Degradation of diclofenac, trimethoprim, carbamazepine, and sulfamethoxazole by laccase from Trametes versicolor: Transformation products and toxicity of treated effluent

Article abstract of DOI:10.1080/10242422.2019.1580268

The degradation of diclofenac (DCF), trimethoprim (TMP), carbamazepine (CBZ), and sulfamethoxazole (SMX) by laccase from Trametes versicolor was investigated. Experiments were conducted using the pharmaceuticals individually, or as a mixture at different initial concentrations (1.25 and 5 mg/L each). The initial enzymatic activity of all the treated samples was around 430–460 U_(DMP)/L. The removal of the four selected pharmaceuticals tested individually was more effective than when tested in mixtures under the same conditions. For example, 5 mg DCF/L was completely removed to below its detection limit (1 μg/L) within 8 h in the individual experiment vs. after 24 h when dosed as a mixture with the other pharmaceuticals. A similar trend was visible with other three pharmaceuticals, with 95 vs. 39%, 82 vs. 34% and 56 vs. 49% removal after 48 h with 5 mg/L of TMP, CBZ, and SMX tested individually or as mixtures, respectively. In addition, at the lower initial concentration (1.25 mg/L each), the removal efficiency of TMP, CBZ, and SMX in mixtures was lower than that obtained at the higher initial concentrations (5 mg/L each) during both the individual and combined treatments. Four enzymatic transformation products (TPs) were identified during the individual treatments of DCF and CBZ by T. versicolor. For TMP and SMX, no major TPs were observed under the experimental conditions used. The toxicity of the solution before and after enzymatic treatment of each pharmaceutical was also assessed and all treated effluent samples were verified to be non-toxic.

Full text of DOI:10.1080/10242422.2019.1580268

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: https://www.tandfonline.com/loi/ibab20
Degradation of diclofenac, trimethoprim,
carbamazepine, and sulfamethoxazole by laccase
from Trametes versicolor: Transformation products
and toxicity of treated effluent
Sultan K. Alharbi, Long D. Nghiem, Jason P. van de Merwe, Frederic D. L.
Leusch, Muhammad B. Asif, Faisal I. Hai & William E. Price
To cite this article: Sultan K. Alharbi, Long D. Nghiem, Jason P. van de Merwe, Frederic D. L.
Leusch, Muhammad B. Asif, Faisal I. Hai & William E. Price (2019): Degradation of diclofenac,
trimethoprim, carbamazepine, and sulfamethoxazole by laccase from Trametesꢀversicolor:
Transformation products and toxicity of treated effluent, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2019.1580268
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2019.1580268
RESEARCH ARTICLE
Degradation of diclofenac, trimethoprim, carbamazepine, and
sulfamethoxazole by laccase from Trametes versicolor: Transformation
products and toxicity of treated effluent
a,b
c,d
e
e
Sultan K. Alharbi , Long D. Nghiem , Jason P. van de Merwe , Frederic D. L. Leusch ,
c
c
a
Muhammad B. Asif , Faisal I. Hai and William E. Price
a
Strategic Water Infrastructure Laboratory, School of Chemistry, University of Wollongong, Wollongong, NSW, Australian;
b
c
Department of Chemistry, Taibah University, Madinah, Saudi Arabia; Strategic Water Infrastructure Laboratory, School of Civil,
d
Mining and Environmental Engineering, University of Wollongong, Wollongong, NSW, Australia; Centre for Technology in Water and
Wastewater, School of Civil and Environmental Engineering, University of Technology Sydney, Sydney, NWS, Australia; Australian
e
Rivers Institute and School of Environment and Science, Griffith University, Brisbane, QLD, Australia
ABSTRACT
ARTICLE HISTORY
The degradation of diclofenac (DCF), trimethoprim (TMP), carbamazepine (CBZ), and sulfameth-
oxazole (SMX) by laccase from Trametes versicolor was investigated. Experiments were con-
ducted using the pharmaceuticals individually, or as a mixture at different initial concentrations
Received 4 August 2018
Accepted 29 January 2019
KEYWORDS
(
4
1.25 and 5 mg/L each). The initial enzymatic activity of all the treated samples was around
30–460U(DMP)/L. The removal of the four selected pharmaceuticals tested individually was
Laccase; T. versicolor;
pharmaceutical degradation;
transformation product;
toxicity assessment;
wastewater treatment
more effective than when tested in mixtures under the same conditions. For example, 5 mg
DCF/L was completely removed to below its detection limit (1 mg/L) within 8 h in the individual
experiment vs. after 24 h when dosed as a mixture with the other pharmaceuticals. A similar
trend was visible with other three pharmaceuticals, with 95 vs. 39%, 82 vs. 34% and 56 vs. 49%
removal after 48 h with 5 mg/L of TMP, CBZ, and SMX tested individually or as mixtures, respect-
ively. In addition, at the lower initial concentration (1.25 mg/L each), the removal efficiency of
TMP, CBZ, and SMX in mixtures was lower than that obtained at the higher initial concentrations
(5 mg/L each) during both the individual and combined treatments. Four enzymatic transform-
ation products (TPs) were identified during the individual treatments of DCF and CBZ by T. versi-
color. For TMP and SMX, no major TPs were observed under the experimental conditions used.
The toxicity of the solution before and after enzymatic treatment of each pharmaceutical was
also assessed and all treated effluent samples were verified to be non-toxic.
1
. Introduction
ecotoxicological impacts (Santos et al. 2010; Hai et al.
018), and effects on endocrine systems of some
2
Pharmaceutically active compounds (PhACs) such as
diclofenac (DCF), trimethoprim (TMP), carbamazepine
aquatic organisms (Hoeger et al. 2005; Fent et al. 2006).
Several advanced oxidation processes (e.g. ozonation,
UV photolysis, and UV/H O ) have been explored to
(CBZ), and sulfamethoxazole (SMX) have been regularly
2
2
reported in wastewater effluent, ground water, surface
water, and even drinking water (Kolpin et al. 2002;
Morasch et al. 2010; Santos et al. 2010; Vulliet and Cren-
Oliv ꢀe 2011; Al Aukidy et al. 2012; Ratola et al. 2012;
Padhye et al. 2014). Their occurrence has been attrib-
uted to their inefficient removal by wastewater treat-
ment plants (WWTPs) (K u€ mmerer 2009; Mi ꢁe ge et al.
degrade these PhACs. However, a number of toxic trans-
formation products (TPs) were reported in case of some
advanced oxidation processes (Alharbi et al. 2017).
Therefore, the removal of PhACs from water without
production of toxic TPs during the treatment processes
is important to improving water quality (Alharbi et al.
2017; Alharbi and Price 2017).
2
009; Luo et al. 2014). Although found in trace concen-
The degradation of PhACs using enzymes secreted
by white rot fungi (WRF) has recently attracted interest
as a method to obtain high removal efficiency and
non-toxic effluents (Yang et al. 2013). WRF comprises
a group of fungi which secret extracellular enzymes
trations, the occurrence of PhACs in the environment
has attracted much attention recently due to possible
undesirable effects including development of bacterial
resistance in the environment (Zhang et al. 2009),
CONTACT Faisal I. Hai
faisal@uow.edu.au
Strategic Water Infrastructure Laboratory, School of Civil, Mining and Environmental Engineering,
University of Wollongong, Wollongong, NSW, Australia
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
S. K. ALHARBI ET AL.
In the limited studies on the degradation of the
two antibiotics TMP and SMX by enzymatic treatment,
TMP was reported to be poorly removed by laccase
from T. versicolor (Touahar et al. 2014; Arca-Ramos
et al. 2016). Contradictory reports on the ability of lac-
case from T. versicolor to degrade SMX can be noted
in the literature. For example, 77% of 50 mg SMX/L
was removed by laccase from T. versicolor after 1 h of
incubation (Rahmani et al. 2015), whereas in a separ-
ate study, laccase from T. versicolor did not degrade
SMX (at 20–25 mg/L) within 72 h of incubation (Margot
et al. 2015). However, the addition of a redox-mediator
was able to achieve a complete elimination within 6 h
of incubation. Therefore, it is important that the deg-
radation of DCF, TMP, CBZ, and SMX by laccase from
T. versicolor and also the identification of any poten-
tially toxic TPs be more thoroughly investigated.
Figure 1. Chemical structures of the four PhACs selected.
Diclofenac, trimethoprim, carbamazepine and sulfamethoxazole
is denoted by DCF, TMP, CBZ and SMX, respectively.
The main aim of this work was to critically assess
the ability of laccase from T. versicolor to degrade four
selected PhACs, namely, DCF, TMP, CBZ, and SMX. This
investigation included the degradation of each PhAC
individually and in a mixture at different PhAC concen-
trations. A particular aim was to investigate the forma-
tion and fate of TPs during the enzymatic treatment,
including confirmation of the molecular structure of
the previously identified TPs using their m/z ratio and
mass fragmentation patterns. In addition, the relative
toxicity of the treated mixtures with the original com-
pounds was compared using a typical bacterial toxicity
test. The combined investigation of the TPs and the
toxicity of the test solution following enzymatic deg-
radation address a significant gap in the literature.
capable of degrading lignin, a complex natural com-
pound protecting softwood in trees. WRF secret lignin-
modifying enzymes, namely peroxidases, for example,
lignin peroxidases and manganese-dependent peroxi-
dases and laccase (Reddy 1995). Unlike peroxidases,
laccase can catalyse pollutant degradation using oxy-
gen dissolved in the reaction mixture.
Laccase secreted by different fungi may have differ-
ent redox potential (Piscitelli et al. 2011). The degrad-
ation of the four selected PhACs, namely, DCF, TMP,
CBZ, and SMX by laccase from Trametes versicolor has
been previously investigated (Marco-Urrea et al. 2009;
Tran et al. 2010; Margot et al. 2015; Ji et al. 2016). In
addition, the use of redox-mediators as the electron
acceptor during enzymatic treatments has been found
to improve biodegradation of some PhACs (Touahar
et al. 2014; Margot et al. 2015). The effectiveness of
DCF degradation by enzymatic treatments has been
reported to vary. Laccase from T. versicolor, either
crude or purified (i.e. commercially available), was
found to achieve high DCF removal (>70%) (Tran
et al. 2010; Margot et al. 2013; Nguyen et al. 2014a,
2. Materials and methods
2.1. PhACs and enzyme
Four PhACs including (DCF, TMP, CBZ, and SMX were
chosen for this study (Figure 1). Analytical grade
PhACs were purchased from Sigma-Aldrich (Castle Hill,
Australia). Commercial laccase powder from T. versi-
color (activity ꢀ0.5 U/mg) was also purchased from
Sigma-Aldrich.
2014b); and the use of a mediator was not found to
affect DCF removal (Tran et al. 2010). However, laccase
from other species was found to be less effective at
PhACs degradation compared to T. versicolor (Lloret
et al. 2010; Nguyen et al. 2014a, 2014b, 2015). CBZ
degradation by laccase from T. versicolor can vary sig-
nificantly, depending on the initial concentration,
enzymatic activity, and incubation time. For example,
about 38% and only 5% of 0.01 and 4.72 mg CBZ/L
were removed by laccase secreted by T. versicolor after
2.2. Enzymatic activity assay
The activity of laccase was measured using a previous
method by Paszczynski et al. (1991). Briefly, the oxida-
tion of a substrate of laccase, namely, 2,6-dimethoxy
phenol (DMP), at 10 mM was monitored for 2 min at
room temperature in 100 mM sodium citrate buffer
(pH 4.5) solution. The change in absorbance at 468 nm
due to degradation of DMP by laccase was monitored
4
and 96 h of incubation, respectively (Tran et al. 2010;
Ji et al. 2016).

BIOCATALYSIS AND BIOTRANSFORMATION
3
by a UV-visible spectrophotometer (DR3900, Hach,
Loveland, CO). Laccase activity (expressed as U(DMP)/L)
was calculated using the molar extinction coefficient
TPs. The Sep-Pak SPE cartridges purchased from
Waters (Rydalmere, NSW, Australia) possessed the fol-
lowing properties: C18, 6 cc; 55–105 mm particle size;
500 mg sorbent per cartridge. Before sample loading
at a flow rate of 1–5 mL/min, the cartridges were pre-
conditioned with (i) 7 mL dichloromethane and metha-
nol mixture (1:1 v/v), (ii) 7 mL of methanol, and (iii)
ꢁ
1
ꢁ1
of 49.6 mM cm
.
2.3. Enzymatic experimental procedure
Stock solutions containing 5 g/L of DCF, TMP, CBZ,
and SMX were separately prepared in pure methanol,
and then stored in amber bottles at ꢁ18 C prior to
7
mL of Milli-Q water. The TPs extracted were then
eluted from the cartridges using methanol (3 mL) at a
flow rate of 1–5 mL/min. This corresponded to a con-
centration factor of approximately 17.
ꢂ
use. The degradation of 5 mg/L of DCF, TMP, CBZ, and
SMX, by laccase from T. versicolor was first investigated
for the PhACs individually. Following this, the degrad-
ation of each PhAC in a mixture with the others was
investigated at different initial concentrations of the
individual PhACs (5 and 1.25 mg/L). These PhACs have
been detected in WWTPs at concentrations ranging
from 0.001 to 0.1 mg/L (Luo et al. 2014). In this study,
a relatively high concentration (i.e. 1.25 and 5 mg/L) of
each PhAC was selected in order to investigate the
formation of TPs. To generate TPs at detectable level,
it is a common practice to work with higher initial
concentrations of contaminants than environmentally
relevant (Hata et al. 2010; Jelic et al. 2012; Ji
et al. 2016).
2.4. Analytical methods
The analysis of the parent PhACs and any TPs were
performed by a liquid chromatography-electrospray
ionisation-mass spectrometer (LC-ESI-MS) (Shimadzu
single quadrupole LC-MS 2020). A series of standard
solutions for each parent compound was prepared for
calibration purposes at 1, 10, 50, 100, and 200 lg/L.
The sample injection volume was 20 lL. The separ-
V
R
ation of the compounds was carried out by a Kinetex
Pentafluorophenyl 100 A column (100 mm ꢃ 3 mm,
.6 lm) purchased from Phenomenex (Lane Cove,
2
NSW, Australia). The column temperature was main-
tained at 31 C. The mobile phase comprised of 0.1%
formic acid in Milli-Q water (A) and acetonitrile (B). It
was passed at a flow rate of 500 lL/min in the follow-
ing gradient elution (%B): 0.01 min (5%), 5 min (10%),
To investigate degradation efficiency, commercially
available laccase from T. versicolor (0.08 g) was added
to ultrapure Milli-Q water (50 mL) in aluminum foil
covered 250 mL beakers to achieve an initial enzym-
atic activity of ca. 430–460 U(DMP)/L. When the targeted
enzymatic activity was achieved, the selected PhAC
concentrations were added to the enzyme solution in
individual beakers. The beakers were incubated on a
ꢂ
2
0 min (45%), 23 min (90%), 28.1 min (90%), 29 min
(10%), 33 min (10%), and 35.01 min (controller stop).
Both positive and negative ion modes were used to
collect mass spectrometric data from m/z 150 to 700.
In addition, selected scan mode was used to look for
the particular m/z of the previously reported TPs of
DCF and CBZ after enzymatic treatment. All of the
detector parameters were held constant during ana-
lysis. These include interface temperature (350 C), des-
olvation line (DL) temperature (250 C), heat block
temperature (200 C), dry gas flow (3 L/min), and nebu-
lizing gas flow (1.5 L/min).
ꢂ
rotary shaker for 48 h at 80 rpm and 25 C. The pH of
the reaction media was between 6.8 and 6.9 at the
start of each experiment. The experiments were car-
ried out in duplicate. Sub-samples were collected after
1
0
, 2, 4, 8, 12, 24, and 48 h of incubation by taking
.5 mL and adding 0.5 mL of methanol to deactivate
ꢂ
ꢂ
the laccase to arrest further degradation. The treated
samples were then diluted to fit in the concentration
range of the calibration curve prior to analysis by LC-
MS to calculate the removal efficiency over time.
For the detection of TPs, 5 mg/L of DCF, TMP, CBZ,
and SMX was added to the laccase solution (activity of
ꢂ
2
.5. Toxicity assessment
At the end of the incubation period, the treated and
untreated solutions were collected and compounds
were extracted using SPE cartridges (Section 2.3) and
eluted in 2 mL of methanol. This corresponded to a
SPE concentration factor of 25. Samples were stored in
4
30–460 U(DMP)/L) in individual beakers, and incubated
as described above for 48 h. At the conclusion of incu-
bation, the treated effluent was collected and solid
phase extraction (SPE) was used to extract the com-
pounds prior to analysis by LC-MS (Alharbi et al.
ꢂ
2
016). Pre-concentration with SPE cartridges increased
2 mL amber vials and kept in a ꢁ18 C freezer prior to
the sensitivity of the LC-MS analysis to identify the
toxicity analysis.

4
S. K. ALHARBI ET AL.
contain one or more EDGs (Lloret et al. 2010; Nguyen
et al. 2014b, 2015). Enzyme-catalysed reactions gener-
ally follow the Michaelis-Menten kinetic model, thus, it
is also possible that degradation of PhACs by laccase
may have attained an equilibrium after certain extent
of removal (Berg, 2002). All the PhACs selected for this
study were non-phenolic and their removal by laccase
from T. versicolor during individual and combined
treatments are discussed in the following sections.
1
00
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
DCF
TMP
CBZ
SMX
3.1.1. Degradation of individual PhACs
The individual degradation of 5 mg/L of each PhAC by
laccase from T. versicolor was evaluated over an incu-
bation period of 48 h. The individual degradation of
the four selected PhACs by laccase appears to be
influenced by their molecular properties. Experiments
showed different reactivity of the four selected PhACs
toward the applied enzymatic treatment (Figure 2).
0
4
8
12 16 20 24 28 32 36 40 44 48
Time (h)
Figure 2. The individual degradation of 5 mg/L of each
pharmaceutical (DCF, TMP, CBZ, and SMX) by laccase from
T. versicolor.
The bacterial luminescence toxicity screen (BLT-
Screen) developed by van de Merwe and Leusch
2015) was used to assess the toxicity of samples
before and after the treatment. An aliquot of naturally
bioluminescent bacteria, Photobacterium leiognathi,
was incubated with a serial dilution of the sample
extracts in a phosphate buffered saline solution. After
ꢁ
4
ꢁ1 ꢁ1
The initial degradation rates (10 mgL
s ) of the
four PhACs were calculated during the first 4 h of the
incubation time. The maximal degradation rates of the
(
ꢁ
4
ꢁ1 ꢁ1
four PhACs (10
mgL
s ) were 5.0 ± 0.2 (DCF),
3
.9 ± 0.3 (TMP), 2.8 ± 0.1 (CBZ), and 2.55 ± 0.07 (SMX).
Five mg DCF/L was degraded to below the experi-
mental detection limit, that is, 1 mg/L within 8 h of incu-
bation, in agreement with a previous study (Margot
et al. 2013). The chemical structure of DCF includes an
aromatic amine (an EDG) which may enhance its oxida-
tion by laccase (Nguyen et al. 2014b). For CBZ and
TMP, fast degradation rates were initially observed
(0–4 h of incubation time) during individual treatments
of 5 mg/L of each PhAC. Then, both PhACs were grad-
ually degraded from 4 to 48 h of incubation time to
achieve total elimination of 95 and 82% for TMP and
CBZ, respectively. This finding confirms the capacity of
laccase from T. versicolor to efficiently degrade CBZ
without using a redox mediator (Tran et al. 2010). This
result somewhat contradicts previous literature findings
on the capacity of laccase from T. versicolor to degrade
CBZ. While previous studies have indicated that the
presence of the electron withdrawing functional group
3
0 min, luminescence was measured on a Fluostar
Omega plate reader (BMG Labtech, Ortenberg,
Germany). The luminescence inhibition was calculated
relative to a negative control. The concentration of
the sample required to inhibit bacterial luminescence
by 20% was denoted IC20. It was computed for each
sample by linear regression of the response between
0
and 40% inhibition. Results are presented as a rela-
tive toxicity unit (rTU), which is the reciprocal of
the IC20.
3
. Results and discussion
3
.1. Laccase-catalysed degradation of PhACs
Laccase-catalysed degradation of a compound com-
prises the transition of an electron from the com-
pound to laccase in presence of a co-factor (i.e.
dissolved oxygen) (Yang et al. 2013; Asif et al. 2017;
‘
amide’ could generate an electron deficiency and con-
sequently may reduce the susceptibility of CBZ to
enzymatic oxidation (Nguyen et al. 2014b), our results
indicate that susceptibilities do exist, and provides new
insight into CBZ degradation by laccase from
T. versicolor.
For SMX, only 56% of the initial concentration was
removed within 48 h. It could be concluded that DCF
was highly susceptible to the oxidation by laccase
from T. versicolor, whereas SMX was the most resistant
2
018). However, the extent of degradation by laccase
is significantly affected by the molecular properties of
the target compounds such as the presence of
aromatic or heterocyclic rings, electron donating
groups (EDGs), coupled bonds, and/or readily oxidized
substitutions (Tran et al. 2010). In addition, laccase can
efficiently degrade phenolic PhACs, while the degrad-
ation of non-phenolic PhACs is only possible if the
redox-potential of laccase is high enough or PhACs

BIOCATALYSIS AND BIOTRANSFORMATION
5
1
00
100
90
80
70
60
50
40
30
20
10
0
(
a) DCF
(b) TMP
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
0
4
8
12 16 20 24 28 32 36 40 44 48
0
4
8
12 16 20 24 28 32 36 40 44 48
1
00
100
90
80
70
60
50
40
30
20
10
0
(
c) CBZ
(d) SMX
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
0
4
8
12 16 20 24 28 32 36 40 44 48
0
4
8
12 16 20 24 28 32 36 40 44 48
Time (h)
Time (h)
Initial concentrations of each pharmaceutical
5 mg/L
1.25 mg/L
Figure 3. The effect of initial concentrations during degradation of (a) DCF, (b) TMP (c) CBZ, and (d) SMX in mixtures by laccase
from T. versicolor.
PhAC during individual treatments under the
applied conditions.
treatments. This observation is consistent with that in
a previous study which reported complete removal of
DCF by laccase (Tran et al. 2010). For TMP, CBZ, and
SMX, better removal efficiency was achieved during
treatment of the mixture at a higher initial concentra-
tion (5 mg/L each). Our results are consistent with
those in a previous study where the efficiency of
removal of several non-phenolic PhACs by laccase-
enzymatic membrane reactor increased at a higher ini-
tial concentration (Nguyen et al. 2016).
3.1.2. Degradation of PhACs in mixtures
The effect of different initial concentrations (i.e. 1.25
and 5 mg/L) of the four selected PhACs in mixtures
during treatments by laccase from T. versicolor was
also investigated (Figure 3). The degradation of 5 mg/L
ꢁ
4
of each PhAC in mixture showed initial rates (10
ꢁ
1
ꢁ1
mgL
.46 ± 0.05 (CBZ), and 1.7 ± 0.2 (SMX). In addition, at
initial PhAC concentrations of 1.25 mg/L each, the ini-
s ) of 2.23 ± 0.05 (DCF), 0.7 ± 0.1 (TMP),
0
3
.1.3. Effect of incubation time on degradation
ꢁ
4
ꢁ1 ꢁ1
tial degradation rates (10
were 1.41 ± 0.03 (DCF), 0.17 ± 0.01 (TMP), 0.15 ± 0.02
CBZ), and 0.22 ± 0.04 (SMX). Independent of the initial
mgL
s
) in mixture
In this study, degradation of PhACs by laccase was
evaluated over an incubation period of 48 h, and sam-
ples were collected after regular intervals to investi-
gate the significance of incubation time. It is clear
from Figure 2 that individual PhAC degradation by lac-
case was fast initially, and the majority of degradation
occurred within first 8 h. For DCF, a complete removal
was achieved within the first 8 h of incubation time
when DCF was tested individually as well as in a mix-
ture of PhACs at an initial concentration of 1.25 mg/L
(Figures 2 and 3(a)). On the other hand, the degrad-
ation of CBZ, SMX, and TMP in mixtures was observed
to be rapid during the first 12 h of incubation time
(
PhAC concentrations, the results confirmed that the
removal efficiency during the mixture treatments was
not as effective as the individual treatments (Figures 2
and 3). The degradation of the four selected PhACs
during combined treatments are likely affected by the
molecular properties of each PhAC, the total PhAC
concentrations, and the competition between the four
PhACs to be oxidized by laccase from T. versicolor.
DCF was highly degradable by laccase from T. versi-
color during both the individual and combined

6
S. K. ALHARBI ET AL.
using their particular m/z. A total of four enzymatic
TPs (two for DCF and two for CBZ) were detected at
low abundance during the degradation by laccase. On
the other hand, there were no TPs observed during
degradation of TMP and SMX in the MS spectrum in
either positive or negative scan mode.
For DCF, two isomer TPs with molecular ions of m/z
3
10 in a negative scan mode were detected (TP #1
and #2; Figure 4). These TPs were eluted through the
LC-MS column at 18.5 and 17.8 min, respectively,
before DCF elution at 21.5 min. Based on their polar-
ities, TP #1 was formed by hydroxylation of the ben-
zene ring that contains the two chlorine atoms, and
had the highest abundance. The fragment ions
obtained by MS of DCF TP #1 confirmed the previ-
ously identified structure in the literature: it has a
molecular weight of 311 g/mol, with molecular formula
C H Cl NO . Collision-induced dissociation of the
Figure 4. The two isomers of DCF TPs identified in this study.
Fragmentation of TP #1 is shown in red.
14
11
2
3
[
M–H]- molecular ion of TP #1 produced ions at m/z of
66.1 and 230.1. Mass losses from the [M–Hþ] ion
were then calculated to be 44 (CO ) and 80 (CO and
2
2
2
HCl) g/mol, respectively (Figure 4). These TPs were
also identified during the oxidation of DCF by ozone,
but at a higher abundance (Alharbi et al. 2016). They
were formed mainly via hydroxylation at the para pos-
ition of the amine group of the benzene rings, includ-
ing 4’-OH diclofenac and 5-OH diclofenac (Marco-Urrea
et al. 2010). Thus, their molecular structures differ by
one extra oxygen atom with respect to DCF, without
any changes in the benzene ring equivalence and
double bond. One of them was produced at a rela-
tively high abundance compared to the other.
However, once the DCF was completely removed,
both TPs were no longer detected, which indicates
that they were also highly degradable by laccase.
For CBZ, two TPs (TP #3 and #4; Figure 5) were
detected at low abundance during degradation of CBZ
by laccase. These TPs eluted at 14.6 and 15.4 min on
the reverse-phase column and had molecular ions at
m/z 253 and 196 in positive scan mode, respectively.
Therefore, their molecular weights were determined to
be 252 and 195 g/mol, with molecular formula of
Figure 5. CBZ TPs identified in the current study.
(Figures 3(b–d)). Then, their degradation by laccase
slowed down from 13 to 24 h of incubation, and
almost stopped for the remaining duration. This cease
of degradation is probably due to the inactivation of
laccase and/or kinetic limitations (Khlifi et al. 2010;
Ashe et al. 2016). Therefore, incubation time is an
important design criterion for determining the size of
the bioreactor during the development of a treatment
process. It should be carefully selected as overesti-
mation could result in increasing the capital cost of
the treatment process.
(C15H N O ) and (C13H NO), respectively. The forma-
12 2 2 9
tion of both TPs during degradation of CBZ by laccase
has been documented previously (Hata et al. 2010;
Jelic et al. 2012; Ji et al. 2016). TP #3 was identified as
11-epoxy-carbamazepine and consists of one extra
oxygen atom with respect to molecular formula of
CBZ. TP #4 was identified as (9(10H)-acridone). Two
other TPs with molecular weights of 253 and 180 g/
mol, were reported during degradation of CBZ in
another study (Jelic et al. 2012). However, none of
3
.2. Formation of TPs
The formation of TPs during the degradation of DCF,
TMP, SMX, and CBZ by laccase from T. versicolor was
investigated using LC-MS in both negative and posi-
tive scan mode (range, m/z 150 to 700). In addition, a
selected ion mass mode was also used to detect the
previously reported TPs during enzymatic treatments

BIOCATALYSIS AND BIOTRANSFORMATION
7
Table 1. Comparison of degradation of 5 mg/L of each PhAC by different oxidation processes.
Treatment process
CBZ
DCF
SMX
TMP
Reference
Ozonation
Dose (mg/L)
Removal (%)
1.6
100
7
2.3
100
11
4.5
100
13
2.8
100
15
Alharbi et al. (2016)
#
TPs
Solution toxicity
UV photolysis
Exposure time (min)
Removal (%)
Toxic
Toxic
Non-toxic
Non-toxic
60
25
13
8
100
15
8
100
12
90
71
5
Alharbi et al. (2017)
#
TPs
Solution toxicity
UV/H
Exposure time (min)
dose (g/L)
Removal (%)
TPs
Toxic
Toxic
Non-toxic
Non-toxic
2 2
O
60
0.12
99
12
Non-toxic
20
0.12
100
NA
Non-toxic
NA
NA
NA
NA
NA
90
0.12
97
2 2
H O
#
8
Solution toxicity
Non-toxic
Purified laccase from T. versicolor
Incubation time (h)
Enzymatic activity (U/L)
Removal (%)
48
430–460
82
8
430–460
100
48
430–460
56
48
430–460
95
This study
#
TPs
2
2
0
0
Solution toxicity
Non-toxic
Non-toxic
Non-toxic
Non-toxic
NA: data not available; TPs: transformation products.
these products was detected in the current study
using both negative and positive selected ion mode,
which indicated that they were not formed under the
applied experimental conditions. TP #3 has been previ-
ously identified during the degradation of CBZ by UV
with H O , whereas TP #4 was detected during both
TMP, there has not been any study to date on the for-
mation of TPs by laccase treatment.
3
.3. Solution toxicity assessment
Advanced oxidation processes, namely, UV photolysis,
UV/H , and ozonation may transform the PhACs to
2
2
direct UV photolysis and also UV with only a small
amount of H O (Alharbi et al. 2017).
O
2 2
a number of potentially toxic and/or resistant TPs
(Alharbi et al. 2016, 2017). To check if this was simi-
larly the case with enzymatic degradation, the toxicity
of the four PhACs (DCF, TMP, CBZ, and SMX), their lac-
case-treated solution mixtures, and the pure laccase
solution were evaluated. All the tested samples were
found to be non-toxic in the BLT-Screen (all <2.5 rTU)
at the PhAC concentrations tested. To the extent of
our knowledge, this is the first report on the toxicity
of TMP to bacteria before and after laccase treatment.
The data from this study indicate that the trans-
formation of the selected PhACs by laccase from T.
versicolor did not lead to the formation of any signifi-
cantly toxic TPs. A few previous studies have also
reported that laccase-mediated degradation of DCF,
CBZ, and SMX did not give rise to toxicity in the
treated effluent (Marco-Urrea et al. 2010; Margot et al.
2015; Ji et al. 2016). However, by simultaneously moni-
toring TPs and effluent toxicity, in this study, we are
able to infer further that the TPs detected were at
very low abundance (Section 3.2) which may not have
the ability to affect the toxicity level.
2
2
In conclusion, the laccase-mediated degradation of
DCF, TMP, CBZ, and SMX by laccase did not produce
any major TPs under the experimental conditions used
and LC-MS parameters employed. In addition, the
detection of minor TPs was challenging due to the for-
mation of many minor peaks in the LC chromatogram
from the enzyme matrix. Most of these peaks were
also observed after the treatment of each PhAC by lac-
case and had m/z ranging from 150 to 700 in either
negative or positive MS scan mode. Consequently, the
detection of new minor PhAC TPs could not be readily
achieved. For DCF and CBZ, the detection of minor
TPs was achieved by using their previously reported
m/z in the literature. On the other hand, there was
insufficient data in literature regarding the production
of TPs during degradation of SMX and TMP by T. versi-
color. For SMX, all the previous studies have treated
SMX in the presence of other PhACs and/or mediators
in order to improve SMX degradation (Shi et al. 2014;
Margot et al. 2015). Therefore, the reported TPs were
not fully transformed from SMX alone as there were
other chemical compounds involved in the applied
treatment. Additionally, in previous studies, the chem-
ical structures of some identified SMX TPs consisted of
the chemical structures of the mediators used. For
It is interesting to compare the enzymatic degrad-
ation of the four selected PhACs with that by
advanced oxidation processes (i.e. ozonation, UV pho-
tolysis, and UV/H O2 treatment) in terms of
2

8
S. K. ALHARBI ET AL.
degradation of parent PhAC, formation of TPs, and
toxicity assessment of treated solutions (Table 1).
These experiments were carried out under the same
experimental conditions (i.e. initial PhAC concentra-
tions, individual PhAC treatment, and solution media)
and using the same toxicity assessment assay (Alharbi
et al. 2016, 2017). Complete elimination of DCF, TMP,
CBZ, and SMX was achieved after the reaction with
DCF and CBZ by laccase from T. versicolor. For SMX
and TMP, TPs were not detected under the applied
treatment conditions and LC-MS parameters. All efflu-
ent samples treated by laccase from T. versicolor were
non-toxic based on the bacterial luminescence toxicity
assay. The advanced oxidation processes (AOPs) such
as ozonation and UV-H O may often be more effect-
2 2
ive than laccase-based treatment systems. However,
the treated effluent produced by AOPs can be more
toxic than the parent PhACs. Enzyme-based treat-
ments, therefore, offer great potential as an alternative
approach to the treatment of pollutants in wastewater
effluent. Future studies to verify these important find-
ings in continuous-flow enzymatic bioreactors oper-
ated under non-sterile conditions are recommended.
2.3, 2.8, 1.6, and 4.6 mg/L of ozone, respectively
(Alharbi et al. 2016). In addition, UV photolysis was
able to completely remove DCF and SMX within a
short time of UV exposure (8 min) (Alharbi et al. 2017).
On the other hand, TMP and CBZ were reported to be
resistant to direct UV photolysis, with approximately
71 and 25% removal after 90 and 60 min of UV expos-
ure, respectively. However, the combination of UV
with 0.12 mg H O /L prior UV exposure significantly
2
2
Acknowledgements
improved the removal of TMP and CBZ up to 97 and
9%, respectively. Therefore, laccase from T. versicolor
PhD Scholarship support from Taibah University, Saudi
Arabia, to Sultan Alharbi is gratefully acknowledged.
9
was able to achieve higher removals for TMP (95%)
and CBZ (82%) than that achieved by direct UV pho-
tolysis under the same initial PhAC concentrations.
A wide range of resistant TPs was reported during
degradation of the four PhAC by ozone, UV photolysis,
and UV/H O treatment. In addition, UV photolysis
caused an increase in toxicity when applied to DCF,
CBZ, and SMX (Alharbi et al. 2017). Similarly, ozonation
of CBZ and DCF resulted in an increase in the toxicity
of the treated solutions. This was ascribed to the for-
mation of several major TPs that are more toxic than
the original PhACs. Therefore, non-toxic effluent and
more efficient degradation of TMP and CBZ by laccase
than by direct UV photolysis indicate the relative
benefit of enzymatic degradation.
Disclosure statement
No potential conflict of interest was reported by the authors.
2
2
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