimers Shi et al.
(2016)
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E2 Laccase synthetic E2 dimers Xia et al.

(2014)
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Lignin peroxidase synthetic E2 coupling products of 2-3 building Mao et al.

units (2010)
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Cross-coupling products with NOM

IPU Laccase – ABTS synthetic hydroxy-isoproturon, coupling products, Margot et al.
ABTS transformation products (2015)
Laccase – HBT synthetic trimers and dimers Zeng et al.
HBT reaction products (2017)
KCZ Laccase synthetic 1-(4-{4-[2-(2,4-dichloro-phenyl)-2- Yousefi-
imidazol-1-ylmethyl-[1,3]-dioxolan-4 Ahmadipour
-ylmethoxy]-phenyl}-4-oxy-piperazin- et al. (2016)
1-yl)-ethanone,
1-(4-{4-[2-(2,4-dichloro-phenyl)-2-
imidazol-1-ylmethyl-[1,3]-dioxolan-4-
ylmethoxy]-phenyl}-3-hydroxy-

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piperazin-1-yl)-ethanone

NPX Chloroperoxidase synthetic desmethylnaproxen → Li et al.

phosphorylated and hydroxylated (2017)
Enzyme extract synthetic Dimers, trimers, tetramers Cabana et al.
from C. polyzona (2007)
STL Horseradish synthetic Aldehyde, Aldehyde and loss of Stadlmair et
peroxidase sulfonylmethane al. (2017)

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SMZ Laccase – AS synthetic DMBQ Margot et al.
and other coupled products (2015)

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SDM Laccase from T. synthetic 5 different degradation products Liang et al.
versicolor (2017)

TC Laccase from T. synthetic oxytetracycline de Cazes et

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versicolor anhydrotetracycline al. (2014)
synthetic oxidized, bi-demethylized products Yang et al.
(2017)

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synthetic 3 different transformation products, one Llorca et al.
with smaller molecular weight (2015)
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Laccase from P. synthetic 6 types: OTC, hydroxylated, quinone-like, Sun et al.
ostreatus dehydration, demethylation and (2017)
deamination products
TCS Enzyme extract synthetic Dimers, trimers, tetramers Cabana et al.
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from C. polyzona (2007)

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4.3 Environmental effects of the effluents

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Understanding the difficulties of measuring the concentration of each individual transformation

product, other solutions have to be explored to evaluate the success of enzymatic treatment organic
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micropollutants. A reasonable option would be to measure toxicity to describe the extent of

environmental effects. While several articles dealt with the ecotoxicity or the estrogenic activity
changes it would seem that including ecotoxicological tests in the research plan was not always self-
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evident. In this subsection the results of ecotoxicity or estrogenic activity assays are discussed. Since
these experiments were done in different matrices, for the sake of simplicity these will be referred to
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as effluent regardless whether it was a solution of one component or synthetic wastewater containing
several compounds or a sample originating from the discharge of a real wastewater treatment plant.
The information collected on the toxicity and estrogenic activity of effluents is summarized in Table
S2 and explained in detail in the following subsections. Toxicity assessments provided information in
a rather diverse way, especially in terms of the expression of toxicity before and after the treatment.
Most references give exact EC50 values, others express toxicity as raw data with regard to growth
inhibition, without calculating EC50. Other use the term Toxic Unit (TU, 1/EC50). Change of toxicity
or residual toxicity is expressed by some references as % of the initial toxicity, without giving exact
values. Nonetheless, the overall conclusion is that in several cases the enzymatic process mitigated the
adverse environmental effects.

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4.3.1 Toxicity tests
De Freitas et al. (2017) measured the ecotoxicity of untreated and laccase treated BPA samples using
Microtox®. This test is based on the bioluminescence inhibition of the marine bacterium Vibrio
fischeri and follows the ISO 11348-3:2007 protocol. Although the species was renamed Aliivibrio
fischeri (Urbanczyk et al., 2007), most standards and even recent literature still apply the V. fischeri
name. The reduction of light intensity is proportional to the toxicity of the sample and can be
measured photometrically. In this study, BPA was treated with two different laccases, derived from
Pleurotus ostreatus and P. pulmonarius. Initial toxicity expressed as % bioluminescence inhibition

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was app. 85%, which was reduced to 4.65% using P. ostreatus, but a discrete though non-significant
increase was experienced when the P. pulmonarius laccase was used suggesting that the latter might
be inadequate for bioremediation purposes.

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Rahmani et al. (2015) evaluated the ecotoxicity reduction of laccase-treated SMZ and sulfathiazole
(STH) solutions based on the growth inhibition on different bacterial strains (Gram-negative
Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Haemophilus influenza ATCC

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49247, and Salmonella enterica ATCC 19430 and Gram-positive Staphylococcus aureus ATCC 6538
and Streptococcus pneumoniae ATCC 49619). Growth was assessed by recording the OD600 (Optical
Density) values of the sample vs. the control (Wiegand et al., 2008). In case of SMZ, growth

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inhibition of tested species varied between 34.0 and 57.8%, H. influenza showing the lowest
inhibition, while for STH the range was between 23.2 and 51.4%, S. aureus showing the lowest
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inhibition. In both treatments, P. aeruginosa exerted the highest sensitivity. Bioactivity reduction of
TCS solution was evaluated with HRP by Melo and Dezotti (2013), based on the growth inhibition of
E. coli K12 broth cultures. A 95% reduction in both the growth inhibition and TCS concentration was
experienced, suggesting that the residual inhibition was induced by the non-converted TCS.
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In the study of Suda et al. (2012), treatment efficiency of TC, chlortetracycline (CTC), doxycycline
(DC) and OTC was assessed. Each sample consisted of the test compound, partially purified
ligninolytic enzyme (MnP), malonate buffer, MnSO4 and glucose (25 mM). Treatment resulted in a
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complete loss of growth inhibition in case of test bacteria (gram-negative E. coli NBRC 14249 and
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gram-positive Bacillus subtilis NBRC 3134) after 0.25 h and 1 h reaction time. Growth inhibition was
calculated using OD620 values. Ecotoxicity reduction was also assessed using the green alga
Pseudokirchneriella subcapitata which is a standard test organism, prescribed by OECD 201 algal
growth inhibition test guidelines. Initial 72 hour EC50s for TC, CTC, DC and OTC were 0.47, 0.84,
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0.14 and 1.2 µM, respectively. No growth inhibition was detected after the enzymatic treatment. This
result was underlined by the works of Sun et al. (2017) who used laccase-HBT system and Yang et al.
(2017) with pure laccase reported a significant decrease in the growth inhibition of E. coli studying
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the same compound.

P. subcapitata was used by Margot et al. (2015) to follow toxicity changes in SMZ and IPU solutions
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in laccase-mediator systems and reported distinct residual toxicity differences between the effluents.
Initial concentration of SMZ was 1.9 mg/l, resulting in 90% of growth inhibition in the test
population. This toxicity was reduced during the laccase mediated treatments by 61% in presence of
SA mediator, 77% in presence of AS and 100% in presence of ABTS. Similar tendency was
experienced in case of 103 µg/l IPU solution where initial growth inhibition was 68% and that was
reduced by more than 95% by the end of the treatment in the laccase-ABTS system. Degradation and
detoxification of IPU solution by the laccase-HBT system were also studied by Zeng et al. (2017)
using P. subcapitata. Significant toxicity reduction was experienced, from the initial 94.3% growth
inhibition to 10.2%. Using another freshwater unicellular green alga, Chlorella pyrenoidosa, Li et al.
(2017) evaluated toxicity reduction of DCF and NPX. While conversion efficiency of the parent
products was 100%, complete removal of toxicity could not be achieved. However, growth inhibition

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decreased: initial EC50 increased from 0.14 to 0.29 mg/l in case of DCF and from 0.21 to 0.33 mg/l in
case of NPX.
Yousefi-Ahmadipour et al. (2016) employed a test battery to follow toxicity of laccase-treated KCZ.
The test battery included the alga P. subcapitata, the fungi Candida albicans, Cryptococcus
neoformans, and Saccharomyces cerevisiae. Growth inhibition tests showed a decrease in the toxicity
of the tested solutions, but complete removal could not be achieved. Also, different test species
showed different sensitivity. For C. albicans and C. neoformans the initial toxicity expressed as EC50
of growth inhibition was 32% which decreased to 48 and 46% after 72 hours, respectively (please
note that as EC50 is the calculated concentration which causes 50% of ecotoxic effect, higher EC50

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values reflect lower toxicity). S. cerevisiae expressed higher sensitivity, with the initial EC50 of 16%,
but EC50 still remained 28% after 72 hours. Finally, algal growth inhibition was reduced to
approximately 23%. Gust et al. (2012) examined the immunotoxicity of antibiotics (ciprofloxacin,

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erythromycin (ERYC), novobiocin, OTC, sulfamethazole and trimethoprim) on the immune
parameters of Elliptio complanata and found out that the examined antibiotics, both alone and as
mixtures, modified the immune responses of E. complanata mussels during in vitro exposure at

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environmentally relevant concentrations.
Inoue et al. (2010) compared MnP, laccase and LMS regarding the removal efficiency and
detoxification of TCS, achieving the best result with MnP. They reported 94% removal rate in 30

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minutes and complete loss of bacterial (E. coli and B. subtilis) and algal (P. subcapitata) growth
inhibition. Tests using Scenedesmus obliquus confirmed that the treatment of antimicrobial additives
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of cosmetics (CP and DCP) with laccase, successfully detoxified the solution, though several different
coupled transformation products were formed (Shi et al., 2016).
It should be noted that while using redox mediators enhance the efficiency of the transformation, they
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could also produce more toxic effluent (Kim and Nicell., 2006). A bioluminescence inhibition-based
test was employed in the study of Nguyen et al. (2014a), using the ToxScreen3 assay (CheckLight
Ltd, Israel) with the bacteria Photobacterium leiognathi. A mixture of 30 trace organic contaminants
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(TrOCs) was used as model sample, including e.g. pharmaceuticals, pesticides, steroid hormones,
industrial chemicals and phytoestrogens. Toxicity of the sample was measured before and after
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treatments, in order to compare the potential contribution of mediators to residual toxicity. Results
were not unambiguous: the presence of different mediators caused elevated toxicity. The addition of
SA at different concentrations (0.1 – 1 mM) resulted in a >1000-fold increase expressed as TU. The
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presence of HBT caused a slight increase only at the highest concentration (1 mM) and there was even
a slight decrease in toxicity with the crude extract on its own. While identifying each metabolite was
impossible, this suggested that the by-products might be non-toxic in the last case. Elevated residual
toxicity in the presence of HBT was also reported by Nguyen et al. (2014c), using a similar model
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mixture and the P. leiognathi bioluminescence inhibition assay. When treating mixture containing
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CBZ, DCF, SMZ and ATZ in EMR the efficiency was enhanced substantially by adding SA it also
increased the toxicity of the effluent based on the result of the ToxScreen3 assay (Nguyen et al.,
2014b). Weng et al. (2013) compared the toxicity of SDM and SMM solutions treated by LMS using
Microtox®, and experienced toxicity increase after treatment with laccase-ABTS and laccase-SA, and
no change when laccase-VLA and laccase-HBA was used. Becker et al. (2016) received the same
results: high transformation efficiency of a mixture of 38 antibiotics was achieved by using laccase
and mediator SA in an EMR, slight increase in toxicity was measured by Microtox assay and
nonspecific toxicity was induced. Further treatment of the solution may solve this problem. Asif et al.
(2018) tested the treatment of 30 trace organic compounds with a laccase containing enzymatic
bioreactor coupled with membrane distillation and reported that, although the mediators enhanced the
degradation, they also increased the toxicity of the mixture in the reactor. However, due to the
separation step the toxicity of permeate was below detection limit.

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4.3.2 Estrogenic activity

In terms of xenobiotic chemicals, estrogenic effect may be critical. In order to evaluate this effect,
Yeast Estrogen Screen Assay (YES) can be used, developed by Routledge and Sumpter (1996). In the
original protocol, S. cerevisiae was used, but other taxa can be employed such as Cryptococcus spp.
and Candida spp. The principle of the test is that the test organism hosts the DNA sequence of the
human estrogen receptor (hER) and expression plasmids carrying estrogen-responsive sequences

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(ERE) integrated into its genome. The expression plasmids carry the reporter gene lac-Z (encoding the
enzyme β-galactosidase). In the presence of estrogens, β-galactosidase is synthesized and secreted
into the medium. The presence of estrogenic compounds is indicated by the appearance of red colour

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and can be assessed colorimetrically. Further improvements are reviewed by Xu et al. (2014).
Treatment of industrial intermediers octylphenol (OP), NP with laccase containing beads in fluidized
bed reactor resulted in the complete removal and YES test indicated loss of estrogenic activity, too

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(Catapane et al., 2013). Since BPA poses severe adverse effects, especially on the reproductive
system, the removal of estrogenic activity is one of the most important goal of the treatment. Tsutsumi
et al. (2001) found that treatment of BPA with laccase resulted in the complete removal of estrogenic

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activity after 6 hours. Evaluation of the treatment of different mixtures containing estrogenic
compounds and industrial chemicals with tests regarding estrogenic activity established that laccase
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can effectively remove the endocrine activity of EDCs (Becker et al., 2017).
In some cases of evaluating the enzymatic treatment in real wastewater samples Microtox test was not
able indicate any change in toxicity, so using other methods like inhibition of V. fischeri
luminescence, E-SCREEN test and the luciferase-transfected human breast cancer cell line (MELN)
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gene-reporter assay is recommended (Spina et al., 2015). In the E-SCREEN cell-proliferation

bioassay, MCF-7 breast cancer cells are used. In the presence of estrogen more cells are generated
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(Soto et al., 1995). The MELN cell line is obtained by transfecting MCF-7 cells (ERα-positive breast
cancer cells) with an estrogen-regulated luciferase gene (Balaguer et al., 1999). This gene is induced
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when hormone-like compounds bind to estrogen receptors. Spina et al. (2015) using the above-
mentioned methods investigated the change in the estrogenic activity in a laboratory scale enzymatic
treatment of xenobiotics (4-t-butylphenol, 2-hydroxybiphenyl, 4-n-octylphenol, BHA and E1) found
in the effluent of a wastewater treatment plant. Despite that the rates of the reaction were influenced
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by the matrix of the treated wastewater, relatively high conversions were achieved (>72%, except in
case of 2,4-DCPh and BPA) and all methods of toxicity evaluation indicated substantial
detoxification.
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5. Discussion
Extensive research was done on the treatment of organic micropollutants with oxidoreductase
enzymes. It was concluded that though many publications use the term ‘degradation products’ for the
products of the enzymatic reaction, complete degradation, i.e. mineralisation of organic
micropollutants cannot be achieved with enzymatic treatment. The products of the enzymatic
treatment with oxidoreductases are mostly oligomers that might form polymers and/or insoluble
components, therefore ‘transformation product’ is a more accurate term. That being said, it has to be
pointed out that in some cases, due to bond cleavage, compounds with smaller molecular weight can
form during the enzymatic reaction.

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Regarding the concentration change of the input materials, relatively high removal percentages could
be achieved in synthetic wastewater under optimal conditions with or without mediators for the
majority of organic micropollutants. The differences between reaction times (e.g. 1 vs. 8 h), enzyme
concentration (expressed in either activity unit or mass), sources of enzymes (e.g. laccase from T.
versicolor vs. Myceliophthore thermiphila) etc., while crucial to define optimal conditions for the
treatment, complicate the comparison of different experiments without knowing the kinetics of each
individual process. The efficiency may be influenced by which oxidoreductase was used in the
process. For example, TC removal varied between 16-100% with different types of laccases under
various conditions, 72-94 % with MnP and 45% with HRP. In some cases, the peroxidases

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outperformed laccase but the latter has the advantage of requiring only oxygen for the oxidation.
Laccases can be declared with great certainty to be the most researched enzymes for treating
micropollutants. Nonetheless there is still lack of information about the kinetics and inhibitory effects

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in environmental matrices for most of the micropollutants which would be essential for upscaling the
biocatalytic treatment and to create a viable technology.
Another barrier is the short lifetime of the enzymes. Several immobilisation solutions were tested and

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most of them achieved higher stability, longer lifespan or broadened the circumstances in which the
enzyme would function effectively. In some cases, the immobilisation caused higher removal
efficiency. Nonetheless the inactivation of the enzyme is a real risk from the point of view of viability

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which should be avoided. Information on the possible adsorption of compounds, encapsulation
material or on the CLEAs should also be collected to understand the mechanisms of micropollutant
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removal processes.
Despite the efficient removal of numerous micropollutants, some recalcitrant compounds e.g. IBU
(painkiller), ATZ (herbicide), CBZ (anticonvulsant) and several of the antibiotics could not be
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removed or their toxicity levels reduced. The reason for that lies in the chemical structure of the
pollutant, if it has a strong electron drawing functional group or its redox potential is not suitable, the
reaction will not take place. To a certain extent this can be overcome by using mediators, though for
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example CBZ remained recalcitrant. Notwithstanding the efficiency improvement achievable by

involving mediators, the type and amount should be carefully chosen because certain mediators or
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their transformation products remaining in the water after treatment may generate more toxic
effluents.
Unfortunately, the information on reaction products are scarce even for experiments carried out with
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synthetic solutions. The situation is even worse for environmental samples. From the articles
presented in Table S1, only eight used wastewater in their experiments, in two of those, information
on products were provided, while three had toxicity results. Based on the research with synthetic
solutions it can be concluded that coupling products provide the majority of the compounds after
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treatment which can be separated from the fluid, and the use of (synthetic) mediators may result in
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unwanted by-products. Other molecules in the wastewater effluent may modify the structure of the
mixture or even inhibit the catalysis itself. In other circumstances NOM may act as mediator, but in
any case product identification and/or ecotoxicity measurements are needed to evaluate the efficiency
of the enzymatic treatment of waterborne micropollutants.
Therefore, the investigation of enzymatic treatment of organic micropollutants should ideally include
the identification of reaction products as well as the evaluation of treatment efficiency from the
viewpoint of ecotoxicity. Determining the products poses challenges to the researchers at higher
concentrations if the composition of the sample is not fully known. Additionally, in the
environmentally relevant concentration range sophisticated sample preparation and measurement
techniques are needed even for quantifying the micropollutant let alone its metabolites. The NOM and
other non-toxic components in the matrix may also cause problems in the separation and/or the
measurement itself.

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Evaluating the toxicity of the pollutants and their products are not so straightforward, either. As it was
shown in section 4.3, several types of toxicity assays can be used depending on the sensitivity of the
organism to the compound. In some cases, the Microtox test, which is a common choice for
toxicological studies, does not show any change but it does not necessarily mean that the substance or
mixture is harmless for the environment. To evaluate ecotoxicity efficiently, several organisms have
to be tested to find one that is especially sensitive to the micropollutant in question. The situation is
even more complicated if chiral compounds are considered but only a few articles were found that
examined micropollutants at an enantiomer level. On the other hand, the measurement of chronic
toxicity and adverse effects at population level was rare in this context which could provide further

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information on the desired degree of treatment.
While more information on the residual ecotoxicity of the enzymatic treatment of micropollutants are
required, results have shown that in most cases lower levels of ecotoxicity and decrease in the

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estrogenic activity could be achieved after the treatment. Should membrane separation be added to the
process, the toxic compounds as well as the products with larger molecular weight can be retained
from the filtrate.

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6. Concluding remarks

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In order to progress from laboratory scale experiment to commercially available and feasible enzyme
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catalytic wastewater treatment technology, several obstacles have yet to be overcome. Several
experiments were done with synthetic wastewater but more information is required on the method
under environmentally relevant conditions. In realistic circumstances a mixture of different organic
compounds, including a vast variety of micropollutants would be found in the effluent sample. That
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also means kinetic measurements and evaluation of removal efficiencies in the concentration range
reported in wastewater effluents and other water bodies instead of using spiked samples.
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Understanding the difficulties of product identification in real wastewater, where the enzymes can
react with NOM beside the target compounds, the authors suggest using ecotoxicological result to
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assess the efficiency of the treatment and to monitor the polishing steps of wastewater treatment or
water reclamation process. For this, protocols using various test organisms sensitive to key
micropollutants should be developed so that misinterpretation due to insensitivity of an assay to
certain components could be avoided. Cost-effective enzyme production, along with efficient
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immobilisation and separation techniques are also required that are able to achieve economical
treatment around neutral pH and ambient water temperature.
Based on the findings of this review the following suggestions are made to close the knowledge gap
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that hinders the upscaling of the technology:

● Limitations of enzymatic treatment such as inactivation and inhibition have to be
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acknowledged and a combination of methods should be used. This could mean combining a
set of different oxidoreductases, using crude enzymes instead of purified, improving stability
and reusability by different immobilization techniques as well as implementing enzymatic
treatment to other technologies.
● Kinetic parameters should be defined for real life conditions to be able to design pilot-scale
and later commercial systems.
● Information of transformation products of enzyme catalytic processes are scarce regarding
municipal or industrial wastewater; thus more experiments should focus on identifying the
products to help understand the mechanism of the processes in more complex systems.
● Efficiency of treatment may be assessed by measuring toxicity or estrogenic activity instead
of determining the concentration decrease of input matter.

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● Research regarding formation of enantiomers in the treatment process is required with special
focus on their toxicity and other environmental effects.

Acknowledgements
The financial support of Széchenyi 2020 under the GINOP-2.3.2-15-2016-00016 is greatly
acknowledged. The authors would like to extend their appreciation for their help in collecting
informations for the manuscript to Hassaan Ali, Mohammed Muktar Nono and Richmond Addiah,

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Stipendium Hungaricum awardees and environmental engineer master students of the University of
Pannonia.

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