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Cytotoxicity and mutagenicity evaluation of gamma radiation and hydrogen peroxide treated textile effluents using bioassays
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JECE 683 1–6 Journal of Environmental Chemical Engineering xxx (2015) xxx–xxx
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Cytotoxicity and mutagenicity evaluation of gamma radiation and hydrogen peroxide treated textile effluents using bioassays
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Munawar Iqbal
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National Centre of Excellence in Physical Chemistry, University of Peshawar, Peshawar 25120, Pakistan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 February 2015 Accepted 10 June 2015
Cytotoxicity and mutagenicity of textile effluents, treated by gamma radiation in combination hydrogen peroxide was investigated. The Allium cepa, heamolytic, brine shrimp bioassays were used for cytotoxicity evaluation, whereas mutagenicity was tested using Ames tests. Textile effluents were irradiated to the gamma radiation absorbed doses of 5 kGy, 10 kGy and 15 kGy in combination with 20 mM hydrogen peroxide. Before treatment, textile effluents showed a significant cytotoxicity and mutagenicity signs and reduced significantly after treatment. A. cepa showed reduction in cytotoxicity 50%, whereas 56–59% in case of heamolytic test and up 93% reduction in cytotoxicity was recorded by brine shrimp assays. The mutagenicity of gamma radiation treated effluents reduced up to 59% and 54% in case of TA98 and TA100, respectively. Results revealed that the gamma radiation in combination with hydrogen peroxide can be implemented for the detoxification of textile effluents. ã2015 Published by Elsevier Ltd.
Keywords: Absorbed dose Hydrogen peroxide Textile wastewater Bioassays Mutagenicity Cytotoxicity
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Introduction Textile industries produce huge volumes of water from wet processing units and resultantly, dyes along with other chemicals are discharged into water sheds. The presence of even very low amount of dyes in the effluent is highly visible and undesirable. Million ton of dye-stuffs of more than 100,000 types are produced and consumed annually [1]. Degradation of textile dye effluent does not occur when treated aerobically and over 90% of 4000 dyes tested in the Ecological and Toxicological Association of Dyes and Organic Pigments Manufacturers (ETAD) survey showed LD50 values greater than 2000 mg/kg [2]. There is evidence that some reactive azo dyes cause contact dermatitis, allergic conjunctivitis, rhinitis, occupational asthma or other allergic reactions in workers [1]. Many dyes are difficult to decolorize due to their complex structure and synthetic origin. Various methods have been applied for decolorization of textile dyes [3–7] and the conventional treatment systems and biological treatment were found to be inefficient in the removal of these dyes and others environmentally problematic [2,8,9]. Hence, advanced oxidation processes (AOPs) are needed for dyes efficient removal [10,11]. In advanced oxidation treatment, strong oxidizing specie like hydroxyl radical (OH radical) is produced in situ, which break down the complex organic molecule into harmless substances such as CO2, H2O and inorganic ions through a chain reactions [7,12]. The OH can be generated by
E-mail address: [email protected] (M. Iqbal).
radiation alone/ in combination with O3, H2O2/electron beam irradiation/or Fenton’s reagent [13]. The treatment of wastewater effluents by radiation has advantages such as it degrade non biodegradable organic substances without causing secondary Q2 pollution and are considered eco-friendly [7,14]. Bioassays have been used to evaluate toxicity levels of target contaminants and complex aqueous matrices. Generally, the organism used includes microorganisms, plants and algae, invertebrates and fishes [7,15,16]. Among higher plants, Allium cepa, Vicia faba, Zea mays, Tradescantia, Nicotiana tabacum, Crepis capillaris and Hordeum vulgare species are regarded as more favorable to assess toxicity [16,17]. Similarly, brine shrimp and heamolytic are suitable bioassays for cytotoxicity evaluation [10]. The Ames test, which measures the reversion of the bacterial mutants, is a reference test in chemical mutagenicity testing and was extensively validated and serves as a quick and convenient way to estimate the mutagenic potential of a target compound [18]. Unfortunately, the partial oxidation of organic contaminants produce more toxic intermediates than parent compounds and monitoring of treated effluents using toxicity assays is helpful in understanding the biological efficiency of treatment method. Literature showed that AOPs such as O3, TiO2/UV, sunlight irradiation, electro-Fenton, wet-air oxidation, UV/electro-Fenton, Photo-Fenton, O3/UV, TiO2 based photocatalysis, H2O2/UV and TiO2/H2O2/UV (Table 1) [15] showed considerable toxicity reduction. To best of our knowledge, the textile wastewater toxicity, treated by gamma radiation has not been reported previously using A. cepa, heamolytic, brine shrimp and Ames test. Therefore, it
http://dx.doi.org/10.1016/j.jece.2015.06.011 2213-3437/ ã 2015 Published by Elsevier Ltd.
Please cite this article in press as: M. Iqbal, Cytotoxicity and mutagenicity evaluation of gamma radiation and hydrogen peroxide treated textile effluents using bioassays, J. Environ. Chem. Eng. (2015), http://dx.doi.org/10.1016/j.jece.2015.06.011
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Table 1 Reported bioassay used in evaluating toxicity of wastewater/simulated solutions treated by AOPs.
Q8
Bioassay
AOPs investigated
Tested system
V. fischeri Ankistrodesmus braunii and S. capricornutum D. magna,P. subcapitata and A. salina P. subcapitata, Cyclotella meneghiniana, Synechococcus leopoliensis S. leopoliensis, Brachyonus calyciflorus
O3 (1 mmol L1, 0.38 L min1 flowrate) O3 (2.0% per volume, 36 dm3 h1 flowrate).
Bezafibrate (0.2–0.5 mmol L1) Carbamazepine (3.3 106 mmol dm3) Diclofenac (5–80 mg L1) Lyncomicin (0.5 mM)
TiO2/UV (0.2–1.6 g L1) Sunlight irradiation, UV/H2O2 (254 nm low-pressure lamp) and O3
O3 (0.42 mM, 36 dm3 h1 flowrate), H2O2/UV (5 and 10 mM of H2O2) and TiO2/UV (suspended or immobilized TiO2, 0.3 g L1, Degussa, 300 W sunlight simulator, up to 48 h contact time) P. putida UV/TiO2, electro-Fenton, wet-air oxidation, and UV/electro-Fenton V. fischeri, D. magna and S. capricornotum Solar driven photo-Fenton and TiO2 photocatalysis pilot plant D. magna and Bacillus subtilis sp. Photo-Fenton (2 L reactor, three 6 W Philips black-light fluorescent lamps (I = 5 106 Einstein s1), controlled temperature (25 C) D. magna, Photobacterium phosphoreum, O3 and O3/UV (40 mg L1 O3dosage, 20 and 40 min treatment), H2O2 and H2O2/UV umu (genotoxicity) test (6 mL L1 of 30% H2O2) V. fischeri (toxicity) andSalmonella Ozonation (applied ozone 2.5–8.0 mg L1, 2–30 min treatment) typhimurium(mutagenicity) D. magna, P. subcapitata, L. sativum TiO2 photocatalysis, catalyst loading in the range of 0.2–0.8 g L1, 125 W black-light fluorescent lamp, 120 min maximum irradiation time FELST with rainbow trout (Oncorhynchus Ozonation (maximum applied ozone concentration 1 mg O3/mg DOC) mykiss) D. longispina. Photo-Fenton process before and after biological treatment by three species of fungi
Mixture of six pharmaceuticals
Reactive Red 120 (20–100 mg L1) Methomyl (50 mg L1) Imidacloprid (100 mg L1) Mixture of municipal and industrial wastewater pre-treated by MBR Effluent from secondary biological treatment Effluent from secondary biological treatment Effluent from secondary biological treatment plant Diluted and undiluted wastewater samples from olive mill plant Centrifuged wastewater sample
P. subcapitata and phytotoxicity to seeds Ozonation, solar photolysis, solar modified photo-Fenton, solar modified photoofR. sativus, C. sativusand L. sativa Fenton–ozonation. H2O2/UV, TiO2/H2O2/UV and TiO2/UV in a continuous operated annular reactor (15 W Coagulated/settled wastewater A. salina UV-lamp, 1 g TiO2 L1, 1 h)
57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
81 80 82 83 84 85 86 87
was hypothesized that gamma radiation treated textile effluents toxicity can be evaluated using bioassays. The textile wastewaters collected from different industries were treated by gamma radiation in the combination with hydrogen peroxide and Q3 cytotoxicity and mutagenicity was evaluated using standard bioassays. Material and methods Sample collection Three textile industries were selected for sampling from the industrial city, Faisalabad, Pakistan and denoted as TIW I, II and III. Q4 The samples were collected using standard method (Eaton et al., 2005). Briefly, the plastic gallon were pre-cleaned by soaking in nitric acid 1% (v/v) for 24 h and rinsed with ultra pure water (MilliQ system, Millipore). Triplicate samples were collected from each industry and stored at 4 C until experimentation. Sample irradiation Cesium-137 gamma radiation source was used for irradiation of wastewater samples at Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, Pakistan. The dose rate at the time of sample irradiation was 1.25 kGy h1 which was calibrated using Fricke dosimeter (Eq. (1)). The samples were irradiated to the absorbed doses of 5 kGy, 10 kGy and 15 kGy in combination with Q5 20 mM hydrogen peroxide. N DA 100 (1) D¼ e r GðFeðIIIÞÞ where D is absorbed dose, e is the molar extinction coefficient of ferric ion (0.2205 M1 cm1 at 304 and 25 C, DA is representing absorbance difference of irradiated and un-irradiated samples, N is Avogadro’s number (6.02 1023), r is the density of Fricke solution (1.024 g/cm3 for 0.4 M H2SO4 and G (Fe3+) is the number of Fe3+ ions produced/100 ev of absorbed energy which is 15.6 for Fricke solution.
Bioassays
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The bioassays such as A. cepa test (ACT), heamolytic, brine shrimp (cytotoxic tests) and Ames tests (mutagenic test) were performed precisely as reported previously [16]. Before toxicity evaluation, the un-reacted hydrogen peroxide was removed from treated samples by adding 0.80 mg MnO2/mL of solution [19], mixed for 1 h, filtered and subjected to the toxicity tests. All samples were run in triplicate except A. cepa test (5 repetitions) and data, thus obtained was averaged and results were reported as mean SD.
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Results and discussion
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Physicochemical characteristics of textile wastewater
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Preliminary water quality assurance test performed, showed that the water quality parameters values were very high and beyond the permissible limits. The water quality parameter e.g. pH, COD, BOD, DO, TDS and TSS values were in the range of 9.17–11.8, 1766–2100 mg/L, 800–874 mg/L, 1.2–1.5 mg/L, 1530–1590 mg/L, 447–505 mg/L respectively. The lmax was recorded to be 539 nm, 625 nm and 486.5 nm for TIW I, II and III, respectively (Table 2) and UV–vis spectra of treated and untreated textile effluents can be seen in Fig. 1A–C. The pH of textile wastewater was alkaline in nature. The bleaching agents and chemicals NaOCl, NaOH, surfactants and sodium phosphate used in the processes, might be the reasons for high alkalinity of wastewater [16,20]. A low DO in TIW indicated highly polluted nature of effluent contaminated with organic matter and previous finding also highlighted that DO might be very low of textile effluents due high organic load and [20] reported nil DO value. A slight change in pH values of TIW II and III might be due to biodegradation of organic matter present in wastewater. BOD is the most important parameter which in true sense determines the pollution load of an effluent and is expressed as a measure of the quantity of oxygen consumed by microorganisms in the degradation of organic matter. The BOD recorded values were higher than previously
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Table 2 Physiochemical characteristics of textile wastewater. Wastewater
pH
COD (mg/L)
BOD (mg/L)
DO (mg/L)
TDS (mg/L)
TSS (mg/L)
lmax
TIW I TIW II TW III
11.8 10.1 9.17
2100 1792 1766
800 895 874
1.5 1.3 1.2
1590 1665 1530
505 495 447
539 625 486.5
TIW I, textile wastewater units I, II and III.
(A)
3.0
Before treatment After treatment
Absorbance (A)
2.5
2.0
1.5
1.0
0.5
0.0 300
450
600
750
900
Wavelength (nm) (B)
3.0
2.5
Absorbance (A)
Before treatment After treatment 2.0
1.5
1.0
0.5
0.0 200
300
400
500
600
700
800
900
Wavelength (nm)
(C) 3.0 2.5
Absorbance (A)
Before treatment After treatment 2.0
1.5
1.0
0.5
0.0 200
300
400
500
600
700
800
900
Wavelength (nm) Fig. 1. UV–vis spectra of untreated and gamma radiation treated textile effluents (A = TIW I; B = TIW II and C = TIW III).
reported [21,22] for textile effluents and lower than Garg and Kaushik [23] and this different is might be due to different nature of wastewater coming out from respective industries. The COD determines the oxygen required for the chemical oxidation of organic matter and non biodegradable matter present in wastewater. COD is also an important pollution indicator which reflects the chemical quality of effluent. In the present investigation, the COD values were recorded to be very high versus previous reports [22–24] which indicates the presence of high concentration of matter in textile wastewater investigated. TDS content in water is a measure for salinity. A large number of salts are dissolved in waters during processing, the common ones are carbonates, bicarbonates, chlorides, sulphates, phosphates, and nitrates of calcium, magnesium, sodium, potassium, iron and manganese and are responsible of higher TDS values and this trend to similar to previous reports [20,22]. The TSS comprises total suspended solids and in contrast to our study, very high TSS content in textile wastewater has been reported by Ajao et al. [25]. However, TSS value reported by Rohit and Ponmurugan [26] were lower. Higher TSS values may be due to colored effluents which eventually use different dye stuffs [24].
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Cytotoxicity
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The untreated and treated effluents were tested for toxicity. Table 3 summarizes the results of cytotoxicity of textile untreated effluents. Then, wastewater was treated by gamma radiation to the absorbed doses of 5 kGy, 10 kGy and 15 kGy in combination with 20 mM hydrogen peroxide. The measured values showed that effluents were highly contaminated with microbes, cytotoxic and mutagenic agents. The total bacterial count and total coliform were recorded to be >1 106 CUF and >1 105 CUF, respectively. The root count and root length, human RBC, bovine RBC and shrimp nauplii showed 41%, 54.44%, 19%, 29% and 32% lower values than control. Previous studies showed that the textile wastewater has the potential to cause toxicity as most of the dyes stuffs are toxic in nature [27–30]. The microbial load and cytotoxicity results of treated effluents are shown in Table 4. The total bacterial count and total coliform reduced to zero in effluents irradiated to the absorbed dose of 5 kGy in the presence of 20 mM hydrogen peroxide. The A. cepa, heamolytic (human RBC and bovine RBC) and shrimp tests also revealed a significant reduction in cytotoxicity. The root count and root length increased by 41.17% and 41.12%, respectively for absorbed dose of 5 kGy and by increasing the absorbed doses to 10 kGy and 15 kGy, the root count increased up to 44.44% and 47.36%, respectively. However, the increase in root length was insignificant and similar trend was observed for effluents collected TIW I and TIW II. Regarding heamolytic test, the reduction in RBC cell deaths were 53.08% (human) and 53.52% (bovine) for the absorbed dose of 5 kGy, whereas 54.32% and 59.15% for the absorbed dose of 10 kGy and 61.72% and 63.38% for 15 kGy, respectively. Brine shrimp test revealed that the shrimp nauplii death reduced linearly with absorbed dose and up to 94.44%, 94.44% and 100% shrimp nauplii reductions in death rate were observed for the absorbed doses of 5 kGy, 10 kGy and 15 kGy, respectively. On average basis, the effluents cytotoxic effect reduced by 100%, 100%, 50.02%, 47.07%, 56.23%, 59.40% and 92.89% for total bacterial count, total coliform, root count, root
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Table 3 Microbial load, cytotoxicity and mutagenicity profile of textile wastewater before treatment. Bioassays
Types
Units
Wastewater TIW I
TIW II
TIW III
Positive
Negative
Mic. load
TBC TC
CFU CFU
>1 106 >1 105
>1 106 >1 105
>1 106 >1 105
— —
0.000 0.000
Cytotoxicity
ACT I) RC II) RL
counts cm
10 0.15 4.1 0.06
9 0.25 3.9 0.04
11 0.20 3.8 0.05
17 0.26 9.0 0.15
08 0.20 2.5 0.04
% %
81 1.6 71 0.9 68 0.80
78 1.8 69 1.5 64 0.85
72 1.9 75 1.3 54 0.81
100 0.0 100 0.0 100 0.0
0.000 0.000 0.000
counts counts
62 2/96 68 2/96
59 2/96 63 2/96
60 1/96 61 3/96
19 2/96 21 2/96
0.000 0.000
heamolytic I) Human II) bovine Shrimp Ames test I) TA98 II) TA100
Mutagenicity
Control
TIW, textile industry wastewater; TBC, total bacterial count; TC, total coliform; ACT, Allium cepa test; RC, root count; RL = root length; —, not measured; ND, not detected; PC, positive control; NC, negative control, for heamolytic test, PC and NC were TritonX-100 (0.1%) and phosphate buffer saline, respectively, for ACT, PC and NC were distilled water and methyl methanesulfonate (MMS) (10 mg/L), respectively, for shrimp test, PC and NC were cyclophosphamide (10 mg/mL) and sea water, respectively, for Ames test, PC for TA98 and TA100 were K2Cr2O7 (0.01 g/L) and NaN3 (0.5 mg/100 mL), respectively and background (without standard and tested compound) was used as NC.
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length, human RBC, bovine RBC and brine shrimp. Results revealed that gamma radiation treatment in combination with hydrogen peroxide is highly efficient for the removal of microbial population and cytotoxic agents from textile wastewater. Recently, Punzi et al. [31] studied the degradation of a textile azo dye using biological treatment followed by photo-Fenton oxidation as well as toxicity and microbial load was evaluated as a function of treatment and found that the acute toxicity was higher in the biologically treated than in the untreated effluent. Photo-Fenton oxidation successfully
reduced the toxicity and the final effluent was non-toxic to Artemia salina and Microtox. Author declared that using advanced oxidation after biological treatment is an effective way to degrade the organic compounds and for the removal toxicity of textile effluents. Similarly, Jadhav et al. [32] evaluated the toxicity of biologically treated remazol red. As a result of biodegradation, >97% dye was degraded and toxicity evaluated by A. cepa, Phaseolus mungo and Sorghum vulgare showed less toxic nature of metabolites formed after dye degradation and Gomes de Moraes
Table 4 Microbial load, cytotoxicity and mutagenicity profile of textile wastewater after gamma radiation treatment. Bioassays
Types
Units
TIW I
TIW II
TIW III
Ave
5 kGy
10 kGy
15 kGy
5 kGy
10 kGy
15 kGy
5 kGy
10 kGy
15 kGy
Microbial load
TBC TC
CFU CFU
ND ND
— —
— —
ND ND
— —
— —
ND ND
— —
— —
— —
Cytotoxicity
ACT I) RC I) RL
count cm
17 1.0 7.0 0.2
18 0.5 7.1 0.2
19 0.5 7.4 0.2
16 0.4 6.9 0.4
20 0.5 7.5 0.4
26 0.7 8.0 0.5
18 0.6 —
21 0.8 8.0 0.2
23 0.9 8.1 0.5
— — —
% %
38 2.0 34 1.5 01 0.0
37 3.0 29 1.3 01 0.0
31 2.0 26 2.2 0.0 0.0
42 1.0 — —
— 36 2.5 2 0.0
34 3.0 29 2.0 1 0.5
39 1.0 33 2.6 3 1.00
29 1.5 26 1.1 2 1.0
22 1.0 21 1.1 1 0.33
— — —
Ames test I) TA98 II) TA100
count count
29 1.00 33 0.33
23 1.5 27 0.5
22 0.5 25 2.0
30 1.33 34 0.33
27 2.0 28 1.0
24 0.33 —
— —
28 1.0 30 0.3
21 0.3 25 0.5
— —
TBC TC
% %
100 100
100 100
100 100
100 100
100 100
100 100
100 100
100 100
100 100
100 100
ACT I) RC I) RL
% %
41.17 41.42
44.44 42.25
47.36 44.59
43.75 43.47
55 48
65.38 51.25
44.44 —
52.38 52.5
56.26 53.08
50.02 47.07
% %
53.08 53.52 94.44
54.32 59.15 94.44
61.72 63.38 100
46.15 — —
— 47.82 89.47
56.41 57.97 94.73
45.83 56 85
59.72 65.33 90
69.44 72 95
56.23 59.40 92.89
% %
53.22 51.47
66.17 60.29
67.64 63.23
49.15 46.03
54.23 55.55
59.32 —
— 42.62
53.33 50.81
65 59
58.51 53.63
heamolytic I) Human II) bovine Shrimp Mutagenicity
Reduction (%) Mic. load
Cytotoxicity
heamolytic I) Human II) bovine Shrimp Mutagenicity
Ames test I) TA98 II) TA100
Explanations as given in Table 3.
Please cite this article in press as: M. Iqbal, Cytotoxicity and mutagenicity evaluation of gamma radiation and hydrogen peroxide treated textile effluents using bioassays, J. Environ. Chem. Eng. (2015), http://dx.doi.org/10.1016/j.jece.2015.06.011
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et al. [33] also reported 50% toxicity reduction of textile effluent by combined photocatalytic and ozonation processes. It was observed that as the reaction proceeded, the toxicity reduced continuously, implying that all the generated products were less toxic than the original dye dyes in the effluents. The similar results were reported by Neamtu et al. [34] who observed that the toxicity of azo dye C.I. Reactive Yellow 84 kept on decreasing during homogeneous photo-Fenton process, although higher toxic intermediates were formed in the initial stage of UV/H2O2 treatment process. de Luna et al. [35] also suggested that all the generated products of Orange II were less toxic than the original dye during homogeneous photoFenton process. Similar to present study, various advanced oxidation processes such as O3, TiO2/UV, sunlight irradiation, UV/H2O2 and O3, electro-Fenton, wet-air oxidation, UV/electroFenton, solar driven photo-Fenton and TiO2 photocatalysis on pilot plant, O3/UV, Photo-Fenton process before and after biological treatment, TiO2/H2O2/UV have been employed successfully for toxicity evaluation of different systems (Table 1) and this study also revealed efficiency for toxicity evaluation of gamma irradiated textile effluents.
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Mutagenicity
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The untreated and treated effluents were tested for mutagenicity using TA98 and TA100 strains. Table 3 summarizes the results of mutagenicity of textile untreated effluents. The Ames test revealed the presence of mutagenic agent in effluents, 60 (37.5%) and 61 (36.45%) micro plates were affected out of 96 before treatment. The effluents treated by gamma radiation, to the absorbed doses of 5 kGy, 10 kGy and 15 kGy in combination with 20 mM hydrogen peroxide were tested for mutagenicity. After gamma radiation treatment, mutagenicity reduced considerably and up to 53.22% (TA98) and 51.47% (TA100) for the absorbed dose of 5 kGy of TIW I. The mutagenicity reduced linearly with gamma radiation absorbed dose and reduction reached to 67.64% and 63.23% in case of TA98 and TA100 for the absorbed dose of 15 kGy, respectively. The mutagenicity reeducation trend was similar for TIW II and TIW III (Table 4). The studies regarding mutagenicity testing of gamma radiation treated textile effluents are rare. However, our previous study indicated that the gamma radiation without hydrogen peroxide was not effective for the degradation of mutagenic agent and better efficiency can be achieved at low gamma radiation absorbed dose using small amount of hydrogen peroxide. Previous studies also indicated that the oxidant can enhance the gamma radiation efficiency [36,37]. Based on the toxicity test, it can be concluded that gamma radiation in combination with hydrogen peroxide is a suitable method for detoxification of textile effluents.
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Conclusion
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The results of this study suggest that gamma radiation treatment is an effective way to degrade the dye and remove toxicity from textile effluents. The employed toxicity test showed that the bioassays were better tools to study the effluent to evaluate the biological efficiency of method used for treatment of effluents. The gamma radiation treatment efficiency can be enhanced using small amount of hydrogen peroxide. It is concluded that the textile effluents are cytotoxic and mutagenic; however, these can be detoxified through degradation by the gamma radiation in combination with hydrogen peroxide and due to its high capacity, relatively low reaction times, this treatment method might be an interesting alternative, especially for the degradation of effluents contaminated with cytotoxic and mutagenic agents.
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Acknowledgements
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Higher Education Commission (HEC) of Pakistan is highly Q6 acknowledged for financial support under the start-up Research Project Program (Grant No: 21-194/SRGP/R&D/HEC/2014) and Dr. Muhammad Shahid, UAF for assistance in toxicity studies.
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Cytotoxicity and mutagenicity evaluation of gamma radiation and hydrogen peroxide treated textile effluents using bioassays
3 Q1
Munawar Iqbal
4
National Centre of Excellence in Physical Chemistry, University of Peshawar, Peshawar 25120, Pakistan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 February 2015 Accepted 10 June 2015
Cytotoxicity and mutagenicity of textile effluents, treated by gamma radiation in combination hydrogen peroxide was investigated. The Allium cepa, heamolytic, brine shrimp bioassays were used for cytotoxicity evaluation, whereas mutagenicity was tested using Ames tests. Textile effluents were irradiated to the gamma radiation absorbed doses of 5 kGy, 10 kGy and 15 kGy in combination with 20 mM hydrogen peroxide. Before treatment, textile effluents showed a significant cytotoxicity and mutagenicity signs and reduced significantly after treatment. A. cepa showed reduction in cytotoxicity 50%, whereas 56–59% in case of heamolytic test and up 93% reduction in cytotoxicity was recorded by brine shrimp assays. The mutagenicity of gamma radiation treated effluents reduced up to 59% and 54% in case of TA98 and TA100, respectively. Results revealed that the gamma radiation in combination with hydrogen peroxide can be implemented for the detoxification of textile effluents. ã2015 Published by Elsevier Ltd.
Keywords: Absorbed dose Hydrogen peroxide Textile wastewater Bioassays Mutagenicity Cytotoxicity
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Introduction Textile industries produce huge volumes of water from wet processing units and resultantly, dyes along with other chemicals are discharged into water sheds. The presence of even very low amount of dyes in the effluent is highly visible and undesirable. Million ton of dye-stuffs of more than 100,000 types are produced and consumed annually [1]. Degradation of textile dye effluent does not occur when treated aerobically and over 90% of 4000 dyes tested in the Ecological and Toxicological Association of Dyes and Organic Pigments Manufacturers (ETAD) survey showed LD50 values greater than 2000 mg/kg [2]. There is evidence that some reactive azo dyes cause contact dermatitis, allergic conjunctivitis, rhinitis, occupational asthma or other allergic reactions in workers [1]. Many dyes are difficult to decolorize due to their complex structure and synthetic origin. Various methods have been applied for decolorization of textile dyes [3–7] and the conventional treatment systems and biological treatment were found to be inefficient in the removal of these dyes and others environmentally problematic [2,8,9]. Hence, advanced oxidation processes (AOPs) are needed for dyes efficient removal [10,11]. In advanced oxidation treatment, strong oxidizing specie like hydroxyl radical (OH radical) is produced in situ, which break down the complex organic molecule into harmless substances such as CO2, H2O and inorganic ions through a chain reactions [7,12]. The OH can be generated by
E-mail address: [email protected] (M. Iqbal).
radiation alone/ in combination with O3, H2O2/electron beam irradiation/or Fenton’s reagent [13]. The treatment of wastewater effluents by radiation has advantages such as it degrade non biodegradable organic substances without causing secondary Q2 pollution and are considered eco-friendly [7,14]. Bioassays have been used to evaluate toxicity levels of target contaminants and complex aqueous matrices. Generally, the organism used includes microorganisms, plants and algae, invertebrates and fishes [7,15,16]. Among higher plants, Allium cepa, Vicia faba, Zea mays, Tradescantia, Nicotiana tabacum, Crepis capillaris and Hordeum vulgare species are regarded as more favorable to assess toxicity [16,17]. Similarly, brine shrimp and heamolytic are suitable bioassays for cytotoxicity evaluation [10]. The Ames test, which measures the reversion of the bacterial mutants, is a reference test in chemical mutagenicity testing and was extensively validated and serves as a quick and convenient way to estimate the mutagenic potential of a target compound [18]. Unfortunately, the partial oxidation of organic contaminants produce more toxic intermediates than parent compounds and monitoring of treated effluents using toxicity assays is helpful in understanding the biological efficiency of treatment method. Literature showed that AOPs such as O3, TiO2/UV, sunlight irradiation, electro-Fenton, wet-air oxidation, UV/electro-Fenton, Photo-Fenton, O3/UV, TiO2 based photocatalysis, H2O2/UV and TiO2/H2O2/UV (Table 1) [15] showed considerable toxicity reduction. To best of our knowledge, the textile wastewater toxicity, treated by gamma radiation has not been reported previously using A. cepa, heamolytic, brine shrimp and Ames test. Therefore, it
http://dx.doi.org/10.1016/j.jece.2015.06.011 2213-3437/ ã 2015 Published by Elsevier Ltd.
Please cite this article in press as: M. Iqbal, Cytotoxicity and mutagenicity evaluation of gamma radiation and hydrogen peroxide treated textile effluents using bioassays, J. Environ. Chem. Eng. (2015), http://dx.doi.org/10.1016/j.jece.2015.06.011
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Table 1 Reported bioassay used in evaluating toxicity of wastewater/simulated solutions treated by AOPs.
Q8
Bioassay
AOPs investigated
Tested system
V. fischeri Ankistrodesmus braunii and S. capricornutum D. magna,P. subcapitata and A. salina P. subcapitata, Cyclotella meneghiniana, Synechococcus leopoliensis S. leopoliensis, Brachyonus calyciflorus
O3 (1 mmol L1, 0.38 L min1 flowrate) O3 (2.0% per volume, 36 dm3 h1 flowrate).
Bezafibrate (0.2–0.5 mmol L1) Carbamazepine (3.3 106 mmol dm3) Diclofenac (5–80 mg L1) Lyncomicin (0.5 mM)
TiO2/UV (0.2–1.6 g L1) Sunlight irradiation, UV/H2O2 (254 nm low-pressure lamp) and O3
O3 (0.42 mM, 36 dm3 h1 flowrate), H2O2/UV (5 and 10 mM of H2O2) and TiO2/UV (suspended or immobilized TiO2, 0.3 g L1, Degussa, 300 W sunlight simulator, up to 48 h contact time) P. putida UV/TiO2, electro-Fenton, wet-air oxidation, and UV/electro-Fenton V. fischeri, D. magna and S. capricornotum Solar driven photo-Fenton and TiO2 photocatalysis pilot plant D. magna and Bacillus subtilis sp. Photo-Fenton (2 L reactor, three 6 W Philips black-light fluorescent lamps (I = 5 106 Einstein s1), controlled temperature (25 C) D. magna, Photobacterium phosphoreum, O3 and O3/UV (40 mg L1 O3dosage, 20 and 40 min treatment), H2O2 and H2O2/UV umu (genotoxicity) test (6 mL L1 of 30% H2O2) V. fischeri (toxicity) andSalmonella Ozonation (applied ozone 2.5–8.0 mg L1, 2–30 min treatment) typhimurium(mutagenicity) D. magna, P. subcapitata, L. sativum TiO2 photocatalysis, catalyst loading in the range of 0.2–0.8 g L1, 125 W black-light fluorescent lamp, 120 min maximum irradiation time FELST with rainbow trout (Oncorhynchus Ozonation (maximum applied ozone concentration 1 mg O3/mg DOC) mykiss) D. longispina. Photo-Fenton process before and after biological treatment by three species of fungi
Mixture of six pharmaceuticals
Reactive Red 120 (20–100 mg L1) Methomyl (50 mg L1) Imidacloprid (100 mg L1) Mixture of municipal and industrial wastewater pre-treated by MBR Effluent from secondary biological treatment Effluent from secondary biological treatment Effluent from secondary biological treatment plant Diluted and undiluted wastewater samples from olive mill plant Centrifuged wastewater sample
P. subcapitata and phytotoxicity to seeds Ozonation, solar photolysis, solar modified photo-Fenton, solar modified photoofR. sativus, C. sativusand L. sativa Fenton–ozonation. H2O2/UV, TiO2/H2O2/UV and TiO2/UV in a continuous operated annular reactor (15 W Coagulated/settled wastewater A. salina UV-lamp, 1 g TiO2 L1, 1 h)
57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
81 80 82 83 84 85 86 87
was hypothesized that gamma radiation treated textile effluents toxicity can be evaluated using bioassays. The textile wastewaters collected from different industries were treated by gamma radiation in the combination with hydrogen peroxide and Q3 cytotoxicity and mutagenicity was evaluated using standard bioassays. Material and methods Sample collection Three textile industries were selected for sampling from the industrial city, Faisalabad, Pakistan and denoted as TIW I, II and III. Q4 The samples were collected using standard method (Eaton et al., 2005). Briefly, the plastic gallon were pre-cleaned by soaking in nitric acid 1% (v/v) for 24 h and rinsed with ultra pure water (MilliQ system, Millipore). Triplicate samples were collected from each industry and stored at 4 C until experimentation. Sample irradiation Cesium-137 gamma radiation source was used for irradiation of wastewater samples at Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, Pakistan. The dose rate at the time of sample irradiation was 1.25 kGy h1 which was calibrated using Fricke dosimeter (Eq. (1)). The samples were irradiated to the absorbed doses of 5 kGy, 10 kGy and 15 kGy in combination with Q5 20 mM hydrogen peroxide. N DA 100 (1) D¼ e r GðFeðIIIÞÞ where D is absorbed dose, e is the molar extinction coefficient of ferric ion (0.2205 M1 cm1 at 304 and 25 C, DA is representing absorbance difference of irradiated and un-irradiated samples, N is Avogadro’s number (6.02 1023), r is the density of Fricke solution (1.024 g/cm3 for 0.4 M H2SO4 and G (Fe3+) is the number of Fe3+ ions produced/100 ev of absorbed energy which is 15.6 for Fricke solution.
Bioassays
88
The bioassays such as A. cepa test (ACT), heamolytic, brine shrimp (cytotoxic tests) and Ames tests (mutagenic test) were performed precisely as reported previously [16]. Before toxicity evaluation, the un-reacted hydrogen peroxide was removed from treated samples by adding 0.80 mg MnO2/mL of solution [19], mixed for 1 h, filtered and subjected to the toxicity tests. All samples were run in triplicate except A. cepa test (5 repetitions) and data, thus obtained was averaged and results were reported as mean SD.
89
Results and discussion
98
Physicochemical characteristics of textile wastewater
99
Preliminary water quality assurance test performed, showed that the water quality parameters values were very high and beyond the permissible limits. The water quality parameter e.g. pH, COD, BOD, DO, TDS and TSS values were in the range of 9.17–11.8, 1766–2100 mg/L, 800–874 mg/L, 1.2–1.5 mg/L, 1530–1590 mg/L, 447–505 mg/L respectively. The lmax was recorded to be 539 nm, 625 nm and 486.5 nm for TIW I, II and III, respectively (Table 2) and UV–vis spectra of treated and untreated textile effluents can be seen in Fig. 1A–C. The pH of textile wastewater was alkaline in nature. The bleaching agents and chemicals NaOCl, NaOH, surfactants and sodium phosphate used in the processes, might be the reasons for high alkalinity of wastewater [16,20]. A low DO in TIW indicated highly polluted nature of effluent contaminated with organic matter and previous finding also highlighted that DO might be very low of textile effluents due high organic load and [20] reported nil DO value. A slight change in pH values of TIW II and III might be due to biodegradation of organic matter present in wastewater. BOD is the most important parameter which in true sense determines the pollution load of an effluent and is expressed as a measure of the quantity of oxygen consumed by microorganisms in the degradation of organic matter. The BOD recorded values were higher than previously
100
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Table 2 Physiochemical characteristics of textile wastewater. Wastewater
pH
COD (mg/L)
BOD (mg/L)
DO (mg/L)
TDS (mg/L)
TSS (mg/L)
lmax
TIW I TIW II TW III
11.8 10.1 9.17
2100 1792 1766
800 895 874
1.5 1.3 1.2
1590 1665 1530
505 495 447
539 625 486.5
TIW I, textile wastewater units I, II and III.
(A)
3.0
Before treatment After treatment
Absorbance (A)
2.5
2.0
1.5
1.0
0.5
0.0 300
450
600
750
900
Wavelength (nm) (B)
3.0
2.5
Absorbance (A)
Before treatment After treatment 2.0
1.5
1.0
0.5
0.0 200
300
400
500
600
700
800
900
Wavelength (nm)
(C) 3.0 2.5
Absorbance (A)
Before treatment After treatment 2.0
1.5
1.0
0.5
0.0 200
300
400
500
600
700
800
900
Wavelength (nm) Fig. 1. UV–vis spectra of untreated and gamma radiation treated textile effluents (A = TIW I; B = TIW II and C = TIW III).
reported [21,22] for textile effluents and lower than Garg and Kaushik [23] and this different is might be due to different nature of wastewater coming out from respective industries. The COD determines the oxygen required for the chemical oxidation of organic matter and non biodegradable matter present in wastewater. COD is also an important pollution indicator which reflects the chemical quality of effluent. In the present investigation, the COD values were recorded to be very high versus previous reports [22–24] which indicates the presence of high concentration of matter in textile wastewater investigated. TDS content in water is a measure for salinity. A large number of salts are dissolved in waters during processing, the common ones are carbonates, bicarbonates, chlorides, sulphates, phosphates, and nitrates of calcium, magnesium, sodium, potassium, iron and manganese and are responsible of higher TDS values and this trend to similar to previous reports [20,22]. The TSS comprises total suspended solids and in contrast to our study, very high TSS content in textile wastewater has been reported by Ajao et al. [25]. However, TSS value reported by Rohit and Ponmurugan [26] were lower. Higher TSS values may be due to colored effluents which eventually use different dye stuffs [24].
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Cytotoxicity
142
The untreated and treated effluents were tested for toxicity. Table 3 summarizes the results of cytotoxicity of textile untreated effluents. Then, wastewater was treated by gamma radiation to the absorbed doses of 5 kGy, 10 kGy and 15 kGy in combination with 20 mM hydrogen peroxide. The measured values showed that effluents were highly contaminated with microbes, cytotoxic and mutagenic agents. The total bacterial count and total coliform were recorded to be >1 106 CUF and >1 105 CUF, respectively. The root count and root length, human RBC, bovine RBC and shrimp nauplii showed 41%, 54.44%, 19%, 29% and 32% lower values than control. Previous studies showed that the textile wastewater has the potential to cause toxicity as most of the dyes stuffs are toxic in nature [27–30]. The microbial load and cytotoxicity results of treated effluents are shown in Table 4. The total bacterial count and total coliform reduced to zero in effluents irradiated to the absorbed dose of 5 kGy in the presence of 20 mM hydrogen peroxide. The A. cepa, heamolytic (human RBC and bovine RBC) and shrimp tests also revealed a significant reduction in cytotoxicity. The root count and root length increased by 41.17% and 41.12%, respectively for absorbed dose of 5 kGy and by increasing the absorbed doses to 10 kGy and 15 kGy, the root count increased up to 44.44% and 47.36%, respectively. However, the increase in root length was insignificant and similar trend was observed for effluents collected TIW I and TIW II. Regarding heamolytic test, the reduction in RBC cell deaths were 53.08% (human) and 53.52% (bovine) for the absorbed dose of 5 kGy, whereas 54.32% and 59.15% for the absorbed dose of 10 kGy and 61.72% and 63.38% for 15 kGy, respectively. Brine shrimp test revealed that the shrimp nauplii death reduced linearly with absorbed dose and up to 94.44%, 94.44% and 100% shrimp nauplii reductions in death rate were observed for the absorbed doses of 5 kGy, 10 kGy and 15 kGy, respectively. On average basis, the effluents cytotoxic effect reduced by 100%, 100%, 50.02%, 47.07%, 56.23%, 59.40% and 92.89% for total bacterial count, total coliform, root count, root
143
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Table 3 Microbial load, cytotoxicity and mutagenicity profile of textile wastewater before treatment. Bioassays
Types
Units
Wastewater TIW I
TIW II
TIW III
Positive
Negative
Mic. load
TBC TC
CFU CFU
>1 106 >1 105
>1 106 >1 105
>1 106 >1 105
— —
0.000 0.000
Cytotoxicity
ACT I) RC II) RL
counts cm
10 0.15 4.1 0.06
9 0.25 3.9 0.04
11 0.20 3.8 0.05
17 0.26 9.0 0.15
08 0.20 2.5 0.04
% %
81 1.6 71 0.9 68 0.80
78 1.8 69 1.5 64 0.85
72 1.9 75 1.3 54 0.81
100 0.0 100 0.0 100 0.0
0.000 0.000 0.000
counts counts
62 2/96 68 2/96
59 2/96 63 2/96
60 1/96 61 3/96
19 2/96 21 2/96
0.000 0.000
heamolytic I) Human II) bovine Shrimp Ames test I) TA98 II) TA100
Mutagenicity
Control
TIW, textile industry wastewater; TBC, total bacterial count; TC, total coliform; ACT, Allium cepa test; RC, root count; RL = root length; —, not measured; ND, not detected; PC, positive control; NC, negative control, for heamolytic test, PC and NC were TritonX-100 (0.1%) and phosphate buffer saline, respectively, for ACT, PC and NC were distilled water and methyl methanesulfonate (MMS) (10 mg/L), respectively, for shrimp test, PC and NC were cyclophosphamide (10 mg/mL) and sea water, respectively, for Ames test, PC for TA98 and TA100 were K2Cr2O7 (0.01 g/L) and NaN3 (0.5 mg/100 mL), respectively and background (without standard and tested compound) was used as NC.
177 178 179 180 181 182 183 184 185
length, human RBC, bovine RBC and brine shrimp. Results revealed that gamma radiation treatment in combination with hydrogen peroxide is highly efficient for the removal of microbial population and cytotoxic agents from textile wastewater. Recently, Punzi et al. [31] studied the degradation of a textile azo dye using biological treatment followed by photo-Fenton oxidation as well as toxicity and microbial load was evaluated as a function of treatment and found that the acute toxicity was higher in the biologically treated than in the untreated effluent. Photo-Fenton oxidation successfully
reduced the toxicity and the final effluent was non-toxic to Artemia salina and Microtox. Author declared that using advanced oxidation after biological treatment is an effective way to degrade the organic compounds and for the removal toxicity of textile effluents. Similarly, Jadhav et al. [32] evaluated the toxicity of biologically treated remazol red. As a result of biodegradation, >97% dye was degraded and toxicity evaluated by A. cepa, Phaseolus mungo and Sorghum vulgare showed less toxic nature of metabolites formed after dye degradation and Gomes de Moraes
Table 4 Microbial load, cytotoxicity and mutagenicity profile of textile wastewater after gamma radiation treatment. Bioassays
Types
Units
TIW I
TIW II
TIW III
Ave
5 kGy
10 kGy
15 kGy
5 kGy
10 kGy
15 kGy
5 kGy
10 kGy
15 kGy
Microbial load
TBC TC
CFU CFU
ND ND
— —
— —
ND ND
— —
— —
ND ND
— —
— —
— —
Cytotoxicity
ACT I) RC I) RL
count cm
17 1.0 7.0 0.2
18 0.5 7.1 0.2
19 0.5 7.4 0.2
16 0.4 6.9 0.4
20 0.5 7.5 0.4
26 0.7 8.0 0.5
18 0.6 —
21 0.8 8.0 0.2
23 0.9 8.1 0.5
— — —
% %
38 2.0 34 1.5 01 0.0
37 3.0 29 1.3 01 0.0
31 2.0 26 2.2 0.0 0.0
42 1.0 — —
— 36 2.5 2 0.0
34 3.0 29 2.0 1 0.5
39 1.0 33 2.6 3 1.00
29 1.5 26 1.1 2 1.0
22 1.0 21 1.1 1 0.33
— — —
Ames test I) TA98 II) TA100
count count
29 1.00 33 0.33
23 1.5 27 0.5
22 0.5 25 2.0
30 1.33 34 0.33
27 2.0 28 1.0
24 0.33 —
— —
28 1.0 30 0.3
21 0.3 25 0.5
— —
TBC TC
% %
100 100
100 100
100 100
100 100
100 100
100 100
100 100
100 100
100 100
100 100
ACT I) RC I) RL
% %
41.17 41.42
44.44 42.25
47.36 44.59
43.75 43.47
55 48
65.38 51.25
44.44 —
52.38 52.5
56.26 53.08
50.02 47.07
% %
53.08 53.52 94.44
54.32 59.15 94.44
61.72 63.38 100
46.15 — —
— 47.82 89.47
56.41 57.97 94.73
45.83 56 85
59.72 65.33 90
69.44 72 95
56.23 59.40 92.89
% %
53.22 51.47
66.17 60.29
67.64 63.23
49.15 46.03
54.23 55.55
59.32 —
— 42.62
53.33 50.81
65 59
58.51 53.63
heamolytic I) Human II) bovine Shrimp Mutagenicity
Reduction (%) Mic. load
Cytotoxicity
heamolytic I) Human II) bovine Shrimp Mutagenicity
Ames test I) TA98 II) TA100
Explanations as given in Table 3.
Please cite this article in press as: M. Iqbal, Cytotoxicity and mutagenicity evaluation of gamma radiation and hydrogen peroxide treated textile effluents using bioassays, J. Environ. Chem. Eng. (2015), http://dx.doi.org/10.1016/j.jece.2015.06.011
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et al. [33] also reported 50% toxicity reduction of textile effluent by combined photocatalytic and ozonation processes. It was observed that as the reaction proceeded, the toxicity reduced continuously, implying that all the generated products were less toxic than the original dye dyes in the effluents. The similar results were reported by Neamtu et al. [34] who observed that the toxicity of azo dye C.I. Reactive Yellow 84 kept on decreasing during homogeneous photo-Fenton process, although higher toxic intermediates were formed in the initial stage of UV/H2O2 treatment process. de Luna et al. [35] also suggested that all the generated products of Orange II were less toxic than the original dye during homogeneous photoFenton process. Similar to present study, various advanced oxidation processes such as O3, TiO2/UV, sunlight irradiation, UV/H2O2 and O3, electro-Fenton, wet-air oxidation, UV/electroFenton, solar driven photo-Fenton and TiO2 photocatalysis on pilot plant, O3/UV, Photo-Fenton process before and after biological treatment, TiO2/H2O2/UV have been employed successfully for toxicity evaluation of different systems (Table 1) and this study also revealed efficiency for toxicity evaluation of gamma irradiated textile effluents.
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Mutagenicity
216
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The untreated and treated effluents were tested for mutagenicity using TA98 and TA100 strains. Table 3 summarizes the results of mutagenicity of textile untreated effluents. The Ames test revealed the presence of mutagenic agent in effluents, 60 (37.5%) and 61 (36.45%) micro plates were affected out of 96 before treatment. The effluents treated by gamma radiation, to the absorbed doses of 5 kGy, 10 kGy and 15 kGy in combination with 20 mM hydrogen peroxide were tested for mutagenicity. After gamma radiation treatment, mutagenicity reduced considerably and up to 53.22% (TA98) and 51.47% (TA100) for the absorbed dose of 5 kGy of TIW I. The mutagenicity reduced linearly with gamma radiation absorbed dose and reduction reached to 67.64% and 63.23% in case of TA98 and TA100 for the absorbed dose of 15 kGy, respectively. The mutagenicity reeducation trend was similar for TIW II and TIW III (Table 4). The studies regarding mutagenicity testing of gamma radiation treated textile effluents are rare. However, our previous study indicated that the gamma radiation without hydrogen peroxide was not effective for the degradation of mutagenic agent and better efficiency can be achieved at low gamma radiation absorbed dose using small amount of hydrogen peroxide. Previous studies also indicated that the oxidant can enhance the gamma radiation efficiency [36,37]. Based on the toxicity test, it can be concluded that gamma radiation in combination with hydrogen peroxide is a suitable method for detoxification of textile effluents.
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Conclusion
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The results of this study suggest that gamma radiation treatment is an effective way to degrade the dye and remove toxicity from textile effluents. The employed toxicity test showed that the bioassays were better tools to study the effluent to evaluate the biological efficiency of method used for treatment of effluents. The gamma radiation treatment efficiency can be enhanced using small amount of hydrogen peroxide. It is concluded that the textile effluents are cytotoxic and mutagenic; however, these can be detoxified through degradation by the gamma radiation in combination with hydrogen peroxide and due to its high capacity, relatively low reaction times, this treatment method might be an interesting alternative, especially for the degradation of effluents contaminated with cytotoxic and mutagenic agents.
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5
Acknowledgements
256
Higher Education Commission (HEC) of Pakistan is highly Q6 acknowledged for financial support under the start-up Research Project Program (Grant No: 21-194/SRGP/R&D/HEC/2014) and Dr. Muhammad Shahid, UAF for assistance in toxicity studies.
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References
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