Journal of Hazardous Materials 292 (2015) 52–60

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Combined anaerobic–ozonation process for treatment of textile wastewater: Removal of acute toxicity and mutagenicity Marisa Punzi a,∗ , Filip Nilsson b , Anbarasan Anbalagan a,1 , Britt-Marie Svensson c , Karin Jönsson b , Bo Mattiasson a , Maria Jonstrup a,2 a

Department of Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Water and Environmental Engineering at the Department of Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden c School of Education and Environment, Kristianstad University, SE-291 88 Kristianstad, Sweden b

h i g h l i g h t s • • • • •

COD and UV absorbance were effectively reduced. The treated effluents were non-toxic to Artemia salina and Vibrio fischeri. The real textile wastewater was mutagenic. Mutagenicity persisted after bio treatment and even more after a short ozonation. Higher ozone doses completely remove mutagenicity.

a r t i c l e

i n f o

Article history: Received 27 November 2014 Received in revised form 6 March 2015 Accepted 7 March 2015 Available online 11 March 2015 Keywords: Ozone Biological treatment Azo dye Textile wastewater Mutagenicity

a b s t r a c t A novel set up composed of an anaerobic biofilm reactor followed by ozonation was used for treatment of artificial and real textile effluents containing azo dyes. The biological treatment efficiently removed chemical oxygen demand and color. Ozonation further reduced the organic content of the effluents and was very important for the degradation of aromatic compounds, as shown by the reduction of UV absorbance. The acute toxicity toward Vibrio fischeri and the shrimp Artemia salina increased after the biological treatment. No toxicity was detected after ozonation with the exception of the synthetic effluent containing the highest concentration, 1 g/l, of the azo dye Remazol Red. Both untreated and biologically treated textile effluents were found to have mutagenic effects. The mutagenicity increased even further after 1 min of ozonation. No mutagenicity was however detected in the effluents subjected to longer exposure to ozone. The results of this study suggest that the use of ozonation as short post-treatment after a biological process can be beneficial for the degradation of recalcitrant compounds and the removal of toxicity of textile wastewater. However, monitoring of toxicity and especially mutagenicity is crucial and should always be used to assess the success of a treatment strategy. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Textile production is one of the largest industrial sectors, accounting for 8% of the world trade in manufactured goods [1]. A major issue associated with textile production is the treatment

∗ Corresponding author. Tel.: +46 46 2223672. E-mail address: [email protected] (M. Punzi). 1 Present address: Department of Energy, Building and Environment, Mälardalen University, SE-721 23 Västerås, Sweden. 2 Present address: VA SYD, 211 20 Malmö, Sweden. http://dx.doi.org/10.1016/j.jhazmat.2015.03.018 0304-3894/© 2015 Elsevier B.V. All rights reserved.

of wastewater. Textile wastewater is rich in salts and organic compounds such as dyes and colorants, surfactants and other substances used to make clothes resistant to physical, chemical and biological agents [2]. One of the most widely used type of dyes, azo dyes, are known to be transformed into toxic and mutagenic compounds, which is why their complete degradation is fundamental [3]. Treatment of textile wastewater is challenging and has received a lot of attention in the last decades. Biological processes are the ideal choice for wastewater treatment because they are environmentally friendly and cost efficient. However, many studies demonstrate that biological treatment alone is not enough to

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pre-treatment, in full scale applications. Moreover, contradictory results have been reported with regards to the consequences of ozonation on the toxicity of textile wastewater [20,22,23]. Thus, it is crucial to evaluate acute toxicity and, especially in the case of effluents containing azo dyes, mutagenicity of the treated water before scaling up a treatment process. In our study, a novel setup composed of an anaerobic biofilm reactor followed by ozonation was used for treatment of artificial and real textile wastewater containing azo dyes. The anaerobic biofilm was chosen because of its ability to adapt to diverse environmental conditions and presence of toxic compounds [24]. A short ozone treatment was applied to break down those molecules recalcitrant to biological degradation. Acute toxicity of the effluent before and after each step was measured using Microtox® and a toxicity tests based of the shrimp Artemia salina. In addition, Ames test was used to evaluate the mutagenicity of the real textile effluent. Fig. 1. The ozonation unit used in the experiments, consisting of 1: O2 (99,5% w), 2: pressure regulator, 3: ozone generator (Primozone GM2), 4: three way valve, 5: ozone gas analyzer (BMT 963), 6: catalytic ozone destructor, 7: ozone contact chamber (8 l total volume, 4 l of sample water).

2. Experimental 2.1. Textile wastewater

detoxify and degrade pollutants in textile effluents and in most cases only partial mineralization is obtained [4,5]. Nevertheless, biological treatment offers important advantages in terms of removal of organics and should not be put aside. A better solution is to combine it with other technologies [6,7]. For example, because of low energy requirements and their ability to handle high organic loads [8], anaerobic processes have been widely used as a first step in such combined treatment processes to remove the color and increase the biodegradability of textile effluents [9,10]. Despite the lower organic content, bio-treated textile wastewater, can be more toxic than the untreated effluent as some dyes are transformed into small organic molecules, e.g., aromatic amines. Consequently, further treatment is required. Particularly promising are the so-called Advanced Oxidation Processes (AOPs), which have the advantage that they can break down conjugated bonds thanks to the oxidative power of hydroxyl radicals generated in situ. Application of AOPs for treatment of textile effluents has been the topic of many research studies [11–13]. Several classes of AOPs can be distinguished. When it comes to textile wastewater, both ozonation [11], photo-Fenton [14] and photo catalysis with TiO2 [15] have been considered as stand-alone treatments or in combination with other processes. Ozonation has the advantage that it does not require addition of chemicals, apart from ozone; moreover, it does not produce sludge and it is simple to operate. These advantages suggest that, among the several AOPs, ozonation is the technology that can more easily be implemented in already existing treatment plants. The molecules of ozone and the hydroxyl radicals generated in situ act together to mineralize organic compounds [16]. However, the oxidation due to the hydroxyl radicals is non-specific, which means that most organic molecules in the water, and also some inorganic ones, will compete for the oxidants. At present, most studies on ozonation have focused on its use as pre-treatment before biological processes [17–19]. This set up is usually justified by the fact that raw wastewater may contain compounds that are toxic for the microorganisms that perform the biological treatment. Thus, the pre-treatment aims at improving the biodegradability of the organic compounds in the wastewater [20]. However, when ozonation is used on untreated wastewater, the biodegradable compounds compete with the recalcitrant ones for the ozone and the hydroxyl radicals, and more ozone is required to obtain high removal of organics [21]. This can lead to high amounts of energy required and excessive costs, which encourages the use of ozonation as post-treatment, rather than

The study was conducted first on artificial and then on real textile wastewater. The anaerobic medium was prepared according to Jonstrup et al. [25] and modified to simulate a real textile effluent. Starch (BDH Chemicals Ltd., England) was used as carbon source at 0.465 g/l, corresponding to 590 mg/l of chemical oxygen demand (COD). The starch was dissolved in water heated at 150 ◦ C for 2 h and hydrolyzed overnight by adding NaOH 1M until reaching pH 12. Moreover, the commercial azo dye Remazol Red (RR) and sodium chloride, 10 g/l, were also added to the prepared effluent. RR was provided from a textile factory in Tirupur, India. According to the manufacturer it is a mixture of azo dyes composed of 50–60% dyestuff, 30–40% inorganic salts and up to 5% functional additives. In the experiment it was used as received, without further purification. The real effluent was received from Ten Cate, The Netherlands and stored at +4 ◦ C.

2.2. Biological treatment Two parallel anaerobic biofilm reactors were set up and fed with the textile wastewater in continuous mode. The reactors were made of glass and had each a volume of 0.6 l. Poraver carriers (Dennert Poraver GmbH, Schlüsselfeld, Germany) were used as support for the biofilm and the working volume of the reactors filled with carriers was 0.340 l and 0.345 l for A and B, respectively. The temperature was kept at 37 ◦ C using a water bath connected to the water jacket of the reactors. The content of the reactors was constantly recirculated in an up-flow mode. In the first part of the experiment the artificial wastewater was used and three different dye concentrations were studied: 100, 500 and 1000 mg/l, corresponding to 60, 300 and 600 mg/l of COD, respectively. The hydraulic retention time (HRT) was kept constant at 2.5 days. Each time the dye concentration was changed, the reactors were operated at the new condition for three HRTs to allow stabilization; then the effluent was collected for a minimum of 5HRTs, analyzed and stored. In the second part of the experiment, the reactors were fed with the raw textile wastewater. The HRT was kept constant at 3 days and no additional parameter was modified. Samples were withdrawn regularly from the reactors to measure COD and absorbance in the UV-visible range and the biologically treated effluent was analyzed for nutrient composition before storage at +4 ◦ C.

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Table 1 Ozone added and consumed for each ozone dose used.

2.4. Toxicity tests

Ozonation (min)

Ozone added (g/l)

Ozone consumed (g/l)

1 2 4 6

0.13 0.26 0.52 0.78

0.12 0.25 0.37 0.38

2.3. Ozonation Ozonation was used to further degrade the aromatic amines and other organic pollutants that were recalcitrant to biological treatment or were formed as product of the biotransformation. Fig. 1 shows the ozone unit used in the experiments. A pressure regulator controlled the transfer of oxygen to the ozone generator (Primozone GM2, Löddeköpinge, Sweden), which converted part of the oxygen into ozone (Fig. 11–3). The gas was fed into the ozone gas analyzer (Fig. 15) until a stable ozone concentration was reached (203 g O3 /m3 ). Ozone concentration was measured with a UV photometer (Ozone Photometer 963, BMT Messtechnik, Germany) Thereafter, the three-way valves (Fig. 14) were opened to allow the ozone-containing gas (2.56 lpm) to reach the ozone contact chamber (Fig. 17). Ozone was bubbled through the sample water with a diffuser stone having pores of 80 ␮m diameter. The ozone that did not dissolve and react with the sample water was fed (2.56 lpm) into the ozone gas analyzer and finally into the catalytic ozone destructor (Fig. 16). To test the effect of different ozone doses on the degradation of the biologically treated water, the ozonation was carried out for different time lengths: 1, 2 and 4 min for the synthetic effluents; 1, 2, 4, and 6 min for the real textile effluent. Values for the consumed amount of ozone and hence a dosage of ozone was obtained by calculating the difference between the amount of ozone going into and leaving the chamber. The concentration of ozone fed to the reactor, which was constant, was used to calculate the amount of ozone going in (Eq. (1)). As shown in Fig. 2a, the concentration of ozone going out of the contact chamber rose rapidly and reached a plateau after 3 min. The ozone concentration increased linearly during the first 3 min (Fig. 2b) and this relation was used to calculate the amount of ozone consumed in the first 3 min (Eq. (2)). The concentration after 3 min was considered to be constant and the amount of ozone leaving the reactor was calculated (Eq. (3)). Thus, depending on the time length of the ozonation, the Eqs. (1)–(4) were used to calculate the amount of ozone consumed: Ingoing amountO3 = QO3 × [O3 ]in × t



Outgoing amountO3 , 0 − 3 min=

t

(1)





QO3 × f [O3 ]out, t



dt

(2)

0

Outgoing amountO3 , 3 − 6min=QO3 × [O3 ]out × (t − 3)

(3)

ConsumedO3 = Ingoing amountO3 − Outgoing amountO3

(4)

where QO3 = flow of ozone-containing gas ((m3 O2 )/min); [O3 ]in = concentration of ozone into the contact chamber ((g O3 )/(m3 O2 )), constant; [O3 ](out,t) = concentration of ozone out of the contact chamber ((g O3 )/(m3 O2 )), function; [O3 ]out = concentration of ozone out of the contact chamber ((g O3 )/(m3 O2 )), constant; t = ozonation time (min). Table 1 summarizes the amounts of ozone added and consumed in all experiments, which was calculated using these equations considering that the flow was 2.56 lpm, the ingoing concentration 203 g O3 /m3 and the outgoing concentration after 3 min 198 g O3 /m3 .

The acute toxicity of the wastewater before and after treatment was evaluated using two toxicity tests targeting organisms which belong to different trophic levels. Microtox® , which evaluates changes in bioluminescence of the bacteria Vibrio fischeri in response to the exposure to a contaminant for 5 and 15 min, was performed according to the “81.9% Basic Test” protocol as described in the manual of the Microtox Model 500 analyzer (Modern 276 Water Inc., New Castle, DE, USA). The other test targets the brine shrimp A. salina and was chosen for its tolerance to different salt conditions. The immobility rate of a population of 2 days old shrimps exposed to a contaminant for 48 h was measured. Cysts of A. salina were hatched in artificial seawater (ASW) and used for the test following the protocol for the standardized Artemia nauplii test (ARC-test) [26,27]. For both tests the effective concentration (EC20 ) was calculated, which is the concentration of contaminant or wastewater at which 20% of the target organisms gives the described response. A solution of potassium dichromate 100 mg/l was used as positive control in both tests; the culture media for V. fischeri and A. salina were used as negative control in the respective tests. The pH of the sample water was adjusted to 6–8 before the testing if required. All experiments were performed in triplicates. Ames fluctuation test (without metabolic activation) was used to assess the mutagenicity of the textile wastewater before and after treatment. The strain used was Salmonella typhimurium YG7108 [28] received from the Department of Aquatic Ecotoxicology of the Goethe University in Frankfurt am Main, Germany. This strain was chosen because it lacks O6-methylguanine DNA methyltransferases and is thus highly sensitive to mutagenic alkylating agents and nitrosamines that may be formed during ozonation of wastewater [22,29]. The Ames fluctuation test was performed according to the procedure described in Reifferscheid et al. [30]. The strain was inoculated in 10 ml growth medium in presence of 10 ␮g/l kanamycin and 25 ␮g/l chloramphenicol and incubated at 37 ◦ C, 150 rpm for 10 h. A 24 wells micro plate was filled with 3 replicates of water samples, negative (demineralized water) and positive control (0.2% propylene oxide). After addition of the exposure medium and the strain, the plate was incubated at 37 ◦ C, 250 rpm for 100 min. The concentration of the culture in the test was 300 formazin attenuation units [31]. After incubation, the reversion indicator medium was added to each well of the micro plate. From each well, 50 ␮l were transferred to 16 wells in a 384 wells micro plate, which was then incubated at 37 ◦ C for 48 h. Then, the absorbance at 415 nm was read with a micro plate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). Each well having absorbance ≥0.6 was considered to be a revertant (mutant colony able to synthetize histidine). The test was repeated 3 times for each sample. William’s multiple t-test was used to compare each sample to the negative control and a sample was defined as mutagenic when the p-value was lower than 0.05. The free statistic software R was used for the data analysis. Cytotoxicity was assessed in parallel by measuring the cell density before and after exposure of the culture to the water samples. 2.5. Analysis COD, total organic carbon (TOC) and nutrients concentrations were determined using the cuvettes LCK114 and LCI 500 (COD), LCK 385 (TOC), LCK 339 (NO3 -N), LCK 303 (NH4 -N) and LCK 049 (PO4 -P) and analyzed with a LASA 100 spectrophotometer (Hach Lange, Berlin, Germany). The absorbance in UV-visible range was measured with a spectrophotometer. The absorbance in the visible range, at 518 nm for RR and 500 nm for the textile wastewater, was used to quantify the color removal, whereas the absorbance in the UV range, at 269 nm for RR and 254 nm for the textile

M. Punzi et al. / Journal of Hazardous Materials 292 (2015) 52–60

b.

250

Concentration out of the contact chamber (g O3 / m3 O2)

Concentration out of the contact chamber (g O3 / m3 O2)

a. 250

55

200

200

150

150

100

100 50 0 0

100

200 Time (s)

300

400

y = 1.0322x + 10.737 R² = 0.9212

50 0 0

50

100 Time (s)

150

200

Fig. 2. Median concentrations out of the contact chamber from all ozone experiments within this study: (a.) 0–6 min and (b.) 0–3 min.

wastewater, was used to evaluate the removal of aromatic compounds. If necessary, the samples were diluted with deionized water prior to analysis. 3. Results and discussion 3.1. Synthetic textile wastewater 3.1.1. Anaerobic treatment Two parallel anaerobic biofilm reactors were used for treatment of artificial textile wastewater containing the azo dye RR. Fig. 3 summarizes the results. The average color removal was 95% or higher at all the dye concentrations tested. The COD of the artificial textile wastewater was 650, 900 and 1288 mg/l at the dye concentration of 100, 500 and 1000 mg/l, respectively. The COD removal tended to increase with increasing dye concentration. The average COD removal was 70% at 100 mg/l RR for both reactors (final COD: 160 mg/l); 67% and 71% for reactor A and B, respectively, at 500 mg/l RR (final COD: 180 mg/l) and 76% and 74% for reactor A and reactor B, respectively, at 1000 mg/l RR (final COD: 350 mg/l). The positive trend in COD reduction is in contrast to previous studies in which a decrease in COD reduction with the increase of the initial dye concentration has been reported [25,32]. Several factors may have played a role in determining this unusual trend. The presence of starch, which is a more complex carbon source compared to glucose, and high concentration of sodium chloride are likely to have applied a selective pressure on the microbial communities. These communities may have adapted to the high salinity and improved their performance in terms of metabolism of starch and COD removal. Moreover, the fact that the dye was not pure but mixed with additives may have also played a role. The dye itself is not expected to be degraded to inorganic compounds, which means that the COD associated with the dye is not usually removed during biological treatment. The dye additives, however, may be biodegradable and their concentration in the synthetic effluents increased together with the concentration of the dye. Thus, part of the COD reduction may be due to degradation of dye additives. Overall, the anaerobic treatment performed well even at the highest dye concentration and the pH of the effluent was stable between 7.5 and 8 during the entire experiment. 3.1.2. Ozonation of biologically treated artificial textile wastewater The ozonation was performed in batch mode on the anaerobically treated artificial textile wastewater and 3 ozone doses were evaluated (0.13; 0.26 and 0.52 g O3 /l, applied) by changing the ozonation time (1–4 min). Fig. 4 shows the removal of organic compounds and color obtained for each initial RR concentration. Although the effluent

was almost decolorized in the previous biological treatment, the decolorization was monitored also during ozonation. The reduction of absorbance in both visible and UV range increased with higher ozone exposure (Fig. 4). In all cases, very high reduction of absorbance was obtained after 4 min of ozonation, which indicates that the treatment can efficiently degrade the dye molecules and open the aromatic rings. Nevertheless, clear differences can be observed between effluents containing different initial concentrations of RR. Fig. 4a shows that for the effluent containing 100 mg/l of RR, the reduction of absorbance after 1 min of ozonation was 52% and 76% in the UV and visible range, respectively. After 4 min of ozonation, complete color removal was obtained and the reduction of UV absorbance was 88%. Fig. 4b shows the results for the effluent containing 500 mg/l of RR. The ozone treatment resulted in 25% and 43% of reduction of absorbance in the UV and visible range, respectively, after 1 min. Higher reduction was obtained after 4 min of ozonation: 74 and 86% in the UV and visible range, respectively. In Fig. 4c similar results for the effluent containing 1000 mg/l of dye are shown. After 1 min of ozonation, only 30 and 37% reduction of absorbance in the UV and visible range were achieved. Nevertheless, increasing the ozone exposure resulted in improved removal and 65% and 86% of reduction of absorbance in UV and visible range were obtained. The mineralization of the organic compounds was evaluated measuring COD and TOC, and, as shown in Fig. 4, a higher degree of mineralization was obtained at higher ozone exposure. The effluent containing 100 mg/l of RR had COD of 160 mg/l and TOC of 39 mg/l after biological treatment. The COD was further reduced to 100 mg/l already after 1 min of ozonation. Increasing the exposure to ozone from 1 to 4 min resulted in COD of 65 mg/l and TOC 27 mg/l (Fig. 4a). The effluent containing 500 mg/l of RR had COD of 178 mg/l and TOC of 71 mg/l after biological treatment. The effect of 1 min of ozonation was low; nevertheless, after 4 min of ozonation, the COD had been reduced to 115 mg/l and the TOC to 62 mg/l (Fig. 4b). In the case of the effluent containing 1000 mg/l of RR, the COD after biological treatment was 350 mg/l and the TOC 117 mg/l. Only 35% reduction of COD was obtained with a final COD value of 230 mg/l even after 4 min of ozonation. The TOC reduction for this effluent was never above 10%, final TOC of 103 mg/l, and no significant improvements could be seen when increasing the ozone exposure from 1 to 4 min (Fig. 4c). These data highlight the need for a further treatment step, however such a high dye concentration is unusual as common dye content goes between 100 and 700 mg/l [33,34]. The results show that ozonation used as post-treatment of an anaerobic biofilm process can degrade most aromatic compounds and lower the COD achieving an overall reduction between 85 and 90%, with the exception of the effluent containing 1 g/l of dye.

56

4.0

60

3.0

40

2.0 RR 100 mg/l

RR 500 mg/l

1.0

RR 1000 mg/l

0

HRT (Days)

80

20

b.

5.0

100

5.0

80

4.0

60

3.0

40

2.0

Reduction (%)

100

Reduction (%)

a.

20

RR 100 mg/l

RR 500 mg/l

1.0

RR 1000 mg/l

0.0

0

0.0

123456781234567812345678

123456781234567812345678

Cycles (1 cycle = 3 HRTs)

Cycles (1 cycle = 3 HRTs)

Abs 518 nm

COD

HRT (Days)

M. Punzi et al. / Journal of Hazardous Materials 292 (2015) 52–60

Abs 518 nm

HRT

COD

HRT

Fig. 3. COD, color removal and HRT during anaerobic treatment of artificial textile wastewater at different initial dye concentrations in (a.) reactor A and (b.) reactor B. Values are averages of 3 measurements.

Table 2 Composition of the raw textile wastewater before and after biological treatment.

3.2. Real textile wastewater 3.2.1. Anaerobic treatment The same anaerobic reactors were used for treatment of the raw textile effluent. Table 2 summarizes the average composition of the textile effluent before and after biological treatment. It can be noted that together with a reduction in COD, also 45% of the phosphate was consumed. In contrast, the total inorganic nitrogen was higher after biological treatment, probably as a consequence of the release

a. 100

pH Conductivity (mS/cm) PO4 -P (mg/l) NH4 -N (mg/l) NO3 -N (mg/l) COD

b.

90 70

Reduction (%)

Reduction (%)

80 60 50 40 30 20

10 0 1 TOC

COD

2 Ozonation (min) Abs 518 nm

100 90 80 70 60 50 40 30 20 10 0 -10 -20

Biologically treated

10 12.5 16.8 1.7 3.6 1714

7.5–8 15.5 9.2 5.0 3.6 1190

2

1

4

Raw wastewater

4

Ozonation (min)

Abs 267 nm

TOC

COD

Abs 518 nm

Abs 267 nm

Reduction (%)

c. 100 90 80 70 60 50 40 30 20 10 0 -10 TOC

1

COD

2 Ozonation (min) Abs 518 nm

4

Abs 267 nm

Fig. 4. Reduction of TOC, COD and absorbance in the UV and visible range after ozonation of bio-treated artificial textile effluent as a function of time. Three initial concentration of RR were considered: (a.) 100 mg/l; (b.) 500 mg/l; (c.) 1000 mg/l. Values are average of 3 measurements. Error bars represent standard deviation.

M. Punzi et al. / Journal of Hazardous Materials 292 (2015) 52–60

100 90 80 70 Reduction (%)

of some amino-groups from the dye molecules. The pH of the biotreated effluent was between 7.5 and 8. Fig. 5 summarizes the results of the experiment in terms of HRT and COD removal over time. The HRT was set to 3 days and the slight difference in HRT between the two reactors is due to the slight difference in volumes. As can be seen from the figure, the average COD reduction was 30%, which is in line with the performance of the anaerobic treatment carried on at the full scale plant in Nijverdal, the Netherlands [34]. The raw wastewater contained some biodegradable compounds such as starch, polyvinyl alcohol, carboxy-methyl cellulose. However, dyes, detergents, dispersing and degreasing agents also contribute to the COD and are often recalcitrant to biological removal; the dyes, a mixture of vat, azo and reactive-dyes, are estimated to be present at concentrations not higher than 150 mg/l [34]. There was no distinguished peak in the visible range and the water was brownish, but there were blue fibers suspended in the water which might have adsorbed the pigments or were just released from the processed clothes.

60 50 40 30 20 10 0

0 COD - In

3

6 9 Cycles (1 cycle = 3 HRTs)

COD Out - A

COD Out - B

HRT (Days)

COD (mg/L)

10 9 8 7 6 5 4 3 2 1 0

2000 1800 1600 1400 1200 1000 800 600 400 200 0 12 HRT - A

HRT - B

Fig. 5. Concentration of COD in the inlet and effluent of the reactors and measured HRT during anaerobic treatment of raw textile wastewater.

1

2

4

6

Ozonation (min) TOC

3.2.2. Ozonation of biologically treated textile wastewater The effluent of the anaerobic reactors was collected and stored at +4 ◦ C. Once a sufficient volume was available, the effluent was ozonated in 4 batches, each characterized by a different ozone exposure. Fig. 6 clearly illustrates that a higher reduction of absorbance, both in the visible and UV range, was obtained as the exposure to ozone increased. In contrast, the reduction of COD and TOC was similar at the 4 different conditions tested. The reduction of absorbance in the UV range indicates that the ozone and the hydroxyl radicals generated from its decomposition can break down most of the aromatic molecules present in the bio-treated effluents; however, in this study such molecules were only degraded to smaller organic compounds and not mineralized, as shown by the fact that COD (final value 540 mg/l) and TOC (final value 197 mg/l) were only slightly reduced even at the longest ozone exposure (Fig. 6). One of the reasons for the low degree of mineralization obtained is the high organic content of the bio-treated effluent, 780 mg/l and 230 mg/l of COD and TOC, respectively. In addition, most of the organic content is due to compounds like carboxy-methyl cellulose, polyvinyl alcohol and detergents, which are mostly saturated chains and are rich in hydroxyl and amino groups, known radical scavengers [35]. These results are different from those obtained when applying ozone on the synthetic textile wastewater. In that case, a progressive increase in COD reduction was observed as the exposure to ozone increased. This is a further indication that real effluents contain organic molecules with radical scavenging properties, which make the treatment even more challenging.

57

COD

Abs 500 nm

Abs 254 nm

Fig. 6. Reduction of TOC, COD and absorbance after applying different ozone doses on the bio-treated textile effluent.

It should also be observed that the ozone fed into the reactor was not completely consumed, which can be explained by the fact that achievable equilibrium concentrations of ozone in water are in the mg/l range and the highest ozone dose applied in this study are close to the g/l [36]. In this experiment, the consumption of ozone did not increase as expected when the exposure time was increased from 2 to 4 and 6 min, as shown in Table 1. A longer residence time may improve the ozone consumption, and thus, the COD and TOC reduction. These results, in agreement with previous studies, show that ozonation can degrade organic compounds and open aromatic rings [37], but its ability to achieve complete mineralization is still a matter of discussion. The combined anaerobic–ozonation treatment achieved an overall 70% of reduction of COD; in particular, the treated effluent had COD and TOC of 540 mg/l and 200 mg/l, respectively. Increasing the ozone utilized in the treatment may improve COD and TOC reduction, but the energy consumption may become too high and make the process unfeasible [35]. Instead, the treatment can be completed by adding an additional process for example biological filters or reverse osmosis, as suggested also by other researchers in previous studies dealing with ozonation of textile wastewater [18,38]. 3.3. Toxicity tests The acute toxicity of the untreated and treated wastewater was evaluated through Microtox® and A. salina toxicity tests. The results are reported in Fig. 7 as EC20 , the concentration of the effluent (in %) that caused a response in 20% of the population after a predetermined exposure time (5 min for Microtox and 48 h for A. salina). Thus, the higher is the EC20 , the lower the toxicity of the tested water. As can be seen in the figure, Microtox (Fig. 7a) was typically more sensitive than Artemia (Fig. 7b), which detected almost no toxicity for the effluents containing 100 and 500 mg/l of RR, but the two tests show similar trend. The artificial textile wastewater was toxic and its toxicity increased as the concentration of RR increased. The evolution of the toxicity during the different treatment steps showed the same pattern for the wastewater containing 100 and 500 mg/l of RR. The effluent of the anaerobic treatment had higher toxicity than the untreated wastewater, but the ozonation could efficiently reduce the toxicity and a clear relation between

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M. Punzi et al. / Journal of Hazardous Materials 292 (2015) 52–60

RR 1000 mg/l RR 100 mg/l

RR 500 mg/l TE

RR 1000 mg/l RR 100 mg/l

80

80

60

60

EC20 (%)

100

EC20 (%)

100

40

40

20

20

0

0

a.

RR 500 mg/l TE

b.

Fig. 7. Summary of the calculated EC20 for (a.) microtox and (b.) A. salina toxicity tests of untreated and treated artificial and real textile wastewater. EC20 ≥ 100% = not toxic. TE = textile effluent. Error bars represent standard deviation.

exposure to ozone and toxicity reduction was observed. In contrast, the wastewater containing 1000 mg/l of the dye had higher toxicity than the bio-treated effluent, and according to Microtox the toxicity increased dramatically after 1 min of ozonation. Higher exposure to ozone resulted in a reduction of toxicity which was still higher than after the biological treatment alone. The test with Artemia (Fig. 7b) shows a different picture as almost no toxicity was measured already after the biological treatment. During biological treatment the UV absorbance was reduced by 35% and this change, together with the removal of organic compounds, is likely the reason for the lower toxicity of the biologically treated effluent toward Artemia. These results indicate that this toxicity test has lower sensitivity compared to Microtox and support previous findings [39]. The raw textile effluent received from a factory in the Netherlands was also rather toxic. The EC20 was 3.1 and 78.7 according to Microtox and the Artemia toxicity test, respectively. The combined treatment could quite efficiently reduce the toxicity. After 6 min of ozonation, the effluent was 20 times less toxic than the untreated one according to Microtox (Fig. 7a) and non-toxic according to Artemia (Fig. 7b). Despite the fact that the effluent has a high organic content even after 6 min of ozonation, which indicates the requirement for additional treatments, the fact that the toxicity is efficiently lowered shows that the combined anaerobic–ozonation process is a promising alternative for treatment of textile wastewater. Ames fluctuation test was used to determine mutagenic effects of the raw textile wastewater and the effluent after biological treatment and ozonation with different ozone exposures. Fig. 8 shows that mutagenicity was detected in the untreated and biologically treated effluent (p-value 5.407 e−10 and 0.027, respectively) as well as in the effluent after 1 min of ozonation (p-value < 2.2 e−16 ). Interestingly, the latter resulted in higher mutagenic effect than the biologically treated effluent (p-value 0.05). The effluent after longer ozonation, 2, 4 or 6 min, did not exhibit any mutagenic effect. No cytotoxicity was observed (data not shown). Our results support previous findings reporting that azo dyes and the aromatic amines produced as degradation products during the treatment of dyescontaminated water can be mutagenic and carcinogenic [3,40,41]. The fact that the mutagenicity is higher after 1 min of ozonation indicates that several compounds having alkylating functions are produced, since the strain used is particularly sensitive to such molecules. Magdeburg et al. [22] also detected mutagenic effects in ozonated secondary effluents using the strain YG7108. This

finding, together with the results of our study, indicates that ozonation may cause the formation of mutagenic compounds belonging to the family of alkylating agents. Thus, it is fundamental to monitor the toxicity of wastewater before and after ozonation and to optimize the treatment so that the right ozone exposure is applied to remove the mutagenic compounds. Interestingly, many studies report that ozonation is very efficient in removing color and improving the biodegradability of textile wastewater, which can thereafter be treated through activated sludge or other conventional biological processes [17,18,23]. However, as reported by de Souza et al. [19], there is a risk that the toxicity of the wastewater increases as a consequence of ozonation. A further problem is that to use ozone directly on raw wastewater is not economically feasible because the biodegradable fraction will be oxidized together with the recalcitrant compounds and this will

Fig. 8. Mutagenicity of untreated (U), biologically treated (B) and ozonated (O1: 1 min ozonation; O2: 2 min ozonation; O4: 4 min ozonation; O6: 6 min ozonation) textile wastewater compared to negative control (NC) and positive control (PC). The y-axis represents the arcsine-square root of the nominal number of revertants transformed to metric data. The data were plotted with R. * = p-value < 0.05 compared to negative control. # = p-value < 0.05 compared to biologically treated effluent.

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cause increased ozone consumption [20]. Our findings suggest that the use of ozonation as post-treatment after biological degradation, instead of pre-treatment, is a very attractive option for textile wastewater treatment even though less investigated [10,20].

4. Conclusions • The combined anaerobic–ozonation process was successful in removing more than 99% of the color, 85–90% of COD and toxicity from an artificial textile wastewater containing 100–1000 mg/l of the azo dye Remazol Red. • Similar results were achieved for treatment of a real textile effluent where in average 70% of COD reduction was obtained (3 days HRT during biological treatment followed by 6 min ozonation) and the final toxicity was 20 times lower than that of the raw wastewater. • The textile effluent, as well as the biologically treated effluent, was mutagenic. The mutagenicity increased after 1 min of ozonation, but was completely removed after longer exposure to ozone. • Our results strongly suggest that combined processes in which ozonation is used as short post-treatment to degrade recalcitrant compounds should be developed for textile wastewater. • Monitoring of mutagenicity and toxicity through tests that target different organisms is an important tool and should be used to complement conventional analysis, which focuses on removal of nutrients and in particular organic compounds.

Acknowledgments The authors wish to thank Dr. Daniel Stalter from Entox, University of Queensland (Brisbane, Australia) for the information and fruitful discussion regarding the Ames test as well as Dr. Martin Wagner and Andrea Misovic from the Department Aquatic Ecotoxicology, Goethe University (Frankfurt, Germany) for providing the strain YG7108 used in the Ames test. Marc van Coeverden is acknowledged for providing the textile wastewater and sharing precious information about the full scale treatment plant in the Netherlands. The authors gratefully acknowledge the Swedish International Development Cooperation Agency (SIDA) for financial support.

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