Introduction

Novel solutions are thought of which might help to counter the issue of water pollution and scarcity and tapping the Unconventional Water Resources (UWRs) like wastewater can be a significant part of the solution1. However, the nature of wastewater is changing due to the presence of Emerging Contaminants (ECs)2 and it limits the potential of wastewater as a UWR. Hence, the remediation of such ECs becomes inevitable in the present context.

Benzophenone-3 (BP-3), aka Oxybenzone, is a slightly hydrophobic ultraviolet filter having wide utility as a photostabiliser in Personal Care Products, plastics, furniture, toys, food and pesticide additives, etc.3 However, as per the United Nations Globally Harmonised System, it has been assigned the codes H315, H319, H335, H411 and H413, being proven as an irritant and is also considered an environmental hazard, especially for aquatic life4. It has also been recognized as an EC5. BP-3 majorly contaminates the water bodies indirectly through the Sewage Treatment Plants. Other sources are recreational activities6. The maximum reported concentration of BP-3 in wastewater is 3.95 mg L−17. It has bioaccumulating, bioconcentrating and biomagnifying potential and disrupts the endocrine system, showing estrogenic and anti-androgenic effects8. BP-3 and its metabolites are responsible for coral bleaching as well9. Scientists estimate that BP-3 exhibits joint toxicity with microplastics10. This emphasizes the removal of BP-3 from aquatic bodies.

Physicochemical methods of BP-3 degradation, like irradiation11, chlorination12, ozonation13 or reverse osmosis14, are inefficient, either due to inadequacy to remove this contaminant or formation of toxic end-products adversely impacting the surroundings or are not financially viable15. Also, the present methods of secondary and tertiary wastewater treatment are inadequate for the elimination of Emerging Contaminants. Considering the water scarcity problem and the corresponding use of UWRs, the worldwide presence of BP-3 in all aqueous matrices, its adverse impacts on biota and the inadequacy of the present physicochemical methods of water treatment, there is a dire need for eco-friendly solutions for BP-3 remediation. Biochar as a bioadsorbent is a potential alternative for activated carbon in pollution abatement and water treatment applications, as it is cheap, readily available, has better adsorption capacity and is ecologically friendly16,17. It would help in concentrating the contaminants, thereby making further wastewater treatment faster and easier; however, pristine biochar does not degrade the contaminants18. Secondly, phytoremediation using duckweed Lemna minor can be considered a “green clean” technique owing to the high removal and degradation potential of micro-pollutants19 due to its root structure, easy growth, and it does not necessarily add-in xenobiotics into the system20. Lemna minor has shown high removal efficiency (89–100% in 14 days) for multiple Endocrine-disrupting chemicals, Pharmaceuticals and Personal Care Products and Antibiotics such as Bisphenol-A, N,N-diethyl-meta-toluamide, triclosan, estrogens, cefadroxil, metronidazole, etc.21,22 It also provides added advantages like water quality enhancement, feed for livestock and biofuel production23. However, aquatic macrophyte-assisted remediation is a time-consuming process and may hence result in incomplete remediation24. Also, the limitations of previous bioremediation experiments are lab vs. field scale performance25, long time taken for phytoremediation24 and reduced removal in real water matrices21. Hence, a hypothesis was placed that the combined use of biochar and Lemna for bioremediation in water would help to overcome these shortcomings and achieve synergistic remediation of BP-3. Also, Kumar et al. suggest that the integration of biochar with macrophyte-based treatment systems is intended to couple the physicochemical adsorption capacity of biochar with the biological degradation and uptake processes mediated by aquatic plants, thereby enhancing the removal of contaminants, while simultaneously contributing to carbon sequestration and sustainable wastewater treatment26. So far, no work has been done using biochar or Lemna minor, alone or in integration, for the removal of BP-3. Hence, this study was designed to evaluate the bioremediation potential of a combination of biochar and Lemna minor-based system for BP-3 removal from spiked distilled- and waste- water.

Materials and methods

Materials

Analytical grade Benzophenone-3 (CAS No. 131-57-7), 2,4-Dihydroxybenzophenone (2,4-DHB) (CAS No. 131-56-6) and 2,3,4-Trihydroxybenzophenone (2,3,4-THB) (CAS No. 1143-72-2) were obtained from Sigma-Aldrich, India. A stock solution of BP-3 (20,000 mg L−1) was prepared in absolute ethanol and a working standard of 20 mg L−1 was prepared in distilled water. Other chemicals and reagents used for the research were procured from Sigma-Aldrich and Sisco Research Laboratories, Mumbai, India. Glassware and plasticware were procured from Borosil Limited. Effluent municipal wastewater was sampled from the Versova Wastewater Treatment Plant and stored in a refrigerator until use.

Preparation and characterisation of biochar

Raw sugarcane bagasse waste was collected from local juice shops, as it was readily available in ample quantity, and was sun-dried to remove moisture. Bagasse was pyrolyzed at 400 °C with a temperature ramp rate of 4–5 °C min−1 for 1 h and 30 min in an electrical heating kiln, and the biochar was prepared. The conditions of preparation were decided based on previous BP-3 removal screening studies.

The produced biochar was examined for different physicochemical characteristics. Estimation of Cation Exchange Capacity (CEC) was done following Song & Guo27 The carbon, nitrogen, hydrogen and sulphur content of the biochar was estimated using a CHNS Elemental Analyser (Vario MICRO cube, Germany) and functional groups of raw as well as BP-3-impregnated biochar were identified using the Fourier Transform Infrared (FTIR) spectroscopy technique (SHIMADZU, FTIR 4100). Scanning Electron Microscopy (SEM) (SEM, FEI Quanta 200, United States) and Brunauer–Emmett–Teller (BET) Surface Area and Pore Size Analysis (NOVA 1200e, Quantachrome Instruments) of the prepared pristine biochar were done to study the surface morphology and internal surface area. SEM + EDX of the BP-3-impregnated biochar was done post-treatment using a Field Emission Gun-Scanning Electron Microscope (FEGSEM JEOL JSM 7600F).

Culture of Lemna minor

Lemna minor pure culture was procured from the wetlab of ICAR—Central Institute of Fisheries Education, Mumbai. It was initially maintained outdoors in FRP tanks and then cultured indoors in 500 ml Erlenmeyer flasks and plastic trays under controlled conditions in SIS media, 7 days prior to the bioremediation experiment28. The culture conditions in the plant growth incubator were as follows: temperature-24 ± 1 °C; light intensity-30 watts and 24 h illumination. Acid-washed culture vessels and aseptic handling techniques were followed to avoid contamination.

Bioremediation of BP-3 with biochar and Lemna minor in spiked distilled water and wastewater

Integrated removal of BP-3 was studied by first remediating BP-3 using biochar, followed by Lemna minor in BP-3-spiked distilled water and wastewater. The baseline conditions for removal using biochar were standardised based on experimental trials and were as follows: BP-3 concentration-10 mg L−1, Biochar dosage- 0.1%, Contact time-1 h, pH-7.3, Temperature-30 °C, intermediate sampling at every 15 min. This was followed by the preparation of SIS media for the growth of Lemna in the biochar-treated water samples and then BP-3 removal using Lemna minor was checked for 7 days. The standardised baseline conditions were: Media volume : Lemna biomass-100 ml: 0.1 g, Contact time-7 days, pH-6.5, Temperature-24 ± 1 °C. The experiments were conducted in duplicates, accompanied by a biotic and an abiotic control. Procedural blank for BP-3 remediation using biochar was set up in an Erlenmeyer flask with biochar and without BP-3 in water. And abiotic control was set up with 10 mg L−1 BP-3 without biochar. For Lemna-assisted bioremediation, the abiotic control had BP-3 without Lemna, and the biotic control had Lemna without BP-3. BP-3 and its metabolites’ concentrations in the culture water and the plant tissue were quantified, based on method optimisation done using the authentic standards and the retention times thus obtained, following sampling on the 2nd, 4th, and 7th day. Reaction kinetics and isotherm modelling of the removal experiments were studied.

Sample processing and quantification of BP-3

The culture water and plant tissue samples collected were processed prior to the quantification of the analyte.

Aqueous sample preparation for analysis

The water and plant extract samples were processed to extract, purify and concentrate the target compounds, according to Downs et al.29. For this, “10 ml aqueous sample was loaded onto 1 cm3 30 mg Oasis HLB solid phase extraction (SPE) cartridges. The cartridges were then washed with 3 mL of HPLC-grade water and dried under a gentle current of nitrogen. Then, the analytes were eluted with (a) 7.5 mL of ethyl acetate and dichloromethane (1:1, v/v) and (b) 2 mL of dichloromethane. The extracts were completely evaporated with nitrogen gas and then reconstituted with 0.5 mL of HPLC-grade water. This reconstituted sample was transferred to HPLC vials and finally, 10 μl of the extracts were analyzed by the HPLC”.

Lemna extract sample preparation for analysis

The plant samples were processed to extract the analytes from their tissues as per Chen & Schroder30 where “0.5 g of plant material was macerated in a mortar and pestle and extracted with 1.5 mL H2O/acetonitrile (30:70, v/v). The extracted sample was centrifuged for 30 min at 13,000 × g, and the supernatants were further purified with 1 cm3 30 mg Oasis HLB SPE cartridges,” as per Downs et al.29.

Analysis of purified samples

The extracted and concentrated samples were analysed using HPLC (Agilent Technologies 1260 infinity II) and the column used was Eclipse XDB-C18, 5µ, 4.6*250 mm. The mobile phase was methanol : water, and a gradient elution method was used, as shown in Table 1. The flow rate was maintained to be 0.9 ml/min and the total run time was 15 min. The oven temperature was 25 °C. The samples were analysed at a wavelength of 330 nm and the detector used was a Diode Array Detector.

Table 1 Gradient chromatography conditions for HPLC analysis.
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Adsorption kinetic modelling

Adsorption kinetics modelling helps predict solute uptake rate and imparts an understanding of the adsorption mechanism.

Pseudo-second order modelling

The pseudo-second order kinetic model’s linearized form is written as-

$$\frac{t}{{q_{t} }} = \frac{1}{{K_{2} q_{e}^{2} }} + \frac{1}{{q_{e} }}^{{}}$$

where, t = time interval, qt = adsorption capacity at t time, qe = equilibrium adsorption capacity and K2 = rate constant of adsorption.

Intraparticle diffusion modelling

The equation for the intraparticle diffusion model is as follows-

$${q}_{t}= {k}_{1}{t}^{1/2}$$

where (0 ≤ t ≤ t1), qt = adsorption capacity at time t, k1 = model parameter.

Adsorption isotherm

Langmuir isotherm31

BP-3 adsorption data at various concentrations were fitted to the linear equation of the Langmuir isotherm, which can be written as:

$$\frac{{C_{e} }}{{q_{e} }} = \frac{1}{{q_{m} b}} + \frac{{C_{e} }}{{q_{m} }}$$

where Ce = Equilibrium concentration, qe = Equilibrium adsorption capacity, b = Langmuir constant and qm = Maximum sorption capacity.

The dimensionless constant separation factor (RL) is used to calculate the fundamental parameters of a Langmuir isotherm, which is written as:

$$R_{L} = \frac{1}{{1 + bC_{0} }}$$

where C0 = Initial concentration, b = Langmuir constant, type of isotherm depends on the value of RL, such as Favourable: 0 < RL < 1, Unfavourable: RL > 1 and Linear: RL = 1.

Freundlich isotherm32

The Freundlich adsorption isotherm was first suggested by Freundlich to account for simple adsorption and model equation can be written as:

$${log q}_{e}= log {K}_{F}+ \frac{1}{n} log {C}_{e}$$

where qe = equilibrium adsorption capacity, n = measure of deviation from linearity of the sorption, KF = biosorption capacity and Ce = Equilibrium concentration.

Removal efficiency

The removal efficiency was calculated using the following formula at various time intervals:

$$Removal efficiency \left( \% \right) = \frac{{\left( {C_{0} - C_{e} } \right)}}{{C_{0} }} \times 100$$

where, C0 and Ce were the concentrations of BP-3 before and after treatment.

Wastewater quality analysis

Secondary-treated wastewater effluent was collected from Versova Waste Water Treatment Plant, Lokhandwala, Andheri (W), Mumbai. It was characterized to estimate the physicochemical parameters- before and after treatment in the proposed system. Temperature, Dissolved Oxygen (DO) and pH were measured using sensors (PondGuard, Eruvaka Technologies). Alkalinity, Hardness, Total Ammonia Nitrogen, nitrite, nitrate and phosphate were estimated following the methods of the American Public Health Association33. Elements such as Calcium, Sodium and Potassium were analyzed by using a flame photometer (Microcontroller Flame Photometer Labard LIM-204).

Statistical analysis

To confirm the normal distribution of the dataset, it was subjected to the Shapiro–Wilk test prior to analysis. The statistical testing was done using SPSS 16.0. Data was tested using One-way ANOVA and Duncan multiple-range tests (p < 0.05).

Results and discussion

Preparation and characterisation of biochar

The sugarcane bagasse biochar (SBB) prepared at 400 °C was black, coarse, porous and amorphous. The yield obtained was 33%. Characteristics of the prepared biochar are outlined in Table 2.

Table 2 Characteristics of SBB.
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FTIR spectroscopy analysis of SBB

The FTIR spectra of sugarcane bagasse and its biochar reveal the presence of different functional groups on their surface (Fig. 1A, B). The vibrational stretching of the aromatic hydroxyl functional group is observed at a wave number of 3780.48 cm−1. The 3360.93 cm−1 wave no. shows the presence of a non-polymeric hydroxyl group. At 1707 cm−1 wave no. carbonyl and carbonate groups are present. There is a vibrational stretching of secondary amine and alkane at 1586.2 and 754.17 cm−1 wave nos., respectively. Figure 1C shows the presence of BP-3 at 1672 cm−1 wave no. attributed to the vibrational stretching of the carbonyl group of BP-3. Quantitative analysis reveals that the FTIR spectra (Fig. 1C) correspond to a similar trend as that of the BP-3 removal achieved in the BP-3-spiked distilled water and wastewater.

Fig. 1
Fig. 1
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FTIR spectra of (a) Raw sugarcane bagasse, (b) SBB produced at 400 °C and (c) Spent SBB derived at different time intervals (30 min and 60 min) from distilled water (DW) and wastewater (WW).

Scanning electron microscopy analysis of SBB

SEM is an effective technique for visualising the surface morphology of biochar. The SEM micrographs demonstrate a porous structure of the sugarcane bagasse used as a feedstock for the biochar (Fig. 2A). Further, the SEM micrograph at magnification 250× (Fig. 2B) reveals that pyrolysis at 400 °C improved the porous and flaky nature of the prepared SBB, thus aiding in BP-3 adsorption. At magnifications, 2500× and 25000× (Fig. 2C, D), rounded or elongated pores on the biochar surface are clearly visible. BP-3 bound to the biochar by surface binding and pore-filling mechanisms is evident in the SEM images at 2000× and 25000× magnifications (Fig. 2E, F).

Fig. 2
Fig. 2
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SEM micrographs of SBB-400 °C (a) raw sugarcane bagasse, (b) SBB before BP-3 adsorption [X250], (c) SBB before BP-3 adsorption [X2500], (d) SBB before BP-3 adsorption [X25000], (e) SBB after BP-3 adsorption [X2000], (f) SBB after BP-3 adsorption [X25000].

Integrated remediation of BP-3 using biochar and Lemna minor in spiked distilled and wastewater

Removal of 10 mg L−1 of BP-3 by biochar in distilled water and wastewater was studied at optimised conditions as shown in Fig. 3 and its concentration was checked as shown in Table 3. BP-3 removal was a maximum of about 54.36 ± 1.34% at 60 min in BP-3 spiked distilled water, and in wastewater it was 32.25 ± 0.46% at 60 min. The reactions followed the pseudo-second-order rate of reaction and the intraparticle diffusion model (Supplementary Figs. S1, S2). The biochar-treated BP-3-spiked distilled and wastewater was further treated with Lemna minor for 7 days. The total removal by Lemna was 42.63 ± 0.94% and 71.16 ± 1.42% till the 7th day in biochar-treated distilled and wastewater samples, respectively.

Fig. 3
Fig. 3
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Flow chart of the bioremediation process.

Table 3 Concentration of BP-3 in distilled water and wastewater after biochar and Lemna treatment.
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Thus, the integrated remediation yielded a total BP-3 removal efficiency of 73.82 ± 1.65% and 80.46 ± 0.98% in the distilled and wastewater samples, respectively. The reactions followed a pseudo-second-order reaction rate (Supplementary Figs. S1, S2). Isotherm modeling was also done, wherein the Freundlich isotherm model fitted the remediation reaction and the R2 was above 0.9. This indicates that the surface of the adsorbent is heterogeneous and has unevenly distributed adsorptive sites with different adsorptive capacities34. Further, it suggests that there is multi-layer adsorption of the BP-3 molecules onto the biochar. Batch removal experiments of Sulfamethoxazole and Di-methyl Phthalate using biochar as a substrate in vertical flow constructed wetlands also followed pseudo-second order kinetics and intraparticle diffusion models and Freundlich isotherm model and removal of around 99.5% of dimethyl phthalate is reported35 and is comparable with the present study.

BP-3 was metabolised into its constituent products 2,4-DHB and 2,3,4-THB after Lemna treatment in both distilled and wastewater samples and their concentration detected in the water as well as the plant tissues is mentioned in Table 3. This is the first report of the formation of 2,3,4-THB in plants.

In the remediation experiment, the initial concentration of BP-3 was 10 mg L−1, and around 70–80% BP-3 was removed in the integrated system of biochar and Lemna minor. Out of the 80% removal, 4–11% was transformed into 2,4-DHB metabolite, 3–6% was metabolized as 2,3,4-THB, as found in the water and Lemna tissue samples post-treatment. Also, an insignificant proportion of 2–5% contributed to abiotic removal processes. Thus, a significant amount of BP-3 was degraded by Lemna minor.

Overall, the removal of BP-3 was maximum in biochar-treated distilled water samples than in wastewater samples. However, for Lemna treatment, the removal was more in the case of wastewater samples as compared to the distilled water samples. Similarly, the overall removal of BP-3 was greater in the wastewater samples than in the distilled water samples.

Mechanism of BP-3 removal from the water matrix

BP-3 removal mechanism by SBB

Characterisation of the prepared biochar provided meaningful insights to decipher the BP-3 removal mechanisms. Pyrolytic temperature (400 °C) enhanced the biochar’s surface-to-volume ratio and its microporosity as depicted through the SEM images (Fig. 2). The pores on the biochar surface thus acted as a sieve and trapped the BP-3 molecules. Also, the BET surface area of 33.11 ± 1.07 m2 g−1, an average pore diameter of 32.63 ± 1.89 Å and the reaction being a good fit to the intraparticle diffusion model help to conclude that diffusion and pore-filling would be the major removal mechanisms. Secondly, Bilba & Ouensanga36 proved that the structural functionalisation of biochar initiates at 200 °C and progresses vigorously between 300 and 400 °C during pyrolysis, enriching it with more functional groups. As per Fonseca et al.37 and de Moraes Rocha et al.38 bio-adsorbents produced from sugarcane bagasse contain functional groups such as hydroxyl, carboxyl, amine, amide, hydrogen sulfide and methoxy groups which are the potential sites for adsorption of contaminants by various mechanisms like ion exchange, complexation or adsorption. Along similar lines, the FTIR results (Fig. 1), showing the abundance of oxygen-containing functional groups coupled with a higher Cation Exchange Capacity (Table 2) suggested that ion exchange might be another mechanism for BP-3 remediation. Furthermore, the flame photometry readings after biochar treatment revealed that the number of cations (Na+, K+, Ca2+) discharged to the solution as a result of the interaction of these molecules were significantly different (Table 4), additionally confirming the hypothesis.

Table 4 Initial and treated wastewater quality parameters.
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Mechanism of BP-3 degradation by Lemna minor

The major contaminant removal mechanisms in phytoremediation studies are abiotic processes like photodegradation, evaporation, hydrolysis and biotic processes like plant uptake and accumulation and microbial degradation39. Consistent with the earlier reports that BP-3 is inert to abiotic degradation40, abiotic removal of BP-3 seen over all the treatment concentrations was found to be about 2–5% over an experimental period of 7 days. According to Collins et al.41 and Pilon-Smits42, the plant can take up mildly hydrophobic compounds by crossing the lipid bilayer membrane. Likewise, BP-3 could be diffused into the plant. Additionally, Collins et al.41 have quoted that direct plant uptake and accumulation of contaminants are key processes in phytoremediation. Similar mechanisms might prevail for the biodegradation of BP-3 as BP-3 and its metabolites were also recorded in the plant tissues in the present experiments, as also observed by Chen & Schroder6.

Mechanism of comparative removal of BP-3 in distilled and wastewater media by biochar and Lemna minor

The removal of BP-3 through biochar was more in the distilled water than in the wastewater. This may be due to the competitive binding of ions present in wastewater, since the wastewater is a complex mixture of organic and inorganic contaminants. The SEM + EDX data (Fig. 4) of the biochar used for wastewater treatment also explains the mechanism of competitive adsorption of ions and subsequent less adsorption of BP-3. The carbon percentage of raw sugarcane bagasse is 59.93% (Table 2), and carbon and oxygen are the major components of its biochar as well. Other minerals like sodium, chlorine, potassium, calcium and copper could be found in lesser amounts. These two main elements might come from the oxygen-containing carboxyl and hydroxyl functional groups or oxygen-containing carbonate, phosphate, sulphate ions. However, as seen in the EDX data, there is flash adsorption of carbon compounds onto the biochar, thereby increasing to 71.34% after 30 min of introducing biochar into the wastewater. Thus, there are fewer sites available and more competition for the other elements to adsorb. However, after 60 min, it is seen that the carbon percentage of the biochar has reduced to 64.25% due to its competitive exclusion or desorption into the wastewater, making more room for the other elements to adsorb. Thus, the amount of other elements like magnesium and calcium and the BP-3 adsorption on the biochar surface increased during 60 min of biochar treatment. Correspondingly, the O/C ratio has also increased from 0.36 to 0.47 from 30 to 60 min of biochar treatment. Thus, it is contributing to the adsorption of BP-3 from the wastewater onto the biochar surface by providing oxygen-containing carboxyl, carbonate and hydroxyl functional groups. However, the overall removal of BP-3 in wastewater remained less compared to that in distilled water, as wastewater was, in general, rich in other ions competing for adsorption. Similar findings were reported by Kim et al.43 who studied the effect of inorganic salts on the adsorption of 4 micropollutants- Benzophenone, Benzotriazole, Bisphenol A and 17 β-Estradiol. They stated that the CaCl2 salt interfered and competed with the micropollutants for sorption onto the adsorbent surface, largely hindering the micropollutant removal.

Fig. 4
Fig. 4
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SEM + EDX of sugarcane bagasse biochar after (a) 30 min and (b) 60 min of BP-3 removal in wastewater.

However, the removal by Lemna minor was more in the case of wastewater than distilled water. This is attributed to the abundance of nutrients essential for Lemna’s growth in wastewater compared to the distilled water, which was quite evident from the healthy growth of the duckweed in the experimental wastewater setup. No chlorosis, necrosis, or colony breaks were observed in the system. A similar conclusion was drawn by Dosnon-Olette et al.44 that adverse conditions affect the health of the duckweed and hamper the contaminant removal potential of the plant.

Wastewater quality analysis

The wastewater sampled from the Versova Wastewater Treatment Plant was characterised to know its water quality dynamics before and after treatment, and the same is presented in Table 4. A significant increase in DO after biochar treatment could be attributed to multiple reasons, which might be due to the rotational shaking causing enhanced atmospheric diffusion. The larger micropore volume of the biochar also increases the interfacial surface area for atmospheric oxygen diffusion45,46. Also, biochar has endogenous oxygen-containing functional groups, which might impart an increase in the DO of the water47. In terms of DO enhancement by Lemna treatment, comparable findings were obtained by Selvarani et al.23 The significant DO enrichment and negligible pH increase can be attributed to the photosynthesis of the plant during Lemna treatment. The total DO increment after the combined treatment was 88.20%. An insignificant increase in alkalinity of about 3.27% is observed post-treatment, which is consistent with the pH. The increase in total hardness post-biochar treatment is not significant, with an insignificant decrease of 1.93% post-Lemna treatment and this trend is consistent with the calcium concentration during the treatment.

Post-biochar treatment, ammonia, nitrite, and nitrate content decreased in water due to direct adsorption by biochar48. In Lemna treatment, as suggested by Rogers et al.49, nitrogen utilisation by a plant is the major mechanism of its removal, wherein a nitrification–denitrification complex is established. This is well supported by the presence of a nitrification cycle observed in the results of this study (Table 4), wherein there is a significant reduction in the ammonia content with a subsequent increase in the nitrate content. Lemna utilises ammonia as a nutrient for its growth and leaves behind nitrite and nitrate as suggested by Matusiak et al.50 and Tam & Wong.51 The overall decrease in ammonia was 84.01% and nitrate content increased by 57.17%. Phosphorous enrichment in water is observed post-biochar treatment, possibly due to the leaching of endogenous phosphorous in the biochar.48 In terms of Lemna treatment, phosphorous is also an essential nutrient utilised by plants by various mechanisms like plant uptake, immobilisation by microorganisms in the plant detritus, entrapment by sediments and precipitation in the water column.23 Considering the similar results obtained in terms of phosphate reduction in the experiment, the plant uptake mechanism might be contributing majorly to the 73.65% of the total phosphate reduction. The increase in the concentration of cations like sodium, calcium and potassium post-biochar treatment suggests that the adsorption of BP-3 has happened by a cation exchange mechanism due to the CEC of the biochar and conforms with the report of Ding et al.52. The further significant decrease in potassium post-Lemna treatment might be due to its uptake by the plant, as it is also an essential element for plant growth.53

Conclusion

The present study highlights the potential of biochar and Lemna minor for the remediation of an emerging contaminant, BP-3. The results reveal that biochar and Lemna act synergistically when combined together in an integrated system. BP-3 removal by biochar is higher in spiked distilled water, while Lemna contributes significantly in the case of BP-3 spiked wastewater. The total BP-3 removal achieved is 73.82% in spiked distilled water and 80.46% in spiked wastewater. Thus, the overall removal is more in wastewater. Lemna metabolised BP-3 into 2,4-DHB and 2,3,4-THB, which are less potent metabolites of BP-3 and this is the first report of the formation of the latter in macrophytes. The optimised treatment conditions also improved the wastewater quality, making it suitable for further reuse. Thus, biochar acts as an adsorbent and Lemna as a phytoremediator, which metabolises and breaks down the compound and renders it less toxic, aiding in its easy elimination. It also addresses significant wastewater concerns such as DO depletion, ammonia build-up and over-fertilisation. Further research on the regeneration and reusability of the spent biochar for repeated adsorption cycles followed by use as a soil amendment and evaluation of the utility of Lemna for other uses such as biofuel can make the proposed model a sustainable circular model for wastewater treatment (Supplementary Fig. S3). Utilisation of biochar sourced from agro-waste and Lemna as bioremediating agents and making use of natural processes is, thus, a potential eco-friendly system for wastewater treatment. However, joint toxicity of BP-3, along with any other pollutants present in the real water matrices, to Lemna minor, inhibiting its degradation capacity; along with environmental fluctuations, can be a major limitation of this system. Also, the BP-3 adsorption potential of the sugarcane bagasse biochar would depend on the feedstock available and the competing ions; and the week-long bioremediation experiment can limit the on-field applicability of the model. Hence, further research on column removal studies and on-field applications is required to scale up the proposed water treatment model.