A simple and highly efficient approach towards the degradation of methylene blue and study the impact of degraded water on seed germination of cicer arietinum

The scarcity of fresh air, drinking water, and soil is a matter of serious concern worldwide owing to the presence of organic pollutants in the environment. The organic dye, such as methylene blue (MB) have enormous toxic effects on the environment and human health. Therefore, the degradation of non-biodegradable dyes is very important to reduce toxicity in water and-a step towards waste water management systems. This paper focuses on the degradation of non-biodegradable MB dye using carbon quantum dots (CQDs). CQDs were synthesized by a microwave irradiation method using citric acid and L-cysteine as precursor and confirmed by X-Ray Diffraction (XRD), Raman Spectrum, Fourier Transform Infrared Spectroscopy (FTIR) and Energy Dispersive x-ray (EDX) spectroscopy techniques. The optical properties of the synthesized CQDs of 2.56 nm, were investigated by UV-visible spectroscopy technique and the absorption peak appeared at 340 nm which corresponding to n → π* transition. In photoluminescence (PL) spectra analysis, the highest emission peak was obtained at 440 nm when excited at 345 nm. The synthesized CQDs were used for the dye degradation of MB in distilled water and degradation percentage was calculated and found to be 99.17% in 90 min under UV light irradiation. Also, studied the impact of degraded water in seed germination of Cicer arietinum (black gramme) and calculated the seed germination growth rate in degraded water was found to be 15%–20% more than the seed germination growth rate in MB containing dye water.


Introduction
Water pollution is a critical issue developed due to immense population growth and increase in demand of water.To reduce consequences of this issue, new advanced technological resources are being explored to clean the water from organic pollutants, keeping the sustainability of the technique which could be harmless to aquatic health and other resources.These water pollutants leave dreadful effects on health of human beings and also the other living organisms.The studies have found out and measures hundreds of endocrine-disrupting chemicals (EDCs) in living beings.These are so harmful that even small trace of these pollutants are enough to disrupt systems of any organisms and can cause adversities such as abnormalities in gene, infertility, feminization, increase in rate of cancer, they can trigger Alzheimer or other diseases.The microbe's organisms and human beings are in threat because in a large amount dyes are released in natural water sources from textile industries.Above a certain level Methylene blue (MB) dye has harmful effect on the environment and human health due to its carcinogenic, substantial toxicity and non-biodegradable.MB dye ricks on mental health and physical health such as blindness, distress, digestive, abdominal disorders respiratory distress and mental disorders.In the last few years, semiconducting nanoparticles have gained attention in photocatalysis of organic pollutant due to environmental-friendly, cost-effective and easy method for wastewater treatment.This can be achieved by harnessing the natural light as the demand of photoactive materials have drawn attention because of abundance sunlight energy and its potential application in the form of catalyst in hydrogen generation by water splitting technique and for removal of water pollutants .
CQDs are the type of carbon nanomaterials, having size less than 10 nm.Due to high solubility in water, negligible toxic effect, good photoluminescence in visible spectrum range, great biocompatibility, stable nature in photobleaching process and feasible functional properties, these materials are developing as interesting research materials.These properties of CQDs make it a promising candidate for applying in cellular imaging, biosensing, drug delivery and catalysis (Li et al 2012).Nanomaterials of carbon can be synthesized in many shapes, the known among them are: graphene, carbon nanotube (CNTs), which could be multi or single-walled nanodiamonds, Nanofibers, Buckminsterfullerene (C60) (Li et al 2011, Li et al 2012, Rochester 2013, Pires et al 2015), Dao et al 2016, Thambiraj andShankaran 2016), comprising graphitic layered sheets and, the recently developed Carbon Dots (CQDs) (Li et al 2012, Jusuf et al 2018, Dhiman and Singh 2019, Wang et al 2019, Zhou et al 2019, Anand et al 2020).All of these carbon nanomaterials show excellent physicochemical properties, possibly evolving the technology in the near future (Li et al 2011).In comparison to the organic dyes or CQDs, CQDs exhibits excellent photo luminescent in terms of high solubility in aqueous media, chemically inert, functionalization and good biocompatibility (Li et  Recent researchers discovered the fact that, CQDs exhibit PL emission in the spectral region of near-infrared (NIR), whenever excited by the irradiation of NIR light.That notable point is that, PL emission at NIR region, of CQDs, is particularly useful for nanobiotechnology, in vivo due to their body tissues which are transparent in the NIR 'water window'.
It is interesting to know that, PL from CQDs are quenchable effectively by using electron acceptor molecules or the electron donor molecules in acceptor-donor system.This excellent property of CQDs shows great potential in the field of photovoltaic, and light energy conversion devices (Sarkar et al 2020, Ahlawat Dhiman et al 2021).CQDs can also serve as useful candidates in Nano-probes in the field of sensitive ion detection (Thambiraj and Shankaran 2016).The outstanding water solubility, unique optical properties, low economic value, environmental compatibility, environment friendly, chemically less reactive and simple synthesis routes of CQDs make it good candidates for photocatalytic application.CQDs possess unique fluorescence behavior and photoelectron transfer properties for high-performance photocatalyst.The surface functional groups of CQDs are adjusted and their band gaps are decreased, further facilitating the electron transfer among reactions.Advanced CQDs-derived photocatalyst materials for the degradation of dyes are central to the area of the environmental pollution.CQDs-enabled photocatalysis are regarded as one of the most efficient technologies to degrade pollutants in water.By some specific surface modification, the absorbance and PL properties of CQDs are tuned for the specific applications.CQDs can be both, electron acceptors as well as donors leading to good separation of electron and hole.The up conversion effect of CQDs is responsible for the increase in sunlight photo absorption value of wide-band-gap semiconductor materials to visible regions significantly and even for the near infrared region.Overall, CQDs find their application in designing of high-performance photocatalysis and can be used in various advance applications such as electron mediator, spectral converter and photosensitizer.
Moreover, previously reported articles for the degradation of dye used bulk particles, nanosheets, and quantum dots (Cheng et al 2022, Guo et al 2022, Wu et al 2023).Table 1 is summarized the results obtained from the present work to the previously reported research work.The current study shows good photocatalytic activity of CQDs in degrading MB dye from water.Generally, in the previously reported work researcher have used CQDs as supporting ingredients to enhance the photocatalytic performances of their nanomaterials.In present study CQDs itself shows a good photocatalytic activity without using any other materials.

Chemicals
The precursors, citric acid (CA) (C 6 H 8 O 7 ) and L-cysteine, Methylene blue (C 16 H 18 ClN 3 S) were purchased from Sigma-Aldrich, India.Hydrogen peroxide (H 2 O 2 ) was purchased from Qualigens.All the precursors were used as such and prepared the solutions in deionized (DI) water.

Instrumentation
The UV-Vis spectroscopy was performed using the T90 + UV/VIS Spectrometer, while the PL spectra were acquired using the Cary Eclipse Fluorescence Spectrometer.The size of synthesized CQDs was studied using high-resolution transmission electron microscopy (HR-TEM) of JEOL JEM-2200FS, Japan.For a TEM (transmission electron microscopy) study, an evenly dispersed solution of CQDs was prepared using ultra sonication.The amorphous nature of CQDs were recorded using an XRD (Cu Ka radiation (λ = 0.15406 nm), Rigaku Miniflex 600 Diffractometer).The spectra were recorded in range from 10 to 70°(2θ), having a step size of 5°min −1 at RT, and the EVA software was used to assess the procured data.The Raman spectroscopy of CQDs was carried out in the range of 500-2000 cm −1 using EnSpectr R532, laser of WITEC system.The FTIR was carried out using (Perkin Elmer in universal ATR mode) to study the functional groups and chemical bonds available in CQDs.The elemental composition of synthesized CQDs using EDX (JEM-2200 FS, JEOL).
A centrifuge was used for centrifugation of CQDs in order to obtain pure CQDs.Electron spin resonance (ESR) spectra were obtained using a Bruker model A300 ESR spectrometer.The settings for the ESR spectrometer were center field 3450.00G, microwave frequency 9.73 GHz, power 4.84 Mw, and modulation amplitude 4.91.

Synthesis of CQDs
For the synthesis of CQDs, 1 gm of citric acid and 25 mg of L-cysteine were thoroughly mixed in 10 ml of DI until a clear solution was obtained.This mixture was microwaved in oven at 720 Watt for 3 min.After the reaction was completed, the beaker was kept at room temperature for cooling.After that, 10 ml of DI was added into the reaction mixture.Further CQDs purified by centrifugation process at 10,000 RPM for 20 min.The CQDs solution was kept at 4℃ for further use.The concentration of CQDs is 0.093 gm in 1 ml solution.

Experimental procedure for photocatalysis of MB dye
For dye degradation application, 10 ppm solution of MB was prepared in DI water to study catalytic and photocatalytic properties of CQDs.250 μLof CQDs were added to the solution of dye and ultrasonicated for 30 min to get adsorption/desorption equilibrium on the surface of CQDs.Then, constantly stirred the solution under dark environment at the rate of 300 rpm, and then added H 2 O 2 to the reaction.All the dye solution were studied under three different conditions, first, under the closed and dark chamber, second, under visible light exposure i.e. wavelength (λ) > 400 nm (using 100-Watt Philips bulb) and third, under Ultra-Violet light i.e. wavelength (λ) < 400 nm (using 300-Watt Osram Lamp) irradiation.Light intensity calculated as 9.5 * 10 3 watt m −2 .The parameters which were maintained constantly during all these process was, the distance between beaker and bulbs kept at 15 cm and stirring speed at 300 rpm.The reaction solution was took ff at different time intervals of 0, 10, 20, 30, 45, 60, 90 min and recorded the absorbance spectra of the solution.

Seed germination assay
The study of seed germination of Cicer arietinum in the presence of DI, MB dye and final degraded dye solution was carried out with the slight modification in previously reported method (Sajjadi et al 2019).The final dye Degraded water shows the exert cytotoxic and anti-proliferative effects on seed germination of black gramme (Cicer arietinum).The Cicer arietinum seeds were taken because they are reasonable and easy to grow in a lab.As plants have nuclei, which makes them analogous to human and animal cells, the main objective of this research is to provide an alternative to the animal or human bioassay models.

Optical studies of CQDs
The UV-absorbance and PL spectroscopy were used to study the optical properties of CQDs.Figures 1(a

Surface morphology and particle size analysis
The TEM images of CQDs is exhibited in figure 3

CQDs concentration study
In CQDs concentration study, the concentration of MB dye i.e. 10 ppm was fixed with varying the concentration of CQDs from 100 to 400 μl(100 μl, 150 μl, 200 μl, 250 μl, 300 μl, 400 μl) in 30 ml of MB solution.UV-Vis absorption spectra of various prepared concentrations of GQDs MB were taken in a different intervals of time from 0 to 90 min (0 min, 30 min,60 min,90 min) in presence of H 2 O 2 .Figure 4, illustrate the UV-Vis spectrum of CQDs concentration in MB solution, clearly shows that as by increasing the CQDs concentration from 100 to 250 μl, absorption intensity decreases and by further increasing the concentration of CQDs, absorption peak intensity get saturated which indicates that there is no further radicals are formed which responsible for degradation of MB dye.Therefore, 250 μl was the optimum concentration used for the further experiments for MB dye degradation in water.

pH study of MB dye
For pH study, 30 ml of MB dye solution was taken in beaker added few drops of nitric acid (HNO 3 ) of 0.1 M for the acidic medium and few drops of NaOH of 0.1 M were added for basic medium.Figure 5(a) shows that UVvisible spectrum of MB dye in acidic medium no significant change in absorbance intensity in acidic medium whereas UV-Vis absorption spectrum in basic medium shown in figure 5(b) illustrates that there is more adsorption taking place and degraded the MB in a long period of time i.e. approximately (150 min) to but degraded in more time.So, we did the degradation of MB dye in neutral medium which is more efficient (90 min) than the results obtained with the degradation of MB dye in basic pH (150 min).Therefore, it is observed that MB dye is degraded in a lesser minute 90 min in neutral pH as compare to acidic and basic medium.Therefore, further experiments were performed for the degradation of dye at neutral pH.

Control study
In this control study, The absorption spectra of MB in the presence of CQDs, for catalytic and photocatalytic degradation accomplished in the dark, visible light irradiation, and UV light exposure as shown in figures 6 (A) (a), (b), and (c) respectively.Figures 6(B) (a), (b) and (c) demonstrtes the absorption spectrum of MB with H 2 O 2 UV-Vis range, for catalytic and photocatalytic performance in dark, visible exposure and UV light irradiation.It was observed that there was no significant change when CQDs treated with MB dye and also when MB dye treated with H 2 O 2 separately.Then, finally UV-vis absorbance spectra of MB dye with CQDs in presence of H 2 O 2 .Figure 8 shows the photocatalytic dye degradation mechanism of MB using CQDs.The absorbance peak of MB appeared at 668 nm and after the dye degradation process, even the absorbance peak position appeared at the same wavelength (668 nm) under the UV-light.Some of the authors have explained that with light irradiation, the electrons excite to the conduction band

Length profiling assay of cicer arietinum
To Seedlings were placed in water overnight to assess vitality before the experiment began.The immersed seeds are washed with DI after 24 h.Thereafter, 23 seeds were placed on filter sheets in 9 cm petri dishes, and 20 ml of both the degradation solution and non-degradation solution were added to each dish shown in figure 10.Dishes were stored in the dark at (26±2) °C for 48 h.DI was used as a control.Three times the experiment was run.Lastly, a thread was used to measure the sprout length, and the results were represented as a percentage growth of inhibition (PIG).

Conclusion
The carbon quantum dots (CQDs) were prepared by the microwave process using citric acid and L-cysteine as precursors and size, shape were investigated by spectroscopy and microscopy techniques.The highly intense fluorescence peak found by florescence technique.The broad peak obtained at 340 nm is because of the transition of n → π * in the CQDs.In PL spectra analysis, the highest emission was obtained at 440 nm when excited at 345 nm.The spherical shape of CQDs have average particle size around 2.56 nm which was determined using TEM technique.Further, synthesized CQDs were utilized for degradation of MB dye.The percentage of degradation in the dark, visible and under the UV light, were calculated as 5, 35 and 99.17%, respectively.The percentage of degradation was high under UV light which was calculated as 99.17% in 90 min.
Here, mineralization process was dominated process among all the other processes.The pseudo first-order rate constant of this reaction was obtained as 0.017 min −1 .The percentage length of seed germination Cicer arietinum increased from 15 to 20% when compared to polluted dye solution.
) and (c) depicts UV-Vis spectrum andemission spectrum of as prepared CQDs.The broad peak obtained at 340 nm is due to the transition of n → π * in the CQDs.As reported previously the peak obtained at 310 nm is shifted to 340 nm (Ahlawat et al 2023).The Band gap of CQDs was calculated as 3.18 eV, by Tauc's plot technique (shown in Inset, figure 1(b)) (Ahlawat et al 2023).These CQDs exhibited high PL emission and shows maximum emission intensity at 440 nm when excited at 345 nm (Sarkar et al 2020, Ahlawat et al 2021, Sangeetha et al 2021, Kujur et al 2022) as shown in figure 1(c).
Figure 2(a)  shows the X -Ray diffraction (XRD) pattern of the CQDs.A single broad peak appeared at 2θ = 27.70°,which is consistent with the (002) lattice spacing of carbon-based materials with abundant sp 3 disorder.Also, the interlayer spacing CQDs was calculated using Braggs law and found to be of 3.21 A˚indicates lesser crystallization with more amorphous in nature(Vikneswaran et al 2014, Wei, Zhang et al 2014, Ramanan et al (2016).

3. 3 .
Raman spectroscopy and FTIR spectrum studies Figure2(b) illustrates The Raman spectrum of the CQDs exhibits two peaks at 1350 cm −1 and 1574 cm −1 , corresponding to the D and G bands, respectively.The D band is associated with the vibrations of carbon atoms with dangling bonds in the termination plane of the disordered graphite.The G band is associated with the vibration of sp 2 carbon atoms in a two-dimensional (2D) hexagonal lattice.The ratio of ID/IG is 0.93, which is characteristic of the disorder extent and the ratio of sp 3 /sp 2 carbon, implying that there are plenty of structural defects in the CQDs(Ramanan et al 2016).Further, the obtained ratio clearly depicts that the nanoparticles formed are amorphous CQDs and not graphene quantum dots.FTIR spectrum of CQDs depict in figure 2(c) illustrates the surface functionalities and vibrational bondings.The band appeared at 3390 cm −1 is attributed to the O-H stretching vibrations of the surface hydroxyl groups, whereas weak stretching can be found in the broad absorption centered at 2075 cm −1 .The band at 1633 cm −1 and the weak one at 1384 cm −1 arise due to C=O and C-O stretching vibrations of carboxylic ester group, respectively(Mehta et al 2014, Sachdev andGopinath 2015).

Figure 1 .
Figure 1.(a) corresponds to UV-Visible absorption spectrum (b) Inset shows the Tauc plot derived from UV -Visible spectra, (c) PL spectra of CQDs with various excitation from 300 to 360 nm.
(a).The TEM images show that CQDs were uniformly distributed and spherical in shape (Peter et al 2017, Ahlawat Rana et al 2021, Ahlawat et al 2023).The particle size and surface morphology of CQDs were analyzed with HR-TEM technique.For the current work, the histogram was plotted to calculate the average size of 2.56 ± 0.03 nm as shown in figure 3(b).The elemental analysis of CQDs, as shown in figures 3(c), (d), was carried out using EDX technique.It confirmed the presence of oxygen, carbon and nitrogen atom in CQDs composition.

Figure 3 .
Figure 3. (a) Transmission electron microscope image of CQDs (b) Size distribution histogram from TEM image, (c) EDX spectra of CQDs (d) shows the elemental composition of synthesized CQDs.

Figures 7
Figures 7(a), (b), and (c) show the absorption spectra of MB dye in presence of CQDs and H 2 O 2 for catalytic and photocatalytic degradation accomplished in the dark, visible, and UV illumination.No major changes were
(CB) from valence band (VB), causing generation of holes in the VB (Kujur and Singh 2020, Ahlawat, Rana et al 2021, Ahlawat et al 2023).The excited electrons of CQDs get displaced from CB of CQDs and interacted with MB molecule due to electro negativity differences and reduction of O 2 to O 2 -take place.Besides this, holes of VB of CQDs oxidize OH to OH • and simultaneously O 2 molecule interacts with H 2 O to obtain OH • radical.These OH • radicals then further react with MB which break down it into H 2 O and CO 2 molecules.This process is known as mineralization process.The electron paramagnetic resonance (EPR) spin-trap technique is applied to reveal the reactive oxygen species on the photo

Figure 5 .
Figure 5. UV-Vis spectra of MB dye solution treated using CQDs under (a) acidic medium (b) basic medium.

Figure 6 .
Figure 6.(A) UV-Vis spectra of MB dye solution using CQD only in (a) Dark environment (b) under visible light exposure(c) under UV light exposure and (B) UV-Vis spectra of MB dye solution treated using UV-Vis spectra H 2 O 2 in (a) Dark environment (b) under visible light exposure (c) under UV light exposure.

Figure 7 .
Figure 7. (a) UV-Vis spectra of MB in dark, (b) spectra in visible light exposure, (c) spectra under UV-Vis light exposure, and (d) graph corresponds to the C/C 0 versus time plot.(c), the inset picture of dye solution demonstrate the change in color of the dye solution for stock solution under light irradiation.
LN stands for the average length in the degradation and non-degradation solutions, and LC stands for the average length in the control solution (Komilis et al 2005, Chaudhary et al 2018).The length was obtained 66.4%, 85.2%, 100% for MB, MB degraded solution and control solution shown in figure 11.

Figure 10 .
Figure 10.Cicer arietinum seed germination test in the dye, control, and dye-degraded solution.
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Table 1 .
Previous research work compared with present work.