This is the accepted version of the article: Hu, Kaidi; Peris, Andrea; Torán, Josefina; [et al.]. «Exploring the degradation capability of Trametes versicolor on selected hydrophobic pesticides through set- ting sights simultaneously on culture broth and biological matrix». Chemosphere, Vol. 250 (July 2020), art. 126293. DOI 10.1016/j.chemosphere.2020.126293 This version is available at https://ddd.uab.cat/record/238002 under the terms of the license Exploring the degradation capability of Trametes versicolor on selected hydrophobic pesticides through setting sights simultaneously on culture broth and biological matrix Kaidi Hu1, Andrea Peris2, Josefina Torán1, Ethel Eljarrat2, Montserrat Sarrà1, Paqui Blánquez1, Gloria Caminal3* 1Departament d’Enginyeria Química, Biològica i Ambiental, Escola d’Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain 2Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA), Spanish Council for Scientific Research (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain 3Institut de Química Avançada de Catalunya (IQAC), CSIC. Jordi Girona 18-26, 08034 Barcelona, Spain. *Corresponding author: Institut de Química Avançada de Catalunya, Spanish Council for Scientific Research, Barcelona 08034, Spain. Tel: + 34-935812144; E-mail: gcsqbp@iqac.csic.es 1 1 Keywords: White-rot fungi; adsorption; micropollutant removal; metabolites 2 3 1. Introduction 4 Over the last few decades, micropollutants such as pesticides, pharmaceutically active 5 compounds, personal care products, industrial chemicals and their by-products had been 6 introduced into aquatic and terrestrial environments due to anthropogenic activities, evoking 7 global concerns even in small traces (Mir-Tutusaus et al., 2018; Bilal et al., 2019a; Bilal et al., 8 2019b; Bilal et al., 2020). Particularly, pesticides usage shows a sustainable growing tendency 9 because of the exponential increase in human population that actually further stresses the necessity 10 for augmenting food production. Although substantial improvements have been obtained in crop 11 yields by controlling pests, weeds and disease, the risk of environmental pollution resulted from 12 pesticides drifting among hydrosphere, lithosphere and biosphere could not be ignorable 13 (Carvalho, 2017; Ullah et al., 2018; Bilal et al., 2019b). Virtually, pesticides are considered as the 14 main trigger of environmental deterioration, owing to their persistence, recalcitrance and 15 multi-faceted toxicity. 16 Hydrophobic pesticides, such as chlorpyrifos, dicofol, and cypermethrin usually demonstrate 17 better persistence because of their lower bioaccessibility. Specifically, chlorpyrifos and dicofol 18 correspond to organophosphorus and organochlorine pesticides, respectively, while cypermethrin 19 belongs to the family of synthetic pyrethroids. All of them are used extensively throughout the 20 world in public health, agricultural, and domestic applications since they are highly effective at 21 low doses. However, accumulating evidence has shown that the occurrence of those contaminants 22 in environment is posing a severe threat to human as well as other non-target organism in several 2 23 aspects (Burr, 2014; Koshlukova and Reed, 2014; Aznar-Alemany et al., 2017; Carvalho, 2017; 24 Ullah et al., 2018; Bilal et al., 2019b). Besides, they have been restricted by the Stockholm 25 Convention and classified as possible human carcinogens by the Environmental Protection Agency 26 (EPA) of USA. Hence, although some proper managements or strategies should be practiced to 27 minimize release amount (Ullah et al., 2018), seeking for elimination techniques targeting such 28 pollutants from environment is strongly encouraged and urgent. 29 Compared with chemical and physical methods, friendly to environment, efficient and low-cost 30 features are the major advantages of biodegradation, which is widely applied in pesticide 31 elimination (Mir-Tutusaus et al., 2018). Indeed, different microorganisms have been reported 32 harbor the capacity to degrade chlorpyrifos (Singh et al., 2004; Aswathi et al., 2019), dicofol 33 (Osman et al., 2008; Lu et al., 2019), and cypermethrin (Deng et al., 2015; Tang et al., 2018), 34 among which degradation pathways has also been proposed (Singh et al., 2004; Deng et al., 2015; 35 Tang et al., 2018). However, limited attention has been paid to white-rot fungi (WRF) that are 36 metabolically versatile and are capable of degrading a wide spectrum of xenobiotics due to their 37 nonspecific lignin-degrading enzymes (Mir-Tutusaus et al., 2018; Bilal et al., 2019b). On the other 38 hand, in any case, pesticide degradation effectiveness was basically evaluated according to the 39 difference in concentration of culture medium between initial and final (Chen et al., 2012; Deng et 40 al., 2015; Tang et al., 2018). No study so far has paid attention to the sorption from fungal biomass 41 by means of direct measurement of these compounds in the solid phase, namely biological matrix. 42 The purpose of the present study was to explore the capacity of WRF Trametes versicolor to 43 degrade selected hydrophobic pesticides corresponded to different families, and contributions 44 from either degradation or adsorption were thoroughly assessed through mass balance evaluation. 3 45 Meanwhile, metabolites of chlorpyrifos and dicofol were identified because this part of work for 46 cypermethrin has been already done in previous research (Mir-Tutusaus et al., 2014). 47 2. Materials and Methods 48 2.1. Microorganisms and media 49 T.versicolor ATCC 42530 was acquired from American Type Culture Collection, and 50 maintained by subculturing every 30 d on 2% (w/v) malt extract plates (pH 4.5) at 25 °C. 51 T.versicolor blended mycelial suspension and pellets were prepared as the method of Blánquez et 52 al. (2004). 53 Defined medium consisted of (per liter) glucose 8 g, 3.3 g ammonium tartrate, 1.68 g dimethyl 54 succinate, 10 mL micronutrients and 100 mL macronutrients (Kirk et al., 1978). pH was adjusted 55 to 4.5. 56 2.2. Chemicals and reagents 57 Analytical standards (purity, > 99%) of chlorpyrifos, dicofol and cypermethrin were purchased 58 from Sigma-Aldrich (Barcelona, Spain). HPLC-grade methanol was obtained from Merck 59 (Darmstadt, Germany). All other chemicals and solvents were of analytical grade. Stock solutions 60 (5 mg mL−1) of pesticides were prepared in methanol and stored at – 20 °C. 61 2.3. Degradation experiments 62 Degradation experiments were conducted in 500 mL Erlenmeyer flasks containing 100 mL of 63 fresh defined medium with mixed pesticides at 5 μg L−1. Briefly, pellets were transferred into as 64 inoculum, thereby achieving a concentration of approximately 3.3 g dry weight (DW) L−1. Then, 65 the cultures were incubated at 25 °C under shaken (135 rpm) and dark condition for 14 d. Abiotic 66 (uninoculated) as well as heat-killed culture (121 °C, 30 min) were used as controls, and triplicate 4 67 replications were set for each experiment. Samples were taken at specific intervals during 68 degradation by totally sacrificing each flask culture. Pellets and culture medium were separated 69 firstly through 0.45 μm glass microfiber filter (GF/A), and liquid phase was centrifuged (17, 700 × 70 g, 15 min). Afterward, 100 μL of deuterated standards pesticides (d10-chlorpyrifos, 71 d10-chlorfenvinphos and phenoxy-d5-fenvalerate) solution (0.5 μg mL−1, dissolved in methanol), 72 used as internal standards for quantification purposes, was added to obtained supernatant up to a 73 final volume of 100 mL, followed by transferring into amber vial. Solid samples, namely the fugal 74 pellets, were kept in aluminum foil. Both of them were stored at – 20 °C before subsequent 75 pretreatments as follows: 76 (1) Liquid samples: based on Feo et al. (2010), 30 mL of sample was ultrasonically extracted for 77 15 min. Then, 1 mL of chloroform was added and the samples were centrifuged (2, 200 × g, 5 min) 78 after 5 min. The organic phase was recovered and the aqueous phase was washed once more with 79 1 mL chloroform. The organic phases were pooled and evaporated to dryness with nitrogen. The 80 residue was reconstituted with 50 μL ethyl acetate and then subjected to Gas 81 chromatography-tandem mass spectrometry (GC-MS-MS) analysis (Feo et al., 2011). 82 (2) Fungal pellet samples: the freeze-dried samples were mixed with 15 ng of internal standards 83 mentioned above and they were left overnight at 4 °C. Then samples were extracted by 84 pressurized liquid extraction, using a 350 ASE system (Dionex, USA). Fungal pellet samples and 85 2 g of Florisil were loaded into an ASE extraction cell (22 mL) previously filled with 6 g of 86 Florisil. After that, the cell was completely filled with hydromatrix. Hexane and dichloromethane 87 (1:1, v/v) were used as extraction solvent. Temperature and pressure were settled at 100 °C and 88 1650 psi, respectively. After 2 cycles of 10 min extraction, extracts were evaporated with nitrogen 5 89 to dryness and the residue was reconstituted with 50 μL ethyl acetate prior to GC-MS-MS 90 analysis. 91 2.4. Metabolites identification 92 Given high degradation rate and the different properties of their probable metabolites, research 93 on transformation products (TPs) were performed individually. In brief, pellets were carefully 94 transferred into 100 mL of fresh define medium separately spiked each pollutant at final 95 concentration of 1 mg L−1 and then incubated under same conditions described in section 2.3 for 7 96 d. Abiotic was prepared as control and each experiment was conducted in triplicate. Samples were 97 taken at specific intervals during incubation by filtrating with 0.45 μm glass microfiber filter. 20 98 mL of filtrate was mixed with 50 μL of internal standard methanol solution (d10-chlorpyrifos or 99 d7-oxadiazon, 100 mg L−1), followed by adding filtrate up to 50 mL. Afterward, samples were kept 100 at – 20 °C prior to analysis. 101 2.5. Analytical methods 102 2.5.1. Biomass quantification 103 Biomass were determined by the dry weights of mycelia, which were obtained through filtering 104 with glass microfiber filter (0.45 μm), followed by drying at 100 °C to constant weight. 105 2.5.2 Glucose 106 Samples were filtrated with nylon filter (0.45 μm) and then measured by biochemistry analyzer 107 (2700 select, Yellow Springs Instrument, USA). 108 2.5.3. Laccase 109 Laccase activity was assayed through the oxidation of 2,6-dymetoxyphenol (DMP) by the 110 enzyme as described elsewhere (Wariishi et al., 1992). Activity units per liter (UA L−1) are defined 6 111 as the amount of DMP in μM which are oxidized per minute. 112 2.5.4. Pesticide analyses 113 Residual pesticide concentrations were determined through GC-MS-MS (7890B/7000C, Agilent, 114 USA) equipped with a DB-5MS capillary column (30 m × 0.25 mm, 0.25 µm). The operating 115 conditions were as follows: the column was held initially at a temperature of 80 °C for 2 min, then 116 raised at 25 °C min−1 to 180 °C for 6 min, at 5 °C min−1 to 240 °C for 5 min, at 10 °C min−1 to 117 280 °C for 5 min, and finally at 30 °C min−1 to 325 for 2 min. The temperature corresponding to 118 the transfer line and the ionization source were 300 °C and 280 °C, respectively. The collision 119 energy was 70 eV. The injection volume was 2 µL at 280 °C. Helium was used as carrier gas at a 120 flow rate of 1 mL min−1. 121 2.5.5. Metabolite analyses 122 The sample was subjected to separation by an Acquity ultra performance liquid chromatography 123 (UHPLC) system (Waters, USA) equipped with a Purospher STAR RP-18 endacapped (2 µm) 124 Hibar HR 150-2.1 UHPL column (Merck, Germany). Methanol and water both containing 0.1% 125 formic acid (v/v) were used as mobile phases A and B, respectively. A gradient elution program 126 was started with 80% (v/v) B from 0 min to 1 min, decreasing to 5% at 8 min and held until 13 127 min. Then, the percentage of B was further reverted to 80% by 13.5 min and maintained it until 15 128 min. The follow rate was 200 µL min−1 with an injection volume of 10 μL. The UHPLC system 129 was coupled to a hybrid quadrupole-Orbitrap mass spectrometer Q-Exactive (Thermo Fisher 130 Scientific, USA) equipped with a heated-electrospray ionization source HESI II, which was 131 operated in positive ionization mode under the following conditions: spray voltage, + 3.0 kV; 132 sheath gas, 40 arbitrary units; auxiliary gas, 10 arbitrary units; sweep gas, 2 arbitrary units; 7 133 capillary temperature, 350 °C and vaporizer temperature, 300 °C. Nitrogen (> 99.98%) was 134 employed as sheath, auxiliary and sweep gas. The mass spectrometer performed a Fourier 135 Transform Mass Spectrometry (FTMS) scan event of 50-700 m/z at a resolution of 70, 000 and a 136 subsequent MS/MS scan event acquired at a resolution of 35, 000. Xcalibur software (Thermo 137 Fisher Scientific, USA) was employed for instrumental control and data processing. 138 In the case of post-acquisition MS data processing, the total ion current (TIC) chromatograms 139 acquired at different sampling time were compared using Compound Discoverer (Thermo Fisher 140 Scientific, USA), which allows performing differential analysis among selected sets of samples 141 comparing simultaneously thousands of MS spectra, to identify all potential TPs. Accurate mass of 142 the potential TPs was then extracted in Xcalibur to confirm their presence. Identification of the 143 potential TPs was based on their accurate mass, mass error, molecular formula and degree of 144 unsaturation of the parent ion and product ions. 145 2.5.6. Data analysis 146 Degradation percentage was calculated using the following equation: M0－M 147 Degradation percentage (%) = ×100 M0 148 where M0 corresponds to initial amount of contaminant (ng), and C represents the residual 149 amount of contaminant (ng). 150 The mean and standard deviation (SD) of data were calculated and subjected to analysis of 151 variance (ANOVA). Statistical significance was determined using SPSS V22.0. 152 3. Results and discussion 153 3.1. Degradation of mixed pesticides 154 Taking into account that pesticides detected from environment are usually present in the range 8 155 of ng L−1 to μg L−1 (Zheng et al., 2016; Moreno-González and Leon, 2017; Tang et al. 2018), 156 degradation experiments were running at 5 μg L−1, along which time-course in glucose 157 consumption and laccase activity were also investigated. As shown in Figure 1, glucose was 158 totally utilized after 7 d incubation. With respect to laccase, a continuous increase in activity was 159 observed within 7 d, thereby achieving the maximum value at 48 AU L−1 and then maintained at 160 this constant level until 14 d, although the elimination occurred largely before first sampling time. 161 Similar performances were observed in different treatments using T.versicorlor (Blánquez et al., 162 2004; Mir-Tutusaus et al., 2014). 60 8 50 6 Laccase (AU L ) 40 -1 Glucose (g L ) -1 30 4 20 2 10 0 0 0 2 4 6 8 10 12 14 Time (d) 163 164 Figure 1 Profiles of laccase and glucose during pesticides degradation by T.versicolor. Filled 165 inverted triangles, laccase; filled circles, glucose. Values are means of three replicates with 166 standard deviation. 167 On another side, the pesticide residues in liquid phase were determined in the beginning (Figure 168 2) as most cases did (Chen et al., 2012; Deng et al., 2015; Tang et al., 2018), showing that the 169 pollutants in abiotic control maintained at relatively constant levels, although the final 170 concentration of cypermethrin was slightly less than initial. Besides, no statistical differences were 171 observed along treatment. Conversely, considerable removal of added chlorpyrifos, dicofol and 9 172 cypermethrin occurred in both experimental and killed control flasks, by which it is not easy to 173 determine whether degradation occurred. Therefore, focus was switched to biological matrix, in 174 order to seek for the explanation for this phenomenon. Interestingly, results indicated that 175 pesticides in killed control accumulated along incubation and then essentially achieved a 176 stabilisation (p < 0.05), whereas much less amounts of pollutants were detected when it comes to 177 experimental treatment, and it displayed an apparent decreasing tendency (p < 0.05). These 178 profiles correspond to the time-course glucose consuming and laccase activity, because no 179 increase in laccase was detected since 7 d, which perhaps means laccase was involved in this 180 particular biochemical process. But intracellular enzymes (Mir-Tutusaus et al., 2018) should also 181 be further investigated. Nevertheless, it’s feasible to suppose that adsorption not only played an 182 important role but also happened in the first place during pesticides elimination by T.versicolor, 183 which corroborates that observed by Blánquez et al. (2004). Accordingly, decolorization process 184 was started by the initial adsorption of the dye, which was then absorbed and degraded as the 185 result of enzyme, followed by final release of metabolites. And it could also explain why the 186 results were expressed as C/C0. Because this physical adsorption process occurred immediately 187 once the pollutant was added, resulting in the initial concentration was not lower than 5 μg L−1. 188 Simultaneously, in respect of liquid phase, more efficient elimination towards dicofol and 189 cypermethrin were observed in killed control than in experimental treatment. The reasonable 190 explanation is that active metabolic processes somehow negatively affect adsorption (Fomina and 191 Gadd, 2014). 10 Chlorpyrifos Dicofol Cypermethrin 1.5 1.2 Liquid sample 0.9 C/C0 0.6 0.3 0.0 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 3000 2500 Solid sample C (ng g DW ) -1 2000 1500 -1 1000 500 0 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 192 Time (d) 193 Figure 2 Pesticides degradation by T.versicolor. C represents the residual concentration of 194 pesticide in sample (μg L−1), and C0 corresponds to the initial concentration of pesticide in sample 195 (μg L−1); Blue lines with filled squares, experimental; red lines with filled triangles, killed control; 196 green lines with filled circles, abiotic. Values are means of three replicates with standard deviation 197 Values are means of three replicates with standard deviation. 198 In order to ascertain the contributions from either biodegradation or sorption, by which the 199 selected pesticides were eliminated, mass balances during incubation were proposed and the 200 results are summarized in Table 1. Clearly, pesticides were effectively removed in experimental 201 sets, but most of them were still remained in the killed control, especially dicofol, demonstrating 202 even more amounts than initial. The most probable reason for this fact could be the systematic 203 error, likely the inoculum differences in terms of dry weight, which could also explain the high 204 standard deviations between samples. Lucas et al (2018) evaluated the contribution of sorption 205 process in the elimination of pharmaceuticals during the fungal treatment of wastewater, from 206 which a mean value as 7% was obtained. By contrast, much higher average adsorption was 207 observed in present study, yielding more than 90%, probably because of that the selected 208 pesticides possess higher hydrophobicities (Fomina and Gadd, 2014). Anyway, there is no doubt 209 that T.versicolor demonstrated efficient capabilities in degrading chlorpyrifos, dicofol and 11 210 cypermethrin at concentrations in the range of μg L−1, resulting in 94.7%, 87.9% and 93.1% of 211 removal, respectively. 212 Table 1 Mass balance profile of pesticides in Erlenmeyer flasks according to the residues in both 213 liquid and solid phase Amounts (ng) Pesticide Set up Time (d) 0 3 7 14 Experimental 235.24 ± 18.75* 45.62 ± 4.18* 6.08 ± 0.63* 12.55 ± 1.83* Chlorpyrifos Killed control 312.75 ± 80.76 194.82 ± 77.41 ND 249.30 ± 98.47 Experimental 335.68 ± 95.41* 90.54 ± 56.07* 12.64 ± 2.31* 40.75 ± 19.11* Dicofol Killed control 417.98 ± 135.30 412.90 ± 209.72 ND 603.26 ± 235.28 Experimental 144.64 ± 17.34* 38.67 ± 4.20* 6.92 ± 0.84* 9.98 ± 0.95* Cypermethrin Killed control 371.71 ± 64.79 291.17 ± 185.91 ND 290.73 ± 111.27 214 Note: Means and standard deviation of triplicate are shown; ND, not data; *statistically different 215 compared along the time (p < 0.05) 216 Although there are numerous literatures documenting degradation of selected compounds by 217 microorganisms (Chen et al., 2012; Deng et al., 2015; Tang et al., 2018; Aswathi et al., 2019; Lu et 218 al., 2019), few sight have been thrown into adsorption attributed to biological matrix. Because it’s 219 involved in elimination process at different degree, especially remarkable in the cases of 220 hydrophobic xenobiotics (Margot et al., 2015; Liu et al., 2019). To address this gap, this present 221 work is actually first time to evaluate degradation capability of T.versicolor on degradation 222 chlorpyriphos, cypermethrin and dicofol based on analysis of residual concentrations both in 223 culture medium and biological matrix combined with mass balance evaluation. So, it essentially 224 offered a strong proof and better understanding of the elimination mechanism. In the meantime, it 225 also worth to emphasis that comparing the obtained efficacy with previous studies is not that 226 feasible. Because not only should the differences in term of substrate concentration be taken into 227 consideration, but also culture conditions such as temperature, pH and medium can be effect 228 factors. Anyway, this particular species displays promising potential in real bioremediation. 12 229 3.2. Metabolites of chlorpyrifos and dicofol by T.versicolor 230 Owing to pesticides removals contributed from both degradation and adsorption took place so 231 fast, higher concentration was applied into metabolites identification experiments, in order to 232 catch the TPs more easily. As summarized in Table 2, three compounds were captured and 233 identified as metabolites within 7 d incubation according to the results of UHPLC-MS/MS 234 analysis, of which benzaldehyde is first time to be reported as TP of dicofol. In addition, the 235 compounds 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid and 236 3-phenoxybenzoic acid have been identified as TPs of cypermethrin using T.versicolor by 237 Mir-Tutusaus et al (2014). 238 Table 2 Chromatographic characteristics of the TPs of chlorpyrifos and dicofol by T.versicolor Nominal Retention Measured Mass error Molecular Pesticide Identified TP RDB mass time (min) mass (m/z) (ppm) formula 171 171.0239 – 0.280 C4H12O3PS – 0.5 115 114.9616 2.716 H4O3PS – 0.5 O,O-diethyl 143 4.21 142.9928 1.134 C2H8O3PS – 0.5 thiophosphate 97 96.9510 2.238 H2O2PS 0.5 Chlorpyrifos 81 80.9741 6.089 H2O3P 0.5 155 155.0467 – 0.528 C4H12O4P – 0.5 diethyl 127 127.0156 1.325 C2H8O4P – 0.5 3.10 phosphate 99 98.9846 4.732 H4O4P – 0.5 81 80.9740 4.360 H2O3P 0.5 107 107.0495 3.537 C7H7O 4.5 91 91.0547 5.307 C7H7 4.5 Dicofol benzaldehyde 81 7.47 81.0341 8.005 C5H5O 3.5 79 79.0548 7.630 C6H7 3.5 77 77.0392 8.350 C6H5 4.5 239 Note: Molecular formulas were calculated on the basis of their accurate mass measurements and 240 the observed isotopic patterns. Unsaturation degree was expressed as double bound equivalents 241 (RDB). 242 So apparently, hydrolyzation occurred in degradation process of chlorpyrifos and cypermethrin, 243 keeping in line with most cases described to date (Singh et al., 2004; Tang et al., 2018; Aswathi et 244 al., 2019; Deng et al., 2015). And it is always the first reaction which also play an irreplaceable 13 245 role in detoxification of other organophosphorus and pyrethroids (Sogorb and Vilanova, 2002; 246 Singh and Walker, 2006). On the other side, considering our result and previous findings (Bumpus 247 and Aust, 1987; Bumpus et al., 1993), we speculated that dichlorination occurred firstly during 248 dissipation process of dicofol, transforming it into 2,2-dichloro-1,1-bis-(4-chlorophenyl)-ethanol 249 (FW-152). FW-152 was then subject to successive reductive dichlorinations and oxidative 250 cleavage, resulting in formation of 4,4'-dichlorobenzophenone, which was further degraded into 251 benzaldehyde through ring cleavage reaction probably because of the role of lignin-degrading 252 system (Bumpus and Aust, 1987). 253 4. Conclusions 254 Measuring target compounds in liquid and solid phases simultaneously is a worthwhile strategy 255 to illustrate the elimination mechanism of pollutants. Both biodegradation and adsorption were 256 considerably involved in removal of chlorpyrifos, dicofol and cypermethrin. T.versicolor 257 demonstrated ideal degradation capability on those compounds, showing as 94.7%, 87.9% and 258 93.1% removal percentage respectively. Our findings suggest that the particular microorganism is 259 useful for bioremediation of hydrophobic pesticides contaminated environments. Meanwhile, the 260 related metabolites were identified, indicating that hydrolyzation, dichlorination and oxidation 261 played important roles within degradation process. 262 Acknowledgement 263 This work has been funded by the Spanish Ministry of Economy and Competitiveness (project 264 CTM 2016-75587-C2-1-R) co-financed by European Union through the European Regional 265 Development Fund (ERDF) and partly supported by the Generalitat de Catalunya (Consolidated 266 Research Group 2017-SGR-14). The Department of Chemical, Biological and Environmental 14 267 Engineering of the Universitat Autònoma de Barcelona is member of the Xarxa de Referència en 268 Biotecnologia de la Generalitat de Catalunya. K. Hu acknowledges the financial support from 269 Chinese Scholarship Council. 270 Conflict of interest 271 We declare that no conflict of interest exists in the submission of this manuscript. 272 273 References 274 Aswathi, A., Pandey, A., Sukumaran, R.K., 2019. Rapid degradation of the organophosphate 275 pesticide-chlorpyrifos by a novel strain of Pseudomonas nitroreducens AR-3. Bioresource 276 Technol. 292, 122025. DOI: 10.1016/j.biortech.2019.122025. 277 Aznar-Alemany, O., Giménez, J., de Stephanis, R., Eljarrat, E., Barceló, D., 2017. Insecticide 278 pyrethroids in liver of striped dolphin from the Mediterranean Sea. Environ. Pollut. 225, 279 346-353. DOI: 10.1016/j.envpol.2017.02.060. 280 Bilal, M., Ashraf, S.S., Barceló, D., Iqbal, H.M., 2019a. Biocatalytic degradation/redefining 281 “removal” fate of pharmaceutically active compounds and antibiotics in the aquatic 282 environment. Sci. Total Environ. 691, 1190-1211. DOI: 10.1016/j.scitotenv.2019.07.224. 283 Bilal, M., Iqbal, H.M., Barceló, D., 2019b. Persistence of pesticides-based contaminants in the 284 environment and their effective degradation using laccase-assisted biocatalytic systems. Sci. 285 Total Environ. 695, 133896. DOI: 10.1016/j.scitotenv.2019.133896. 286 Bilal, M., Mehmood, S., Rasheed, T., Iqbal, H.M., 2020. Antibiotics traces in the aquatic 287 environment: persistence and adverse environmental impact. Curr. Opin. Environ. Sci. Health 288 13, 68-74. DOI: 10.1016/j.coesh.2019.11.005. 15 289 Blánquez, P., Casas, N., Font, X., Gabarrell, X., Sarrà, M., Caminal, G., Vicent, T., 2004. 290 Mechanism of textile metal dye biotransformation by Trametes versicolor. Water Res. 38, 291 2166-2172. DOI: 10.1016/j.watres.2004.01.019. 292 Bumpus, J.A., Aust, S.D., 1987. Biodegradation of DDT [1, 1, 1-trichloro-2, 2-bis (4-chlorophenyl) 293 ethane] by the white rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 53, 294 2001-2008. 295 Bumpus, J.A., Powers, R.H., Sun, T., 1993. Biodegradation of DDE (1, 1-dichloro-2, 2-bis 296 (4-chlorophenyl) ethene) by Phanerochaete chrysosporium. Mycol. Res. 97, 95-98. DOI: 297 10.1016/S0953-7562(09)81144-1. 298 Burr, S.A., 2014. Cypermethrin, in: Richardson, R.J. (3rd), Encyclopedia of toxicology. 299 Amsterdam: Elsevier, pp. 1120-1121. 300 Carvalho, F.P., 2017. Pesticides, environment, and food safety. Food Energy Secur. 6, 48-60. DOI: 301 10.1002/fes3.108. 302 Chen, S., Liu, C., Peng, C., Liu, H., Hu, M., Zhong, G., 2012. Biodegradation of chlorpyrifos and 303 its hydrolysis product 3, 5, 6-trichloro-2-pyridinol by a new fungal strain Cladosporium 304 cladosporioides Hu-01. PloS one 7, e47205. DOI: 10.1371/journal.pone.0047205. 305 Deng, W., Lin, D., Yao, K., Yuan, H., Wang, Z., Li, J., Zou, L., Han, X., Zhou, K., He, L., Hu, X., 306 Liu, S., 2015. Characterization of a novel β-cypermethrin-degrading Aspergillus niger YAT 307 strain and the biochemical degradation pathway of β-cypermethrin. Appl. Microbiol. Biot. 99, 308 8187-8198. DOI: 10.1007/s00253-015-6690-2. 309 Feo, M.L., Eljarrat, E., Barceló, D., 2010. A rapid and sensitive analytical method for the 310 determination of 14 pyrethroids in water samples. J. Chromatogr. A 1217, 2248-2253. 16 311 doi:10.1016/j.chroma.2010.02.018. 312 Feo, M.L., Eljarrat, E., Barceló, D., 2011. Performance of gas chromatography/tandem mass 313 spectrometry in the analysis of pyrethroid insecticides in environmental and food samples. 314 Rapid Commun. Mass Spectrom. 25, 869-876. DOI: 10.1002/rcm.4936. 315 Feo, M.L., Eljarrat, E., Manaca, M.N., Dobaño, C., Barceló, D., Sunyer, J., Alonso, P.L., 316 Menéndez, C., Grimalt, J.O., 2012. Pyrethroid use-malaria control and individual applications 317 by households for other pests and home garden use. Environ. Int. 38, 67-72. 318 DOI:10.1016/j.envint.2011.08.008. 319 Fomina, M., Gadd, G.M., 2014. Biosorption: current perspectives on concept, definition and 320 application. Bioresource Technol. 160, 3-14. DOI: 10.1016/j.biortech.2013.12.102. 321 Fujii, Y., Haraguchi, K., Harada, K.H., Hitomi, T., Inoue, K., Itoh, Y., Watanabe, T., Takenaka, K., 322 Uehara, S., Yang, H., Kim, Y., Moon, C., Kim, H., Wang, P., Liu, A., Hung, N.N., Koizumi, A., 323 2011. Detection of dicofol and related pesticides in human breast milk from China, Korea and 324 Japan. Chemosphere 82, 25-31. DOI: 10.1016/j.chemosphere.2010.10.036. 325 Halden, R.U., Tepp, S.M., Halden, B.G., Dwyer, D.F., 1999. Degradation of 3-phenoxybenzoic 326 acid in soil by Pseudomonas pseudoalcali genes POB310 (pPOB) and two modified 327 Pseudomonas strains. Appl. Environ. Microbiol. 65, 3354-3359. 328 Kirk, T.K., Schultz, E., Connors, W., Lorenz, L., Zeikus, J., 1978. Influence of culture parameters 329 on lignin metabolism by Phanerochaete chrysosporium. Arch. Microbiol. 117, 277-285. DOI: 330 10.1007/BF00738547. 331 Koshlukova, S.E., Reed, N.R., 2014. Chlorpyrifos, in: Richardson, R.J. (3rd), Encyclopedia of 332 toxicology. Amsterdam: Elsevier, pp. 930-934. 17 333 Liu, T., Xu, S., Lu, S., Qin, P., Bi, B., Ding, H., Liu, Y., Guo, X., Liu, X., 2019. A review on 334 removal of organophosphorus pesticides in constructed wetland: performance, mechanism and 335 influencing factors. Sci. Total Environ. 651, 2247-2268. DOI: 10.1016/j.scitotenv.2018.10.087. 336 Lu, P., Liu, H., Liu, A., 2019. Biodegradation of dicofol by Microbacterium sp. D-2 isolated from 337 pesticide-contaminated agricultural soil. Appl. Biol. Chem. 62, 1-9. DOI: 338 10.1186/s13765-019-0480-y. 339 Lucas, D., Castellet-Rovira, F., Villagrasa, M., Badia-Fabregat, M., Barceló, D., Vicent, T., 340 Caminal, G., Sarrà, M., Rodríguez-Mozaz, S., 2018. The role of sorption processes in the 341 removal of pharmaceuticals by fungal treatment of wastewater. Sci. Total Environ. 610, 342 1147-1153. DOI: 10.1016/j.scitotenv.2017.08.118. 343 Margot, J., Rossi, L., Barry, D.A., Holliger, C., 2015. A review of the fate of micropollutants in 344 wastewater treatment plants. WIRES Water 2, 457-487. DOI: 10.1002/wat2.1090. 345 Mir-Tutusaus, J.A., Baccar, R., Caminal, G., Sarrà, M., 2018. Can white-rot fungi be a real 346 wastewater treatment alternative for organic micropollutants removal? A review. Water Res. 347 138, 137-151. DOI: 10.1016/j.watres.2018.02.056. 348 Mir-Tutusaus, J.A., Masís-Mora, M., Corcellas, C., Eljarrat, E., Barceló, D., Sarrà, M., Caminal, 349 G., Vicent, T., Rodríguez-Rodríguez, C.E., 2014. Degradation of selected agrochemicals by the 350 white rot fungus Trametes versicolor. Sci. Total Environ. 500, 235-242. DOI: 351 10.1016/j.scitotenv.2014.08.116. 352 Moreno-González, R., Leon, V., 2017. Presence and distribution of current-use pesticides in 353 surface marine sediments from a Mediterranean coastal lagoon (SE Spain). Environ. Sci. Pollut. 354 R. 24, 8033-8048. DOI: 10.1007/s11356-017-8456-0. 18 355 Osman, K.A., Ibrahim, G.H., Askar, A.I., Alkhail, A.R.A.A., 2008. Biodegradation kinetics of 356 dicofol by selected microorganisms. Pestic. Biochem. Phys. 91, 180-185. DOI: 357 10.1016/j.pestbp.2008.03.012. 358 Singh, B.K., Walker, A., 2006. Microbial degradation of organophosphorus compounds. FEMS 359 Microbiol. Rev. 30, 428-471. DOI: 10.1111/j.1574-6976.2006.00018.x. 360 Singh, B.K., Walker, A., Morgan, J.A.W., Wright, D.J., 2004. Biodegradation of chlorpyrifos by 361 Enterobacter strain B-14 and its use in bioremediation of contaminated soils. Appl. Environ. 362 Microbiol. 70, 4855-4863. DOI: 10.1128/AEM.70.8.4855-4863.2004. 363 Sogorb, M.A., Vilanova, E., 2002. Enzymes involved in the detoxification of organophosphorus, 364 carbamate and pyrethroid insecticides through hydrolysis. Toxicol. Lett. 128, 215-228. DOI: 365 10.1016/S0378-4274(01)00543-4. 366 Tang, J., Liu, B., Chen, T., Yao, K., Zeng, L., Zeng, C., Zhang, Q., 2018. Screening of a 367 beta-cypermethrin-degrading bacterial strain Brevibacillus parabrevis BCP-09 and its 368 biochemical degradation pathway. Biodegradation 29, 525-541. DOI: 369 10.1007/s10532-018-9850-0. 370 Tang, W., Wang, D., Wang, J., Wu, Z., Li, L., Huang, M., Xu, S., Yan, D., 2018. Pyrethroid 371 pesticide residues in the global environment: an overview. Chemosphere 191, 990-1007. DOI: 372 10.1016/j.chemosphere.2017.10.115. 373 Ullah, S., Zuberi, A., Alagawany, M., Farag, M.R., Dadar, M., Karthik, K., Tiwari, R., Dhama, K., 374 Iqbal, H.M., 2018. Cypermethrin induced toxicities in fish and adverse health outcomes: its 375 prevention and control measure adaptation. J. Environ. Manage. 206, 863-871. DOI: 376 10.1016/j.jenvman.2017.11.076. 19 377 Wariishi, H., Valli, K., Gold, M.H., 1992. Manganese (II) oxidation by manganese peroxidase 378 from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of 379 chelators. J. Biol. Chem. 267, 23688-23695. 380 Zheng, S., Chen, B., Qiu, X., Chen, M., Ma, Z., Yu, X., 2016. Distribution and risk assessment of 381 82 pesticides in Jiulong River and estuary in South China. Chemosphere 144, 1177-1192. DOI: 382 10.1016/j.chemosphere.2015.09.050. 20