Aerobic inhibition assessment for anaerobic treatment ...

Aerobic inhibition assessment for anaerobic treatment effluent of antibiotic production wastewater Zeynep Cetecioglu

Environmental Science and Pollution Research ISSN 0944-1344 Environ Sci Pollut Res DOI 10.1007/s11356-013-2243-3

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Author's personal copy Environ Sci Pollut Res DOI 10.1007/s11356-013-2243-3

RESEARCH ARTICLE

Aerobic inhibition assessment for anaerobic treatment effluent of antibiotic production wastewater Zeynep Cetecioglu

Received: 20 August 2013 / Accepted: 11 October 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Biological treatment of antibiotic production effluents is an economical approach; however, there are still difficulties to overcome because of the recalcitrant characteristics of these compounds to biodegradation. This study aims to reveal that anaerobic treatment technology can be an option as pretreatment before the activated sludge system treatment to treat antibiotic production effluents. The ISO 8192 method was chosen to test the inhibitory effect of raw and treated antibiotic production effluents in this work. Inhibition tests, which were applied according to ISO 8192, highlighted that the anaerobic treatment effluent is less inhibitory than antibiotic production effluent for activated sludge system. Early EC50 concentrations (30-min values) of raw and treated wastewaters were lower than 180-min values. Also, triple effects (sulfamethoxazole–erythromycin– tetracycline) of antibiotics are more toxic than dual effects (sulfamethoxazole–tetracycline). In light of the experimental results obtained and their evaluation, it can be concluded that anaerobic digestion can be applied as a biological pretreatment method for pharmaceutical industry wastewater including antibiotic mixtures prior to aerobic treatment. Keywords Anaerobic effluent . ISO 8192 . Antibiotic . Activated sludge . Inhibition

Introduction Pharmaceuticals are one of the major micro-pollutants in recent years, and antibiotics are among these compounds. Every year, Responsible editor: Gerald Thouand Z. Cetecioglu (*) Environmental Engineering Department, Civil Engineering Faculty, Istanbul Technical University, 34469, Maslak Istanbul, Turkey e-mail: [email protected]

approximately 100,000–200,000 tonnes of antibiotics are produced and consumed in the world (Wise 2002). Wastewater including antibiotics generally comes from houses, hospitals, pharmaceutical industries, and stock farms. This kind of wastewater cannot be treated in conventional treatment plants and discharged directly to receiving water bodies such as rivers, lakes, and seas. The total concentration of these active compounds and their metabolites/transformation products in water bodies increases gradually, and it causes pathogenic organisms to gain antibiotic resistance (Kümmerer 2009a, b). Sulfamethoxazole, erythromycin, and tetracycline are frequently used antibiotics in the realm of medicine. The action mechanisms of all these antibiotics are bacteriostatic. Sulfamethoxasole is a sulfonamide bacteriostatic antibiotic that is used to treat urinary tract infections. It inhibits the multiplication of bacteria since they are competitive inhibitors of p-amino benzoic acid in the folic acid metabolism cycle. Erythromycin is one of the macrolide antibiotics which directly affect protein synthesis by preventing bacterial growth by binding to the 23S ribosomal RNA. Macrolide antibiotics have a relatively broad-spectrum effect, which inhibits both Gram-positive and Gram-negative bacteria (Sweetman 2009). In the literature, most of the studies have focused on the fate and removal of these compounds in wastewater treatment plants (Drillia et al. 2005; Radjenović et al. 2009; Yang et al. 2011; Cetecioglu et al. 2013a). Tetracycline is generally used for the treatment of respiratory tract infections and has a reversible inhibitory effect. It is a broad-spectrum active compound, which inhibits bacterial protein synthesis by binding to the 30S ribosomal subunit to prevent the association of the aminoacyl-tRNA to the ribosomal acceptor-A site (Chopra and Roberts 2001). In the literature, most of the studies have investigated the fate and removal of tetracycline not only in domestic wastewater plants (Miao et al. 2004; Prado et al. 2010) but also in anaerobic digesters (Cetecioglu et al. 2013b).

Author's personal copy Environ Sci Pollut Res

Previous studies in the literature have generally focused on the biological treatability of these compounds under different electron-accepting conditions (Deng et al. 2012; Yang et al. 2012; Cetecioglu et al. 2013a, b), acute and chronic effects on the mixed microbial culture (Cetecioglu et al. 2012; Pala-Ozkok et al. 2013), and their fate in both wastewater treatment plants and receiving water bodies such as lakes, rivers, and seas (Batt and Aga 2005; Batt et al. 2006; Segura et al. 2009; Zhang et al. 2013). However, there is a gap in the literature with regard to a classical biological treatment strategy for pharmaceutical industry wastewater including antibiotics. Since this kind of industrial wastewater is considered as high-strength wastewater and contains high level of inhibitory compounds, they are suitable for anaerobic treatment. In fact, a few studies revealed that anaerobic treatment is suitable for wastewater produced during antibiotic production (Drillia et al. 2005; Amin et al. 2006; Shimada et al. 2008; Fountoulakis et al. 2008; Cetecioglu 2011, 2013b); however, there is no study investigating aerobic treatment as a second step of the biological treatment. Also, the effect of anaerobic treatment effluents coming from pharmaceutical industry wastewater on activated sludge systems and the ecosystem has been unknown. The activated sludge biomass is a heterogenous complex community in equilibrium that gives the flexibility to operate the system in certain limits. However, inhibitory compounds affect the system negatively, and they may cause lower treatment efficiency and even failure of the system (Gutiérrez et al. 2002). Two different experimental approaches as shortterm and long-term tests are widely used to determine the inhibitory effects of selected chemicals. In the short-term experiment, the biomass, which is not previously exposed to the selected inhibitory compounds, is used as indicator to measure inhibition effects such as substrate utilization rate, oxygen consumption/respiration rate, microbial growth rate, enzymatic activity, bacterial luminescence, metabolic heat production, and biogas generation rate. The best known inhibition tests for aerobic microorganisms are based on the respirometric approach (Ricco et al. 2004). Standardized respirometric tests such as ISO 8192 and OECD 209 have been established as an effective method for assessing the inhibitory effect and/or toxic level of the selected compounds on activated sludge biomass (Gutiérrez et al. 2002; Dalzell et al. 2002; Gendig et al. 2003; Arslan-Alaton and Caglayan 2006; Prado et al. 2010; Diaz et al. 2012). The main goal of this study is to evaluate whether pharmaceutical industry wastewaters generated from antibiotic production are suitable to be treated by an anaerobic–aerobic biological treatment system. For this context, two different kinds of wastewater were assessed in the aerobic inhibition test based on respirometric approach (ISO 8192). Firstly, real antibiotic concentrations mimicking influent of lab-scale anaerobic reactors were fed to the aerobic

batch systems. Secondly, effluent of lab-scale anaerobic reactors was fed to the aerobic batch system to reveal the behavior of aerobic culture within the classical anaerobic– aerobic biological treatment system. This approach was used to find out whether (1) the effluent of anaerobic treatment including the potential unknown antibiotic transformation product is more toxic than the raw substances and (2) anaerobic–aerobic biological treatment strategy is useful for antibiotic production wastewater.

Materials and methods The experimental approach The experiments were essentially designed to evaluate the acute inhibitory impact of anaerobic treatment influent and effluent of pharmaceutical industry wastewater including antibiotics under aerobic condition. The influents and effluents from two anaerobic sequencing batch reactors (ASBRs) run in a daily “fill-and-draw” mode using a synthetic substrate mixture including volatile fatty acids, glucose, and starch with two different combinations of sulfamethoxazole, erythromycin, and tetracycline were assessed with activated sludge inhibition test (ISO8192 1999). The raw and treated wastewaters from different operation phases were fed to the acute respirometric inhibition test set with different concentrations to determine the half lethal concentration (EC50) for each wastewater sample. Operation of anaerobic sequencing batch reactors Two ASBRs with 1.25 L active volume were set up and operated at 35 °C. The reactors were operated with a 24h cycle consisting of filling (10 min), reacting (23 h), settling (45 min), and decanting (5 min). The reactors were mixed continuously using a magnetic stirrer at 90 rpm. The total chemical oxygen demand (COD) of the synthetic substrate used for the reactors was adjusted to 2,500 mg/L; the substrate was mainly composed of starch and glucose: starch, 1,160 mg COD/L; glucose, 750 mg COD/L; acetate, 135 mg/L; butyrate, 183 mg/L; and propionate, 272 mg COD/L. Trace element solution adapted from a previous study(Amin et al. 2006) (FeCl2.4H2O, 2 mg/L; CoCl2.6H2O, 2 mg/L; MnCl2, 0.32 mg/L; CuCl2, 0.024 mg/L; ZnCl2, 0.05 mg/L; H3BO3, 0.05 mg/L; (NH4)Mo7O24.4H2O, 0.09 mg/L; Na2SeO3, 0.068 mg/L; NiCl2.6H2O, 0.05 mg/L; EDTA, 1 mg/L; resazurine, 0.5 mg/L; and HCl (36 %) 0.001 mL mg/L) and vitamin (4-aminobenzoic acid, 0.04 mg/L; D (+)-biotin, 0.01 mg/L; nicotinic acid, 0.1 mg/L; calcium D (+)pantothenate, 0.05 mg/L; pyroxidine dihydrochloride, 0.15 mg/L; thiamine, 0.1 mg/L in NaP buffer (10 mM, pH 7.1), and 0.05 mg/L B12) solutions were added to the


wastewater. Antibiotic mixture combinations (ST reactor: sulfamethoxazole–tetracycline combination; ETS reactor: erythromycin–tetracycline–sulfamethoxazole combination) are given in Table 1 for each operational phase in each reactor. Each phase was operated for 30 days. The solid retention time was 30 days throughout the study for both ASBRs and was calculated based on volatile suspended solid (VSS) loss in the effluent and removed during sampling of the excess sludge. The pH of the reactors at the start of each cycle varied from 6.8 to 7.2 mainly due to the alkalinity level of around 1,000 mg/L CaCO3 for sustaining the operation stability of the anaerobic reactors. Analytical methods Methane content in the biogas and VFA concentrations were measured using a gas chromatograph (Perichrom, France and Agilent Technologies 6890 N, USA, respectively). Suspended solids (SS), volatile suspended solids, total suspended solids (TS), total volatile suspended solids (TVS), and soluble COD were determined according to standard methods (APHA 2005). Antibiotic concentrations were determined in the raw and treated wastewaters. Sulfamethoxazole and tetracycline measurements were carried out according to Karci and Balcioglu (2009). Erythromycin was measured by the protocol proposed previously by Holzgrabe and Deubel (2007). Activated sludge inhibition test Aerobic inhibition tests for raw influent wastewater using the antibiotic concentrations given in Table 1 and also for effluents of the two ASBRs (ST and ETS reactors) were conducted in accordance with the ISO 8192 procedure (ISO8192 1999). This procedure describes the way to determine the inhibition level of a test substance such as a toxic material or wastewater including toxic/inhibitory compounds on the oxygen uptake rate of the aerobic heterotrophic complex biomass in the presence of a defined biodegradable substrate. All the experiments were run under 22±2 °C and pH was adjusted to 7.5±0.5. The activated sludge used in the test was obtained from the aeration tank of a sewage treatment plant and fed with synthetic municipal wastewater as described in ISO 8192 (ISO8192 1999): peptone, 16 g/L; meat extract, 11 g/L; urea, 3 g/L; NaCl, 0.7 g/L; CaCl2.2H2O, 0.4 g/L; MgSO4.7H2O, O.2 g/L; and K2HPO4, 2.8 g/L. Two different sets were conducted: (1) synthetic wastewater including raw antibiotics, which were fed into the ASBRs, and (2) effluents of ASBRs. The active volume of the test selected was 250 mL. The incubation time according to the ISO 8192 guidelines (ISO8192 1999) was 30 and 180 min. The activated sludge concentration in the batch reactors was 3,000 mg/L, and the F/M ratio was 0.16 mg COD/mg MLVSS. During the experiment for each test substance as raw and treated wastewater,

one control batch reactor (F B) to evaluate oxygen uptake of the biomass without test substances and another control batch reactor (F BC) to evaluate physicochemical oxidation of the test substances were run. For the test substance, different dilutions were used to determine the inhibition level and EC50 values. Samples were taken at 30 and 180 min to perform dissolved oxygen measurement for 5 min using a WTW Inolab Oxi Level 2 oxygen meter in a 50-mL airtight vessel. The initial dissolved oxygen concentration in the test vessels was 7–8 mg/L. The percentage of inhibition in the oxygen uptake rate (OUR) was calculated as I OUR ð%Þ ¼

RB −ðRT −RPC Þ 100 RB

Statistical analysis To determine the significant inhibition of antibiotics, COD removal efficiencies of the ASBRs were compared using oneTable 1 Antibiotic concentration in each reactor during operational phases Erythromycin- Tetracycline- SulfamethoxazoleE (μg/L) T (μg/L) S (μg/L) ST reactor

ETS reactor

Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Phase 6 Phase 7 Phase 8 Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Phase 6 Phase 7 Phase 8

–

100

500

–

200

5,000

–

500

5,000

–

500

10,000

–

1,000

10,000

–

1,000

15,000

–

1,500

15,000

–

1,500

20,000

100

100

500

200

200

5,000

500

500

5,000

500

500

10,000

1,000

1,000

10,000

1,000

1,000

15,000

1,500

1,500

15,000

1,500

1,500

20,000


way ANOVA test, which was followed by running a post hoc Dunnett's test and Student's t test, respectively. Also, Student's t test was applied to find out significant decrease in the oxygen uptake rate in inhibition tests. Minitab Release 16 software was used for all statistical analyses.

Results and discussion Operation of anaerobic sequencing batch reactors COD removal efficiency and methane production profiles obtained from ST and ETS reactors are given in Figs. 1 and 2. In both figures, the first 60 days (≈2 SRT) represented the acclimation period. The first antibiotic feeding phase in both reactors was started on the 61st day. After this acclimation period, antibiotic concentration was increased in a stepwise manner, and the reactors were operated in each antibiotic phase for 30 days (≈1 SRT). The antibiotic concentrations added to the reactors in each phase are given in Table 1. As seen in Fig. 1, while COD removal efficiencies of ST and ETS reactors were 91.1 % ± 3.3 and 92.2 % ± 3.2, respectively, in the acclimation period, the removal efficiency showed a decreasing profile with ascending antibiotic concentration. The synthetic wastewater in this study is composed of organic compounds that are totally biodegradable, except for antibiotics, in nature. In the literature, similar studies were conducted with these compounds as single substrates or substrate mixtures; it would be acceptable to assume that under the operation conditions selected for the reactors, they would be totally removed so that low soluble COD level measured in the effluent is essentially residual soluble microbial products generated in the course of biochemical reactions (Amin et al. 2006; Fountoulakis et al. 2008; Cetecioglu et al. 2013b). In phase 1, this value decreased to 79.4 %±6.0 and 83.6 % ±2.3 in the ST and ETS reactors, respectively. Concordantly, Fig. 1 COD removal efficiency in ST and ETS reactors

the produced methane volume decreased from 870±5 mL to 727±1 mL in the ST reactor. Also, there was a 15 % reduction (874±7 mL to 758±12 mL) in methane generation in the first phase of the ETS reactor as seen in Fig. 2. In phases 2 and 3, while COD removal efficiencies of the ST reactor decreased to 73.6 %±1.6 and 70.0 %±5.1, respectively, the decline profile of the ETS reactor changed from 79.4 %±2.8 to 78.3 %±2.9. Finally, the removal efficiencies of the ST and ETS reactors reached 57.3 %±4.2 and 67.7 %±5.6, respectively, in the last phase (phase 8). The produced methane volumes in the same phase were measured as 650± 23 mL and 723± 11 mL, respectively. The statistical analysis showed that the significant inhibition effects in the ST reactor were observed in phases 1 and 8. Also, there is a significant decrease in the COD removal and methane generation of the ETS reactor in phase 1. However, the second significant inhibitory effect was observed in the last phase (phase 8). During the operation period, the COD removal efficiency and methane generation in both reactors decreased; however, dual inhibitory effect of sulfamethoxazole and tetracycline is more effective than the triple effect (erythromycin, sulfamethoxazole, and tetracycline) in each phase.

Activated sludge inhibition tests A respiration inhibition test was applied to compare the effects of raw antibiotics and those after anaerobic treatment on activated sludge. The test periods were 30 and 180 min. While the activated sludge inhibition test described in ISO Method 8192 (ISO8192 1999) has been applied to assess acute inhibitory effects of different pharmaceuticals, especially antibiotics (Kümmerer et al. 2004), it has never been used to determine the changes in short-term toxicity/inhibition of anaerobically treated pharmaceutical industry wastewater. ISO Method 8192 (ISO8192 1999) requires running an anaerobic reactor in which biomass is acclimated to a defined substrate such as peptone–meat extract mixture, acetone,

Author's personal copy Environ Sci Pollut Res Fig. 2 Produced methane volume of the ST and ETS reactors

glucose, etc. In this study, peptone–meat extract mixture is selected as defined substrate because this compound is a model substrate mimicking domestic wastewater, while in the literature, sole carbon source such as acetate and glucose has been used (Ricco et al. 2004; Cokgor et al. 2007; Diaz et al. 2012). However, Cokgor et al. (2007) showed that different microbial species may utilize this mixture and it provides less inhibitory effects on them. Also, Gutiérrez et al. (2002) drew a similar conclusion with respirometric methods vs. Microtox test. The authors speculated that the respirometric methods are more representative and reliable to evaluate the toxic effects of wastewater discharge to the receiving water body and/or latter steps of biological treatment because it contains a natural, mixed microbial culture. So the activated sludge, which was acclimated to this mixture, was used during the aerobic inhibition tests in this study. Prior to the analyses, the validity of the tests was checked by means of 3,5-dicholorophenol as reference substance. EC50 range of the reference substances is given in the method as 5– 30 mg/L. It was reconfirmed in this study and found to be 10 mg/L.

Dual effect of tetracycline and sulfamethoxazole Respirometric test results with antibiotic concentration in the influent and effluent of the ST reactor obtained from each operation phase are given in Tables 2 and 3, respectively. Firstly, the inhibitory effect of the wastewater obtained from the influent of the ST reactor was assessed to manifest biological treatment strategy for pharmaceutical industry wastewater produced during antibiotic production. The results obtained from the influent of the ST reactor revealed that the antibiotic concentration in phase 5 and upper phases has a toxic effect on aerobic systems, as seen in Table 2. No results were obtained from any dilution of the influents in the ISO 8192 method (ISO8192 1999). In the effluent of the ST reactor, no lethal level was detected even in the last phase (phase 8). It can be mentioned that anaerobic treatment has an effective removal capability even in high concentrations of antibiotics. Also, the expectative transformation products of tetracycline and sulfamethoxazole are not more toxic and/or inhibitory than the mother compounds (Arslan-Alaton and Caglayan 2006; Kümmerer 2009a), whereas the results

Table 2 Antibiotic concentrations of the ST reactor influents and their EC50 concentrations within each phase

Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Phase 6 Phase 7 Phase 8

Reactor influent concentration (μg/L)

30-min EC50 (μg/L)

Tetracycline-T

Sulfamethoxazole-S

Tetracycline-T

Sulfamethoxazole-S

Tetracycline-T

Sulfamethoxazole-S

100 200 500 500 1,000 1,000 1,500 1,500

500 5,000 5,000 10,000 10,000 15,000 15,000 20,000

0.374 0.035 0.003 0.001 ND ND ND ND

1.868 0.708 0.034 0.019 ND ND ND ND

2.394 1.087 0.703 0.160 ND ND ND ND

27.178 23.937 7.032 0.801 ND ND ND ND

ND not determined

180-min EC50 (μg/L)

Author's personal copy Environ Sci Pollut Res Table 3 Antibiotic concentrations of the ST reactor effluents and their EC50 concentrations within each phase


Reactor effluent concentration (μg/L)

30-min EC50 (μg/L)

Tetracycline-T

Sulfamethoxazole-S

Tetracycline-T

Sulfamethoxazole-S

Tetracycline-T

Sulfamethoxazole-S

60 160 400 400 800 820 1,300 1,350

320 4,000 4,500 8,300 9,000 13,700 14,000 18,000

63.215 3.342 3.111 2.125 1.262 1.165 0.377 0.227

639.864 70.002 58.490 32.772 29.428 6.731 3.823 2.269

2,971.466 235.753 190.544 74.264 21.141 1.869 0.903 0.025

41,143.382 5,304.437 1,933.666 1,299.623 213.984 25.385 18.690 0.133

showed that short-term effects of the ST reactor influents determined at 30 min were more inhibitory than those at 180 min. Also, for the effluent obtained at the last phase, the inhibitory effect according to exposure time was similar. These results about the exposure time are different from those in the literature. Kümmerer et al. (2004) examined the OECD 209 method, which is similar to ISO 8192 for a variety of pharmaceuticals including antibiotics, and found out that there is no significant difference between 30- and 180-min exposure times. However, the authors also represented results obtained at 20 h and found that inhibition concentrations could depend on exposure time and the type of compound. Their results showed that for short incubation time such as 30 and 180 min, single IC50 values of the three compounds are more than 100 mg/L. These values decreased to 10–100 and 1–10 mg/ L for erythromycin and tetracycline after 20 h, while the IC50 value was in the same range with more than 100 mg/L for sulfamethoxazole at 20 h. The dual inhibitory effects of these compounds are higher than single compound, as seen in Table 2. Also, their dual effect after anaerobic treatment is stronger, as given in Table 3. In another study performed to reveal biodegradability characteristics and inhibitory effects of antibiotics under aerobic conditions by Gartiser et al. (2007a), no toxic and inhibitory effect of these three compounds were determined for a 14-day biodegradability test. A similar study was carried out under anaerobic conditions by Ozbayram (2012). The authors have evaluated dual and triple effects of same compounds on methanogenic activity under short-term exposure time. The obtained EC50 values are closed to those in the study of Alexy et al. (2004). They found that EC50 values of dual compounds sulfamethoxazole and tetracycline were 102 and 52 mg/L on acetoclastic methanogens and all methanogenic groups, respectively, as seen in Table 4. Additionally, Cetecioglu et al. (2012) reported that EC50 values of these compounds in single form are higher than those of dual forms under anaerobic conditions. The authors performed a study similar with that of Ozbayram (2012) with sulfamethoxazole, tetracycline, and erythromycin


and calculated their EC50 values as 198.5, 204.4, and 155.4 mg/ L, respectively. On the other hand, Gartiser et al. (2007b) found the EC50 value of tetracycline to be 37.3 mg/L, while they could not show any inhibitory effect of sulfamethoxazole and erythromycin via anaerobic inhibition test. Triple effect of erythromycin, tetracycline, and sulfamethoxazole The EC50 values of the influent and effluent of the ETS reactor was calculated according to the ISO 8193 method and given in Tables 5 and 6 with the antibiotic concentrations to reveal the triple effect of tetracycline and sulfamethoxazole with erythromycin. The EC50 concentrations reported in this part of the respiration inhibition test were much lower than in the Table 4 Dual and triple acute inhibitory effects of erythromycin, tetracycline, and sulfamethoxazole under anaerobic conditions and EC50 concentrations for acetoclastic methanogens and homoacetogens with methanogens Antibiotic Antibiotic Acetate as sole mixture concentration carbon source (mg/L)

Acetate, butyrate, and propionate as carbon source

ETS ETS

1 10

Inhibition EC50 Inhibition EC50 (mg/L) (%) (mg/L) (%) 3.4 50 3.6 88 8.1 14.8

ETS ETS ETS ST ST ST

25 50 100 1 10 25

16.1 50.1 73.3 14.5 18.4 28.2

ST ST ST

50 100 250

37.5 46.7 99.1

Control

0

After Ozbayram (2012)

102

22.0 28.4 57.0 27.1 31.7 39.3 49.2 57.4 61.0

52

100 200 500 500 1,000 1,000 1,500 1,500

100 200 500 500 1,000 1,000 1,500 1,500

500 5,000 5,000 10,000 10,000 15,000 15,000 20,000

19.214 0.002 0.000 0.000 ND ND ND ND

19.214 0.002 0.000 0.000 ND ND ND ND

Tetracycline-T

40 160 400 430 920 940 1,400 1,420

ND not determined


60 180 430 470 830 900 1,320 1,380

300 4,200 4,200 8,300 8,700 14,000 14,200 18,300

17.113 8.858 7.421 4.709 0.555 0.011 ND ND

Erythromycin-E

Sulfamethoxazole-S

Erythromycin-E

Tetracycline-T

30-min EC50 (μg/L)

Reactor effluent concentration (μg/L)

16.135 8.858 8.720 4.607 0.624 0.017 ND ND

Tetracycline-T

Table 6 Antibiotic concentrations of the ETS reactor influents and their EC50 concentrations within each phase

ND not determined


Erythromycin-E

Sulfamethoxazole-S

Erythromycin-E

Tetracycline-T

30-min EC50 (μg/L)

Reactor influent concentration (μg/L)

Table 5 Antibiotic concentrations of ETS reactor influents and their EC50 concentrations within each phase

223.686 161.405 86.521 42.488 14.556 0.086 ND ND

Sulfamethoxazole-S

192.144 0.023 0.002 0.000 ND ND ND ND

Sulfamethoxazole-S 5.133 0.671 0.328 ND ND ND ND ND

Tetracycline-T

195.509 3.959 2.553 2.337 0.149 ND ND ND

Erythromycin-E

191.259 5.939 2.747 2.553 0.167 ND ND ND

Tetracycline-T


5.133 0.671 0.328 ND ND ND ND ND

Erythromycin-E


1,763.833 50.840 29.695 24.938 3.900 ND ND ND

Sulfamethoxazole-S

102.651 16.773 1.638 ND ND ND ND ND

Sulfamethoxazole-S

Author's personal copy

Environ Sci Pollut Res


dual inhibition test in all phases except for phase 1. The triple inhibition effects of these three compounds in the influent were assessed in the first three phases. In phase 4 and the latter phases, the toxic effects were determined, and a significant result could not be obtained by respirometric measurements. However, the respirometric tests applied to the effluents of the ETS reactor showed that the toxic effect starts after phase 5. Additionally, the exposure time effect on inhibition was similar to that in the dual test; short-term exposure was more inhibitory than the 180-min exposure. However, the COD removal efficiency of the ST reactor was lower than that of the ETS reactor, as given in Fig. 1. The result revealed that the long-term dual effect of sulfamethoxazole and tetracycline was more inhibitory than the triple effects of sulfamethoxazole, tetracycline, and erythromycin. Also Ozbayram (2012) found out a similar result obtained from short-term anaerobic tests. The author showed that dual effects of these compounds on complex microbial community were more inhibitory than triple effects. However, the same study showed that the EC50 concentration of the triple-antibiotic mixture was lower while only acetoclastic methanogens were targeted, as seen in Table 4.

Conclusion Pharmaceutical industry wastewaters produced during antibiotic production are of main environmental concern because of not only their potential toxic effects on microorganisms and recalcitrant properties to biodegradation but also their causing an increase in antibiotic resistance genes in the environment. To overcome this problem, a biological treatment option has been addressed in this study. In light of the experimental results obtained and their evaluation, it can be concluded that anaerobic digestion can be applied as a biological pretreatment method for pharmaceutical industry wastewater including antibiotic mixtures prior to aerobic treatment. Despite activated sludge system being inhibited by low concentration of antibiotic mixture, the same aerobic system can tolerate the higher concentration of the same mixtures after an anaerobic pretreatment. However, according to the available literature, EC50 concentrations of the same antibiotics as single compound are higher than the EC50 concentrations of dual and triple applications. Yet, it should be noted that the ISO 8192 procedure only provides an index value as in many other similar procedures. In the future, a labscale anaerobic–aerobic system could be operated to improve this approach and also as a second step of scale-up.

Acknowledgments This study was supported by the Scientific Research Projects of Istanbul Technical University (project no: 36966).

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