The effect of combined cometabolism and gamma irradiation treatment on the biodegradability of diclofenac and sulfamethoxazole

Journal Pre-proof The effect of combined cometabolism and gamma irradiation treatment on the biodegradability of diclofenac and sulfamethoxazole Anikó Bezsenyi, Gyuri Sági, Magdolna Makó, György Palkó, Tünde Tóth, László Wojnárovits, Erzsébet Takács PII:

S0969-806X(19)31117-X

DOI:

https://doi.org/10.1016/j.radphyschem.2019.108642

Reference:

RPC 108642

To appear in:

Radiation Physics and Chemistry

Received Date: 27 August 2019 Revised Date:

4 December 2019

Accepted Date: 6 December 2019

Please cite this article as: Bezsenyi, Anikó., Sági, G., Makó, M., Palkó, Gyö., Tóth, Tü., Wojnárovits, Láó., Takács, Erzsé., The effect of combined cometabolism and gamma irradiation treatment on the biodegradability of diclofenac and sulfamethoxazole, Radiation Physics and Chemistry (2020), doi: https://doi.org/10.1016/j.radphyschem.2019.108642. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Author Contribution Statement

Anikó Bezsenyi carried out the measurements, evaluated the results. Gyuri Sági took part in conceptualization. Magdolna Makó consulted on the evaluation of the results. György Palkó consulted on the evaluation of the results. Tünde Tóth took part in the evaluation. László Wojnárovits took part in evaluation of the results and writing and reviewing the manuscript. Erzsébet Takács took part in evaluation of the results and writing and reviewing the manuscript.

The effect of combined cometabolism and gamma irradiation treatment on the biodegradability of diclofenac and sulfamethoxazole Anikó Bezsenyi1,2, Gyuri Sági3, Magdolna Makó1, György Palkó1, Tünde Tóth3, László Wojnárovits3, Erzsébet Takács3 1

Budapest Sewage Works Pte Ltd., H-1087 Asztalos Sándor str. 4, Budapest, Hungary

2

Óbuda University, H-1034 Bécsi út 96b, Budapest, Hungary

3

Institute for Energy Security and Environmental Safety, Centre for Energy Research, H-

1121, Konkoly-Thege Miklós út 29-33, Budapest, Hungary

Corresponding author: Erzsebet Takacs, E-mail: [email protected] Abstract Diclofenac and sulfamethoxazole are poorly degradable when using activated sludge or biofilm biomass in biological wastewater treatment. However, a biological phenomenon called cometabolism can improve the removal efficiency, especially in activated sludge treatment. In the case of cometabolism an easily degradable substrate is added to the wastewater under treatment. Such easily degradable molecules (e.g. methanol, acetic acid, ethylene glycol) also form in the anaerobic wastewater treatment steps and are utilized as substrates. The rate of oxidative biodegradation of pharmaceuticals (measured by using oxygen uptake rate) was shown to greatly increase in the presence of easily degradable substrates in activated sludge treatment. However, in biofilm treatment the rate of cometabolism remained low, because biofim biomass has much narrower bacterial diversity than activated sludge. The oxidation rate on biofilm was found to increase considerably by using an advanced oxidation process, ionizing radiation treatment, before cometabolism. This combined treatment, irradiation and cometabolism is recommended for the degradation of recalcitrant organic compounds on biofilm.

Highlights •

In activated sludge treatment cometabolism increases the biodegradation rate 1

•

The efficiency of cometabolism depends on the type of co-substrate

•

The biodegradability of SMX and DCF was enhanced by irradiation

•

The efficiency of the cometabolism can be enhanced by 0.5 kGy irradiation

Keywords Biodegradability; diclofenac; sulfamethoxazole; cometabolism, ionizing radiation

1. Introduction Hardly biodegradable micro-pollutants (xenobiotics), such as pharmaceuticals, often pass through the conventional wastewater treatment systems in unchanged form and enter the surface waters (Joss et al., 2006; Lin et al., 2009; Sim et al., 2010; Jelić et al., 2012). These micro-pollutants may have short-term and long-term toxicity, depending on their persistence, endocrine disrupting effects or induce the formation of (antibiotics) resistant pathogens (Fent et al., 2006; Pruden et al., 2006). The decomposition of xenobiotics can be enhanced by the stimulation of natural processes. One of these methods is cometabolism (co-oxidation), where a complex non-biodegradable molecule (co-substrate) can be degraded in the presence of an easy-to-degrade substrate (growth substrate, nutrient molecule or forming substrate). The products formed during cometabolism are not necessarily the same as those metabolically formed (Evangelista et al., 2010). There may be a significant difference between cometabolic intermediates, too, depending on the pathway of cometabolism. For sulphamethoxazole in metabolic pathway 3amino-5-methylisoxazole and 4-hydroxysulfamethoxazole form, in the latter the p-amino group is replaced by a hydroxyl group. In cometabolic pathway N-acetylsulfamethoxazole and sulfanilic acid form. The latter, together with 3-amino-5-methylisoxazole, indicates hydrolytic cleavage of the sulfonamide group (Müller et al., 2013; Majewsky et al., 2014). Simple, easily biodegradable organic compounds are available in wastewater, as they form at the sewage treatment plants. These compounds form by hydrolysis and microbiological fermentation from complex organic compounds. Microbiological processes produce methanol, ethanol, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, lactic acid, ethylene glycol, etc. in the wastewater. Depending on its composition and diversity the microbiota can successfully utilize the above-mentioned alcohols and organic 2

acids. Acids and alcohols as external carbon sources are frequently used for feeding the bacterial community. There are several types of commercially available external carbon sources (such as methanol, ethanol, acetic acid, sodium acetate, glucose), but they are generally expensive to use. For this reason, many industrial by-products are utilized in practice as alternative external carbon sources. Distillery by-products, such as fusel oil, syrup, reject water, brewery spent grain extract, corn silage derivative, yeast and whey. Spent sulphite liquor which is the main by-product of acid sulphite pulping and the glycerol phase from biodiesel production are also usable (Monteith et al., 1980; Skrinde and Bhagat, 1982; Czerwionka et al., 2012). The efficiency of cometabolism may also vary with the chemical nature of the easily degradable organic substrate added. Several technological innovations are under development in order to eliminate xenobiotics. Among these, advanced oxidation processes (AOPs) are the most promising technologies due to their highly effective xenobiotic removal performance (Huber et al., 2003; Neafsey et al., 2010; Pérez-Moya et al., 2010; Moreira et al., 2016; Tokumura et al., 2016; Zhang et al., 2016). During AOPs, organic molecules are oxidized in their reactions with hydroxyl radicals (•OH) (von Sonntag, 2008; Krzemińska et al., 2015). This process ultimately leads to the formation

of

simpler

organic

compounds

that

are

biodegradable

(available

to

microorganisms). The raw wastewater contains also harmless organic matter (e.g., humic and amino acids) and inorganic ions (e.g. carbonate and bicarbonate) in large quantities. These act as scavengers of hydroxyl radicals (Ribeiro et al., 2015). Therefore, AOPs are recommended as post-treatment, following the biological purification stage (Oller et al., 2011; Cesaro et al., 2013). The fate of pharmaceuticals in wastewater treatment plants (WWTPs) depends on the operating conditions including oxygen supply (Zwiener et al., 2002), organic loading, or residence time (Pérez et al., 2006), but also depends on the metabolic type of wastewater bacteria. The biomass of each wastewater treatment plant has a special composition, so its removal efficiency and biotransformation capability for micro-pollutants are also different (Helbling et al. 2012; Xia et al. 2012). Particularly in the removal of polar micro-particles, the microbiota of the activated sludge plays a significant role, and the physicochemical elimination procedures are less effective (Reemtsma and Jekel, 2006). The purpose of this work was to improve the removal efficiency of the biological treatment for two non-biodegradable pharmaceuticals, sulfamethoxazole (SMX, antibiotic) and diclofenac (DCF, analgesic, non-steroidal anti-inflammatory drug), applied as model 3

compounds. In a series of multi-stage experiments, two processes, AOP and cometabolism, as well as their combination were tested. Both compounds are present in WWTP’s effluents at concentrations ≤1.0 µg/L (Luo et al. 2014) and in the effluent they are emitted to the surface waters and remain there for a long time. The concentration of diclofenac decreased only by 42% after 57 days by aerobic bacterial culture. By biodegradation the concentration of sulfamethoxazole decreased by 33% after 58 days under aerobic conditions (Poirier-Larabie et al. 2016). In fact, SMX can persist in the environment for more than a year (Dirany et al. 2010). The phenomenon of cometabolism was studied by the addition of simple substrates (methanol, acetic acid, ethylene glycol), and γ radiation was used as an advanced oxidation process at three different doses. The change in the biodegradability of the pharmaceuticals was followed by respiratory tests on wastewater activated sludge and biofilm cultures.

2. Experimental 2.1. Chemicals, materials, pre-treatment and irradiation procedure The pharmaceutical compounds, SMX and DCF, were purchased from Sigma Aldrich, while all the other chemicals from VWR. The activated sludge and the heterotrophic biofilm biomass culture were collected from the South-Pest Wastewater Treatment Plant's biofilter unit (belonging to the Budapest Sewage Works) and was used for testing freshly without storage. As an advanced oxidation method, γ irradiation was used with 0.5, 1.0 and 2.0 kGy doses. Irradiations were performed by a

60

Co (1.85 PBq) SSL-01 panoramic type gamma source,

with 9.4 kGy h-1 dose rate. During the treatment the samples containing pharmaceuticals in 0.1 mmol L-1 concentration were continuously aerated and kept at room temperature. After the irradiation procedure 0.25 g L-1 manganese(IV) oxide was added to the samples to eliminate the considerable H2O2 content generated during the treatment. H2O2 elimination was carried out at pH 10.0 (stirred at 20 °C for 10 minutes). After filtration with 0.22 µm regenerated cellulose membrane filter the solutions were neutralized. By removing hydrogen peroxide, we can eliminate its toxic effect and prevent distortion in respiration tests data caused by elevated oxygen levels.

4

2.2. Experimental methods 2.2.1. Organic matter content This parameter was expressed as chemical oxygen demand (COD) determined according to ISO 6060:1989. In this technique the organic matter is oxidized by sulfuric acid and a known excess of potassium dichromate (K2Cr2O7) at high temperature (2 h, 170°C). The remaining unreduced K2Cr2O7 is titrated with ferrous ammonium sulfate. 2.2.2. Activated sludge and biofilm respiration inhibition test The respiratory tests were carried out with two types of wastewater bacterial cultures: activated sludge and biofilm biomass. They come from two different technological units of the same wastewater plant. Methanol is used as an external carbon source to maintain the biofilm in the WWTP. This fundamentally affects the species composition of biomass. Therefore, the cometabolic efficiency of the two bio-cultures may also be highly different. The biofilm culture and the activated sludge were treated in the same way. Biomass of the biofilm was collected during routine technological biofilm washing. Biochemical biomass is present in concentrated form in non-chemical wash water. Thus, the biofilm is processed in a form equivalent to activated sludge. The biomass used was washed several times. The respiration intensity is expressed in oxygen uptake rate (OUR, mg O2 L–1 h–1). OUR is suitable for monitoring and characterize the activated sludge and biofilm systems. The experiments were conducted and evaluated on the basis of ISO 8192:1986 standard. The dissolved oxygen uptake is increasing in the presence of biodegradable compounds, and it is reduced in the presence of toxic substances. The cometabolic effect, in which a nonbiodegradable molecule (co-substrate) is degraded by the microorganism when a nutrient is present, is achieved by the addition of simple organic substrates. Oxygen consumption of the culture was measured in 300 mL Karlsruher bottles with an FDO® 925 oxygen sensor at 20 °C. In order to obtain comparable results, the same load levels were applied in each bottle: 25 mg dry matter of biomass (activated sludge or biofilm biomass), 150 mg O2 L–1 COD-equivalent substrate, filled up with tap water to give 150 mL volume. 150 mL of 0.1 mmol irradiated (with different doses) pharmaceutical solution was added to the bottles to give a final concentration of 0.05 mM. The blank test mixtures contained only biomass and substrates, while the control mixtures were obtained by mixing irradiated pharmaceutical samples (with different doses) and substrates without biomass. Each 5

measurement was performed in triplicate. The two pharmaceutical compounds, DCF and SMX, were tested separately in triplicate assays. All data used to evaluate the results were corrected for endogenous OUR values (absence of pharmaceutical and substrate) and control values (pharmaceutical and substrate without biomass). 3. Results 3.1. Assessing the cometabolic potential The aim of these experiments was to select a poorly, a medium and a highly effective substrate and the appropriate bacterial culture and to test how cometabolism is working under these conditions. The effective substrate increases the ability of the bacterial culture to decompose the pharmaceutical compounds. The efficiency of cometabolism, which is working well in the case of SMX degradation by heterotrophic organisms (Fernandez-Fontaina et al., 2016), may vary with the type and amount of easily degradable organic substrate added. The following ten substrates were tested: methanol (Meth), ethanol (Eth), formic acid (Form Ac), acetic acid (Ac Ac), propionic acid (Prop Ac), butyric acid (But Ac), valeric acid (Val Ac), caproic acid (Cap Ac) (or hexanoic), lactic acid (Lac Ac), and ethylene glycol (Eth Gly). With the exception of the latter, all the compounds are also produced by the microorganisms during normal operation in the anaerobic stages of wastewater treatment (Bitton, 2005). However, external carbon source is often used in wastewater treatment technology for proper nitrogen removal, such as acetic acid, formic acid, methanol, ethanol, glucose or ethylene glycol (Du et al., 2017, Li et al., 2017, Liwarska-Bizukojć et al., 2018). These compounds as industrial by-products are available at reasonable price. The applicability of organic materials as an external carbon source was also an important consideration in the selection of the substrates for the further studies. Fig. 1 shows the effect of the substrate on OUR. The activated sludge was taken from three different basins at three different times (AS1, AS2, AS3) to test the data comparability, and no considerable differences were found. Activated sludge can utilize all substrates to a greater or lesser extent. The lowest OUR values were measured in the presence of methanol, formic acid and ethylene glycol. However, biofilm (which has a relatively narrow bacteria diversity) can only use methanol, ethanol, ethylene glycol, formic acid and acetic acid. In biofilm the socalled methylotrophic bacteria, which are dominant in its biomass, are adapted to the one carbon atom containing methanol. These microorganisms have the ability to metabolize 6

beside methanol, also methane, formic acid as their only carbon and energy source. Facultative methanotrophs could also utilize two-carbon atom containing compounds (e.g., acetate and ethanol) (Chistoserdova et al., 2009, Dedysh and Dunfield, 2011). Alcoholoxidizing enzyme of the facultative methylotroph has broad specificity for primary alcohols and also capable of oxidizing secondary alcohols. A potential substrate for methanol-grown alcohol dehydrogenase is ethylene glycol (Bellion and Wu, 1978).

5

AS1 AS2 AS3 Biofilm

OUR, mg O2 L-1 h-1

4

3

2

1

0

Meth Eth

Val Ac Lac Ac Form Ac Prop Ac Ac Ac Cap Ac Eth Gly But Ac

Fig. 1. The oxygen uptake rate (OUR) of activated sludge from three different cultures (AS1, AS2, AS3) and biofilm biomass, using various substrates.

7

Relative OUR, (Cometabolism/substrate respiration)

24

SMX

20

AS Biofilm

16 12 8 4 0

Meth Eth

Val Ac Lac Ac Form Ac Prop Ac Ac Ac Cap Ac Eth Gly But Ac

Fig. 2. Relative oxygen uptake rate (OUR) of activated sludge and biofilm biomass in the presence of sulfametoxazole and various substrates. The thick black line drawn at value 1 indicates no effective cometabolism, or inhibition of oxidation (below 1) with co-substrate (pharmaceutical) dosing. Fig. 2 shows the relative OUR data measured in solutions containing both substrates and SMX (co-substrate), and the same is shown in Fig. 3 for DCF. For the evaluation a special form of OUR (relative OUR) was used, which is the ratio of cometabolic respiration to substrate respiration. Cometabolic respiration is the oxygen consumption measured in the presence of the pharmaceutical molecule (co-substrate) and the substrate. Higher values of the ratio show more effective cometabolism. The thick black line drawn at value 1 indicates no effective cometabolism, or inhibition of oxidation (below 1) with co-substrate dosing.

8

Relative OUR, (Cometabolism/substrate respiration)

24

DCF

22 20 18 16 14

AS Biofilm

12 10 8 6 4 2 0

Meth Eth

Val Ac Lac Ac Form Ac Prop Ac Ac Ac Cap Ac Eth Gly But Ac

Fig. 3. Relative oxygen uptake rate (OUR) of activated sludge and biofilm biomass in the presence of diclofenac and various substrates. The thick black line drawn at value 1 indicates no effective cometabolism, or inhibition of oxidation (below 1) with co-substrate dosing. For SMX, the relative OUR ratio (cometabolism/substrate respiration) increased in the following order: formic acid (2.41), propionic acid (2.87), methanol (3.29), lactic acid (3.39), ethylene glycol (3.68), ethanol (4.87), caproic acid (4.90), butyric acid (7.01), acetic acid (9.95), valeric acid (10.75). For DCF in increasing order: methanol (1.29), ethanol (1.82), butyric acid (1.97), caproic acid (2.05), lactic acid (2.54), valeric acid (2.67), propionic acid (4.31), ethylene glycol (6.43), acetic acid (7.35), formic acid (18.22). The biofilm experiments showed only cometabolic effects with DCF. With the addition of ethanol, methanol, butyric acid, caproic acid, the special OUR ratio ranged from 1.39 to 1.75. Only ethylene glycol produced a significant change, increasing the respiratory rate by 5.15-fold. It is clear from Fig. 2 and Fig. 3 that the microorganism community in the activated sludge is more successful in the cometabolic process than the community in the biofilm. Biofilm works under special conditions. Non-aerated biomass, fed with nitrate rich wastewater and methanol, is characterized by a less diverse community. The wastewater that feeds the activated sludge has a composition of organic matter and aerated, non-aerated sections alternate in the basins. As a result, a species-rich and functionally diverse community can develop. 9

Since activated sludge is efficient in cometabolism alone, the effect of irradiation treatment is further tested on the biofilm, as the efforts increasing the efficiency in this case may be more spectacular. For the further work we selected a weak, a medium, and a highly effective substrate that worked similarly for both pharmaceuticals: methanol, ethylene glycol and acetic acid. The OUR data for the selected three substances are summarized in Fig.4.

b

Relative OUR, Cometabolism/substrate respiration

SMX

Methanol Ethylene glycol Acetic acid

8

6

4

2

0

Relative OUR, Cometabolism/substrate respiration

a 10

10

DCF

Methanol Ethylene glycol Acetic acid

8

6

4

2

0

Activated sludge

Biofilm

Activated sludge

Biofilm

Fig. 4. Relative OUR values for SMX (a) and DCF (b) with the three selected substrates using activated sludge and biofilm biomass. During normal operation in the anaerobic stages of wastewater treatment in largest amount acetic acid is formed from all the substrates tested. As the results show it strongly contributes to cometabolism.

3.2. Combination of irradiation and cometabolism

10

We continued the experiments with biofilm biomass, because the activated sludge alone showed effective cometabolism as mentioned above. As Fig. 5 shows the untreated pharmaceuticals were not eliminated by the biofilm bacterial culture (0 mg L-1 h-1, i.e. no metabolic activity). However, in the irradiated samples bioavailability of both compounds was observed and the effect was enhanced with the increasing dose. At 0.5, 1.0 and 2.0 kGy absorbed doses for SMX solutions 0.16, 0.27 and 0.35 mg L-1 h-1, while for DCF 0.01, 0.14 and 0.31 mg L-1 h-1 oxygen consumption rates were measured, respectively. The chemical oxygen demand (COD) (Fig. 5, inset) decreased due to the irradiation treatment from 54 (not irradiated), to 52, 37 and 30 mg L-1 at 0.5, 1.0 and 2.0 kGy, respectively, for SMX, and from 48 to 40, 39 and 34 mg L-1 in the case of DCF (Fig. 5 inset). This indicates radiation induced degradation of the molecules.

1.2

0.8

COD, mg dm-3

OUR, mg O2 L-1 h-1

1.0

SMX DCF

SMX DCF

55 50 45 40 35 30

0.6

0.0

0.5

1.0

2.0

Dose, kGy

0.4 0.2 0.0 0

0.5

1.0 Dose, kGy

2.0

Fig. 5. The effect of irradiation on oxygen uptake rate and chemical oxygen demand values (inset) of two pharmaceutical compounds (SMX and DFC).

11

a

4.19

4

4.1 3.71

3.59

3 2

2.13

2.35

2.24

2.04

1 0

0

0.5

1

2

OUR Increment, mg O2 L-1 h-1

OUR Increment, mg O2 L-1 h-1

SMX, methanol DCF, methanol

5

SMX, acetic acid DCF, acetic acid

5 4 3 2 1 0.05

0

0.47 0.53

0.33

0

0.5

OUR Increment, mg O2 L-1 h-1

Dose, kGy 5

b

0.57 0.3

0.54 0.16

1

2

Dose, kGy SMX, ethylene glycol c DCF, ethylene glycol

4 3

2.05

2 1 0

1.71

1.6 0.77

0.72 0.68

0

0.5

1

1.45 1

2

Dose, kGy

Fig. 6. The effect of irradiation on oxygen uptake rate (OUR) increment in the presence of pharmaceuticals (SMX and DCF) and substrate (methanol, acetic acid and ethylene glycol) with the values measured for the substrate subtracted. The post-irradiation OUR increment values in solutions containing co-substrates are shown in Fig. 6. The values measured for the substrates were subtracted. The OUR increment values of the figure show the effect of both cometabolism and irradiation. The higher the OUR values, the more pronounced the effect of irradiation on cometabolism. The combination of the two methods revealed that the efficiency of the cometabolism can be enhanced by 0.5 kGy dose. At this absorbed dose, the increase in co-oxidative respiration for SMX was 0.47 with acetic acid, 1.60 with ethylene glycol and 2.24 mg L-1 h-1 with methanol. For DCF the increase was 0.53 with acetic acid, 2.05 with ethylene glycol and 4.19 mg L-1 h-1 with methanol. No improvement was observed at higher doses (Fig. 6). This can be explained by the considerable depletion in the organic material (Fig. 5, inset) above 0.5 kGy. If acetic acid was added to the SMX solution irradiated by 0.5 kGy, the OUR was almost ten times higher than without irradiation (0.05 to 0.47 mg L-1 h-1). Ethylene glycol mixed with 12

irradiated DCF solution resulted in a triple respiration intensity relative to the untreated cometabolic effect (0.68 to 2.05 mg L-1 h-1). The effect of treatment on the methanol as substrate is barely detectable (SMX: from 2.13 to 2.24 mg L-1 h-1, DCF: from 3.59 to 4.19 mg L-1 h-1). Based on these results, significant improvement can be achieved in the case of the inherently weak substrate. In the case of the highly effective substrate, methanol, irradiation does not result in significant improvement in the efficiency.

5. Summary Using activated sludge and biofilm biomass the biodegradation of sulfamethoxazole and diclofenac was stimulated separately by the two tested methods: cometabolism and advanced oxidation treatment (by high-energy ionizing radiation). From the 10 organic molecules tested, methanol, acetic acid and ethylene glycol were selected as substrates for cometabolism. Both pharmaceuticals showed cometabolic effect, but it was really significant for activated sludge treatment. One of the highly effective substrates was acetic acid, a compound which is able to increase the relative respiration intensity (OUR) by ~10 times during the degradation of sulfamethoxazole. In the case of biofilm biomass with narrow bacterial diversity originally fed with methanol, the substrates were much less effective. They contributed only to the degradation of diclofenac (the ratios were: 1.47 for methanol, 5.15 for ethylene glycol, 1.06 for acetic acid). Further tests were performed on biofilm biomass, as activated sludge alone was sufficiently effective in cometabolism without any additional treatment. The biodegradability of sulfamethoxazole and diclofenac in 0.1 mmol L-1 aqueous solutions was enhanced by irradiation. A further increase was observed when the two methods, cometabolism and irradiation were combined. The effect of irradiation with 0.5 kGy dose was significant. No further improvement was observed at higher doses most possibly due to the depletion of organic molecules by irradiation. In samples irradiated by 0.5 kGy acetic acid added to the sulfamethoxazole almost ten times, the ethylene glycol mixed with diclofenac three times increased the respiration intensity as compared to the values obtained without irradiation. The use of advanced oxidation techniques combined with cometabolic technology based on the intensification of natural processes can be a successful and cost effective for the removal 13

of persistent micro-pollutants. The two methods can be combined at the end of the wastewater treatment technology as a fourth stage. Acknowledgement This work was financially for supported by the International Atomic Energy Agency (IAEA) (Coordinated Research Project F23034, Contract No: 23754) and by the National Office for Research and Development, Hungarian-Chinese Industrial Research and Development Cooperation Project (Contract No. 2017-2.3.6.-TÉT-CN-2018-00003).

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: