Effective bioremediation of clarithromycin and diclofenac in wastewater by microbes and Arundo donax L

Abstract

Bioremediation
of pharmaceuticals has gained large research efforts, but there is still a need to improve the performance of bioremediation systems by selecting effective organisms. In this study, we characterized the capability to remove clarithromycin (CLA) and diclofenac (DCF) by the bacterium Streptomyces rochei, and the fungi Phanerochaete chrysosporium and Trametes versicolor. The macrolide antibiotic CLA and the non-steroid anti-inflammatory DCF were selected because these are two of the most frequently detected drugs in water bodies. Growth and content of the PhCs and a DCF metabolite (MET) by the energy crop Arundo donax L. were also evaluated under hydroponic conditions. The removal rate (RR) by S. rochei increased from 24 to 40% at 10 and 100 µg CLA L−1, respectively, averaged over incubation times. At 144 h, the RR by P. chrysosporium was 84%, while by T. versicolor was 70 and 45% at 10 and 100 CLA µg L−1. The RR by S. rochei did not exceed 30% at 1 mg DCF L−1 and reached 60% at 10 mg DCF L−1, whereas approached 95% and 63% by P. chrysosporium and T. versicolor, respectively, at both doses. Root biomass and length of A. donax were strongly affected at 100 µg CLA L−1. CLA concentration in roots and shoots increased with the increase of the dose and translocation factor (TF) was about 1. DCF severely affected both shoot fresh weight and root length at the highest dose and concentration in roots and shoots increased with the increase of the dose. DCF concentrations were 16–19 times higher in roots than in shoots, and TF was about 0.1. MET was detected only in roots and its proportion over the parent compound decreased with the increase of the DCF dose. This study highlights the potential contribution of A. donax and the tested microbial inoculants for improving the effectiveness of bioremediation systems for CLA and DCF removal.

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Environmental Science and Pollution Research (2023) 30:77193–77209
https://doi.org/10.1007/s11356-023-27660-4
RESEARCH ARTICLE
Effective bioremediation ofclarithromycin anddiclofenac
inwastewater bymicrobes andArundo donax L
LauraErcoli1· RudyRossetto1· SabrinaDiGiorgi2· AndreaRaaelli1· MarcoNuti1· ElisaPellegrino1
Received: 12 January 2023 / Accepted: 11 May 2023 / Published online: 30 May 2023
© The Author(s) 2023
Abstract
Bioremediationof pharmaceuticals has gained large research efforts, but there is still a need to improve the performance of
bioremediation systems by selecting effective organisms. In this study, we characterized the capability to remove clarithro-
mycin (CLA) and diclofenac (DCF) by the bacterium Streptomyces rochei, and the fungi Phanerochaete chrysosporium and
Trametes versicolor. The macrolide antibiotic CLA and the non-steroid anti-inflammatory DCF were selected because these
are two of the most frequently detected drugs in water bodies. Growth and content of the PhCs and a DCF metabolite (MET)
by the energy crop Arundo donax L. were also evaluated under hydroponic conditions. The removal rate (RR) by S. rochei
increased from 24 to 40% at 10 and 100µg CLA L−1, respectively, averaged over incubation times. At 144h, the RR by P.
chrysosporium was 84%, while by T. versicolor was 70 and 45% at 10 and 100 CLA µg L−1. The RR by S. rochei did not
exceed 30% at 1mg DCF L−1 and reached 60% at 10mg DCF L−1, whereas approached 95% and 63% by P. chrysosporium
and T. versicolor, respectively, at both doses. Root biomass and length of A. donax were strongly affected at 100µg CLA
L−1. CLA concentration in roots and shoots increased with the increase of the dose and translocation factor (TF) was about
1. DCF severely affected both shoot fresh weight and root length at the highest dose and concentration in roots and shoots
increased with the increase of the dose. DCF concentrations were 16–19 times higher in roots than in shoots, and TF was
about 0.1. MET was detected only in roots and its proportion over the parent compound decreased with the increase of the
DCF dose. This study highlights the potential contribution of A. donax and the tested microbial inoculants for improving
the effectiveness of bioremediation systems for CLA and DCF removal.
Keywords 4’-Hydroxydiclofenac· Bioremediation· Clarithromycin· Diclofenac· Giant reed· Phanerochaete
chrysosporium· Streptomyces rochei· Trametes versicolor
Introduction
Over recent decades, the presence of pharmaceuticals com-
pounds (PhCs) and their metabolites in the aquatic environ-
ment has been documented worldwide (e.g., Fatta-Kassinos
etal. 2011; Wilkinson etal. 2022) at concentrations rang-
ing from few ng L−1 to hundreds μg L−1 (Castiglioni etal.
2006; Gros etal. 2010; Hughes etal. 2012; Petrie etal. 2015;
Wilkinson etal. 2022). Post-consumption excretion of PhCs
through urine and feces by humans and animals represents
the main and widespread source of PhCs released into the
environment, while pharmaceutical industries are the sec-
ondary point source (Jjemba 2006; Wilkinson etal. 2018).
The final concentrations of PhCs and/or their metabolites
in surface water is mainly affected by the removal of the
sewage treatment plants, breakdown in surface water, and
dilution by river flows and rainfall (Castiglioni etal. 2006;
Verlicchi etal. 2012). Although PhCs and their metabolites
are often present in low concentrations in water bodies,
bioaccumulation and biomagnification processes lead to an
increase in detectable biologically active molecules, with
toxic effects for both fauna and flora (Christensen 1998). In
addition, the presence of antibiotics at low concentration in
the wastewater leads to the improvement of bacteria resist-
ance against the existing antibiotics (Baquero etal. 2008).
Responsible Editor: Gerald Thouand
* Elisa Pellegrino
elisa.pellegrino@santannapisa.it
1 Crop Science Research Center (CSRC), Scuola Superiore
Sant’Anna, Piazza Martiri Della Liberta 33, 56127Pisa, Italy
2 Ministero Della Salute, Direzione Generale perl’Igiene e la
Sicurezza degli Alimenti e della Nutrizione, Rome, Italy
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77194 Environmental Science and Pollution Research (2023) 30:77193–77209
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The removal or transformation of PhCs and their metab-
olites by conventional wastewater treatment plants is only
partially achieved, as they were designed with the princi-
pal aim of removing easily or moderately biodegradable
compounds (Verlicchi etal. 2012; Garcia-Rodríguez etal.
2014). Additional technologies (i.e., ozonation, reverse
osmosis, advanced oxidation processes) can be included in
the depuration process, but their high cost limits the wide-
spread application (Göbel etal. 2005; Castiglioni etal.
2006; Grandclément etal. 2017). Therefore, it is impera-
tive to develop nonconventional technologies with low
operation and maintenance cost that are effective in PhC
decontamination.
One promising technique is bioremediation, using natural
biological activity (i.e., plants and microorganisms). This
technology has been applied to remove/transform toxic com-
pounds located in soils, sediments, groundwater, and surface
water with varying degrees of success according to pollutant
and environmental conditions (Juwarkar etal. 2010; Adams
etal. 2015). Among bioremediation techniques, constructed
wetlands (CWs) have been accepted as an attractive and eco-
nomic alternative for the improvement of the overall effluent
quality prior to discharge into surface waters (Verlicchi etal.
2013; Carvalho etal. 2014; Carvalho 2020). In these nature-
based systems, macrophytes, invertebrates, and microorgan-
isms can uptake, metabolize, or sequester PhCs and nutrients
(Li etal. 2014; Ilyas and van Hullebusch 2019). However,
despite the effective degradation of PhCs by CWs, one of
their main limitations is the low efficiency of removal since
they typically require low hydraulic loading rates, and there-
fore large surface areas. In addition, they need relatively long
retention times, as the length of time the water is in contact
with the substrate, biofilm, and plant roots affects the extent
to which the removal or biotransformation of the PhCs can
occur (Carvalho 2020).
Therefore, there is a strong need to improve the design
of these systems for their better performance by a proper
selection of the most efficient organisms, plants, and micro-
organisms, which tolerate the potential toxic effects of the
wastewaters, and grow and uptake/degrade the toxic con-
taminants. An additional criterion for the choice of plants
is the delivery of an economic income which can originate
from the utilization of their biomass as raw materials for
energy production or green chemistry (Pandey etal. 2016).
The most widely used plants for remediation purposes are
common reed (Phragmites australis) (Carvalho etal. 2012;
Hijosa-Valsero etal. 2010), Typha spp. (Dordio etal. 2010),
and giant reed (Arundo donax) (Elhawat etal. 2014; Coppa
etal. 2020; Zhang etal. 2021). However, to our knowledge,
A. donax, selected for this study, was not previously tested
for the removal or biotransformation of PhCs. This plant spe-
cies can be grown as energy crops and for phytoremediation
purposes and has shown interesting yields of lignocellulosic
biomass from shoots under low fertility conditions, along
with a preferential allocation of contaminants in rhizomes
that can be harvested at the end of the phytoremediation
program (Fagnano etal. 2020; Zhang etal. 2021).
Among the PhCs most frequently detected in the aquatic
environment (e.g., Gavrilescu etal. 2014; Verlicchi etal.
2012), clarithromycin (CLA), an antibiotic, and diclofenac
(DCF), a non-steroidal anti-inflammatory drug, were
selected as the target molecules in the present study. CLA
is a macrolide antibiotic and one of the most prescribed
drugs in human medicine to treat upper and lower respira-
tory tract infections, as well as skin and mycobacterial infec-
tions (Kummerer and Henninger 2003; Pereira etal. 2013;
Cardini etal. 2021). The presence of CLA in wastewater
effluents has been reported in various studies at concentra-
tions ranging from 12 to 536ng L−1 worldwide (Miao etal.
2004; Miege etal. 2009; Fatta-Kassinos etal. 2011; Verlic-
chi etal. 2012; Michael etal. 2013). Li etal. (2014), in a
review summarizing the state of research activities on the
application of CWs for removing PhCs from wastewater,
showed that the removal efficiency of CLA was from 18%
in conventional waste-water treatment plants (WWTPs) to
31% in CWs. Similarly, Verlicchi and Zambello (2014) esti-
mated removal efficiency of CLA ranging from 10 to 60% in
CWs, acting as secondary and tertiary steps. DCL is widely
used in human and veterinary medicine to reduce inflam-
mation and pain (Caracciolo etal. 2015). It is one of the
most commonly detected PhCs in effluents of WWTPs, at
concentrations ranging from < 1 to 4110ng L−1 (Miege etal.
2009; Gavrilescu etal. 2014; Luo etal. 2014). The removal
efficiency of DCF from conventional WWTPs widely var-
ies from 17% (Heberer 2002) to 75% (Daughton and Ternes
1999), and higher efficiencies are associated to the addi-
tion of advanced procedures, such as chemical degradation
assisted by specialized microorganisms, or UV light action.
Zhang etal. (2014) in a review, summarizing the PhCs and
personal care products removal performance in different
aquatic plant-based systems, estimated a removal efficiency
of DCF up to 87% in hybrid CW systems applied as alterna-
tive secondary treatments, and up to 98% in hybrid CWs
applied as tertiary treatments.
Trametes versicolor and Phanerochaete chrysosporium
are white-rot fungi able to produce extracellular enzymes
(i.e., lignin peroxidases and manganese peroxidases) that
can degrade lignin and are also able to mediate oxidation
of a wide variety of organic pollutants and heavy metals,
as a result of the non-specificity of their enzyme system
(Baldrian 2008; Cameron etal. 2000). For PhCs, some
experiments have demonstrated the degrading ability of
white-rot fungi. Marco-Urrea etal. (2009) investigated the
degradation ability of ibuprofen, clofibric acid and car-
bamazepine by four white-rot fungi (T. versicolor, Irpex
lacteus, Ganoderma lucidum, and P. chrysosporium).
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77195Environmental Science and Pollution Research (2023) 30:77193–77209
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Whereas ibuprofen was extensively degraded by all the
fungi tested, T. versicolor was the only species able to
degrade either cloficric acid and carbamazepine, although
the latter was also degraded by G. lucidum. Marco-Urrea
etal. (2010), investigating the degradation of DCF by T.
versicolor, observed in liquid media almost a complete
removal (≥ 94%) in about 30’ at a concentration of 10mg
L−1 and 45μg L−1. Trametes versicolor was reported to
be able also to degrade CLA (≥ 96%) at an initial concen-
tration of 76ng L−1 in inoculated sterile and not-sterile
membrane biological reactor sludge (Llorens-Blanch etal.
2015).
The aim of this study was to characterize the microbial
and plant bioremediation of CLA and DCF from aquatic
media. This study presents results from two experiments.
The first experiment was carried out to assess the capabil-
ity of the bacteria Streptomyces rochei, and two fungi, P.
chrysosporium and T. versicolor, to remove CLA and DCF.
The second experiment aimed to evaluate plant growth and
uptake of CLA and DFC by A. donax L. grown in mesocosm
under hydroponic solution. The microbial strains used in
the first experiment were S. rochei DSM 41,732, P. chrys-
osporium DSM 1556 and T. versicolor DSM 11,309, which
have been considered good candidates for bioremediation
because they use a wide range of C sources and are natu-
rally occurring microorganisms in the rhizosphere of plants
(Bumpus etal. 1985; Pointing 2001). The second experi-
ment with A. donax L. was performed in mesocosm under
hydroponic conditions to avoid the potential interference of
soil or other wetland substrate particles that could adsorb
the tested PhCs (Liu etal. 2013). Moreover, considering
the potential utilization of the aerial plant part for energy
purposes, the partitioning of CLA and DCF among plant
parts has been assessed. The achieved results would provide
solutions for the improvement of the design of effective and
cost-efficient nature-based depuration plants to remove PhCs
from wastewaters.
Materials andmethods
Microbial degradation ofCLA andDCF
Microbial strain propagation
Streptomyces rochei DSMZ 41,732 stock cultures were
maintained at 28°C in a GYM Streptomyces medium
(DSMZ, medium 65), whereas P. chrysosporium DSMZ
1556 and T. versicolor DSMZ 11,309 and stock cultures
were maintained at 25°C and 35°C, respectively, in
malt extract peptone agar (medium 90) (DSMZ) (Fig.1)
(DSMZ n.d.).
Set‑up oftheexperiment ofCLA andDCF microbial
degradation
A batch experiment was set up for assessing the efficacy
of degradation of CLA and DCF by S. rochei, P. chrys-
osporium, and T. versicolor. For S. rochei, a spore sus-
pension was prepared from the stock plates. The culture
surface of each plate was gently scraped with a sterilized
spatula, and spores were suspended in 9mL of sterile water
(Kieser etal. 2000). The suspension was filtered through
a syringe with a non-absorbent cotton wool sterile filter
(Termo Fisher Scientific, USA), and then, 9mL of glycerol
solution (40% v/v) were added to each tube containing the
spore filtrate. The tubes were shaked in a vortex mixer to
homogenize the solution and stored at − 20°C until further
use. For each suspension, additional plates on agar medium
were set up in order to determine the colony forming units
(c.f.u.) per mL of suspension by the method of serial dilu-
tions. An average of 1 × 109 c.f.u. of S. rochei per mL of
solution was detected. Then, two pre-inoculum Erlenmeyer
flasks (150mL) were prepared by the addition of 1mL of
the spore suspension to 99mL of the DSMZ medium 65.
Since S. rochei tends to grow forming a rather compact
masses or pellets in liquid medium (Hobbs etal. 1990), 50
glass beads were inserted in each flask to encourage the dis-
persed growth of the bacterial biomass (Kieser etal. 2000).
Flasks were covered with aluminum foils and incubated at
28°C in an orbital shaker (IKA KS 4000 I Control Shaker,
Germany) at 110rpm. The pre-inoculum of S. rochei was
kept in the orbital shaker for 48h. For the preparation of the
fungal pre-inocula of P. chrysosporium and T. versicolor,
an agar disk (ca. 5mm in diameter) taken from the exter-
nal portion of a stock culture was transferred in each flask
containing 30mL of liquid medium (DSMZ medium 90).
Two flasks per each fungus were set up and incubated in the
orbital shaker at 30°C and 110rpm for 4days, with steel
beads in order to homogenize the fungus.
The degradation experiment with the bacterial/fungal
inoculum was carried out in Erlenmeyer flasks of 125mL.
The bacterial/fungal mycelium was collected from the pre-
inoculum flasks, centrifuged at 3000rpm for 10min, washed
in sterilized UHQ water, and divided in four aliquots that
were then resuspended in 50mL of the appropriate medium.
Two solutions were used for the determination of the con-
centration of the inoculum (oven-dry weight at 50°C), while
two solutions were used as inoculum. The pre-inoculum
concentration was about 2g L−1 on a dry weight basis. An
inoculum of ca. 10mg of mycelium was taken from the
pre-inoculation flasks and applied to the flask utilized to
the degradation experiment. This corresponds to a concen-
tration of mycelium in the inoculated flask of 0.1g L−1.
CLA was added to the flasks to give a final concentration
of 10 e 100µg L−1 (Merck, Darmstadt, Germany), while
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77196 Environmental Science and Pollution Research (2023) 30:77193–77209
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DCF (sodium salt, Cayman Chemical, Michigan, USA) was
added to a final concentration of 1mg L−1 and 10mg L−1.
To summarize, for the three microorganisms, each flask was
inoculated with 5mL of inoculum, spiked with the differ-
ent doses of PhCs and filled up to 100mL of the appropri-
ate growth medium. Thus, the experiments of CLA/DCF
microbial degradation were set-up following a full-factorial
completely randomized design with two concentrations of
the PhC (CLA doses: 10 and 100µg L−1; DCF doses: 1 and
10mg L−1) and five sampling time (Tinc: 0, 24, 48, 72, and
144h). To evaluate the possible adsorption of the PhCs by
dead microorganisms, the experiment included also killed
controls, which consisted of inoculated flasks that were then
autoclaved at 120°C for 20min before the addition of the
PhCs. Each treatment was replicated four times (n = 4); thus,
a total of 48 flasks were set-up for each PhCs. All the cul-
tures were grown at 28°C in the orbital shaker at 110rpm.
Moreover, to exclude the possible influence of light on DCF
and CLA stability, all the replicates were maintained in the
dark using aluminum foil.
Sampling andanalyses
Samples were collected from each flask at 0, 24, 48, 72, and
144h for the determination of CLA/DCF concentration. Two
mL were sampled from each flask and then centrifuged at
17,000rpm for 20min. Finally, the supernatant was trans-
ferred in amber HPLC vials, without moving the bacterial
pellet. The LC–MS/MS analyses were performed on a PE
Sciex API 365 triple quadrupole mass spectrometer (AB
Sciex LLC, Framingham, MA, USA) equipped with a Turbo
Ionspray source, interfaced to an Agilent 1100 HPLC system
with binary pump and auto-sampler (Agilent, Santa Clara,
CA, USA) (McArdell etal. 2003; Pierattini etal. 2018).
The separation was carried out by a Phenomenex Synergi
Fusion 2 × 75mm column, 5µm particle size (Phenomenex,
Torrance, CA, USA) using the following chromatographic
conditions: mobile phase A, acetonitrile with 0.1% formic
acid, mobile phase B, water with 0.1% formic acid; gradi-
ent: flow rate, 400 µL/min; 0–1min, A 10%; 1–8min, A to
95%; 8–10min, A 95%. The analytes (CLA and DCF) were
Fig. 1 Streptomyces rochei DSMZ 41,732 (a, b) stock bacterium cul-
ture maintained in a GYM Streptomyces medium; Phanaerochete
chrysosporium DSMZ 1556 (c, d) and Trametes versicolor DSMZ
11,309 (e, f) and stock fungal cultures maintained in malt extract pep-
tone agar. Examples of the experimental set-up utilized for studying
the Arundo donax growth and uptake of clarithromycin (CLA) and
diclofenac (DCF) at different pharmaceutical doses and time of expo-
sure(g, h)
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77197Environmental Science and Pollution Research (2023) 30:77193–77209
1 3
determined using the SRM (selected reaction monitoring)
technique, monitoring two fragmentations for each compo-
nent. SRM details and retention times of the analytes are
reported in the TableS1. After the LC–MS/MS analysis, the
removal rate of CLA/DCF was calculated for each sampling
time as [100– ([CLA/DFC]x/([CLA/DFC]0) × 100]. A cali-
bration curve for DCF (Sigma-Aldrich, Germany) was built:
R2 = 0.993 and concentration range 20–10,000ng mL−1
DCF. Similarly, a calibration curve for CLA (Sigma-Aldrich,
Germany) was built: R2 = 0.989 and concentration range
1–10,000ng mL−1 CLA. Data were normalized according
to matrix effect, calculated as peak area of the sample spiked
after extraction/peak area of the standard, and recovery per-
centages of 99.7% and 98.9%, respectively, calculated as
peak area of the sample spiked before extraction/peak area of
the sample spiked after extraction. Limit of detection (LOD)
of DCF and CLA was 1ng mL−1 and 0.5ng mL−1, respec-
tively, while limit of quantification (LOQ) of DCF and CLA
was 3.18ng mL−1 and 1.60ng mL−1.
Plant growth anduptake ofCLA andDCF byArundo
donax
Plant material andset upofthemicrocosm experiment
Plants of A. donax from invitro cultures (micropropa-
gated plants) were acclimatized for 5weeks in a growth
chamber under controlled environmental conditions
(23:18°Cday:night temperature, 65/70% relative humidity,
16h photoperiod at 400µmol m-2 s-1 photosynthetic photon
flux density supplied by fluorescent lights). During the accli-
mation process, 15 plants were grown in steel containers
of 3 L in Hoagland nutrient solution (a total of 30 contain-
ers), continuously aerated by aquarium pumps (a total of
450 plants). Plants were held in place in the lids of the pots
by a layer of polystyrene. The nutrient solution was replaced
every 2weeks.
After 5weeks of acclimation (T0), when new roots and
leaves had developed, plants of same size (approximately
10cm height) were selected and transferred to steel contain-
ers of 3 L (8 plants per container), which contained aerated
Hoagland nutrient solution spiked respectively with 10 and
100mg L-1 of CLA and 1 and 10mg L-1 of DCF. The con-
trol plants for each PhC were not spiked. Plants were grown
in the growth chamber maintaining the controlled environ-
mental conditions applied for acclimatation. For CLA, each
treatment was replicated six times (n = 6), whereas for DCF,
it was replicated four times (n = 4). The containers were
arranged in a completely randomized design. An example of
the experimental set-up is reported in Fig.1g, h. To exclude
the effect of root-associated microbial communities in the
degradation of CLA/DCF, the plants roots were sterilized
in NaClO solution (8%) before PhC application and sterility
checked by plating onto Nutrient agar plates. For CLA, 18
containers were set up (3 doses × 6 replicates), whereas for
DCF, 12 containers were set up (3 doses × 4 replicates). In
order to avoid photodegradation of CLA and DCF, the con-
tainers were covered with aluminum foils. Moreover, abiotic
controls were set up to exclude other dissipation mechanisms
like volatilization, photooxidation, or adsorption.
Plant physiological measurements: growth parameters
andchlorophyll content
Four acclimated plants per replicate were sampled at the
beginning of the experiment (T0). Then, in the CLA exper-
iment, four plants were sampled after 18 and 30days of
growth (T18 and T30, respectively), whereas in the DCF
experiment, four plants were samples after 18days of growth
(T18). At each sampling, plants were carefully washed with
deionized water and separated into roots and shoots (stems
and leaves) for fresh weight (FW) and dry weight (DW)
determination (oven dried at 70°C to constant weight). In
addition, stem and leaf number was recorded, as well as the
occurrence of any visual symptom of injury. After image
capture of leaves by 16 MP Samsung SM-A520F mobile
phone camera, the total leaf area was determined using the
open-source image processing program ImageJ (https://
imagej. net/ downl oads), while root length was measured
using the semi-automated digital image analysis tool HyL-
ength (Cardini etal. 2020). Chlorophyll concentration in
leaves was determined by a SPAD meter (SPAD-502 chlo-
rophyll meter, Konica Minolta, Osaka, Japan).
Extraction andquantification ofCLA/DCF fromplant tissues
At the sampling times T18 and T30 in the CLA experiment
and at T18 in the DCF experiment, 0.5g of fresh roots and
shoot were collected for the analysis and stored at − 80°C
until extraction for the analysis of CLA and DCF concentra-
tion in plant tissues. The four plants from individual contain-
ers were pooled in the sample collection. Plant organs (roots
and shoots) were finely grounded in liquid N2, transferred
into tubes and then weighted. Two milliliter of methanol
was added to each tube. The extracts were sonicated for
5min and centrifuged at 17,000 × g for 10min. After cen-
trifugation, the supernatant was filtered through a 0.45-µm
syringe cellulose acetate membrane filters (Sigma-Aldrich,
Germany) and stored at − 20 °C until further analysis.
Quantification of CLA/DCF was made by LC–MS/MS, as
described above. In addition to the analytes CLA and DCF,
4’-hydroxydiclofenac was determined. A calibration curve
for DCF (Sigma-Aldrich, Germany) was built: R2 = 0.997
and concentration range 10–10,000ng mL−1 DCF. Similarly,
a calibration curve for CLA (Sigma-Aldrich, Germany) was
built: R2 = 0.994 and concentration range 1–10,000ng mL−1
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77198 Environmental Science and Pollution Research (2023) 30:77193–77209
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CLA. Data were normalized according to matrix effect,
calculated as peak area of the sample spiked after extrac-
tion/peak area of the standard, and recovery percentages of
99.5% and 99.1%, respectively, calculated as peak area of
the sample spiked before extraction/peak area of the sam-
ple spiked after extraction. LOD of DCF and CLA was
0.7ng mL−1 and 0.4ng mL−1, while LOQ of DCF and CLA
was 2.20ng mL−1 and 1.27ng mL−1.
Plant uptake of CLA/DCF was calculated by multiplying
CLA/DCF concentrations in the plant tissues by DW. The
partitioning of CLA/DCF in roots and shoots was calculated.
The ability of A. donax plants to accumulate the studied
PhCs from the nutrient solution in roots and shoots during
the experiment was estimated using the bioaccumulation fac-
tor (BAF), which was calculated as the ratio of CLA/DCF
concentration in roots and shoots (in fresh weight basis) and
in the nutrient solution. The translocation factor (TF) was
calculated as the ratio of CLA/DCF concentration in the
shoots and the concentration in roots.
Statistical analysis ofresults
Data on the removal rates of CLA and DCF collected in the
experiment of PhC microbial degradation were analyzed by
a two-way analysis of variance (ANOVA), using dose and
time of incubation as fixed factors. For the A. donax experi-
ment, a two-way ANOVA was performed using CLA dose
and time of exposure as fixed factors. Similarly, for the A.
donax experiment, a one-way ANOVA was performed using
the DCF dose as fixed factor. Data were ln- and arcsine-
transformed when needed to fulfil the assumptions of the
ANOVA. Differences between means were assessed by a
post-doc Tukey B test. Means and standard errors given in
figures and supplementary tables are for untransformed data.
All these analyses were performed in SPSS version 21.0
(SPSS Inc., Chicago, Illinois, USA).
Results
Microbial degradation ofCLA
In the batch experiments with S. rochei and P. chrys-
osporium, the removal rate of CLA was significantly affected
by the main effect of PhC concentration (dose) and time of
incubation (Tinc), whereas with T. versicolor, the removal
rate was affected by the interaction of the two factors (Fig.2;
Supplementary TableS2). The removal rate by S. rochei
increased from 24% at 10µg CLA L−1 to 40% at 100µg L−1,
and increased during incubation time until 72h, after that it
was unchanged (ca. 47%). Conversely, the removal rate by
P. chrysosporium decreased from 55% at 10µg CLA L−1 to
46% at 100µg L−1, and during incubation it raised to 42% at
24 and 48h and to 84% at 72 and 144h. Trametes versicolor
Fig. 2 Effect of clarithromycin (CLA) concentration (dose) and time
of incubation (Tinc 0, 24, 48, 72, and 144h) on the removal rate of
CLA by Streptomyces rochei and Phanaerochete chrysosporium
and of the interaction between dose and Tinc on the removal rate of
CLA by Trametes versicolor. Effect of the interaction between dose
and Tinc on the removal rate of diclofenac (DCF). Concentrations in
the nutrient medium were for CLA 10 and 100µg L−1 and for DCF
1 and 10mg L.−1. The Tinc were 0, 24, 48, 72, and 144h. Data are
mean ± SE (n = 4). Different letters indicate significant differences at
P ≤ 0.05. Details about the two-way ANOVAs are given in Supple-
mentary TableS2
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77199Environmental Science and Pollution Research (2023) 30:77193–77209
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progressively degraded CLA with the increase of the incuba-
tion time, but the rates of increase were higher at 10 than at
100µg CLA L−1. As a consequence, at 144h, the removal
rate was 70% at 10µg CLA L−1 and 45% at 100µg CLA L−1.
Microbial degradation ofDCF
The removal rate of DCF varied according to the interaction
between dose and time of incubation (Fig.2; and Supple-
mentary TableS2). At all incubation times, S. rochei was
less effective at the lower than at the higher concentration
(1 and 10mg L−1, respectively). At 1mg DCF L−1, the
removal rate was lower than 20% up to 48h and did not
exceed 30% at 72 and 144h. At the higher concentration
(10mg DCF L−1), already after 24h, the removal rate was
60%, and remained unchanged until 144h. Conversely, P.
chrysosporium was highly effective at both concentrations
since the removal rate at 144h approached 97% and 93% at
1 and 10mg L−1, respectively. However, at the intermediate
incubation time of 72h, the removal rate was higher at 1mg
DCF L−1 than at 10mg L−1 (82% vs 42%). Finally, T. versi-
color progressively degraded DCF with the increase of incu-
bation time up to 62–63% of the initial DCF concentration at
both concentrations, but with the lowest DCF concentration,
the highest removal rate was achieved already at 72h (ca.
60%), while with the highest DCF concentration, the highest
removal rate was achieved at 144h (ca. 63%).
Growth anduptake ofCLA byArundo donax
Plants harvested just before the addition of CLA (T0) had an
average of 1.2 culms per plants and 8 green leaves per plant,
total fresh weight was 1.64g plant−1 (shoot plus roots) and
plant height was 10cm (data not shown). Root fresh weight
and root length were significantly modified by CLA concen-
tration (dose) in the growth medium and differed over time
(Fig.3c–f; Supplementary TableS3). Averaged over time,
root fresh weight at 10µg CLA L−1 did not change compared
to the untreated control, while it significantly increased by
48% at 100µg CLA L−1 (Fig.3c). Root length showed an
opposite pattern as it was unchanged at 10µg CLA L−1
respect to the untreated control and significantly decreased
by 27% at 100µg CLA L−1 (Fig.3e). Thus, the morphol-
ogy of the root system of A. donax changed in response to
CLA supply: in plants treated with 10µg CLA L−1 the ratio
root length/root fresh weight did not change compared to
Fig. 3 Effect of clarithromycin (CLA) concentration in the nutrient
medium (0, 10, and 100µg L.−1) (dose) on fresh weight of shoots (a)
and roots (c), and root length (e) of Arundo donax; effect of time of
exposure (18 and 30days: T18 and T30) on fresh weight of shoots
(b) and roots (d) and root length (f) of Arundo donax; effect of the
interaction between CLA concentration and time of exposure on the
SPAD of leaves(g). Data are mean ± SE (n = 6). Different letters indi-
cate significant differences at P ≤ 0.001. Details about the two-way
ANOVAs are given in Supplementary TableS3
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77200 Environmental Science and Pollution Research (2023) 30:77193–77209
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the untreated plants (819.2 vs 787.0cm g−1), whereas in
plants treated with 100µg CLA L−1, the ratio dropped to
391.4cm g−1. Averaged over CLA dose, root fresh weight
and root length increased by 215% and 37% from T18 to T30
(Fig.3d, f). Moreover, visual observations showed that in
plants treated with the highest dose of CLA, the root system
consisted of only short and thick main roots.
Shoot fresh weight, leaf area, and number of stems were
not affected by CLA dose and did not vary across time
(Fig.3a, b; Supplementary TableS3), whereas the number
of leaves increased from T18 to T30 (13 vs 16 leaves) (Sup-
plementary TableS3). Leaf SPAD readings were affected
by the interaction between CLA dose and time: at T18, they
slightly increased with the increase of CLA concentration
in the medium, whereas at T30 values, they were higher at 0
and 10µg CLA L−1 and slightly decreased (− 6%) at 100µg
CLA L−1 (Fig.3g; Supplementary TableS3).
CLA concentration and content in roots were affected
by the interaction between CLA dose and time of expo-
sure (Fig.4a, c; Supplementary TableS4). CLA was not
detected in roots of the untreated plants, while concen-
tration of CLA in roots increased with the increase of
CLA dose at T18 up to 1.8µg g−1, whereas at T30 val-
ues at both doses, it did not exceed 1.8µg g−1 (Fig.4a).
CLA content in roots showed a different pattern: values
increased with the increase of CLA dose, but at T30, the
values were significantly higher than at T18 at both CLA
doses (Fig.4c). CLA concentration and content in shoots
were affected only by CLA dose and they did not show
statistically significant differences between T18 and T30
(Fig.4b, d; Supplementary TableS4). Averaged over sam-
pling times, CLA was not detected in the shoots of the
untreated plants, while shoot CLA concentration increased
with the increase of the dose from 0.91 to 1.50µg g−1
(Fig.3b). Similarly, CLA content in shoots increased
with CLA dose, but differences between the doses 10 and
100µg L−1 were statistically not significant (on average
4.5µg plant−1) (Fig.4d). Overall, at T30, CLA content
was higher in shoots than in roots at both CLA doses,
about three-fold and over two-fold at 10 and 100µg CLA
L−1, respectively (Supplementary TableS4). CLA content
at T30 in the whole plant increased from 4.6 to 6.3µg
plant−1 with the increase of CLA dose from 10 to 100g
L−1, and it was partitioned for 75% and 71% into shoots,
respectively (Supplementary TableS4).
The bioaccumulation factor (BAF) of roots/shoots,
calculated as the ratio of CLA concentration in roots/
shoots (fresh weight basis) and in the nutrient solution,
was significantly affected by the interaction between
CLA dose and time of exposure (Fig.5; Supplementary
Fig. 4 Effect of the interac-
tion between clarithromycin
(CLA) concentration in the
nutrient medium (0, 10, and
100µg L.−1) (dose) and time of
exposure (18 and 30days: T18
and T30) on concentration (a)
and content (c) of CLA in roots;
effect of CLA concentration on
concentration (b) and content
(d) of CLA in shoots. Data are
mean ± SE (n = 6). Different
letters indicate significant differ-
ences at P ≤ 0.05. Details about
the two-way ANOVAs are given
in Supplementary TableS4
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77201Environmental Science and Pollution Research (2023) 30:77193–77209
1 3
TableS5). Root BAF at both sampling times was sig-
nificantly higher at 10µg CLA L−1 compared to 100µg
CLA L−1 (ca. 708 vs 66), but the highest value was
reached at T30 (ca. 814). Indeed, root BAF increased
over time at the lower CLA dose, and decreased over
time at the higher CLA dose, although differences in
time of exposure at this dose were statistically not sig-
nificant (Fig.5a). Shoot BAF at both sampling times
was higher at 10µg CLA L−1 compared to 100µg CLA
L−1, decreased over time at the lower CLA dose, and it
did not change with at the higher CLA dose (Fig.5b).
The translocation factor (TF) of CLA, calculated as the
ratio of CLA concentration in shoots and roots was not
affected by CLA dose and time of exposure (Supplemen-
tary TableS5). On average, TF of CLA was 1.0.
Growth anduptake ofDCF byArundo donax
Similar to the experiment carried out to evaluate plant
growth and CLA uptake, plants harvested just before the
addition of DCF (T0) had an average of 1.2 culms per
plants and 7.6 green leaves per plant, fresh weight was
1.05g plant−1 (shoot plus roots) and plant height was
10cm. After 18days of exposure to DCF, shoot fresh
weight, root length, and leaf area were affected by DCF
and this effect varied according to its concentration in
the medium (Fig.6; Supplementary TableS6). Shoot
fresh weight, root length, and leaf area at 1mg DCF L−1
did not change compared to the untreated control, while
they decreased at 10mg DCF L−1 by 63%, 67%, and
55%, respectively. Conversely, root fresh weight, number
Fig. 5 Effect of the interaction
between clarithromycin (CLA)
concentration in the nutrient
medium (10 and 100µg L.−1)
(dose) and time of exposure (18
and 30days: T18 and T30) on
the bioaccumulation factor (BF)
of CLA roots (a)and shoots(b)
of Arundo donax L. Data are
mean ± SE (n = 6). Different
letters indicate significant differ-
ences at P ≤ 0.05. Details about
the two-way ANOVAs are given
in Supplementary TableS5
Fig. 6 Effect of diclofenac (DCF) concentration in the nutrient
medium (0, 1, and 10mg L.−1) (dose) on fresh weight of shoots and
roots (a), root length (b), and leaf area (c) of Arundo donax. Data are
mean ± SE (n = 4). Different letters indicate significant differences at
P ≤ 0.05. Details about the one-way ANOVAs are given in Supple-
mentary TableS6
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77202 Environmental Science and Pollution Research (2023) 30:77193–77209
1 3
of leaves, and stems and leaf SPAD readings were not
affected by DCF exposure at both doses (Supplementary
TableS6). The morphology of the root system of A. donax
changed in response to DCF supply: in plants treated with
1mg DCF L−1, the ratio root length/root fresh weight
decreased by 80% compared with the untreated plants
(from 1289 to 262cm g−1), while in plants treated with
10mg DCF L−1, the ratio was unchanged (230cm g−1)
(data not shown). Visual observations confirmed the effect
of DCF at both doses on root architecture.DCFconcen-
tration and content in roots and shoots were affected by
DCF concentration in the nutrient medium (Fig.7; Sup-
plementary TableS7). DCF was not detected in roots and
shoots of the untreated plants, while concentration of DCF
in roots and shoots increased with the increase of DCF
dose (Fig.7a, b). The increase from 1 to 10mg DCF L−1
was about eightfold and over tenfold in roots and shoot,
respectively. However, DCF concentration was much
higher in roots than in shoot (about 16–19 times higher).
Similarly, DCF content in roots and shoots increased due
to the increase of DCF dose (4.6-fold in roots and 5.9-fold
in shoot), but due to the higher biomass allocation to shoot
compared with roots, DCF content in shoots was much
higher than in roots (Fig.7d, e). DCF content in the whole
plant increased from 3.4 to 23µg plant−1 with the increase
of DCF dose from 1 to 10mg L−1, which was partitioned
for 79 and 82% into shoots, respectively, at the lower and
higher DCF dose (Supplementary TableS7).metabolite
concentration (MET: 4’-hydroxydiclofenac, 4’-OH DCF)
was high in roots of plants treated with both DCF doses
and was almost not detected in the shoots (Fig.7c; Sup-
plementary TableS7). However, no significant difference
was detected between DCF doses (on average 48.9µg g−1)
(Fig.7c) and values were similar to the DCF concentration
observed in roots at the higher DCF dose (ca. 59.7µg g−1)
(Supplementary TableS7).
The bioaccumulation factor (BAF) of roots and shoots
was not modified by DCF dose and was on average 59.3
and 3.1, respectively (Supplementary TableS8). Similarly,
the translocation factor of DCF did not vary according to
DCF dose and was on average 0.06, indicating that DCF
concentration in shoots was ten times lower than in roots.
Fig. 7 Effects of diclofenac (DCF) concentration in the nutrient
medium (0, 1, and 10mg L.−1) (dose) on DCF concentration and con-
tent in roots (a, d) and shoots (b, e) and on DCF metabolite (MET,
4′-hydroxydiclofenac) concentration and content in roots (c, f). Data
are mean ± SE (n = 4). Different letters indicate significant differences
at P ≤ 0.05. Details about the one-way ANOVAs are given in Supple-
mentary TableS7
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77203Environmental Science and Pollution Research (2023) 30:77193–77209
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Discussion
Microbial degradation ofCLA andDCF
In our batch experiments with the selected microorgan-
isms, the experimental set-up allowed to exclude or mini-
mize volatilization and photooxidation, and differences
in removal efficiency can be exclusively attributed to the
differential ability of the microorganisms to tolerate high
CLA and DCF concentration and to use these compounds
as C and energy sources or degrade them as a result of
co-metabolism. In addition, the axenic growing condi-
tions applied in our experiment prevent the occurrence of
limitations to microbial growth due to competition with
other microorganisms. Indeed, the growth of the ligni-
nolytic basidiomycetes, such as P. chrysosporium and T.
versicolor, in most soils is limited due to the low amount
of available C and N, as some soil microbes are very effi-
cient competitors at low resource availabilities (Baldrian
2008). Moreover, we tested higher concentrations of both
CLA and DCF compared to the ones generally recorded
in water bodies, since one of the key questions posed by
the scientific community is whether PhCs could induce
toxic effects to the microbial community putatively able to
degrade contaminants and thus potentially able to reduce
microbial degradation.
Overall, the highest removal efficiency of CLA, clas-
sified as a recalcitrant compound, was recorded with P.
chrysosporium: the PhC was almost completely degraded
(ca. 80%) already at 72h. Conversely, the degradation
ability of T. versicolor was lower, although lower rates
were recorded at the higher CLA concentration. In com-
parison with these fungi, the bacterium S. rochei showed
a lower degradation ability, not exceeding 50% of CLA at
both doses. These results suggest that the different ability
of the tested microorganisms to degrade CLA might be
related to the capacity of producing significant amounts of
extracellular laccases (Sutaoney etal. 2022). Accordingly,
Margot etal. (2013) found that laccase activity produced
by T. versicolor was more than 20 times greater than four
strains of the bacterial genus Streptomyces (S. cyaneus, S.
ipomoea, S. griseus and S. psammoticus).
The reduction of the degrading ability of P. chrys-
osporium at the highest CLA dose could be attributed to
the toxicity of the compound to the fungus. Although in
the present study we could not determine the degrada-
tion products of CLA, it is important to highlight that
degradation could not result in a complete disappearance
of toxicity, as some oxidative treatments of CLA yield a
number of products, some of which (e.g., 14-hydroxy(R)-
clarithromycin) have pharmacological activity, and thus,
a putative toxicity for the microorganisms (Baumann etal.
2015). However, Zeng etal. (2021) studied the metabolic
mechanism of CLA in waste activated sludge from anaero-
bic digestion system and demonstrated that CLA could be
degraded into the macrolide antibiotic oleandomycin with
lower antimicrobial activity and into other metabolites
without antimicrobial activity. This indicates that effec-
tive degradation and reduced potential environment risk
induced by CLA could be attained.
DCF is classified as a recalcitrant or poor removal com-
pound following biological wastewater treatment processes
(removal efficiency < 30%) (Matamoros and Bayona 2006;
Luo etal. 2014). This behavior was attributed to the molecu-
lar properties of DCF, and specifically to the absence of
electron donating groups in the molecule (Tran and Gin
2017). In our study, DCF removal was observed in all
experiments, but the removal efficiency was highly variable
(from 30 to 97%), according to the degrading microorgan-
ism and DCF concentration. Removal efficiency by S. rochei
at the lower DCF concentration (ca. 30%) is similar to the
values recorded by Tran and Gin (2017) and other authors
(e.g., Zhang etal. 2008) in activated sludge processes, where
microorganisms mineralize or transform pollutants. How-
ever, when DCF was applied at the higher concentration,
the removal efficiency of S. rochei increased, but did not
exceed 60%.
We can therefore assume that the high degrading abil-
ity of S. rochei at the higher CLA and DCF concentrations
could result from bacterial metabolism using PhCs as C
and energy sources. This hypothesis is supported by the
high ability to remove DCF from liquid cultures by Act-
inobacteria endophytes (i.e., Streptomyces, Microbacterium,
and Glycomyces) isolated from the roots and rhizomes of
Miscanthus × giganteus plants (Sauvêtre etal. 2020). The
highest DCF removal rates were observed for the isolates
DS24 (41%) and DS4 (35%) both identified as Streptomyces
griseorubiginosus, which were able to use DCF as a sole
C source. Conversely, higher degrading ability of T. versi-
color (Badia-Fabregat etal. 2015) and bacteria was recorded
in activated sludge from municipal wastewater depuration
plant with nutrient addition (Muter etal. 2017), supporting
the general agreement that most of the pollutants are co-
metabolically degraded (Harms etal. 2011).
Similar to the results obtained with CLA, degradation abil-
ity of DCF increased with T. versicolor, and even more with
P. chrysosporium compared to S. rochei, although with the
higher concentration of DCF in nutrient media both fungi
required more time to achieve the same percentage of removal.
In many environmental conditions and applications, bac-
teria are chosen or self-established because they outperform
fungi. Compared to fungi, bacteria tolerate a broader range
of habitats, use higher specificity biochemical reactions,
more often they productively degrade contaminants (leading
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77204 Environmental Science and Pollution Research (2023) 30:77193–77209
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to independence from auxiliary organic substrates), grow
faster, and are more mobile in aqueous environments (Harms
etal. 2011). Additionally, failure of filamentous fungi in
remediation schemes (namely, land farming and soil reac-
tors) have been reported, due to lack of supply of organic
substrates, oxygen starvation, and mechanical disturbance
sensitivity that prevents fungi from developing mycelia
(Lamar etal. 1994). However, bacteria might be disadvan-
taged if substrates contain rare mineral elements, have a low
bioavailability, contain little energy, or occur permanently
at low concentrations. In addition, fungi possess other char-
acteristics that could make their use more attractive than
bacteria, such as long-range transport, production of many
intracellular, and extracellular enzymes involved in chemi-
cal catabolism that lacks substrate specificity (Harms etal.
2011).
Growth anduptake ofCLA andDCF byArundo
donax
In CWs, plants mainly contribute to PhC removal through
direct uptake, absorption, and sequestering of contaminants,
promoting microbial growth around the roots, and control-
ling water movement (Ravichandran and Philip 2021). The
use of non-conventional water resources, such as treated
wastewater for irrigation purposes, is a consolidated practice
worldwide, and the evidence of the occurrence of PhCs in
waters has promoted research activity in order to elucidate
the uptake and bioaccumulation of PhCs in the edible parts
of food crops and fodders and their subsequent entry into
the food chain. While food and feed crops were extensively
tested for uptake and accumulation of contaminants (Chris-
tou etal. 2019), A. donax used as biomass plants, i.e., non-
food crop plants grown for energy production, has not yet
been tested for PhCs removal or biotransformation, despite
the fact that energy crops pose no intake risks for humans
and animals.
In our hydroponic experiments, we evaluated the ability
of A. donax to uptake CLA and DCF from nutrient media
and to PhCs partition the into shoots and roots. The experi-
mental set-up allowed to exclude or minimize volatiliza-
tion and photooxidation. The growth pattern of A. donax
indicates that this species expresses a toxicity response to
CLA only at the maximum dose of 100µg L−1. Although
shoot biomass production was not affected by CLA expo-
sure at either dose, root growth and length were signifi-
cantly affected (48% increase in weight and 27% decrease
in length, compared to control, corresponding to more than
50% decrease of root length/weight ratio) with 100µg CLA
L−1. Therefore, this CLA dose dramatically changed root
morphology, with short and thick main roots and the inhibi-
tion of fine lateral roots. To our knowledge, this is the first
report of CLA producing a severe root length reduction and
stunted roots formation.
CLA was found in roots and shoots at increasing con-
centrations with increasing CLA dose, but the values
were similar in both roots and shoots for any dose (roots:
0.89–1.82µg g−1; shoots: 0.77–1.21µg g−1), and TF was
consequently about 1. However, due to the different growth
pattern of the plant parts, CLA content was much higher in
shoots than in roots, suggesting that the plant is able not only
to uptake CLA from the growth medium but also to trans-
locate and accumulate it into the shoots. In previous stud-
ies, carried out in hydroponic culture, CLA was detected at
concentrations up to 1.63 and 5.0µg g−1 in lettuce leaves and
roots, respectively (Tian etal. 2019). These higher values
compared to our results could be determined by the small
size and fast growth of lettuce, leading to the quick uptake
and accumulation of CLA in plant tissues. However, in the
study of Manasfi etal. (2021), the accumulation of CLA in
lettuce leaves irrigated with spiked treated wastewater with
10μg CLA L−1 was small (126.6ng g−1 d.w., correspond-
ing to 1.6μg L−1 on fresh basis assuming 8% d.w. of leaves)
and similar to our results. Furthermore, they were unable
to detect any of the known metabolites of CLA, probably
because they were present, but at values below the detection
threshold. Conversely, in the work of Tian etal. (2019), eight
metabolites of CLA were detected in both lettuce leaves and
roots after 18days of exposure, and their proportion to the
parent compound was estimated to be greater than 70%,
indicating that most of the CLA was metabolized in plant
tissues. Although in the present research we did not deter-
mine degradation products of CLA in tissues of A. donax,
we cannot exclude their presence.
DCF treatments affected plant growth only at the high-
est dose, with which shoot fresh weight and root length
were severely reduced. In contrast to the results with CLA
treatments, shoots showed reduced growth and toxic effect,
although morphological modification of the root system with
many short and stunted roots occurred also with DCF exposure
at both doses. DCF was found in roots and shoots at increasing
concentrations with increasing DCF dose, but the concentra-
tions were much higher in roots than in shoots (16–19 times
higher) and the values were similar in roots and shoots for
each rate (roots: 6–60µg g−1, shoots: 0.3–3.6µg g−1), and
TF was consequently about 0.1. A higher DCF concentra-
tion in roots than in shoots agrees with the results of Ravi-
chandran and Philip (2021) in Cannabis indica and Cannabis
zizanioides and of Zhang etal. (2013) in Scirpus validus. In
the present study, the 4′-OH DCF metabolite was detected in
roots and not in shoots and its proportion to the parent com-
pound varied according to the DCF dose. We estimated that
4′-OH DCF was approximately 88% and 39% of the sum of
DCF and 4′-OH DCF metabolite content, at 1 and 10mg DCF
L−1, respectively. These results suggest that most of the DCF
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77205Environmental Science and Pollution Research (2023) 30:77193–77209
1 3
was metabolized in root tissues, and as expected, the rate of
degradation was higher at the lower dose. Similar accumula-
tion of the metabolites 4′-OH DCF and DCF-OH glucose was
detected in roots by Ravichandran and Philip (2021).
Previous studies have shown reduced plant growth due
to DCF exposure (e.g., Ziółkowska etal. 2014; Schmidt and
Redshaw 2015; Podio etal. 2020). Shortening of root and
shoot lengths was detected on three legume plants (pea, lupin,
and lentil) when the DCF concentration increased from 17.8
to 3560mg L−1 and the negative effect increased with the
increase of the DCF dose (Ziółkowska etal. 2014). Schmidt
and Redshaw (2015) found a negative effect of DCF at 1mg
L−1 on the ratio root to aerial part biomass of Raphanus sati-
vus. Conversely, Pierattini etal. (2018) observed no differ-
ences in leaves number, shoot length, and fresh or dry weight
of poplar plants (Populus alba, L. Villafranca clone) exposed
to 0 and 1mg L−1 of DCF in hydroponic solution. Similarly,
Podio etal. (2020) did not found differences in phenotypes of
chicory plants exposed to 0 and 1mg DCF L−1 during seed
germination and early growth stages, but found a negative
effect of DCF on the concentration of photosynthetic pig-
ments and an activation of the plant detoxification system.
Other authors reported induction of other enzymes following
DCF exposure (i.e., catalase, glycosyltransferase and glu-
tathione S-transferase) (Bartha etal. 2014; Huber etal. 2016;
Majewska etal. 2018; Pierattini etal. 2018). Overall, these
studies showed that the effect of DCF on plants depends on
DCF dose and sensitivity can change according to plant spe-
cies, and suggested that high doses can cause oxidative stress,
mainly in roots, generating phenotypic changes and activating
endogenous antioxidant defense mechanisms.
Plant uptake is thought to be strongly dependent on the
physicochemical characteristics of the compound, including
Henry’s Law constant, water solubility, and octanol–water
partition coefficient (hydrophobicity; Kow). Dissociation
constants are important because they can describe whether
a compound is neutral or ionizable at environmentally rel-
evant pH values. Previous research demonstrated that plant
uptake of dissociated species of an ionizable compound is
lower compared to a neutral compound (Malchi etal. 2014).
There are separate models for predicting chemicals uptake
in both of these forms (Trapp 2000; Trapp etal. 2010). This
is because the uptake of a neutral compound can be mainly
related to octanol–water partition coefficient, whereas the
uptake of an ionic compound depends on pH in the external
solution and the transport depends also on ion trapping in
the phloem and electrostatic interactions with cell walls. In
this study, CLA and DCF are expected to be ionizable in the
hydroponic solution (pKa equal to 8.99 and 4.15, respec-
tively). Therefore, these PhCs are expected to exist in the
cation and anion form, respectively, in the environment at
pH values from 5 to 9 (Pubchem 2022). Organic chemi-
cals with log Kow > 4 are expected to have high potential
for root retention and low translocation capacity. The lower
log Kow of CLA (3.16) compared to the log Kow of DCF
(4.5) could explain why CLA is more easily translocated and
accumulated mainly in leaves following the water transpira-
tion current (Duarte-Davidson and Jones 1996). However,
these results disagree with the general belief that cationic
compounds (in our study CLA) had significantly higher
accumulation in roots and significantly lower accumulation
in leaves than anionic compounds (DCF) (Wu etal. 2013;
Dodgen etal. 2015; Miller etal. 2016).
The low translocation factor is a favorable character for
edible plants, except for crops yielding roots (i.e., sugarbeet,
carrot, radish), since PhCs are mainly concentrated in roots
that are not harvested and remain in soil as residues. Con-
versely, the low translocation factor is an unfavorable char-
acter for energy crops whose aerial plant part is harvested
for energy production and the PhCs eventually accumulated
in shoots do not interfere with the transformation process
into gaseous/liquid fuels and are easily degraded during
these processes. In perennial crops, the accumulation of
PhCs into rhizomes at concentrations above the toxicity
thresholds could hinder regrowth in the following year.
Conclusions
This study demonstrates that S. rochei, T. versicolor, and
especially P. chrysosporium are able to break down CLA
and DCF in liquid media to complete elimination, suggesting
that these microorganisms are suitable candidates for appli-
cation in the remediation of CLA and DCF-contaminated
waters. Furthermore, according to the obtained results, A.
donax was shown to behave as accumulator for CLA and
DCF. In accordance with the physicochemical characteris-
tics of the PhCs, CLA was mainly translocated from roots
and concentrated in leaves, while DCF was mainly accumu-
lated in roots. Therefore, the association of A. donax with
microbial inoculants promises to improve the efficiency of
bioremediation systems. However, to improve the design of
cost-effective degradation systems for their better perfor-
mance, we would need to better clarify which transformation
products are formed, which are the microbial- and plant-
mediated biotransformation pathways, and if the microbial
processes are driven by co-metabolism and/or catabolism.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s11356- 023- 27660-4.
Acknowledgements We thank Alice Cheli, Dr. Gaia Piazza, and Dr.
Valentina Ciccolini for their support during the experiments. Moreover,
the authors wish to thank Prof. Anna Maria Puglia and Prof. Giuseppe
Gallo of the University of Palermo (Italy) for giving comments and
recommendations for Streptomyces rochei cultivation.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

77206 Environmental Science and Pollution Research (2023) 30:77193–77209
1 3
Author contribution Laura Ercoli and Elisa Pellegrino contributed to
the study conception and design. Material preparation, data collection,
and analysis were performed by Elisa Pellegrino, Rudy Rossetto, Sab-
rina Di Giorgi, and Andrea Raffaelli. The first draft of the manuscript
was written by Laura Ercoli and Elisa Pellegrino, and all authors com-
mented on previous versions of the manuscript. Marco Nuti revised
it critically for important intellectual content. All authors read and
approved the final manuscript.
Funding Open access funding provided by Scuola Superiore Sant'Anna
within the CRUI-CARE Agreement. This work was supported by the
Ministry of Foreign Affairs and International Cooperation General
Directorate for Political Affairs and Security Italian Republic under
the project named “Removal of PHARMaceuticals from treated waste-
waters in the Soil–WAter-Plant continuum in the MEDiterranean basin-
PHARM-SWAP MED” (Rep. 353 of 2015) within the Israel-Italian
Call for Proposals on Scientific and Technological Cooperation (Call
for Proposal for 2015–2017).
Data availability Supplementary material is available. Raw data will
be made available on request.
Declarations
Ethics approval This manuscript is only submitted to this journal. We
declare that we have no human participants, human data, or human tissues.
Consent to participate Not applicable.
Consent for publication Authors consent to publish this work.
Competing interests The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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