Mycoremediation of textile effluent: A toxicological evaluation and its possible correlation with COD

Globally, textile industries are one of the major sectors releasing dye pollutants. This is the first report on the 15 positive correlation between toxicity and COD of textile effluent along with the proposed pathway for enzymatic 16 degradation of acid orange 10 using Geotrichum candidum within a very short stretch of time (18h). Removal 17 efficiency of this mycoremedial approach after 18 h in terms of color, dye concentration as well as reduction of 18 chemical oxygen demand (COD) and biological oxygen demand (BOD) in the treated effluent reached to 89%, 87%, 19 98.5% and 96.3% respectively. FT-IR analysis of the treated effluent confirmed biodegradation. The LC-MS 20 analysis showed the degradation of acid orange 10, which was confirmed by the formation of two biodegradation 21 products, 7-oxo-8-iminonapthalene-1,3-disulfonate and nitrosobenzene, which subsequently undergoes stepwise 22 hydrogenation and dehydration to form aniline via phenyl hydroxyl amine as intermediate. The X-ray diffraction degradation study of acid orange 10 by G. candidum . The genotoxicity, phytotoxicity and microbial toxicity analyses proved the raw effluent is harmful, whereas the treated effluent is less toxic. Relationship between effluent 379 COD and TU 50 showed that an increase in effluent COD resulted in increase in wastewater toxicity. There was a clearly defined correlation between toxicity and COD. It was evident that toxic effects of the textile effluent were 381 significantly reduced upon treatment with G. candidum. The major relationships between toxicity and COD will 382 provide directions for more efficient control of textile dyeing effluents. This is the first report on the positive correlation between toxicity and COD of textile effluent using G. candidum within a very short stretch of time (18h). Therefore, the findings of this study have shown that the treated effluent was safer to be released in regard to physicochemical parameters and toxicity unit (TU 50 ). The correlation between conventional indicators and toxicity may provide assistance in effluent management.


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Urbanization and industrialization have paved the path for development of the many industries, including the textile 57 industries. Clothing and textiles, after agriculture, is the basic requirement of human being. While the textile 58 industry contributes worldwide economically, the environmental effects are due to high volumes of water use and 59 the diversity and quantities of chemicals that are used in all manufacturing phases of textiles. The untreated effluent 60 when disposed in the water bodies seriously impacted the people in the area (Agrawal and Verma 2019). Rivers and 61 drainage bodies get loaded with precarious textile effluents that impact on the water quality, the aquatic organisms

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To assess the performance of wastewater treatment facilities, the influent and treated water samples after each 51 69 treatment phase (physical, chemical and biological) should be tracked. In general, microbial degradation is known to 70 be a safe, natural, inexpensive and effective pollutant removal technique in the world (Mishra et

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Geotrichum sp. out of the few fungi have been observed for degradation of large quantity of artificial colors and 84 molasses (Kim andShoda 1998, 1999; Chen and Zhao 2008; Shintani and Shoda 2013). Since, Geotrichum sp have 85 not been explored much, therefore it is being used in the current study for the biodegradation and detoxification of 86 textile effluent. The analysis of conventional parameters of textile effluent (before and after mycoremediation) and 87 the inter-relationship between toxicity and COD have been carried out in this study. In an attempt to validate the 88 non-toxicity of treated effluent, the biodegradation analysis such as FT-IR, LC-MS and XRD have been conducted.

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Furthermore, bioassays such as genotoxicity, phytotoxicity and microbial toxicity assays have also been carried out 90 so that the toxicity level of raw and treated textile effluent can be assessed.

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In this part of the analysis, the G. candidum culture was used to biodegrade the textile effluent. A conical flask 110 containing raw textile effluent (25 ml) was inoculated with fungal culture (5%, v/v), followed by incubation at 35°C,

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Pt-Co color scale was used to measure color. Dye concentration of the sample was determined using colorimeter 116 (Systonic, S-912). Every experiment has been carried out in triplicates and standard deviation has been presented 117 with the average data.

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The following equation (Eq. (1)) was used to quantify degradation as a percentage reduction of COD: 119 120 The following equation (2) has been used to determine the specific growth rate of G. candidum.

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The expression for growth yield (Y) is

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The equation 3 can be rewritten as follows (

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The metabolites formed in textile effluent after decolorization and degradation were obtained by same volume with 149 ethyl acetate. The extract was dried over anhydrous sodium sulfate and evaporated to dryness in a rotary evaporator.

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The resulting crystals were dissolved in small volumes of methanol (HPLC grade), and then used for analysis such 151 as FT-IR (Fourier-transform infrared spectroscopy), LCMS (Liquid chromatography-mass spectrometry) and XRD 152 (X-ray Diffraction).

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The FTIR analysis of the effluent was performed with Attenuated total reflectance-Fourier transform infrared 154 spectroscopy (ATR-FTIR, FT/IR-4600, JASCO, Japan). A drop from each sample was placed on a Zinc selenide 155 (ZnSe) frame, and the spectra were documented with an average of 32 scans between the 4000 and 600 cm -1 spectral 156 ranges.

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The raw and treated samples were also analyzed using LC-MS (Waters Micromass Q-Tof Micro) and the flow rate 159 and temperature were maintained at 0.2 ml min -1 and 35°C, respectively. The running time was 41 minutes. Two

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X-ray diffraction patterns before and after biodegradation of textile effluent were recorded using RIGAKU-

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The metals were identified with powder diffraction standard file (JCPDS, Joint Committee on Powder Diffraction

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The phytotoxicity analysis was performed using Phaseolus mungo (at room temperature) on both raw and treated 172 effluent. Simultaneously, the control set was conducted using water. After 7 days, toxicity of the raw and treated

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The Pearson's correlation coefficient at the significance level of 0.05 was used to assess the correlation between 218 toxicity and conventional indicators of the raw and treated textile effluent, and the impact of COD on toxicity was 219 analyzed by means of linear regression. The significance level of the regression analysis (p) and R 2 illustrated the 220 extent of toxicity variance caused by COD.

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The conventional indicators of raw and treated textile effluent have been listed in Table 1. It was evident that the

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Agriculturally valuable seeds i.e. P. mungo were used for phytotoxicity assessment of raw and treated textile 301 effluent. The analyzed parameters were germination percentage, plumule and radical length. The seeds treated with 302 water were considered as positive control and the test samples were significantly compared with each other, a 303 maximum germination of 100% was recorded in P. mungo with treated textile effluent indicating it as less 304 phytotoxic. Seeds treated with raw textile effluent showed a minimal germination of 33% (Fig. 5). Though the seeds 305 exposed to raw textile effluent germinated, they couldn't grow further, exhibiting maximum phytotoxicity. The 306 results shown in Table 2 indicated that the germination (%) and length of plumule and radicle of P. mungo seeds 307 were less with the untreated as compared to treated effluent. This study shows that the metabolites formed after 308 effluent biodegradation are less harmful than the compound present in the raw textile effluent. It is evident that the 309 seed germination and average plumula and radical development were unaffected by decolored textile effluent. Thus, 310 the comprehensive results indicated that the textile effluent treated with G. candidum was not harmful to plant 311 germination and growth Similar kind of phytotoxicity studies can be seen reported for several times in the literature.

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This ensures that treated effluent could be used for agriculture or recycled.  Values are mean of three experiments, SD (±), significantly different from the control (seeds germinated in water) at p> 0.05 (One-way analysis of variance, ANOVA).

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The short-term toxicity of the raw and treated textile effluent was assessed using the well-established bacteria E. coli 322 and the results are shown in Fig. 6. The raw textile effluent was highly toxic, while treated textile effluent presented 323 a low toxicity. Fig. 6 shows that after 30 minutes of exposure to raw textile effluent, the number of bacterial cells 324 declined drastically by approximately 41%, indicating acute toxicity to E coli. This was plausibly due to the 325 enormous amount of ionic and acid dyes entering the wastewater during the textile processing. Ionic and disperse 326 dyes discharged from textile processing and dyeing were usually particularly toxic, and some were mutagenic and The A. cepa study is a standard test to assess the genotoxicity of any toxic substance. The test was carried out to 338 identify MI and chromosomal aberrations in the root cells (Table 3)

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significantly different from the control (roots germinated in water), *P < 0.05, **P < 0.001 by one-way analysis of variance (ANOVA) with TukeyeKramer comparison test.

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The decrease in colour, COD and BOD might have lead to the minimization in the toxicity of textile effluent. This 352 study indicates that the metabolites produced after biodegradation are less toxic than the compounds present in raw 353 effluent.

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Pearson's correlation analysis for the textile effluent indicated a significantly positive correlation between COD and

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COD was one of the most widely used water quality monitoring metrics and also was an important measure for the

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The competent Geotrichum candidum culture involved in the current work biodegraded the toxic textile effluent.

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The analysis of conventional parameters such as COD, BOD and color were indicative of the decreased toxicity of

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COD and TU50 showed that an increase in effluent COD resulted in increase in wastewater toxicity. There was a 380 clearly defined correlation between toxicity and COD. It was evident that toxic effects of the textile effluent were 381 significantly reduced upon treatment with G. candidum. The major relationships between toxicity and COD will 382 provide directions for more efficient control of textile dyeing effluents. This is the first report on the positive 383 correlation between toxicity and COD of textile effluent using G. candidum within a very short stretch of time (18h).

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Therefore, the findings of this study have shown that the treated effluent was safer to be released in regard to 385 physicochemical parameters and toxicity unit (TU50). The correlation between conventional indicators and toxicity 386 may provide assistance in effluent management.