Bioremediation of Acetaminophen and Hydroxychloroquine by Kosakonia cowanii JCM 10956(T) with ecotoxicity studies

Acetaminophen and hydroxychloroquine are widely used drugs during COVID situations. Residual concentrations of acetaminophen and hydroxychloroquine have been detected in pharmaceutical industry wastewater, effluent treatment plants, and surface water. The present study was carried out on the bioremediation of acetaminophen (paracetamol) and hydroxychloroquine by using the bacterial isolate Kosakonia cowanii JCM 10956(T) (GenBank: OQ733302.1). Identification of the isolate was done using the 16S rRNA sequencing technique. The LC50 values for bacteria were determined for acetaminophen and hydroxychloroquine as 2186.70 and 1735.13 ppm, respectively. Isolate was found to degrade acetaminophen (1500 ppm) into hydroquinone after five days of incubation with an 81% biodegradation rate. Hydroxychloroquine (1000 ppm) was found to be degraded into oxalic acid with 7-chloroquinoline-4-amine and 4-aminoquinoline-7-ol as intermediates. After 15 days of incubation, 60% of hydroxychloroquine was found to be degraded. Acetaminophen and hydroxychloroquine biodegradation followed a first-order kinetic model with a rate constant of 0.339 d− 1 and 0.0618 d− 1, respectively. Half-lives for acetaminophen and hydroxychloroquine were found to be 2.05 and 11.2 days, respectively. Based on the analytical techniques of UV-visible spectra, HPLC, mass spectra, and proton nuclear magnetic spectroscopy (1H NMR) studies, biodegradative metabolites were identified. Ecotoxicological testing of the parent drug and degradative product was done using algal inhibition and shrimp lethality assays. The biodegradative product of acetaminophen, hydroquinone, has more algal toxicity and less toxicity against shrimp as compared to the parent drug. Whereas for the hydroxychloroquine biodegradative product, oxalic acid has less algal toxicity and more toxicity against shrimp compared to the parent drug. Industrial applications of hydroquinone and the metal leaching role of oxalic acid will give new insight into the bioconversion of expired paracetamol and hydroxychloroquine into value-added products.


Introduction
Overuse of the personal care products and pharma products, are leading some environmental concerns.
During Covid 19 pandemic situation, antiviral drugs (Remdesivir, hydroxychloroquine, Azithromycin) and analgesic drugs were used for the treatment of hospitalized patients. Pharmaceutical drugs usage has been increased with the increase in the population and diseases. Pharmaceutical micropollutants are leading to the development of antibiotic and antiviral drug resistance and the possibility of transfer of this resistance genes among other microorganisms During covid 19 pandemic situation, for the treatment of patients and post vaccine, antiviral and painkiller drugs were generally used [1][2]. Pandemic situations have the impact on environmental reactions [3]. As per the report available, paracetamol has risk quotient which is more than 1 [4].
As per the water quality report of different countries, plastic, SARS CoV2 RNA, heavy metals, chemical pollutants drugs were reported in the surface, ground, and waste water [3]. Consumption rate of acetaminophen is huge in US countries where 6% adults reported to have more than 6 gm of acetaminophen per day [5]. Global production of acetaminophen is more in India and China [6]. In the end of the 2021 year, estimated market size of acetaminophen was nearly $10 billion [7]. Production of Hydroxychloroquine has been reported to be 300 metric tons and 20% of consumption rate in India during 2018. For domestic supply, India was manufacturing 60 metric tons which was increase to meet the demand during covid 19 pandemic situation [8].
When treating covid 19, paracetamol served as the rst line analgesic and antipyretic medication [9]. Due to its presence in the environment, paracetamol (acetaminophen; N-acetyl-para-aminophenol) is developing as a pharmaceutical pollutant [10]. According to the Environment Agency (EA) of England and Wales' planned pollutant ranking system, paracetamol is ranked fth [11].
Aryl acylamidase, deaminase, and hydroquinone 1,2-dioxygenase catalyse the biodegradation of paracetamol, producing 4 aminophenol and hydroquinone as the product [18][19][20]. Acetaminophen and its degradative products, has been reported to be ecotoxic for bacteria, aquatic organisms, and plants when the concentration surpasses the detoxi cation potential of exposed organisms. Impact of acetaminophen on bacterial cell membrane, cytoplasmic contents and even on genes has been studied. Although acetaminophen is not having antimicrobial action but it could assist in the spreading of antimicrobial resistance genes by assisting the conjugation process. In the presence of acetaminophen, bacteria showed increased conjugation rate of gene transfer by inducing changes in the cell membrane permeability, reactive oxygen species production, supported conjugation bridge and SOS mechanism [21].
Sometimes rather than the parent compound, its degradative metabolite has more toxicity as reported for the photo-exposed Acetaminophen with EC50 value of 29.46 mg/L for luminescent bacteria [22].
Phytotoxicity of acetaminophen on two aquatic plant species, Lemna gibba and Lemna minor has been studied where this drug showed impact on the reduction in the number of fronds and proline content with EC50 of 446.6 mg/L [23]. Oxidative stress imposed by drug was found to be different for the organism with species differences.
A 4-aminoquinoline derivative having an aromatic ring and a basic side chain is hydroxychloroquine (HCQ). It is an immunomodulatory medicine that reduces the production of cytokines and Toll-like receptors (TLR), which is why it is used to treat autoimmune illnesses [24][25]. Hydroxychloroquine has antiviral activity by inducing changes in Ph of endosomes for the inhibition of viral entry and act as glycosylation inhibitor of cellular receptor of SARS-CoV, angiotensin-converting-enzyme 2 (ACE2) [26][27][28]. In order to lower the mortality rate, hydroxychloroquine was administered over the course of treatment to hospitalised patients, either alone or in combination with azithromycin [29].
Hydroxychloroquine persists in the environment due to its bio accumulative properties in vegetation through the polluted soil and groundwater [30][31][32]. It also has been reported to cause depletion of ozone [33]. Even the natural degradative products of hydroxychloroquine have more toxicity due to their bio resistance [34].
2.2 Isolation, Identi cation, and screening of bacteria for laccase and amidase production: E uent wastewater sample was collected from the site of industrial discharge. The collected sample was stored at 4 o C until use. Sewage sample was used for the isolation of acetaminophen and hydroxychloroquine tolerating bacteria in separate study [10]. At the National Centre for Microbial Resource's (NCMR) sequencing facility in Pune, Maharashtra, India, the isolates were identi ed. The phenol/chloroform extraction procedure was used to isolate genomic DNA, and then the 16S rRNA gene was ampli ed using universal primers 16F27 [35]. According to the manufacturer's instructions, the ampli ed 16S rRNA gene PCR product was puri ed by PEG-NaCl precipitation and immediately sequenced on an ABI®3730XL automated DNA sequencer (Applied Biosystems, Inc., Foster City, CA). Essentially, additional internal primers were used to perform sequencing from both ends, reading each place at least twice. Assembly was carried out with the Lasergene package, and identi cation was done with the EzBioCloud database [36].
Laccase enzyme has been reported in the detoxi cation of painkiller and cytostatic drugs, whereas amidase has been reported to be potential candidate for the degradation of drugs like paracetamol. Hence, isolate was screened for laccase production by growing the culture on soil extract agar containing 0.04% Guaiacol and formation of brown ring around was considered as positive test for laccase production [37].
Mineral salt medium with 2% Acetamide was used as screening medium for amidase where pink colour formation surrounding the bacterial growth was considered as amidase positive [38].  [39]. In a different experiment, acetaminophen (100-3000 ppm) and hydroxychloroquine were added to nutritional broth (100-1200 ppm). The inoculated broth contained 10 7 CFU/ml. OD was measured at 600 nm after 48 hours of incubation at 30 o C. The acquired values of OD600 in a culture with drug over OD600 in a control culture were used to compute the percentage of bacterial viability. To get the LC50 value, the online aatbio calculator was used to plot the bacterial viability percentage (%) with the logarithm of the paracetamol concentration (mg/L) [46].

Acetaminophen Bioegradation studies
After studies of inhibitory concentration of acetaminophen for bacterial growth, concentration was optimised for the degradation experiment. Bushnell Haas medium with a pH of 7, an inoculum of 1%, a temperature of 30, and an amount of acetaminophen (1500 ppm) were used for the fermentation, which was done stationary. Cell concentration was also assessed every 10 hours of incubation by measuring absorbance at 600 nm. The pH of cell free broth was determined using pH meter. Residual concentration of acetaminophen was determined by taking absorbance of cell free supernatant at 244.4 nm [40].
Standard dose response of acetaminophen (1-25 µg.ml -1 ) was used for the determination of residual concentration of acetaminophen after every 10 h of incubation.
Eq. 1 was used to determine the degradation percentage (R) of APAP: R = (C0-Ct)/C0 100 (1) Ct is the absorbance at time 't' following incubation, where C0 is the absorbance at the starting concentration of APAP. Degradation kinetics was evaluated using Computer Assisted Kinetic Evaluation (CAKE) program [41].

Hydroxychloroquine biodegradation studies
Following the nding of the hydroxychloroquine IC50, a stationary fermentation experiment was conducted utilising Bushnell Haas medium with pH 7.0, inoculum 1%, temperature 30oC, and 1000 ppm of hydroxychloroquine. Cell concentration was also assessed every 15 hours of incubation by measuring absorbance at 600 nm. Using a pH metre, the pH of cell-free broth was calculated. By measuring the cell free supernatant's absorbance at 343 nm, the residual content of hydroxychloroquine was discovered [43].
The residual concentration of hydroxychloroquine was calculated after every 10 hours of incubation using the standard dosage response of hydroxychloroquine (1-25 µg.ml-1). Hydroxychloroquine biodegradation kinetics was evaluated using Computer Assisted Kinetic Evaluation (CAKE) program [41]. Equation 2 was used to calculate the hydroxychloroquine degradation percentage (R): (2) Ct = absorbance following incubation at time 't'. C0 = absorbance at the starting concentration of HCQ. Cell free supernatant was used for the estimation of laccase enzyme concentration using 10 mM guaiacol (substrate) and 100 mM acetate buffer as per the protocol [42]. Enzyme activity was calculated using formula given below 2.4 Characterization of Acetaminophen and Hydroxychloroquine Biodegradative Product

Characterization of Acetaminophen Biodegradative Product
Analysis of paracetamol and its bacterial transformed product was determined by using HPLC and NMR spectroscopy. Reverse phase HPLC method was done as per the modi ed protocol of Zhang et al. 2012 [44]. Five days old fermented broth was centrifuged at 10000 rpm for 30 min. Broth was concentrated and extracted with methanol. The organic phase was collected and evaporated using rota evaporator.
Reverse phase HPLC with isocratic mode, JASCO (250 × 4.6 mm, packed with 5 µ) was used to elute paracetamol. A 1 ml/min ow rate of methanol: water (15:85) was utilised as the solvent system, which was seen at a wavelength of 240 nm. JASCO column, methanol: water (20:80) solvent system in isocratic mode, 1 ml.min-1 ow rate, and wavelength monitoring at 280 nm were used in reverse phase HPLC for the measurement of hydroquinone.
The semi-puri ed acetaminophen biodegradative product underwent further processing for NMR-based structural elucidation. Spectroscopic Nuclear Magnetic Resonance (NMR) Analysis Using DMSO as the solvent, proton nuclear magnetic resonance (1H-NMR) spectra were captured using a Varian 500 NMR spectrometer running at 300 MHz. The sample was kept at a constant temperature of 25°C.

Characterization of Hydroxychloroquine Biodegradative Product
The reverse phase HPLC technique and Mass spectrometry were employed for the analysis of hydroxychloroquine biodegradative products. A 15-day-old fermented broth was centrifuged for 30 minutes at 10,000 rpm. Methanol was used to extract the degradative metabolite from the concentrated broth. The rota evaporator was used to evaporate the organic phase. Reverse phase HPLC was used to elute hydroxychloroquine using a JASCO (250x 4.6 mm, packed with 5 µ) column, a solvent system of Methanol: Acetonitrile (50:50): Water (pH 3 corrected with Orthophospheric acid) (75:25), 1.5 ml/min ow rate, and wavelength monitoring at 343 nm.
The partially puri ed degradation product underwent additional processing for identi cation using mass data. The X-Bridge C18 column (1504.6mm3.5m) was used for MS. A 1000 ppm concentration of methanol was used to prepare the sample. 2 µl of injection volume, a 25°C column oven, and a temperature-controlled autosampler were used for the analysis. A gradient solvent system (5mm ammonium acetate and acetonitrile) was employed.

Algal Inhibition Assay
With slight changes, the algal inhibition assay was carried out using the technique described by Bährs et al. in 2013[72]. By cultivating algae in bold basal medium (BBM) with a 10% inoculum (OD at 620 nm = 0.2), the Chlorella vulgaris culture was preadapted to the laboratory environment [73]. The culture was cultivated in a 50 ml media at a temperature of 25-28°C with a light intensity of 1500-2000 lux. Algal growth was monitored by measuring optical density at 620 nm as described by Wang [45].
Using concentrations of 0.01, 0.1, and 1 ppm, the effects of the medication and its biodegradative product were further examined for their impact on the algal pigment's chlorophyll a, b, and total carotenoids. According to the procedure outlined by Lichtenthaler et al. in 1987, the pigment content of algae was estimated [47]. In a nutshell, a 10 ml algal culture treated with the tested chemicals was centrifuged for 15 minutes with 10,000 rpm speed at 4 o C. Pure methanol was added to the pellet, which was then heated at 45 0 C for 24 hours at 150 rpm in a shaking incubator. At 470 nm, 652.4 nm, and 665.2 nm, absorbance readings were collected. Following formulae were used for the calculation.
The phototropic nauplii were capillary harvested from the lighter side after 48 hours and utilised for bioassay. The Bioassay experiment was carried out in accordance with the steps outlined by Meyer et al. in 1982 [48].Ten shrimp were transferred to a sample vial containing 4.5 ml of brine solution (exact amount brine and yeast suspension) after being counted in the stem of the glass capillary under a lit background. Nauplii were collected in the water-lled glass capillary. Each experiment involved adding 0.5 ml of the solution to 4.5 ml of the brine solution with the indicated concentrations. 4.5 ml of synthetic seawater and 0.5 ml of synthetic seawater mixed with 0.2% DMSO were placed in the control vial.
After 48 hrs the phototropic nauplii were collected by capillary from the lighter side and used for bioassay. Nauplii were collected in a glass capillary containing water, and ten shrimps were shifted to sample vial with 4.5 ml brine solution (precise volume brine and yeast suspension) after they were counted in the stem of capillary against lighted background. In each experiment, 0.5 ml of the solution was added to 4.5 ml of brine solution of mentioned concentrations. In control vial ,4.5 ml of arti cial sea water and 0.5 ml arti cial sea water was mixed with 0.2% DMSO. After 24 hrs. Survivors were counted under 3x magni cation and in front of a lit background, and the percent deaths was calculated by using formula 7 [49].

Isolation and Identi cation of Microorganism
The isolation of microorganisms that degrade drugs has been done using pharmaceutical waste water or activated sludge [10,39]. In this work, microorganisms that break down acetaminophen and hydroxychloroquine were isolated from a sample of industrial wastewater. Based on the morphological and 16s rRNA sequencing methods, bacteria were identi ed. Gram negative rod-shaped motile bacteria were discovered to be the isolate. The isolate was identi ed using 16S rRNA sequencing of 1392 bases, a phylogenetic tree, and pairwise similarity results obtained from the GenBank database. Pairwise similarities between strain (query of C-March-213) and the species Kosakonia cowanii strain ranged from 100 to 99%, according to a comparative 16S rRNA gene-based phylogenetic study. Isolate belongs to calss Gammaproteobacteria; Enterobacterales order; Enterobacteriaceae;family and Kosakonia genus.
Kosakonia cowanii JCM 10956(T) with accession number BBEU01000098 and isolate (C-March-213) shared 99.42% similarity with 55.7% G + C contents. The sequence has been submitted in GenBank with OQ733302 accession number [50]. The Neighbour-Joining approach was used to infer the evolutionary history [ 51].The related taxa that gathered in the bootstrap test's 1000 replicates are displayed above the branches in Fig. 1 to represent the ideal tree [52]. With branch lengths that correspond to the evolutionary distances used to infer the phylogenetic tree, the tree is rendered to scale. In MEGA11, evolutionary analyses were prioritised [53].
The order Enterobacterales and family Enterobacteriaceae include Kosakonia cowanii (formerly known as Enterobacter cowanii). A Kosakonia strain has been identi ed as a plant pathogen that causes bacterial wilt [54]. But according to the present research, it has been identi ed as a human pathogen and a cause of acute cholecystitis causative agent [55]. Kosakonia cowanii has been reported to cause bacteremia in a preterm neonate[56]. Kosakonia cowanii was tested using the plate screening method for the production of the enzymes laccase (EC 1.10.3.2) and amidase (EC 3.5.1.4), both of which have been linked to the breakdown of medicines with aromatic rings and amide bonds [57][58]20]. When cultivated on soil extract agar with guaiacol, brown colour development around the bacterial colony indicates that strain is laccase producer (Fig. 2a) [37]. While amidase production potential of the strain is indicated by the pink ring that forms around the bacterial colony on mineral medium containing acetamide (Fig. 2b) [38].
Some strains of Kosakonia have been reported to be involved in the bioremediation of organophosphorus pesticides Profenofos (PF) and Quinalphos (QP) with degradation rate was found to be more than 80% due to the bio lm formation and organophosphate hydrolase (OPH) enzyme potential [59]. Kosakonia oryzae proved to be the suitable in situ candidate for bioremediation of soil polluted by Polyoxyethylene tallow amine (POEA) (nonionic surfactant) [60].

Biodegradation of Acetaminophen and
Hydroxychloroquine by Kosakonia cowanii: 3.3.1 LC50 of acetaminophen and Hydroxychloroquine for bacterial growth According to the Palma et al. 2021 methodology, the inhibitory concentrations of acetaminophen and hydroxychloroquine were determined [4]. To get the LC50 value, the online aatbio calculator was used to plot the bacterial viability percentage (%) with the logarithm of the paracetamol concentration (mg/L). Acetaminophen and hydroxychloroquine were found to have LC50 values of 2186.70 and 1735.13 ppm, respectively. Aiming for concentrations of acetaminophen and hydroxychloroquine of 1500 and 1000 ppm, respectively, was done based on the LC50 value.

Acetaminophen Biodegradation studies of Kosakonia cowanii JCM 10956(T)
The strain was inoculated in Bushnell Haas medium containing acetaminophen (1500 ppm) and hydroxychloroquine (1000 ppm) as sole carbon and energy source. Bushnell Haas is generally used for the bioremediation of xenobiotic compounds. Chopra and Kumar have used Bushnell Haas medium for the degradation of acetaminophen using Bacillus drentensis strainS1 [10]. Biodegradation of acetaminophen was found to be maximum on 5th day of incubation when bacteria were in late log phase. During the degradation pH shifting was observed from 7 to 7.4. The time course effect of biodegradation of acetaminophen by Kosakonia cowanii JCM 10956 (T)is represented in Fig. 3.
Strains isolated from membrane bioreactor, Delftia tsuruhatensis and Pseudomonas aeruginosa showed 99.99% removal of 100 µg APAP L − 1 in 5 days and hydroquinone was identi ed as acetaminophen transformation product [15]. Compared to the reported strains, isolated strain Kosakonia cowanii JCM 10956 (T) could tolerate and degrade acetaminophen in high concentration in 5 days but extracellular laccase production was not observed.

Hydroxychloroquine Biodegradation studies of Kosakonia cowanii JCM 10956(T)
As far as we are aware, this is the rst time anyone has reported the potential contribution of bacteria to the breakdown of hydroxychloroquine. The Bushnell Haas medium was inoculated with the Kosakonia cowanii JCM 10956(T) strain using hydroxychloroquine (1000 ppm) as the only carbon and energy source. Based on the spectrophotometric method, biodegradation of hydroxychloroquine was found to be maximum on 15th day of incubation when microorganism was in late log phase. The time course effect of biodegradation of acetaminophen by Kosakonia cowanii JCM 10956 (T)is represented in Fig. 4. The Hydroxychloroquine biodegradation followed a rst-order kinetic model with a rate constant of 0.0618 and half-life of 11.2 days.
According to the methodology, 10 mM guaiacol was employed as the substrate and 100 mM acetate buffer was used to estimate the concentration of laccase enzyme in the cell-free supernatant [4]. In the presence of hydroxychloroquine, bacteria found to produce 9.4955 U/ml laccase which suggests possible role of laccase in the degradation of hydroxychloroquine. Application of enzyme in the biodegradation of recalcitrant pharmaceutical micropollutants has been extensively studied. Report is available on the degradation of diclofenac and naproxen in short period with the help of commercial Laccase (Lac) from Myceliophthora thermophila [62]. Cell free synthetic systems are proving to be the better option for the remediation of drugs in short time and with high e cacy. After studying the possible role of laccase in the degradation of hydroxychloroquine, laccase based synthetic system can be developed and employed in the removal of drugs from the wastewater within short time and in eco-friendly manner. Laccase based system has been studied for the removal of antibiotic umequine [63]. Applicability of the laccase in detoxi cation can only be achieved by protecting the catalytic site of enzyme. Laccase enzyme can get denature in the urea contaminated water, hence protectants like sorbitol and proline must be used. Research data suggested that proline and sorbitol treated laccase can remove 76.2% and 82.9% of drug levo oxacin even in the presence of urea [64].
Human and veterinary medicine, ketoconazoles biotransformation and detoxi cation has been studied with the help of laccase from Trametes versicolor [65]. Although the laccase enzymes have the potential to detoxify the pharmaceutical pollutants but still the biotoxicity issue cannot be resolved [66]. Hence, current study must be further extended to study the cell based or laccase enzyme-based system for the removal of hydroxychloroquine.

Characterization of Acetaminophen Biodegradative Product
Analysis of paracetamol and its bacterial transformed product was determined by using UV-Visible for the determination of maximum wavelength. Maximum wavelength for the paracetamol and its biodegradative metabolite, found to be 244.4 nm and 289 nm. Biodegradative metabolite was further con rmed by using reverse phase HPLC. Reverse phase HPLC was carried for the quantitation of residual concentration of acetaminophen and its product in fermented broth. Standard acetaminophen was used for reference which showed retention time of 6.7 minutes when solvent system methanol: water (15:85) was used and wavelength was monitored at 240 nm (Fig. 5). Standard hydroquinone was used for reference with methanol: water (20:80) solvent system and 280 nm wavelength. Quantitation of hydroquinone was done using reverse phase HPLC, based on the calculations 85% hydroquinone is generated as product whereas no detectable concentration of 4 aminophenol was found (Figs. 6 and 7). For structural con rmation of degradative metabolite, proton NMR was carried out. 1H-NMR (300 MHz, DMSO-D6) spectra of acetaminophen degradative metabolite by Kosakonia cowanii sp. is given in Fig. 8 where chemical shifts are δ (ppm): 8.63 (s, 2H) ,6.56 (M, 4H). Based on the peak pattern, hydroxyl group and benzene ring are analysed. NMR peak at 2.51 ppm is belonging to the solvent DMSO whereas peak of 3.37 might be the impurity. Characterization of compound was done by using the techniques of UV-Visible spectrophotometry, HPLC, and NMR technique. Based on the research ndings, biodegradative pathway is proposed in Fig. 9. Hydroquinone is one of the reported degradative metabolites of acetaminophen by physical process like ultrasound method ,TiO2/UV system based advanced oxidation [6]. Report is available on the acetaminophen biodegradation by Stenotrophomonas and Pseudomonas into hydroquinone with 4 aminophenol as intermediate [14].But in the current study, 4 aminophenol was not detected showing the use of alternate degradation pathway which must be con rmed by using transcriptomics studies.Hydroquinone has gained importance in the chemical, rubber, photographic, and cosmetic industries. Even hydroquinone has been reported to be naturally occurring substance [67].

Characterization of Hydroxychloroquine Biodegradative Product
Analysis of hydroxychloroquine and its bacterial transformed product was determined by using UV-Visible spectra for the determination of maximum wavelength. Maximum wavelength for the hydroxychloroquine and its biodegradative metabolite, found to be 343 nm and 202 nm. Estimation of hydroxychloroquine using spectrophotometric based method is reported earlier [68]. Reverse phase HPLC was carried for the quantitation of residual concentration of hydroxychloroquine and its product in fermented broth. Standard hydroxychloroquine was used as reference which showed retention time of 1.4 minutes when solvent system Methanol: Acetonitrile (50:50): Water (pH 3 adjusted with Orthophospheric acid) (75:25) was used and wavelength was monitored at 343 nm (Fig. 10). Quantitation of residual hydroxychloroquine was done using reverse phase HPLC, based on the calculations 60% of hydroxychloroquine was found to be degraded into the product with retention time of 2.258,2.767,3.025,3.192,3.308,3.483 minutes (Fig. 11).For the identi cation of biodegradative metabolites mass spectra was determined which showed peaks with m/z of 260. Oxalic and oxamic acid are produced as byproducts of the photolysis method used to break down hydroxychloroquine (1250 ppm, 40% degradation rate) using a boron-doped diamond (BDD) anode [69].
In our research, mass data was used to evaluate the production of oxalic acid. Figure 13 shows the biodegradative process for hydroxychloroquine. The outcomes are consistent with the published electrogenerated oxidant degradative route utilising boron-doped diamond anode [69].
3.5 Ecotoxicological studies 3.5.1 Algal Inhibition Assay Algal inhibition assay was performed on the Chlorella vulgaris for the toxicity evaluation of paracetamol and its biodegradative product Hydroquinone. Chlorella vulgaris is cosmopolitan microalga, used for the toxicity bioassay [70]. EC50 value of paracetamol was calculated to be 460.5782 ppm which was higher as compared to the result of Wang et al. 2015 [45]. To nd the effect of pollutants on the physiological state of green algae, chlorophyll and carotenoids are considered as markers. As per the report with increase in the concentration of acetaminophen, the ratio of carotenoid and chlorophyll gets increased [45]. In this study, with increase in the concentration of acetaminophen, there was substantial increase in the carotenoid content and decrease in chlorophyll pigment. So, the results are in line with the previous reports of algal toxicity of drugs. For hydroquinone, the value of EC50 calculated to be 0.0663 ppm which suggests more ecotoxicity of degradative product as compared to parent compound paracetamol.EC50 value of Hydroxychloroquine and its biodegradative product oxalic acid was calculated to be 31.62 and 100.2657 ppm, suggesting more algal toxicity of parent drug hydroxychloroquine. Effect of parent drug and their degradative metabolite on the algal pigment is given in table 1.  [4]. In our research, we were able to determine that the EC50 value for the Chlorella vulgaris strain was 0.0663 ppm, with an inhibitory effect on chlorophyll pigments and a stimulatory effect on total carotenoids, demonstrating that hydroquinone-induced oxidative stress was present. It was discovered that after treatment with hydroxychloroquine and its nal degradative metabolite, oxalic acid, the total carotenoid pigment of algae had risen. Oxalic acid has a stimulating and inhibiting effect on the chlorophyll pigment of freshwater green algae Pithophora oedogonia and Rhizoclonium hieroglyphicum depending on the concentration [77].

Lethality test for brine shrimp
Brine shrimp lethality assay was performed for the parent drug acetaminophen, hydroxychloroquine and their respective biodegradative product hydroquinone and oxalic acid respectively. Percent mortality and LC50 value was determined using the concentration 10,100,500 and 1000 µg/ml as per the Table 2. LC50 value of acetaminophen biodegradative product, was found to be more compared to the parent drug, which suggests that drug is more toxic to the shrimp even at the lower concentration. On the contrary, for hydroxychloroquine, its biodegradative product found to be more toxic to the shrimp than the parent drug. Hydroquinone was hazardous to aquatic organisms in the environment, although it was less poisonous to bacteria and fungi. Some bacteria, including Alcaligenes sp., Moraxella sp., and Pseudomonas sp., and some fungi, including Tyromyces palustris, Gloeophyllum trabeum, Aspergillus fumigatus, Candida parapsilosis, Phanerochaete, and Penicillium chrysogenum, have been reported to be hydroquinone-tolerant [71]. Soil contaminated with hydroquinone, exhibited low β-glucosidase and dehydrogenase microbial activity [74]. Although Hydroquinone gave negative Ames test using Salmonella typhimurium strain, suggesting its non-mutagenic nature but its role in arresting the cell cycle at the G2/M transition due to the activation of Hog1-Swe1 pathway in Saccharomyces cerevisiae has been reported [75]. (Shiga et al. 2010).
Toxicity of hydroxychloroquine on the free-living marine nematodes has been tested and it has been recorded that hydroxychloroquine affects the species richness and abundance of the nematodes [4]. (Ali et al. 2021).

Conclusion
In summary, a Kosakonia cowanii JCM 10956 (T) strain that can degrade 81% and 60% of acetaminophen and hydroxychloroquine was isolated and identi ed. More signi cantly, to the best of our knowledge, this is the rst study to show hydroxychloroquine's biodegradation. It showed that the degradation capability was attributed to the potential of strain to produce laccase and amidase.
Therefore, the laccase enzyme produced by the strain is the promising agent for the removal of drugs from the polluted site. However, further research such as strain improvement program, and optimization of fermentation conditions are needed to enhance the biodegradation e ciency for hydroxychloroquine.
Meanwhile, more research is needed to identify drug-isolate interactions, and elucidate the mechanisms of drug uptake or degradation, to provide new insights into the pharma micropollutants bioremediation. Industrial application of Hydroquinone and metal leaching role of oxalic acid will give new insight in the bioconversion of expired paracetamol and hydroxychloroquine into value added products. This will be applicable for the recovery of potentially active ingredient from wastewater generation site.  Phylogenetic tree of the isolate using Neighbour Joining method.

Figure 2
Laccase and Amidase plate screening assay for Kosakonia cowanii.