Degradation of Reactive Yellow 18 Using Ionizing Radiation Based Advanced Oxidation Processes: Cytotoxicity, Mutagenicity and By-Product Distribution

Abstract

The degradation of Reactive Yellow 18 (RY-18), induced by gamma radiation in aqueous medium, was carried out as a function of gamma radiation dose (5–20 kGy) and concentration of hydrogen peroxide, the initial dye concentration and pH of the solution were optimized for the maximum degradation efficiency. Gamma radiations alone and in combination with H₂O₂ were used to degrade the RY-18. A degradation rate of 99% was achieved using an absorbed dose of 20 kGy, 0.6 mL H₂O₂ in acidic pH. Variations in the functional groups of untreated and treated RY-18 were determined by FTIR analysis. The LCMS technique was used to determine the intermediates formed during the degradation process. The cytotoxicity and mutagenicity of RY-18 were studied by hemolytic and Ames tests, respectively. There were significant reductions in cytotoxicity and mutagenicity in response to gamma radiation treatment. Cytotoxicity was reduced from 15.1% to 7.6% after treatment with a 20 kGy absorbed dose of gamma radiations with 0.6 mL H₂O₂. Mutagenicity was reduced by 81.3% and 82.3% against the bacterial strains TA98 and TA100 after treatment with a 20 kGy absorbed dose with 0.6 mL H₂O₂. The advanced oxidation process efficiency was evaluated using the byproduct formations, which were low-molecular-weight organic acid units, which through further oxidation were converted into carbon dioxide and water end products. Based on RY-18 degradation, cytotoxicity and mutagenicity reduction, the gamma radiation in combination with H₂O₂ has potential for the removal of dye from the effluents.

1. Introduction

Dye contamination is a major source of water pollution from the textile industries, which is increasing day by day due to advancements in the textile sector. Synthetic dyes are commonly used in various fields [1,2,3]. However, the excessive use of synthetic dyes causes serious environmental problems, because 12% of textile dyes are disposed of into water resources without applying detoxification and treatment processes. Almost 280,000 T/Y of all textile dyes are released into water sheds without treatment worldwide [4]. In industrial effluents, an average dye concentration of 300 mg/L has been reported [5].

According to dyeing applications, dyes can be classified into various categories, such as disperse, acidic, basic and reactive dyes. The most common class of synthetic dyes is reactive dyes, because these are soluble in water and are used to dye cotton fabrics [6]. In the dyeing process, reactive dyes form covalent bonds with cellulose fibers, which have hydroxyl groups for good fastness. Reactive dyes offer strong forces to anchor with fibers [7]. A wide range of reactive dye colors with low costs and good fastness properties are available, such as navies, black, bright shades, etc. [8]. According to the reactive groups present in reactive dyes, these can be classified as vinyl sulphone and triazinyl ring systems. They undergo addition reactions or substitution reactions to react with hydroxyl moieties present in cellulose fibers [9].

Chemical coagulation, catalytic reduction, biological treatment and adsorption are the common traditional methods to degrade dyes [10,11,12]. These methods are inappropriate for degradation processes because these produce some harmful byproducts that require separate treatment processes and cause environmental pollution [13,14]. On the other hand, advanced oxidation processes (AOPs) are successfully used to degrade dyes present in industrial wastewater [15]. The AOP uses strong oxidant-like hydroxyl species, which are produced in situ and start a reaction sequence to degrade the organic molecules into smaller inorganic moieties, which are harmless. AOPs include many techniques to degrade dyes, such as ozonation, Fenton and photo-Fenton processes, gamma radiations, photocatalysis, UV photolysis and sonolysis, etc. [16,17,18,19].

Different high-energy radiations are reported in the literature to decolorize the dyes, but the most suitable alternative to degrade the dyes present in wastewater is the use of gamma radiations [20]. Powerful species such as hydroxyl radicals are produced by gamma radiations, which non-selectively degrade the organic dyes into inorganic moieties, such as CO₂, H₂O and other less harmful substances [21]. During the degradation process with hydroxyl radicals, other different species, such as hydrogen radicals and hydrated electrons (e_aq⁻), are also generated [22]. Hydrogen peroxide is used as an oxidant to enhance hydroxyl radical production and to scavenger the reducing species, such as hydrated electrons and hydrogen radicals [23].

This study aimed to evaluate the effect of gamma radiations on the degradation of RY-18 dye. The effects of oxidant hydrogen peroxide, dye concentration, and absorbed gamma radiation dose on the degradation of RY-18 were also studied. UV–visible and FTIR techniques were used to investigate the progress of the degradation process. LCMS analysis was used to determine the intermediates formed during the degradation process. The efficiency of AOP was assessed by the bases of dye degradation and the toxicity reduction of the RY-18 dye.

2. Material and Methods

2.1. Chemicals

RY-18 (C₂₅H₁₆ClN₉Na₄O₁₃S₄, 906.12 g/mole) was purchased from Khawaja Company of dyes, Faisalabad, Pakistan. The chemical structure of RY-18 is given in Figure 1 and the properties are given in

Table 1. Properties of Reactive Yellow 18.

Name of DyeReactive Yellow 18
Molecular formula	C25H16ClN9Na4O13S4
Molecular weight (g/mole)	906.12
Chemical nature	Anionic yellow 18
Class	Single azo
Color index name	Yellow
Color index number	13245
λmax (nm)	410

. Hydrogen peroxide (30%) was obtained from Sigma Aldrich (Germany). Sodium hydroxide (0.1 M) and hydrogen chloride (0.1 M) were used to adjust the pH of the solution. Distilled water was used to prepare the stock solution and other solutions of different concentrations. Further, 0.45 µm Millipore filter paper was used to filter the dye solution after gamma radiation treatment. All of these chemicals were of analytical grade and were used as received.

2.2. Determination of λ_max of RY-18

A spectrophotometer (UH5300-HITECH, Tokyo, Japan) was used to determine the maximum wavelength at which RY-18 gives the maximum absorption. Analysis of RY-18 was performed in the 200–800 nm wavelength range.

2.3. Preparation of Sample Solution

The RY-18 solution of different concentrations (30–90 mg/L) was prepared by dilution from the stock solution (1000 mg/L). All the concentrations were subjected to gamma radiation treatment to monitor the degradation. Distilled water was used to prepare all the solutions.

2.4. Gamma Treatment of Sample

Gamma radiation treatment was performed at the Nuclear Institute of Agriculture and Biology (NIAB), Faisalabad. Different dye concentrations (30–90 mg/L) were studied. Gamma radiation source Cs-137 was used and the dose rate was 669 Gy/h. Pyrex glass tubes were used to treat samples under gamma radiation. A total of 10 mL of each dye concentration (30–90 mg/L) was taken in Pyrex glass tubes, and irradiation was performed for different absorbed doses (5–20 kGy). This experiment was performed at room temperature. A spectrophotometer (UH5300-HITECH, Tokyo, Japan) was used to measure the absorbance of dye solutions before and after treatment. The effect of pH was studied in the 3–9 range. The effect of hydrogen peroxide (0.2–0.8 mL) in combination with gamma radiation was also studied.

2.5. FTIR Analysis

In order to study the different functional groups present in RY-18, FTIR analysis was performed. The RY-18 analysis was performed using an FTIR spectrometer (U-2001, Schimadzu, Kyoto, Japan) in the range 400–4000 cm⁻¹ [21].

2.6. LCMS Analysis

Liquid chromatography mass spectroscopy was used to analyze the degraded sample of RY-18 at the National Institute of Biotechnology and Genetic Engineering (NIBGE), Faisalabad. A mass spectrometer (Thermo Scientific, Waltham, MA, USA) with a linear ion trap and electro ionization sprayer was used. Before direct insertion into the mass spectrometer, a sample was extracted in methanol. Gamma degraded samples were scanned in the range 0.0–500 m/z ratio. The flow rate for injection of the sample was 10 µL/min, while the temperature and voltage were 280 °C and 4.2 kV, respectively [24].

2.7. Toxicity Analysis

Hemolytic and Ames toxicity tests were performed to check the cytotoxicity and mutagenicity of treated and untreated samples. Before toxicological tests, hydrogen peroxide present in degraded samples was removed by using less than 0.1 mg/mL of magnesium dioxide. Hydrogen peroxide was removed in 45 min and samples were ready for toxicological tests.

Human red blood cells were used to study the hemolytic activity of RY-18 samples, and Triton X-100 and PBS were used as positive and negative controls, respectively. In a Falcon tube, 15 mL of blood was taken and then centrifuged for 5 min at 850× g. Supernatant was removed and pellets were cleaned using phosphate buffer saline (PBS). A total of 180 µL of blood cells and 20 µL of sample were incubated for 30 min at 37 °C. Then, they were centrifuged at 1310× g for 5 min, and 100 µL of supernatant was taken and diluted with 900 µL of PBS [25]. Percentage hemolysis was calculated using the following formula:

$$\mathrm{Hemolysis}\left(\%\right)=\frac{\mathrm{Abs}.\left(\mathrm{sampel}\right)}{\mathrm{Abs}.\left(\mathrm{Triton}\mathrm{X}-100\right)}\times 100$$

Mutagenicity of samples was studied against bacterial strains TA98 and TA100 (Salmonella Typhimurium). NaN₃ (0.5 µg/100 µL) and K₂Cr₂O₇ (0.01 g/L) were used as positive controls, while distilled water was used as a negative control. Agar plates in plastic bags were incubated at 37 °C for 4 days. The total number of wells in blank plates was counted, and then the number of wells in incubated plates was counted. The total number of yellow wells gives the number of affected plates for mutagenicity analysis [26,27].

3. Results and Discussion

3.1. Reactive Yellow 18 Analysis

A spectrophotometer (UH5300-HITECH) was used to monitor the absorbance at 410 nm. The absorbance spectrum is shown in Figure 2. Later on, the absorbance of all the degraded samples was measured at a wavelength of 410 nm, and the percentage degradation was calculated using the following formula:

Percentage degradation = (A₀ − A/A₀) × 100

3.2. Effect of Dye Concentration

To study the influence of the initial concentrations of RY-18, different concentrations (30, 60, and 90 mg/L) were treated. Gamma radiations of different doses, from 5 to 20 kGy, were used. The effect of the initial dye concentration on the degradation of RY-18, under different doses of gamma radiations, is shown in Figure 3. It was observed that as the initial concentration of dye was increased from 30 mg/L to 90 mg/L, the degradation efficiency was decreased from 99% to 70%. The maximum degradation was achieved using 30 mg/L at 20 kGy. At higher concentrations of dye, the optical density increases and light photons cannot penetrate. At higher concentrations of dye, fewer rays can interact with hydrogen peroxide and fewer hydroxyl species are produced, and degradation was decreased. Therefore, as the concentration of dye was increased, the degradation rate decreased. However, the dye degradation increased linearly with the absorbed dose of gamma radiation. These findings are in line with previous studies [28,29].

3.3. Effect of Hydrogen Peroxide

The effect of hydrogen peroxide on the degradation of RY-18 was also studied. Different concentrations (0.2–0.6 mL) of hydrogen peroxide were used to study the degradation efficiency. Degradation was increased by increasing the concentration of hydrogen peroxide. The maximum (99%) degradation was achieved using 0.6 mL H₂O₂ and a gamma radiation dose of 20 kGy (Figure 4). The increase in degradation efficiency was due to the increased production of hydroxyl species on interaction with gamma radiation. As the hydrogen peroxide concentration was increased, more reactive species, such as hydroxyl radicals, were produced, and, as a result, the RY-18 dye degradation was also increased. The degradation efficiency for RY-18 was obtained at 0.6 mL concentration of hydrogen peroxide. These findings are in line with previous studies that found that hydrogen peroxide in combination with gamma radiation has a significant effect on the degradation of dye [30,31].

3.4. Effect of pH

The chemistry of the RY-18 dye solution is strongly affected by changes in pH, which was also evaluated. The degradation of RY-18 was >90% at pH 3, with 0.6 mL H₂O₂ and a 20 kGy absorbed dose of gamma radiation (Figure 5). The effect of pH on degradation was observed in the range 3–9. The pH of pure dye solution was neutral, while for the dye samples treated under gamma radiation, the pH decreased, which is an indication of the formation of acidic species during the degradation process, which are low-molecular-weight organic acid intermediates. This effect was more pronounced when the sample was treated with gamma radiations in combination with hydrogen peroxide. This decrease in pH was due to the conversion of chromophores of RY-18 into low-molecular-weight organic acidic units, which decreased the pH of the solution [32]. At acidic pH, the increase in degradation was due to the increased production of hydroxyl species [33].

3.5. Identification of By-Products

In order to determine the functional groups, FTIR analysis was performed on treated and untreated RY-18 samples. The FTIR spectrum of pure RY-18 reveals a single peak at 1559.9 cm⁻¹ that shows N=N stretching, a peak at 3421.7 cm⁻¹ for N-H stretching, a peak of C=O at 1724 cm⁻¹, C-N bond peak at 792 cm⁻¹, and peaks at 1090 cm⁻¹ and 702 cm⁻¹ show stretching for C-S and C-Cl bonds. The FTIR spectrum of gamma/H₂O₂-treated sample shows no functional peaks for these groups. This confirmed the complete degradation of RY-18 into simple compounds. Characteristic functional groups of RY-18 were diminished after treatment with gamma radiations/H₂O₂ (Figure 6).

LCMS is an effective method to study the intermediates formed during the degradation of dyes. The degraded end products of RY-18 were analyzed by LCMS. The degraded sample was scanned in positive mode, with a mass–to-charge ratio of 00.0−500 for better resolution, and their retention time was 1.28 min. The peaks of degraded products were analyzed with m/z values of 90.0, 130.0, 179, 241.1 and 296.17 for oxalic acid or (Z)-N-(iminomethyl)-formimidoyl chloride, 4-chloro-1,3,5-triazin-2-amine, sodium benzosulfate, sodium-4-chloro-2-diazenylbenzenesulfonate, and sodium 2-aminobenzene-1,4-disulfonate, respectively (Figure 7). The data obtained for the degraded RY-19 dye samples confirmed that the dye breaks down into low-molecular-weight acidic units and, finally, into carbon dioxide and water by an oxidative process. The intermediates recorded along their m/z values are given in

Table 2. Degradation intermediates of Reactive Yellow 18 identified by LCMS.

Sr. No	Structural Formula	Molecular Weight	m/z Value	Status
1		180.16	179.0	Detected
2		84.08	84.0	Not detected
3		130.54	130	Detected
4		90.51	90.0	Detected
5		242.62	241.95	Detected
6		90	90.03	Detected
7		61.47	60.0	Not detected
8		297.22	296.17	Detected

and the proposed mechanism of degradation of RY-18 dye is shown in Figure 8.

The AOP produced a highly reactive hydroxyl radical (^•OH) in situ, which is an oxidizing species. When dyed aqueous solution is irradiated with gamma radiation, it furnishes a significantly higher G-value (radiation yield) (Equation (1)). The G-value refers to the number of species (radicals/ions) generated/1 joule of energy. The ^•OH is an oxidizing species, which degrades the dye in aqueous solution by an oxidative process to low-molecular-weight by-products, especially organic acids (as per LCMS analysis). The mineralization/degradation efficiency during radiation treatment is based on the production of ^•OH. Moreover, to enhance the AOP efficiency, H₂O₂ is applied and, resultantly, the G-value enhanced manyfold (Equation (2)). The ^•OH radical generation is enhanced by the radiolysis of H₂O₂, and due to the enhanced concentration of ^•OH radicals, the dye is degraded with more efficiency (Equation (3)) [34].

H₂O + γ ⟶ ^•OH (0.29), ^•H (0.06), e_aq⁻ (0.28), H₂ (0.047), H₂O₂ (0.07), H₃O⁺ (0.27)

(1)

H₂O₂ + γ ⟶ ^•OH (0.29 + 0.29 = 0.58)

(2)

RH (dye) + ^•OH ⟶ H₂O + ^•R ⟶ CO₂ + H₂O + inorganic ions

(3)

3.6. Effect of Gamma Radiation Treatment on Toxicity

3.6.1. Cytotoxicity

Cell damage or death caused by any process/agent is called a cytotoxic effect. The samples of RY-18 were studied to check the toxicity before and after treatment with gamma radiation in combination with hydrogen peroxide. The percentage of hemolysis of the untreated (30 mg/L) RY-19 dye sample was 15.1%, while the percentage of hemolysis of the RY-19 dye sample treated with gamma/H₂O₂ was reduced to 7.6%. The results showed that there was a significant reduction in toxicity of RY-18 when treated with gamma radiation in combination with hydrogen peroxide, and previous findings revealed that the AOPs had a significant effect on the cytotoxicity reduction of the dye [34,35].

3.6.2. Mutagenicity

The Ames test utilized a bacterium to check mutations in the DNA by a specific chemical/agent. Mutagenicity of 30 mg/L of RY-18 was examined before and after treatment with gamma radiation in combination with hydrogen peroxide. Two bacterial strains, TA98 and TA100, were utilized, which are sensitive for mutagenicity analysis. The untreated RY-18 dye sample showed mutagenicity up to 69% and 73%, while gamma/H₂O₂-treated RY-18 dye samples revealed a reduction in mutagenicity to 81.3% and 82.3%, respectively. These results showed that mutagenicity was reduced significantly when the RY-18 dye sample was treated with gamma radiation in combination with H₂O₂, and previous studies also support these findings [36,37].

4. Conclusions

In view of the promising efficiency of the advanced oxidation process, gamma radiation in combination with hydrogen peroxide was employed for the removal of RY-18 dyes from aqueous solutions. The operational variables, such as the gamma radiation absorbed dose, initial concentration of dye, hydrogen peroxide concentration and initial pH of the solution, significantly affected the RY-18 dye degradation, and up to 99% dye degradation was achieved at 20 kGy of gamma absorbed dose, 0.6 mL hydrogen peroxide at pH 3. The FTIR and LCMS analyses revealed the complete degradation of RY-18 dye into low-molecular-weight acidic units, which, upon further oxidation, are converted into carbon dioxide, water and inorganic ion end products. The bioassay (for toxicity analysis) tests were performed on RY-18 dyes before treatment, and the dyes showed cytotoxic and mutagenic nature. The gamma radiation in combination with hydrogen peroxide reduced the toxicity significantly, which revealed the safer nature of treated RY-18 dye. Based on the findings, it is proved that gamma radiation in combination with hydrogen peroxide is highly effective in removing the RY-18 dye from aqueous solution, along with cytotoxicity and mutagenicity reduction, and this treatment could be employed for the remediation of reactive dyes in textile effluents.