Kinetics, degradation mechanisms and antibiotic activity reduction of chloramphenicol in aqueous solution by UV/H₂O₂ process

Due to lack of proper supervision with high drug administration, the fate and occurrence of bacteria which is resistant towards many antibiotic drugs have been augmented (Brooks et al. 1992). The contamination occurred due to presence of varieties of antibiotic drugs in the range from ng/L to μg/L is measured as a potential threat to the environment (Liang et al. 2013). Chloramphenicol (CHPL) shows its activity in both gram-positive and gram-negative bacteria and is a suspected carcinogen as a broad-spectrum antibiotic (Liang et al. 2013). It shows its action in protein synthesis by the inhibition of microorganisms. CHPL was found to be between 0.001 and 0.031 mg/L in surface water at different places in Singapore and Korea. In effluents of sewage treatment plants (STP), it was detected between 2.08 and 26.6 mg/L in China (Trovo et al. 2013). Conventionally, CHPL is biosynthesized by the soil organism, Streptomyces venezuelae and several other Actinomycetes as well (Aouiche et al. 2012). Conventional wastewater treatment consists of a combination of physical, chemical, and biological processes and operations to remove solids, organic matter and, sometimes, nutrients from wastewater (Trovo et al. 2013). Preliminary treatment is used to remove coarse solids and other large materials often found in raw wastewater and secondary treatment basically eliminates the residual organics and suspended solids. Apart from these different advanced treatment processes like many homogeneous advanced oxidation processes (AOPs) UV, UV/H₂O₂, ozone (O₃), Fenton, Photo-Fenton and heterogeneous AOPs like UV/TiO₂, electro photo-Fenton, UV or solar light assisted bimetal doped catalytic processes have been used to treat the wastewater. Many essential parameters like pH of the drug solution and particulate matter have been described using different specific environmental standards like European Environment Agency (EEA) and National Ambient Air Quality Standards (NAAQS).

The detection of different generic drugs found in the aquatic environment was monitored and confirmed by studying the presence of low levels of CHPL in plants and soil (<1 μg/kg) (Hu et al. 2010). CHPL in Asian countries including China was noticed within the range of 26–2,430 ng/L in influent and 3–1,050 ng/L in STP effluent (Lin et al. 2008). CHPL concentration of 47.4 μg/L in sewage was found as it is a high consumption drug in Guiyang, China (Liu et al. 2009). As expected that CHPL resistant pathogens were found in different water flows (MPEDA 2010). In India, the use of CHPL has been banned in food-generating animals in 2002 (Parsley et al. 2010). Different conventional water treatment techniques like sedimentations, coagulation and filtration, and disinfection are ineffective in eliminating antibiotics dissolved in water (Zhang & Li 2011). The removal efficiency from the aquatic environment is extremely low due to the refractory nature of antibiotics (Boxall et al. 2003). Therefore, some potential treatments are necessary to degrade these antibiotics and minimize their exposure to the environment.

Studies on CHPL have been investigated in the presence of different photocatalytic processes like the Fenton process, metallic nanoparticles, microwave irradiation, and the biochemical system (Liang et al. 2013). Due to their economic efficacy and applicability towards real situations, these above said technologies have not yet been thoroughly evaluated despite having advantages over classical methods. Violet (UV) radiation is a promising water treatment technology and has been used to activate oxidants such as peroxides, chlorine, ozone, and ferrate to produce highly reactive radicals such as HO^• radicals (Deng et al. 2019).

The first reaction is rate-limiting because the rate of this is slower compared to second one. In UVP, a higher initial H₂O₂ concentration produces higher HO^• radical concentration (Equation (1)), which decomposes the target compounds. However, an optimal H₂O₂ concentration exists because the overdosing of H₂O₂ leads to a reaction with HO^• radicals leaving off HO₂^• (Equation (2)). UVP is quite efficient in mineralizing PhACs (De Boxall et al. 2003). UV photolysis showed first-order deterioration kinetics for the decomposition of chloramphenicol drug with rate constant 6.3 × 10⁻² min⁻¹ (Sa Da Rocha 2013).

The main aim of this study is to find out the efficacy of the UVP process to degrade CHPL in an aqueous solution and to explore the reduction of antimicrobial activity which is supported by the proposed CHPL degradation mechanistic pathways. The hydroxyl radicals (HO^•) are the main species responsible for CHPL oxidation.

In order to yield the feasibility of this method for removing CHPL from its synthetic solution, we have also investigated the effect of process parameters (pH, H₂O₂ concentration, reaction time, and UV-irradiation) on degradation kinetics and the influence of foreign ions. To evaluate the toxicity reduction achieved by this process, we determined the antibacterial activity test of the UVP-treated CHPL solutions against E. coli pathogenic bacteria that are highly sensitive towards CHPL. Finally, intermediates obtained under the UVP process were identified, and a proposed mechanism that gave an evidence for reducing the toxicity of both unclevaged CHPL molecule and its daughter products.

CHPL drug with purity >99% (w/w) was procured from Sigma Aldrich (China). Figure 1 shows the chemical structure of CHPH. Acetonitrile (99% v/v purity) and oxalic acid (98% w/w purity) with HPLC grade were purchased from Merck (India). Sulfuric acid (98% v/v purity), NaOH (purity >98% w/w), H₂O₂ (50% v/v purity) and ethanol (purity >98 v/v %) were taken from Merck (India). For the toxicity test, Escherichia coli (E. coli) XL 10GOLD (purity >98.5% w/w) was collected from the Department of Biotechnology, Indian Institute of Technology Guwahati. Tryptone (98% v/v purity) and yeast (99% w/v purity) both were from Himedia (India). All reagents and solutions were prepared using mili-Q water (model: Elix 3, M/s Millipore, USA).

A spectrophotometer (model: UV-2300) was used to analyze UV-vis spectras of CHPL and find out an optimal wavelength with the maximum absorption with an optical path length of 1 cm procured from Thermo Scientific (India). CHPL concentration was determined using high-performance liquid chromatography (HPLC). C₁₈ column as a stationary phase with 25 mm in length and 4.6 ID mm was employed. A UV-Vis detector was placed in the HPLC instrument (model: LC-20AD) of Shimadzu (Japan). A mixture of acetonitrile and oxalic acid (0.01 M) (40:60 v/v) was used as the mobile phase at a flow rate of 0.5 mL/min. CHPL daughter ions identification was carried out using liquid–chromatography-time-of-flight mass spectrometry (LC-TOF-MS) system (Aquity UPLC and Waters Q-Tof Premier). Total organic carbon (TOC) measurements were made with a TOC analyzer (model: 1030C Aurora) of O.I. It was operating at 680 °C furnace temperature. Measurement of TOC by the non-dispersive infrared method, and the analyzer was equipped with an IR detector with an autosampler. pH meter (model: pH/ion 510) was used to measure pH of the solution obtained from Eutech Instruments (Malaysia).

The number of colonies forming unit (CFU) of E. coli bacteria was recorded to measure the toxicity of CHPL and its degradation products. Luria–Bertani (LB) media (yeast extract 5, tryptone ten and NaCl 10 are in g/L) was used for this antimicrobial test. The reaction mixture was autoclaved (model: 7407PAD, Medica Instrument Mfg. Co., India) for 30 min under 39.7 psi (abs) pressure (120 °C) after adjusting the pH at 7.0 using 0.5N NaOH. A mixture of 50 μL initial cultured of E. coli and 5 mL media was incubated for 24 h at 30 °C on an orbital shaker at 250 rpm (model: ORBITEK^R, Scigenics Biotech, India). The residual H₂O₂ present in the CHPL solution was removed after the UVP process using catalase enzyme before performing the toxicity assay (Shen et al. 2016). The cultured cells now made a growth media and explored into three sections. About 1–2 mL diluted growth media was added on the agar plate (APHA 1998) and the number of CFU was counted at 30 °C after 24 h of incubation. The absorption intensity of the second sample was recorded at 621 nm after diluting at different proportions. A calibration curve was then prepared by plotting the number of CFU/mL versus the absorption intensity.

The supernatant obtained from the third sample was rejected after centrifugation at 2,000 rpm for 20 min, and the collected bacterial cells were swept away using deionized water. LB media (100 mL) was used for the suspension of these collected bacterial cells and further diluted to have CFU/mL of ∼2.4 × 10⁸. About 5 mL of the CHPL sample and 1 mL of cultured media withdrawn at different time interval was mixed and incubated for 24 h at 35 °C (Giri et al. 2014). The calibration curve was then used to find out the equivalent absorbance after transforming in terms of CFU/mL unit.

Experiments were performed to investigate the CHPL degradation study under the UVP process maintained at 25 °C. The initial concentration of CHPL with 100 mg/L (TOC, 31.3 mg/L) was taken in this UVP as CHPL has a larger ability to form chelate complexes with different metals at higher concentration (Luis et al. 2009). Usually, in municipal wastewater treatment plants, high concentration of CHPL is not found. The antimicrobial activity, degradation kinetics and its biodegradability index were evaluated with CHPL concentration of 214 mg/L (Zuorro et al. 2014).

A batch experiment with the continuous stirring was explored for the CHPL degradation study. A cylindrical vessel made of borosilicate (Ø 10.5 cm) with a capacity of 1,000 mL was used as the reactor. Solution pH was adjusted by 0.05 N H₂SO₄ prior to addition of H₂O₂ and mixed at 300 rpm for about 10 min on a magnetic stirrer (model: Spinot 6,020; stirring bar: length 40 mm, Ø 0.8 mm) made by Tarson (India), and then pH of the reaction solution was further noted. The predetermined amount of H₂O₂ was added in the calculated amount of CHPL solution to achieve the final volume of 400 mL. After 1 min with the progression of the photoreaction, 10 mL sample was withdrawn, and 1 mL of 0.1 (N) NaOH was immediately added to stop the reaction by quenching of HO^. radicals. The pH was increased with the addition of NaOH to 12.5. Photolysis of H₂O₂ producing HO^. radical (Equation (1)) is tremendously fast at such high pH (Levard et al. 2013; Giri & Golder 2014).

The temperature of clear supernatant was increased to 60 °C to eject residual H₂O₂, if any. 80 μL and 5 mL clear solution was taken out to determine CHPL and TOC concentrations, respectively. 0.45 μm cellulose filter was used to filter the remaining liquid. About a 2 mL sample was used to determine the mass spectra. Likewise, under UV light alone, a blank experiment was also performed without addition of H₂O₂. An UV lamp (intensity: ∼ 9 W and wavelength: 362 nm) obtained from Hong Kong Jie Meng International Lighting Ltd Company (China) was hired for this work. Based on our earlier studies, the UV light intensity was selected in this study (Giri & Golder 2014). The UV-lamp was fixed at 5 cm above from the top of drug solution. The temperature of the reaction mixture was properly controlled using a cooling arrangement (25 ± 2 °C).

UVP treatment was significantly faster than UV process alone upon CHPL degradation (Figure 2). For the maximum amount of CHPL removal, pH, H₂O₂ concentrations and UV-intensity varied from 1.5 to 4, 5 to 20 mM, and 5 to 12 W, respectively (Figs S1 to S3). The preliminary experimental trials were done in the presence of a selected value of UV-intensity with changing pH values from 1.5 to 4 and concentration of H₂O₂ prior to the experiment. After that, UV-intensity and H₂O₂ doses were adjusted at the fixed pH value obtained in the provisional run. Again, pH was changed from 1.5 to 4 with the finest setting of UV-light keeping constant the concentration of H₂O₂ (Figure 2). The initial trial experiments were performed at different pH values from 2 to 4 with an arbitrarily selected value of UV and H₂O₂ concentration but with prior experience. After that, the dose of H₂O₂ and intensity of UV light were tuned at the pH obtained in the trial run. Further, pH was varied 2–4 with the best setting of UV and H₂O₂ concentration obtained before. We have checked at alkaline regions (pH = 11 and 13) for the CHPL removal but didn't get any significant results. Auspiciously, both the experiments at changing pH show the same pH value (Figure 2). Keeping the other two constant parameters, with increasing one of them, CHPL decomposition reached a maximum and then decreased and was ultimately stable at constant removal efficiency. The maximum removal of CHPL obtained in UVP under an optimal condition was 71.4% with UV-irradiation. UVP gave significantly higher CHPL and TOC reductions compared to UV alone (Figure 2). Figure 2 showed that CHPL and TOC removal were found in two discrete rate periods. The transition was sharp and dramatically changed from 2.5 to 10 min for both CHPL and TOC reduction. At 2.5 min, the removal of CHPL was found to be 62.7% and increased to 71.2% in 10 min in the case of UVP. Whereas UV alone shows the shifting from 17.4% to 21.3% in time from 2.5 to 10 min for the same. In the presence of UV light alone the functional groups (=CO, –OH, –NHCOR) presence inside the amide chain of CHPL could disappear (Sykes 1985).

UPV and UV showed the TOC reduction of about 37–48% and 9–11% at 2.5 and 10 min of oxidation in UVP and UV, respectively. Initially, the mineralization of the amide chain in CHPL showed the faster rate of TOC removal. Tan et al. (2017) suggested that the maximum TOC removal of 31.7% was observed in 120 min at an initial concentration of 0.03 mM of CHPL. The second stage, probably after 10 min, was associated with the cleavage of –NO₂ functional group in substituted phenyl ring of CHPL molecule followed by mineralization. Kavitha & Palanivelu (2004) reported that intermediates were usually either fragmented or mineralized to lower molecular weight daughter molecules like oxalic and acetic acid that are mineralized rapidly in the presence of UV-illumination during the initial stage of oxidation.

The COD value was experimentally determined and employed for the determination of MONC. The results are explored in Figure 3. MONC values initially estimated using either of two Equations (3) and (4) were found as 0.367. It indicates that both TOC and COD were determined with rational precision. The MONC was increased faster until <5 min, and then it improved progressively. The MONC elevated to 1.98 and 0.89 from 0.367 as initial value in 45 min of UVP and UV alone, respectively. The result can be verified by the refractory nature of the phenyl ring.

Effects of selected inorganic anions were investigated on UV/H₂O₂ mediated degradation of CHPL (Figure 4). Few inorganic ions such as F⁻, Cl⁻, NH₄⁺, HCO₃⁻ and NO₃⁻ were found to be played significant role during the remediation of CHPL (Li et al. 2020). In this study, some inorganic anions like NO₃⁻, Cl⁻ and HCO₃⁻ were added individually, and their effects on CHPL degradation was explored. The concentration of each anion was 10 mM, and the results are plotted in Figure 4. The addition of ions influenced the degradation of CHPL in the order of Cl⁻ > NO₃⁻ > HCO₃⁻. The fast reactivity of Cl⁻ with HO^. as shown in reaction (Equation (5)), possibly greatly seized the reactivity of HO^. with CHPL and consequently caused more inhibition in CHPL degradation. Li et al. reported a similar statement about the reactivity of Cl⁻ with HO^. for the degradation of ciprofloxacin drug (Li et al. 2020).

Deng et al. showed that NO₃⁻ could hinder the removal efficiency of the micropollutants. NO₃⁻ is strongly sensitive towards UV and acts as HO^. scavenger (H₂O₂) (Equation (8)). But the influence of NO₃⁻ was probably through NO₂⁻ for the drug degradation in the present study (Equations (10) and (11)). NO₃⁻ ions also show an important role in UVP due to conversion to nitrite (NO₂⁻) (Csay et al. 2012). The production of HO^. radicals were also hindered because of NO₃⁻ photolysis (Equations (8) and (9)). Park et al. suggested that the remediation of volatile organic carbon (VOC) occurred by the inhibiting effect of NO₂⁻ during photolysis and HO^. radical depletion occurred due to progressive accumulation of NO₂⁻ (Csay et al. 2012).

The intermediate reaction products originated during UVP process of CHPL were analyzed by the HPLC-MS/MS, LC-QTOFMS, and IC analysis with ESI⁺ mode. The chromatogram for the degradation products is available in the Supplementary Information (Fig. S4). The proposed mechanism is illustrated under the optimal time of 45 min reaction time. There are four mechanistic pathways (paths 1–4) of CHPL oxidation were proposed in Figure 5(a) and 5(b). Thirteen daughter intermediates were found in this study (Figure 4), and their detailed information is given in Table S1. Two asymmetry centers (C₁ and C₂) in CHPL with hydrogen atoms that were weakly bonded to each other. HO^• radical could easily abstract these protons from these centers (Puma & Yue 1991). The presence of the amide group (-CONHR) attached to the C₂ center increased the nucleophilicity of both center (Puma & Yue 1991). Giri et al. stated that the carbonyl carbon (=CO) becomes more nucleophilic in character due to two chlorine (–Cl) atoms in the amide side chain at their geminal position (Levard et al. 2013).

Therefore, the shifting tendency of a lone electron in chlorine atom towards the carbonyl carbon is there. D₃ with m/z 171.13 was yielded for the attack of HO^• radical at C₁ asymmetric center. Therefore, the C₁-C₂ bond in the side chain of the CHPL molecule is broken easily under the attack of HO^. radicals, and then the substituted amino (–NH₂) group is easily oxidized to form p-nitro benzyl alcohol because of the lower C-N bond energy (Nie et al. 2014). Many hydroxylated products were yielded after CHPL degradation because of the addition of hydroxyl (–OH) or carbonyl (=CO) group on the CHPL structure (Aresta et al. 2010).

Thirteen fragments (D₁–D₁₃) were found in mass spectra in 45 min of UPV. From the difference between the proposed and exact masses of daughter ions, mass errors were calculated. Generally, low mass errors were gained from the fragmented molecules (Table S1). Four paths showed the formation of D₁, D₂, D₃, D₄, D₅, D₆, D₇, D₈, D₉, D₁₀, D₁₁, D₁₂, and D₁₃ with molar masses of 157.13, 270.12, 253.30, 237.10, 328.11, 153.11, 305.12, 197.38, 167.21, 242.3, 214.09, 198.21, 340.11 and 283.10, respectively (Figure 5(a) and 5(b)). The molar masses were expressed in terms of mass to charge (m/z) ratio. CHPL molecule is first oxidized at C₂ center to form an intermediate of molar mass 308.12, which produced dichloroacetamide and p-nitrobenzyl alcohol under C-N bond cleavage and hydrolysis followed by decarboxylation (–CO₂), respectively. Dichloroacetamide gets hydrolyzed at pH 3.0 and was converted to dichloromethane (DCM) with the evaluation of NH₃ and CO₂ that leads to evidence for the reduction of both COD and TOC (Figure 5(a)). P-amino phenol (p-AP) (m/z, 124.11) is the reduced product originated from p-nitrobenzyl alcohol through the reduction of p-nitrophenol and evidencing of the reduction of toxicity. Path #1 shows the formation path of D₁ to D₆ with m/z of 157.13, 270.12, 253.30, 237.10, 328.11 and 153.11, respectively. D₁ was yielded in UVP with low mass error (0.06 g/mol) through the amide chain cleavage followed by hydroxylation of CHPL molecule shown in path #1 (Figure 5(a)). D₂ and D₃ are produced from the p-AP through hydroxylation and dehydration reactions, respectively (Figure 5(a)). Hence, D₂ was originated from p-aminophenol (p-AP) with the molar mass of 124.11 by an intermolecular hydrogen bonding between them and was yielded by the reduction of –NO₂ group at p-position. An intermolecular H-bonding between p-AP molecules appeared from the partial charge (δ⁺… δ⁻) separation on oxygen and hydrogen atoms in –OH and –NH₂ groups (Du et al. 2006). D₂ dimer molecule was identified in UVP. UV irradiation could easily break such weak H-bond (Sykes 1985). Again, D₄ and D₅ were hydroxylated products originated from D₃ molecule through hydroxylation reaction. Hence, an electrophilic substitution reaction was occurred effectively into D₃ due to the +R effect of the -NH₂ group that increases electron clouds in the phenyl ring (Tan et al. 2013). In the presence of UV light, the stability of CHPL could lose due to the high capability of light absorption within its functional groups that depends on electron distribution. The order of light absorption capacity of sigma (σ) bonds are very little above 200 nm and falls in the order of C-H > C-C > C-O > O-H), whereas pi (π) bonds (–COOH, = CO) and the ability of the compounds especially in conjugated pi systems are increased to absorb UV light at 254 nm by lowering the net energy difference between ground and excited states (Feiven et al. 2002). Double bond equivalent (DBE) indicates the degree of unsaturation of an organic molecule (Table S1) and signifies the number of molecules of H₂ that would have to be added to a molecule to convert all π-bonds to σ- bond. It was calculated by the summations of the residual ring(s) and the total number of pi bonds (π) (Kavitha & Palanivelu 2004). UVP showed hydroxylated compounds showed higher DBE (Figure 5(a) and 5(b)).

The formation of D₇ and D₈ molecules was shown in path #2. D₇ was yielded due to dehydration of CHPL, and D₈ was originated by hydroxylation followed by decarboxylation (-CO₂) of D₇ molecule along with the formation of DCM. Hence, decarboxylation of the –COOH group of the intermediate with molar mass 251.4 originated D₇ fragment was significantly happened because of the presence of an electron-withdrawing –NO₂ group. D₇ molecule is broken down to form D₉ shown in path #3 with the evaluation of ammonia (NH₃) and CO₂. D₉ (p-nitrobenzoic acid) undergoes further reduction to form p-aminobenzoic acid (p-ABA) and ultimately transferred to CO₂, NO₃⁻ and H₂O, that confirms for an effective COD reduction. In addition, D₁₀ to D₁₄ products were formed through path #4, where CHPL gets cleavage in different routes apart from paths #1, 2, and 3 (Figure 5(b)). D₁₀ originated from CHPL with a series of successive reactions associated with the decarboxylation (–CO₂) reaction followed by hydroxylation along with the formation of DCM. Hence, DCM is originated as the CHPL containing a group (–CHCl₂) that could be decomposed to DCM and chlorine-free radical (Cl^.) under the UV-light (Zhang et al. 2009). D₁₁ molecule was yielded from the intermediate with m/z of 246.03 due to the reduction of –NO₂ group (Figure 5(b)). Under an acidic condition (pH 3.0), –NH₂ group in D₁₁ is protonated and goes to form D₁₂ with the liberation of NH₃. D₁₃ is a direct hydrolyzed product of the CHPL molecule. D₁₄ (AMCl) was originated from CHPL under reduction followed by dehydrohalogenation (–HCl) reaction (Figure 5(b)). Hence, –NO₂ group is reduced to –HN₂ group and Cl atom is removed through homolytic bond cleavage that gives evidence for the antimicrobial activity reduction. Zhang et al. (2009) suggested that D₁₄ (AMCl) is less toxic in nature towards microorganisms and could be easy biodegradable. The reduction of CHPL to D₁₄ would have environmental significance in terms of antibiotic elimination for the generation of antibiotic-resistant bacteria/genes in the environments (Nie et al. 2018). This observation strongly recommended that the final D₁₄ has much less antibacterial activity than CHPL.

CHPL molecules are oxidized by HO^• radicals instantly and act as common oxidants (Equation (12)), and the fragmented products are formed. The reaction temperature of the solution was measured in a small range (23–25 °C). The pH difference was not shown significantly during the degradation of the drug. It was supposed that only active HO^• radical among the other reactive oxidation species (ROS) is involved to cleavage the CHPL drug in the solution.

NO₃⁻ has also strong inhibition effects on CHPL removal, and this effect is increased with increasing NO₃⁻ concentrations. In the presence of NO₃⁻, the rate constant (k_obs) suddenly decreased from 2.93 × 10⁻² min⁻¹ to 1.17 × 10⁻² min⁻¹ in UVP. Tan et al. reported a close value of the rate constant of 0.97 × 10⁻² min⁻¹ on the degradation of chloramphenicol in water by UV/persulfate system (Nie et al. 2018). The similar effect of HCO₃⁻ on the CHPL degradation was found, and the rate constant dropped to 1.67 × 10⁻² min⁻¹ in UVP. Li et al. showed that the pseudo-first-order rate constant was found as 1.55 × 10⁻² min⁻¹ in the presence of HCO₃⁻ on the chloramphenicol degradation during nZVI-heat-activated persulphate oxidation.

The effectiveness of the UVP process was assessed in terms of antibiotic activity removal against E. coli pathogens. The toxicity of CHPL and its decomposition products in UPV to E. coli in LB media (10 g peptone +5 g yeast extract +5 g NaCl +10 g agar) after 24 h of exposure was shown in Figure 7. Pathogen E. coli, as the gram-negative bacteria is highly sensitives towards CHPL (Nie et al. 2018). The activity of E. coli was measured in the CHPL solution in UVP treatment under optimal conditions.

The growth of E. coli was almost inhibited in the presence of 100 mg/L of CHPL entirely was found to be 0.23 × 10⁷ CFU/mL (Figure 7). In toxicity analysis, the growth of E. coli was found 8 × 10⁷ CFU/mL in the presence of LB media and deionized water. The growth of E. coli strain was inhibited after 24 h of exposure in the presence of 32 mg/L CHPL (Liang et al. 2013).

The toxicity in UVP and UVP in presence inorganic ions were comparable. The exposure of toxicity to E. coli was more in UVP in the presence of Cl⁻ even though there was no significant difference between Cl⁻ and NO₃⁻ in UVP as the HO^. radicals scavenging ability of both ions the same. The addition of ions influenced the order of toxicity of CHPL degraded products as Cl⁻ > NO₃⁻ > HCO₃⁻ (Figure 7). About 62.8, 49.7, 47.7, and 53.7% cell death were noticed after 24 h contact of E. coli in the reaction mixture collected after 45 min of UVP, UVP in the presence of Cl⁻, NO₃⁻ and HCO₃⁻, respectively, in comparison to the control condition (Figure 7). It has been found that the product D₁₄, hydroxylamine chloride intermediate (AMCl) lost its antimicrobial activity significantly than pure CHPL due to the reduction of –NO₂ group and the elimination of –Cl^. radical in terms of HCl (Figure 5(b)). Liang et al. showed similar observations for the same intermediate AMCl when CHPL is reduced with biocathode of applied voltage of 0.5 V in a bioelectrochemical system (Liang et al. 2013). Two amine products obtained under CHPL cleavage (D₁₁) and a novel dechlorinated product (D₁₄) were identified in this study (Figure 5(b)) and also showed evidence for the reduction of toxicity. P-nitrophenol and p-nitro benzyl alcohol intermediates are toxic in nature, and the firster one was identified in UVP. Both molecules were mineralized into CO₂, NO₃⁻ and H₂O (Finar 2001; Demir et al. 2010).

The degradation of CHPL was investigated by UV/H₂O₂ photolysis advanced oxidation process. The following conclusions are explored in our study:

1. The maximum CHPL and TOC removal of 72.4% and 51.3%, respectively, were found with initial CHPL concentration of 100 mg/L at optimum conditions.

2. CHPL removal were 21.3% and 71.2% at 45 min when treated in UV alone and UVP, respectively. It dropped to 63.8%, 65.4% and 67.2% in the presence of Cl⁻, NO₃⁻ and HCO₃⁻, respectively.

3. UVP exhibited TOC removal of 47.8% and 11.0% in UVP and UV alone, respectively.

4. The MONC was found to be increased from 0.367 to 2.02 in UVP at the optimal conditions.

5. Measurement of toxicity and the MONC have given the information about the production of low toxic compounds and an idea of the experimental errors between COD and TOC analysis.

6. Thirteen fragmentations with m/z ratio were primarily formed upon CHPL degradation in UVP. Hydroxylation and decarboxylation reactions were significantly found for the CHPL cleavage.

7. A pseudo-first-order kinetic model for the cleavage of CHPL in UVP exhibited good fittings to the experimental data. Rate constant for CHPL degradation was found to be 2.93 × 10⁻² min⁻¹ where as in presence of Cl⁻, NO₃⁻ and HCO₃⁻ ions, k_obs were found as 1.07 × 10⁻² min⁻¹, 1.17 × 10⁻² min⁻¹ and 1.67 × 10⁻² min⁻¹, respectively.

8. The toxicity of intermediates was found to be increased in presence of foreign ions. D₁₁ and D₁₄ products showed decrease in antimicrobial activity due to reduction of –NO₂ group which is present in these molecules.

All relevant data are included in the paper or its Supplementary Information.