Toxicological impacts of antibiotics on aquatic micro-organisms: A mini-review

International Journal of Hygiene and Environmental Health 220 (2017) 558–569

Contents lists available at ScienceDirect

International Journal of Hygiene and Environmental Health journal homepage: www.elsevier.com/locate/ijheh

Toxicological impacts of antibiotics on aquatic micro-organisms: A mini-review Pia Välitalo a,b,∗ , Antonina Kruglova a , Anna Mikola a , Riku Vahala a a b

Aalto University, Department of Built Environment, Espoo, Finland Finnish Environment Institute, Laboratory Centre, Helsinki, Finland

a r t i c l e

i n f o

Article history: Received 29 September 2016 Received in revised form 14 February 2017 Accepted 16 February 2017 Keywords: Antibiotics Ecotoxicity Micro-organisms Effect concentrations

a b s t r a c t Antibiotics are found globally in the environment at trace levels due to their extensive consumption, which raises concerns about the effects they can have on non-target organisms, especially environmental micro-organisms. So far the majority of studies have focused on different aspects of antibiotic resistance or on analyzing the occurrence, fate, and removal of antibiotics from hospital and municipal wastewaters. Little attention has been paid to ecotoxicological effects of antibiotics on aquatic micro-organisms although they play a critical role in most ecosystems and they are potentially sensitive to these substances. Here we review the current state of research on the toxicological impacts of antibiotics to aquatic microorganisms, including proteobacteria, cyanobacteria, algae and bacteria commonly present in biological wastewater treatment processes. We focus on antibiotics that are poorly removed during wastewater treatment and thus end up in surface waters. We critically discuss and compare the available analytical methods and test organisms based on effect concentrations and identify the knowledge gaps and future challenges. We conclude that, in general, cyanobacteria and ammonium oxidizing bacteria are the most sensitive micro-organisms to antibiotics. It is important to include chronic tests in ecotoxicological assessment, because acute tests are not always appropriate in case of low sensitivity (for example for proteobacteria). However, the issue of rapid development of antibiotic resistance should be regarded in chronic testing. Furthermore, the application of other species of bacteria and endpoints should be considered in the future, not forgetting the mixture effect and bacterial community studies. Due to differences in the sensitivity of different test organisms to individual antibiotic substances, the application of several bioassays with varying test organisms would provide more comprehensive data for the risk assessment of antibiotics. Regardless of the growing concerns related to antibiotics in the environment, there are still evident knowledge gaps related to antibiotics, as there is only limited or no ecotoxicological data on many potentially harmful antibiotics. © 2017 Elsevier GmbH. All rights reserved.

Contents 1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559 Antibiotics of high ecotoxicological concern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559 Data variability of effective concentrations of antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561 3.1. Green algae and cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561 3.2. Proteobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563 3.3. Wastewater treatment bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564 Summary and future challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567

∗ Corresponding author at: Aalto University, Department of Built Environment, Espoo, Finland. E-mail address: pia.valitalo@aalto.fi (P. Välitalo). http://dx.doi.org/10.1016/j.ijheh.2017.02.003 1438-4639/© 2017 Elsevier GmbH. All rights reserved.

P. Välitalo et al. / International Journal of Hygiene and Environmental Health 220 (2017) 558–569

1. Introduction Today water security is one of the most pressing global issues due to the growing demand for limited water resources. The release of antibiotics in an aquatic environment at trace concentrations is among the major concerns of water researchers. Antibiotics were invented almost 90 years ago, and since then they have revolutionized human medicine. Today, antibiotics play a crucial role in the management of infectious disease, and they are consumed extensively in human and veterinary medicine and aquaculture. In addition to therapeutic applications, antibiotics are used for nontherapeutic purposes, for example to promote the growth of cattle, hogs, and poultry (Sarmah et al., 2006; Kümmerer, 2009). The use of antibiotics as growth-promoters is prohibited in the EU in 2006, but they are still used in other parts of the world such as China and India (Ronquillo and Hernandez, 2017). Due to their extensive consumption, antibiotics are ubiquitous and they have been detected in various ecosystems from terrestrial to aquatic environments (Yang and Carlson, 2003; Kümmerer, 2009; Martinez, 2009; Leung et al., 2012; Alygizakis et al., 2016). The advantages of antibiotics in healthcare are undisputed; however the bioactive properties of antibiotics and their presence in the environment at trace levels raise concerns about their toxicity to non-target organisms. Fundamentally, antibiotics were designed to be effective towards micro-organisms, due to which they are likely the most antibioticsensitive group of organisms, making them of particular interest (Brandt et al., 2015). According to the reported data based on population estimates, the main origin of environmental pollution by human antibiotics is the diffuse contribution of the general public sewage plants. Antibiotic substances are not fully metabolized in the body, and residues of the antibiotics excreted with urine and feces end up at wastewater treatment plants (Ternes, 1998; Zuccato et al., 2010). Municipal wastewaters are a major source of antibiotics, as only 10–25% of antibiotics consumed by people come directly from hospitals (Kümmerer, 2009). Due to the continuous discharge of antibiotics, they are typically found in the environment in the low ng/L or ␮g/L range (Ternes, 1998; Kümmerer, 2009; Santos et al., 2010). Moreover, some of the antibiotics are poorly biodegradable and thus they can be persistent in the environment, and their toxic properties toward micro-organisms can remain even at trace levels (Kümmerer et al., 2000; Brown et al., 2006). The monitoring of harmful substances is currently based on chemical analytics from collected samples, however the complex nature of environmental samples, low concentrations, the dilution effect, and partial transformation of the parent compounds make the detection of antibiotics challenging. Indirect toxicological methods can provide additional knowledge on water quality and insight on mixture effects (González-Pleiter et al., 2013; Marx et al., 2015). Prokaryotes are likely the most sensitive environmental organisms to antibiotics because antimicrobial agents are efficient inhibitors of bacterial growth (Martinez, 2009; Brandt et al., 2015). The present mini-review focuses on the toxicological impact of antibiotics which are most often passed through municipal wastewater treatment plants (WWTPs) and remain at detectable concentrations in aquatic environments with possible effects on prokaryotic microorganisms and micro-algae. Several methods for evaluating the toxicity of antimicrobial agents are currently available, and different bioassays using representative organisms of aquatic ecosystems have been used to assess the ecotoxicity (Wollenberger et al., 2000; Isidori et al., 2005; Robinson et al., 2005; Kim et al., 2007; González-Pleiter et al., 2013; Yasser and Adli, 2015). Cyanobacteria are an essential group of prokaryotic organisms in the aquatic ecosystems: they represent the majority of phytoplankton mass and contribute largely to the total free oxygen

559

production and carbon dioxide fixation in marine and terrestrial habitats. Many of them are also able to fix atmospheric nitrogen (Mitsui et al., 1986; Berman-Frank et al., 2003). Thereby inhibitory effects of antibiotics on cyanobacteria have been under the scrutiny of the researchers (Baquero et al., 2008; Guo et al., 2016). In addition to cyanobacteria, green algae have been applied to study the toxic effects of antibiotics on aquatic ecosystems (Isidori et al., 2005; Ando et al., 2007; De Liguoro et al., 2012; Kolar et al., 2014; Baumann et al., 2015). Algae are a vital part of the food chain in aquatic environments, forming a substantial share of the total biomass and therefore relevant for ecotoxicological studies. Another important group is proteobacteria. Proteobacteria can be divided into five subgroups (alpha-, beta-, delta-, gamma-, and epsilon-), including marine microorganisms as well as bacteria participating in the wastewater treatment processes, particularly ammonium and nitrite oxidizing and nitrogen fixating bacteria, and also bioluminescent bacteria (Kersters et al., 2006). So far, the majority of the studies have focused on studying different aspects of antibiotic resistance. Additionally the occurrence, fate, and removal of antibiotics from hospital and municipal wastewaters have been studied extensively. Markedly, the ecotoxicological effects of antibiotics on aquatic micro-organisms have received less attention despite their significant role in different ecosystems. This mini-review provides an overview on antibiotics of high ecotoxicological concern based on their toxicological properties to aquatic micro-organisms, frequent detection in environmental samples and high potential for accumulation in natural waters. The existing analytical methods and effect concentrations for the most commonly applied micro-organisms for antibiotic effect studies are summarized and the current state of research is discussed with emphasis on the existing knowledge gaps and future perspectives. The issue of antibiotic resistance is left out of the scope of the study.

2. Antibiotics of high ecotoxicological concern Not all of the antibiotics found in environmental samples are harmful, and the real challenge is to identify the ones that actually pose a risk in the environment from the complex sample mixtures. Some antibiotics are consumed more than others, and penicillins, sulphonamides, macrolides, and quinolones form the largest share of antibiotics consumed by humans globally (Kümmerer, 2009). However, it is very difficult to assess which compounds are potentially toxic, since for several compounds there is no comprehensive ecotoxicological data available, if any. In addition to toxicological data, parameters such as persistence and detection frequency should be considered. Table 1 lists antibiotics that can be considered of high ecotoxicological concern. The compounds were selected based on different parameters: large consumption, frequent detection in environmental samples, persistence, and toxic effects detected at low concentrations. Regardless of the wastewater treatment processes, these antibiotics still enter the environment through effluents because WWTPs are not specifically designed for the removal of these substances (Batt et al., 2007; Watkinson et al., 2007; Luo et al., 2014). Antibiotics from different chemical structure classes were selected. Presented in the table are maximum concentrations (␮g/L) detected in environmental water samples (surface waters or ground water) and wastewater effluents. Median or mean concentrations are presented if this information was given in the reference. Based on the maximum environmental concentrations reported in the literature, the highest concentrations have been measured for enrofloxacin (ENR), ciprofloxacin (CIP), norfloxacin (NOR), sulfamethoxazole (SMX), trimethoprim (TMP), azithromycin (AZM), erythromycin (ERY, ERY-H2 O), chlortetracycline (CTC) and oxyte-

560

P. Välitalo et al. / International Journal of Hygiene and Environmental Health 220 (2017) 558–569

Table 1 Antibiotics of high ecotoxicological concern and maximum concentrations (␮g/L) found in water samples. Median or mean concentrations are presented in parenthesis if this information was given in the reference. Antibiotics family

Name of the substance

Abbreviations

Maximum concentration, (median or mean* concentration), ␮g/L

Type of water

Country of detection

References

Quinolones

enrofloxacin

ENR

ciprofloxacin

CIP

norfloxacin

NOR

ofloxacin

OFL

4.24 (0.21) 1.09 (0.27) 5.93 (0.89) 3.35 (1.05) 1.15 (0.030) 3.7 (2.51*) 7.87 (6.84*) 0.231

river water effluent river water effluent river water effluent effluent river water

China China China China Australia China China France

Wei et al. (2012) Wei et al. (2012) Wei et al. (2012) Wei et al. (2012) Watkinson et al. (2009) Leung et al. (2012) Leung et al. (2012) Dinh et al. (2011)

sulfadiazine

SDZ

sulfapyridine

SPY

sulfamethazine

SMN

sulfadimethoxine

SDM

sulfamerazine

SMR

sulfamethoxazole

SMX

sulfathiazole

STZ

0.002 0.108 (0.004) 0.56 0.378 (0.277*) 0.142 (0.037) 0.360 0.009 0.472 0.002 0.003 0.16 0.0036 0.006 13.765 (1.8) 1.11 3.336 (1.94*) 0.60 (n.d.) 0.006 0.13 0.002

sea water river water effluent effluent river water ground water effluent river water effluent river water river water effluent sea water river water ground water effluent effluent river water river water effluent

Greece Vietnam China UK UK US China US China Luxembourg US China Greece Kenya US Kenya Australia US US US

Alygizakis et al. (2016) Giang et al. (2015) Gao et al. (2012) Kasprzyk-Hordern et al. (2009) Kasprzyk-Hordern et al. (2008a,b) Barnes et al. (2008) Gao et al. (2012) Bartelt-Hunt et al. (2009) Dong et al. (2016) Pailler et al. (2009) Yang and Carlson (2003) Dong et al. (2016) Alygizakis et al. (2016) Ngumba et al. (2016) Barnes et al. (2008) Ngumba et al. (2016) Watkinson et al. (2009) Bartelt-Hunt et al. (2009) Yang and Carlson (2003) Bartelt-Hunt et al. (2009)

0.029 2.65 (0.327) 3.052 (1.152*) 0.0015 0.020 1.89 3.98 2.841 (1.385*) 4.33 (2.82*) 1.547 1.220

sea water river water effluent sea water river water effluent river water river water effluent river water effluent

Belgium Kenya UK Greece Italy Slovakia Spain UK China US Slovakia

Wille et al. (2010) Ngumba et al. (2016) Kasprzyk-Hordern et al. (2009) Alygizakis et al. (2016) Calamari et al. (2003) Biroˇsová et al. (2014) Rodríguez-Gil et al. (2010) Kasprzyk-Hordern et al. (2009) Leung et al. (2012) Bartelt-Hunt et al. (2009) Biroˇsová et al. (2014)

0.128 0.622 (0.149) 1.67 (1.34*) 0.29 0.79

sea water river water effluent river water effluent

Greece UK China Kenya Kenya

Alygizakis et al. (2016) Kasprzyk-Hordern et al. (2008a) Leung et al. (2012) Kimosop et al. (2016) Kimosop et al. (2016)

0.81 (0.27) 1.42 (1.40*) 2.42 (0.32) 0.28 2.20 (0.22) 0.842 (0.53*) 0.4 (n.d.) 0.34

river water effluent river water effluent river water effluent river water effluent

China China China US China China Australia US

Wei et al. (2011) Leung et al. (2012) Wei et al. (2011) Yang and Carlson (2003) Wei et al. (2011) Leung et al. (2012) Watkinson et al. (2009) Yang and Carlson (2003)

0.014 (0.005) 0.421 (0.265*) 0.320 0.249 0.3 (0.003)

river water effluent ground water river water effluent

UK UK US Italy Australia

Kasprzyk-Hordern et al. (2008a) Kasprzyk-Hordern et al. (2009) Barnes et al. (2008) Calamari et al. (2003) Watkinson et al. (2009)

Sulphonamides

sulfachlorpyridazine SCP Trimethoprim

Macrolides

TMP

clarithromycin

CLA

erythromycin erythromycin-H2 O

ERY ERY-H2 O

azithromycin

AZM

amoxicillin

AMOX

ampicillin

AMP

tetracycline

TCN

chlortetracycline

CTC

oxytetracycline

OTC

doxycycline

DXC

Nitroimidazoles

metronidazole

MNZ

Lincosamides

lincomycin

LIN

Penicillins

Tetracyclines

n.d.= not detected.

tracycline (OTC) all exceeding concentrations of 1 ␮g/L in environmental water samples (Table 1). Ofloxacin (OFL), clarithromycin (CLA) and amoxicillin (AMOX) were found in wastewater effluents at concentrations above 1 ␮g/L. CIP, NOR, SMX, TMP, AZM, ERY and OTC are all used for treating several common bacterial infections, such as respiratory tract infections, ear infections, and urinary tract

infections. ENR and CTC are mainly used for veterinary purposes for the treatment of pets and domestic animals. In some cases (e.g., CIP and SMX), the concentrations measured in surface waters were higher than the concentrations measured in wastewater effluent. This phenomenon can be explained by several reasons, such as influent variations followed by antibiotics accumulation (includ-

P. Välitalo et al. / International Journal of Hygiene and Environmental Health 220 (2017) 558–569

ing bioaccumulation in aquatic organisms), desorption processes from activated sludge and biofilms, as well as by the cumulative effect of additional sources (such as agricultural waters) entering the surface waters (Kümmerer, 2009; Watkinson et al., 2009; Na et al., 2013; Dong et al., 2016). The persistence of antibiotics in the environment can vary greatly because half-life times are affected by various environmental parameters, such as geographic location, temperature, and pH. Various studies have analyzed how these parameters affect the persistence of different antibiotics (Andreozzi et al., 2004; Lee et al., 2008; Braschi et al., 2013; Mitchell et al., 2014). For example, Andreozzi et al. (2004) demonstrated that the maximum half-life time for amoxicillin in water was 9 days in winter at 50◦ latitude and the minimum was approximately 2 days in summer at 20◦ latitude. Lee et al. (2008) calculated a half-life time of 3 days for amoxicillin in water and 30 days for erythromycin. Braschi et al. (2013) demonstrated the effects of pH on the persistence of amoxicillin in water, with half-lives ranging from 0.5 to 5.4 days, depending on the pH. Mitchell et al. (2014) studied the effects of pH and temperature on antibiotic hydrolysis. They found that the half-lives at pH 7 and 25 ◦ C for cefalotin, cefoxitin, and ampicillin were 5.3, 9.3, and 27 days, respectively. The biodegradability of the antibiotic substances during the activated sludge process affect how well the substances are removed during wastewater treatment processes (Halling-Sørensen et al., 2000; Dorival-García et al., 2013; Wang et al., 2016). HallingSørensen et al. (2000) showed that the half-life time in activated sludge reactors for trimethoprim was 22–41 days and 1.6–2.5 days for ciprofloxacin. Dorival-García et al. (2013) studied the degradation characteristics of quinolone antibiotics in activated sludge reactors under aerobic, nitrifying and anoxic conditions. They found that sorption on sludge was the most important mechanism in the elimination of quinolone antibiotics. They found that the sorption potential depended on the redox conditions, with highest sorption potential in aerobic sludge and lower potential in nitrifying and anoxic sludge. Wang et al. (2016) studied the removal of fluoroquinolones in activated sludge process. They found that fluoroquinolones were slowly biodegradable, with long half-lives (>4 days). However, they found that biodegradation was enhanced with increasing temperature and under aerobic conditions.

3. Data variability of effective concentrations of antibiotics Biological toxicity tests are used in the risk assessment of harmful substances and there is a large number of methods available for different organisms, ranging from acute tests to chronic assays (Wollenberger et al., 2000; Park and Choi, 2008; Kümmerer, 2009; Brausch et al., 2012). The most commonly applied aquatic microorganisms for the environmental toxicity testing of antibiotics are cyanobacteria (e.g., Ebert et al., 2011), proteobacterium Vibrio fischeri (e.g., Backhaus et al., 2000) and different species of micro-algae and green algae (e.g., Ando et al., 2007; Magdaleno et al., 2015). Additionally, bacteria from wastewater processes and sewage sludge are applied when toxicological effects to the biological wastewater treatment processes have been studied (Katipoglu-Yazan et al., 2013; Tobajas et al., 2016). For many of the organisms listed above, standardized test methods are available (Brandt et al., 2015). So far most of the studies have focused on the toxicity testing of single antibiotic substances, but several mixture studies have also been published (Backhaus et al., 2000; Eguchi et al., 2004; Christensen et al., 2006; Yang et al., 2008; Ghosh et al., 2009; Hagenbuch and Pinckney, 2012; González-Pleiter et al., 2013; Liu et al., 2014; Magdaleno et al., 2015; Yasser and Adli, 2015; Guo et al., 2016). The effect concentrations can vary depending on the test method and organism, suggesting that the harmfulness of

561

antibiotic substances should be assessed with multiple bioassays for a more comprehensive analysis. In the chapters below, the different test methods for different classes of aquatic micro-organisms are compared in relation to the effect concentrations (EC50 ) of antibiotics of high ecotoxicological concern. The EC50 value describes the concentration of the chemical (antibiotic in this case) needed to provoke a response halfway between the baseline and the maximum response.

3.1. Green algae and cyanobacteria Algae and cyanobacteria are a vital part of the food chain in aquatic ecosystems, and even small changes in the algal and cyanobacterial populations could affect the balance of the whole ecosystem. Based on the literature, the most commonly applied species of cyanobacteria for toxicity testing are Microcystis aeruginosa, Anabaena flos-aquae, and Anabaena sp. CPB4337 (Halling-Sørensen et al., 2000; Robinson et al., 2005; Ando et al., 2007; González-Pleiter et al., 2013; Kolar et al., 2014; Baumann et al., 2015). Pseudokirchneriella subcapitata is the most frequently applied green algae species (Eguchi et al., 2004; Ando et al., 2007; Yang et al., 2008; De Liguoro et al., 2012; Kolar et al., 2014; Magdaleno et al., 2015). Other species of green algae applied to antibiotic toxicity testing include Desmodesmus subspicatus, Chlorella vulgaris, Scenedesmus vacuolatus, and Tetraselmis suecica ´ (Eguchi et al., 2004; Białk-Bielinska et al., 2011; Ebert et al., 2011; Seoane et al., 2014). A literary review of studies that have focused on the toxicity of antibiotics to microalgae or cyanobacteria reveal that toxicities have been determined mainly for antibiotics from quinolones, sulphonamides, macrolides, penicillins, and tetracyclines (Table 2 ). Ciprofloxacin, norfloxacin, trimethoprim, erythromycin, amoxicillin, and oxytetracycline are the most studied compounds based on the available toxicity data. The results clearly indicate that cyanobacteria and green algae are sensitive test organisms to antibiotics (Table 2). However, there is a variation between the different species and antibiotics. Cyanobacteria are more sensitive than green algae in most cases. For example, the EC50 values for ciprofloxacin, enrofloxacin and ofloxacin are notably lower for cyanobacteria than green algae. However, there are some exceptions. For example the macrolides, clarithromycin and erythromycin, are equally sensitive to cyanobacteria and algae (Isidori et al., 2005; Ando et al., 2007; Baumann et al., 2015). Many of the antibiotics that have been tested for toxicity to cyanobacteria have EC50 values well below 1 mg/L (e.g., Ando et al., 2007; Ebert et al., 2011). However, some species of cyanobacteria show higher toleration for the same antibiotics than other species, e.g. M.aeruginosa is sensitive to amoxicillin (EC50 0.0037 mg/L) whereas Anabaena sp.CPB4337 can tolerate high concentrations of the same antibiotic (EC50 56.3 mg/L). Fig. 1 summarizes the most toxic antibiotic substances to cyanobacterial species with the highest sensitivity (EC50 <1 mg/L). The most toxic antibiotics are ampicillin, amoxicillin, ciprofloxacin, and clarithromycin. Only trimethoprim is non-toxic to cyanobacteria (EC50 values above 100 mg/L) from the tested substances. The majority of the green algae species show high sensitivity to the macrolides clarithromycin and erythromycin with EC50 values below 1 mg/L (Table 2). Only C.vulgaris appears clearly less sensitive than the other green algae species that have been used in toxicity testing. Macrolides were found hazardous to other test organisms (e.g. Daphnia) as well, indicating that macrolides pose a significant risk to the environment (Gros et al., 2010; Baumann et al., 2015). All of the studies that analyzed amoxicillin from the penicillins indicated that amoxicillin does not affect green algae. This antibiotic is designed to specifically inhibit bacterial cell wall synthesis, which

562

P. Välitalo et al. / International Journal of Hygiene and Environmental Health 220 (2017) 558–569

Table 2 Ecotoxicological effects of antibiotics on cyanobacteria and green algae. Class

Antibiotic

Group of micro-organisms

Species

EC50 (mg/L)

Exposure time (hours)

Reference

Quinolones

ENR

Cyanobacteria

M.aeruginosa A. flos-aquae D.subspicatus P.subcapitata M.aeruginosa A. flos-aquae D.subspicatus P.subcapitata

0.05 0.17 5.57 18.70 0.02 0.01 >8 113 187 39 0.062 5.6

120 72 72 72 120 72 72 72 72 96 144 72

Robinson et al. (2005) Ebert et al. (2011) Ebert et al. (2011) Robinson et al. (2005) Robinson et al. (2005) Ebert et al. (2011) Ebert et al. (2011) Magdaleno et al. (2015) Robinson et al. (2005) Martins et al. (2012) Ando et al. (2007) González-Pleiter et al. (2013)

0.029 10.4 >80 16.6 0.02 12.1 1.44

144 72 72 72 120 72 72

Ando et al. (2007) Eguchi et al. (2004) González-Pleiter et al. (2013) Eguchi et al. (2004) Robinson et al. (2005) Robinson et al. (2005) Isidori et al. (2005)

1.54 0.52 0.146 3.82

24 72 96 24

´ Białk-Bielinska et al. (2011) Isidori et al. (2005) Ferrari et al. (2004) ´ Białk-Bielinska et al. (2011)

112 253 >200 129 83.8 80.3

168 72 144 72 96 72

Lützhøft et al. (1999) Kolar et al. (2014) Ando et al. (2007) Kolar et al. (2014) De Liguoro et al. (2012) Eguchi et al. (2004)

Green algae CIP

Cyanobacteria Green algae

NOR

Cyanobacteria

Green algae

Sulphonamides

Trimethoprim

Macrolides

OFL

Cyanobacteria Green algae

M.aeruginosa P.subcapitata

SMX

Green algae

S.vacuolatus P.subcapitata

SCP

Green algae

S.vacuolatus

TMP

Cyanobacteria

M.aeruginosa A. flos-aquae

Green algae

P.subcapitata

CLA

Cyanobacteria Green algae

A. flos-aquae D. subspicatus P.subcapitata

0.0121 0.0371 0.046 0.002

72 72 72 72

Baumann et al. (2015) Baumann et al. (2015) Yang et al. (2008) Isidori et al. (2005)

ERY

Cyanobacteria

M.aeruginosa Anabaena sp. CPB4337 A. flos-aquae C.vulgaris P.subcapitata

0.023 0.02

144 72

Ando et al. (2007) González-Pleiter et al. (2013)

0.27 33.8 0.35 0.02 0.0366

144 72 72 72 72

Ando et al. (2007) Eguchi et al. (2004) González-Pleiter et al. (2013) Isidori et al. (2005) Eguchi et al. (2004)

M.aeruginosa Anabaena sp. CPB4337 P.subcapitata

0.0037 56.3

168 72

Lützhøft et al. (1999) González-Pleiter et al. (2013)

>50 >2000 >1500 >2000 0.0002 3.3

96 72 72 96 144 144

Andreozzi et al. (2004) Magdaleno et al. (2015) González-Pleiter et al. (2013) Andreozzi et al. (2004) Ando et al. (2007) Ando et al. (2007)

0.09 6.2

72 72

Halling-Sørensen et al. (2000) González-Pleiter et al. (2013)

3.31 0.05 0.207 0.39 2.7 7.05 1.04 0.342

72 72 168 144 72 72 72 72

González-Pleiter et al. (2013) Halling-Sørensen et al. (2000) Lützhøft et al. (1999) Ando et al. (2007) Kolar et al. (2014) Eguchi et al. (2004) Kolar et al. (2014) Eguchi et al. (2004)

Green algae

Penicillins

AMOX

Cyanobacteria

Green algae

Tetracyclines

M.aeruginosa Anabaena sp. CPB4337 A. flos-aquae C.vulgaris P.subcapitata

AMP

Cyanobacteria

M.aeruginosa A. flos-aquae

TCN

Cyanobacteria

CTC OTC

Green algae Cyanobacteria Cyanobacteria

M.aeruginosa Anabaena sp. CPB4337 P.subcapitata M.aeruginosa M.aeruginosa A. flos-aquae

Green algae

C.vulgaris P.subcapitata

might explain why it is not toxic to algae (González-Pleiter et al., 2013). Cyanobacteria are likely more sensitive because they are prokaryotic, making them structurally similar to bacteria and therefore more susceptible to the antibiotics’ mode of action (Robinson 2005). Some antibiotics (e.g. quinolones) can inhibit

protein synthesis (Halling-Sørensen, 2000) or interfere with DNA replication in cyanobacteria (Liu et al., 2011). Additionally, for green algae, some studies have shown that toxic effects caused by antibiotics can be related to the inhibition of the pathways involved in chloroplast and photosynthetic metabolism (Bradel et al., 2000; Pan et al., 2009; Liu et al., 2011). Liu et al. (2011) demonstrated that

P. Välitalo et al. / International Journal of Hygiene and Environmental Health 220 (2017) 558–569

563

Fig. 1. Comparison of effect concentrations (EC50 mg/L) of antibiotics among cyanobacterial species with EC50 values below 1 mg/L. References: 1= Robinson et al., 2005, 2= Ebert et al., 2011, 3=Ando et al., 2007, 4 =Baumann et al., 2015, 6 = Halling-Sørensen et al., 2000,7 = Lützhøft et al., 1999.

many photosynthesis-related processes, primary photochemistry, electron transport, photophosphorylation, and carbon assimilation could be inhibited by erythromycin exposure. Pan et al. (2009) had similar results with significant inhibition of photosynthetic electron transport due to levofloxacin exposure. There are no obvious trends related to the sensitivity of the different green algae species. The results indicate that, depending on the substance, the most sensitive species can vary. The toxic mechanisms of antibiotics to green algae are less known than for cyanobacteria. The toxic effects of antibiotics on green algae are likely related to the interference or inhibition of the pathways related to chloroplast metabolism, especially photosynthesis, disturbing the function of the photosynthetic apparatus and finally affecting cell growth (Liu et al., 2011; Matsumoto et al., 2012; Wang et al., 2015). Some studies with higher plants have shown that antibiotics target chloroplast translation due to its similarity to the prokaryotic translational machinery (Kasai et al., 2004; Wang et al., 2015). Antibiotics can interfere with chloroplast translation by different mechanisms such as repressing the transcription rate of nuclear encoded photosynthesis-related genes (Mulo et al., 2003) or by targeting ribosomal 16S and 23S rRNA (Kasai et al., 2004; Wang et al., 2015). A recent study on tetracyclines demonstrated that antibiotics may also effect mitochondrial translation in plants (Moullan et al., 2015). Another important aspect to consider is the effects of antibiotics on cyanobacterial biofilms. Biofilms are dense communities of micro-organisms supported by a self-produced matrix of extracellular polymeric substances (Flemming et al., 2016). Microorganisms in biofilms pose different properties compared to free-living organisms due to the many characteristic features of the biofilms, such as social cooperation and enhanced resistance to antimicrobial drugs (Stewart and Costerton, 2001; Flemming et al., 2016). Not many studies exist on cyanobacterial biofilms and antibiotics because most of the studies have focused on the effects of antibiotics on the biofilms of health-related micro-organisms such as Escherichia coli or Pseudomonas aeruginosa (Ashby et al., 1994; Olson et al., 2002; Dosler and Karaaslan, 2014; Stewart et al., 2015). However, a recent study by Tan et al. (2016) highlights the relevance of studying biofilms of micro-organisms with ecological importance (such as cyanobacterium) to provide new insight on what kind of ecological effects low-levels of antibiotics may

have in the environment. Tan et al. (2016) demonstrated that in the presence of low levels of kanamycin, the photosynthesis-mediated calcification and biofilm formation of Synechococcus elongatus were enhanced. The results suggest that low levels of antibiotics in the environment may not only influence the formation and functioning of biofilms, but also the biofilm-associated ecological functions of cyanobacteria through, e.g., promoted precipitation of carbonate resulting in increased concentrations of atmospheric carbon dioxide (CO2 ).

3.2. Proteobacteria Proteobacteria are a group of Gram-negative bacteria that is comprised of various genera of bacteria, such as Salmonella, Vibrio, and Yersinia. Due of their prokaryotic nature, these bacteria are expected to be the most sensitive to antibiotics. In the environmental toxicity studies of antibiotics, the bioluminescent marine bacterium Vibrio fischeri has been the primary test species. Some studies have employed a bacterial growth test with another Gramnegative bacterium, Pseudomonas putida (Kümmerer et al., 2000; Tobajas et al., 2016). There are several test methods available for V. fischeri, including a standardized method that is based on the determination of the inhibitory effect of water samples on the light emission of V.fischeri (Microtox) (ISO 11348, 2007). However, the acute test with V.fischeri appears inappropriate for antibiotic toxicity testing based on the high effect concentrations (EC50 ) reported for various antibiotics (Table 3). Similar results were recorded for other pharmaceuticals as well, not only antibiotics (AguirreMartínez et al., 2015). Based on the literature, only NOR, OFL, and a mixture of quinolones cause luminescence inhibition in V.fischeri at concentrations lower than 1 mg/L in the acute tests (Table 3, Backhaus et al., 2000). This might be due to the very short exposure time (15–30 min) in the test rather than the sensitivity of the bacteria (van der Grinten et al., 2010). This is supported by Thomulka et al. (1993), who reported significant harmful effects of several antibiotics on the reproduction of Vibrio harveyi after 5 h of exposure, but obtained little evidence for effects after short-term exposure. Consistent time-dependent effects of antibiotics were reported for V. fischeri as well (Froehner et al., 2000; Kümmerer et al., 2004). Some antibiotics (such as sulphonamides) only slightly interfere with the

564

P. Välitalo et al. / International Journal of Hygiene and Environmental Health 220 (2017) 558–569

Table 3 Toxicological effects on marine proteobacteria. Class

Antibiotic

Species

EC50 (mg/L)

Reference

Quinolones

ENR CIP

V.fischeri V.fischeri V.fischeri P. putida V.fischeri V.fischeri V.fischeri P. putida P. putida V.fischeri

425.0 (5 min), 326.8 (15 min) 11.5 (30 min) 5600 (15 min) 0.080 0.022 0.01359 223 (15 min) 0.010 1 (16 h) 0.237

Park and Choi (2008) Martins et al. (2012) Ji et al. (2013) Kümmerer et al. (2000) Backhaus et al. (2000) Backhaus et al. (2000) Tobajas et al. (2016) Kümmerer et al. (2000) Tobajas et al. (2016) Backhaus et al. (2000)

V.fischeri V.fischeri V.fischeri V.fischeri V.fischeri V.fischeri V.fischeri P. putida V.fischeri V.fischeri V.fischeri V.fischeri V.fischeri

>25 (30 min) 2.7 >50 74.2 (5 min), 78.1 (15 min) 23.3 (30 min) >100 (30 min) 55 (15 min) 477 (16 h) >500 (5 min, 15 min) >1000 (5 min, 15 min) 303 (5 min), 344.7 (15 min) 53.1 (5 min), 26.4 (15 min) >50 (30 min)

´ Białk-Bielinska et al. (2011) García-Galán et al. (2012) ´ Białk-Bielinska et al. (2011) Kim et al. (2007) Isidori et al. (2005) ´ Białk-Bielinska et al. (2011) Tobajas et al. (2016) Tobajas et al. (2016) Kim et al. (2007) Kim et al. (2007) Kim et al. (2007) Kim et al. (2007) ´ Białk-Bielinska et al. (2011)

V.fischeri V.fischeri V.fischeri V.fischeri V.fischeri V.fischeri V.fischeri V.fischeri V.fischeri V.fischeri V.fischeri P. putida V.fischeri

165.1 (5 min), 176.1 (15 min) >100 (15 min) >100 (5, 15 and 30 min) 3.597 (15 min) 2.627 (15 min) 0.0251 29 (15 min) 13 (15 min) 87 (15 min) 64.5 (30 min) 878 (15 min) 2557 (16 h) >100 (30 min)

Kim et al. (2007) Isidori et al. (2005) Hernando et al. (2007) Park and Choi (2008) Park and Choi (2008) Backhaus and Grimme (1999) Tobajas et al., 2016 Park and Choi (2008) Park and Choi (2008) Isidori et al. (2005) Tobajas et al. (2016) Tobajas et al. (2016) Isidori et al. (2005)

NOR OFL

Mixture of 10 quinolonesa Sulphonamides

SDZ SPY SMR SMX

SDM STZ SMN SCP Trimethoprim Macrolides

Penicillins Tetracyclines

TMP CLA ERY AMOX AMP TCN

Nitroimidazoles

CTC OTC DXC MNZ

Lincosamides

LIN

a

NOR, OFL, Cinoxacin, Enoxacin, Flumequine, Lomefloxacin, Nalidixic acid, Oxolinic acid, Pipemidic acid, and Piromidic acid.

biosynthetic pathways of the bacteria, which might also explain ´ the high effect concentrations in some cases (Białk-Bielinska et al., 2011). Therefore, the results given for standard acute bacterial toxicity test may underestimate the risk of antibiotics to environmental bacteria (Kümmerer, 2009). 3.3. Wastewater treatment bacteria Most of the toxicity test protocols are focused on the effects of antibiotics on selected micro-organisms with specific optimal conditions, and thus they are not suitable for studying a diverse group of bacteria such as found in activated sludge. An activated sludge process implicates a large variety of different bacteria capable of fairly rapid population shifts under changing operational conditions, including influent quality (Brandt et al., 2015). This leads to challenges in evaluating the toxic effects and to conflicting resulting data. At the same time, activated sludge offers an interesting environment to assess the effects of antibiotics on environmental microbial communities (Roose-Amsaleg & Laverman, 2016). Biological wastewater treatment features several stages with different operational conditions, including oxygen concentration, temperature, and pH. Changes in the process kinetics and respirometric analysis have been employed so far to examine the inhibitory effects of antibiotics on autotrophic and heterotrophic bacterial processes during wastewater treatment (Halling-Sørensen et al., 2000; Ghosh et al., 2009; Katipoglu-Yazan et al., 2013; Tobajas et al., 2016). The available data on toxicological impacts of selected antibiotics on functional groups of bacteria from wastewater treatment processes are presented in Table 4.

Nitrification is one of the most significant processes in the biological wastewater treatment because nitrogen along with organic matter are two major pollutants of municipal wastewaters. Nitrifying bacteria are also very sensitive to toxins and changes in environmental conditions (Kelly et al., 2004). Schmidt et al. (2012) showed no effect of CIP and SMX on nitrification in up to 40 mg/L concentrations in short experiments (6 h). At the same time, Halling-Sørensen et al. (2000, 2003) showed toxic effects on the activated sludge bacteria already at concentrations less than 1 mg/L, and Ghosh et al. (2009) reported a 20% inhibitory effect on ammonium oxidizing bacteria at 0.5 mg/L of SMX. A similar trend was reported by Louvet et al. (2010) for ERY. These results were explained by the generation times of nitrifying bacteria, which could vary from 8 to 138 h, and thus inhibition of cell division may be observed only after at least several days of the experiment. In addition, some bacteria of activated sludge are able to reserve growth factors (e.g., folic acid) that may prevent the toxic effects of antibiotics, inhibiting folic acid biosynthesis, for example after being exposed to SMX and TMP (Schmidt et al., 2012). Only a few studies have been done under anaerobic conditions (Sanz et al., 1996; Fernandez et al., 2009; Lotti et al., 2012). One of the reasons is that these conditions are more difficult to maintain during the experiments. However, anaerobic processes are an essential part of biological water treatment, and anaerobic digestion in particular is a widely implemented technology for the removal of organic matter. Qin et al. (2016) proposed that anaerobic toxicity should be included in the ecological risk assessment of harmful substances in the environment.

P. Välitalo et al. / International Journal of Hygiene and Environmental Health 220 (2017) 558–569

565

Table 4 Toxicological impact on wastewater treatment process. Antibiotics family

Antibiotic

Bacteria/functional bacterial groups from WWTP processes

Toxicity (effect/inhibition%)

Effective concentration, mg/L

Reference

Quinolones

ENR

Ammonium Oxidizing Bacteria

0.1/0.5

Ghosh et al. (2009)

CIP

Activated sludge bacteria Activated sludge bacteria

Ammonia dependant OUR inhibition >20%/>50% EC50 EC50 0/10 h EC50 (15 min)respirometry

0.61 0.025/0.08 165

Halling-Sørensen et al. (2000) Halling-Sørensen et al. (2003) Tobajas et al. (2016)

EC50

17

Halling-Sørensen (2001)

NOEC

60

Halling-Sørensen (2001)

EC50 0/10 h Ammonia dependant OUR inhibition ∼20% EC50 (15 min)respirometry

15.9/16.8 0.5

Halling-Sørensen et al. (2003) Ghosh et al. (2009)

377

Tobajas et al. (2016)

IC50 (24 h) Inhibition

650 100

Lotti et al. (2012) Lotti et al. (2012)

EC50

17.8

Halling-Sørensen et al. (2000)

Ammonia dependant OUR inhibition >20%/>50%

0.1/0.5

Ghosh et al. (2009)

Ammonia dependant OUR inhibition >20% Inhibition of heterotrophic bacteria Partial inhibition of nitrogen oxidation Total inhibition of nitrogen oxidation

0.1

Ghosh et al. (2009)

0.1

Louvet et al. (2010)

5

Louvet et al. (2010)

50

Katipoglu-Yazan et al. (2013)

IC50 (20% inhibition) EC50

10 4

Sanz et al. (1996) Halling-Sørensen (2001)

partial inhibition of nitrogen oxidation EC50 EC50 (15 min)respirometry Ammonia dependant OUR inhibition >20%/70% EC50 (5 min) EC50 (5 min) EC50

50

Katipoglu-Yazan et al. (2013)

2.2 610 0.1/0.5

Halling-Sørensen (2001) Tobajas et al. (2016) Ghosh et al. (2009)

94 42 0.64

Fernandez et al. (2009)

EC50

0.40

Halling-Sørensen (2001)

IC50 (20%/50% inhibition) EC50

5/40

Sanz et al. (1996)

1.7

Halling-Sørensen (2001)

EC50 EC50 0/10 h

250 0.12/0.27

Campos et al. (2001) Halling-Sørensen et al. (2003)

EC50 IC50 (24 h) IC50 (20% inhibition)

1.2 1.1 8

Halling-Sørensen (2001) Lotti et al. (2012) Sanz et al. (1996)

NOEC

100

Halling-Sørensen et al. (2000)

EC50 (15 min)respirometry Nitrification activity inhibition 17.9% Nitrification activity inhibition 13.8%

537 0.5 4.8

Tobajas et al. (2016) Carucci et al. (2006) Carucci et al. (2006)

OFL Sulphonamides

SDZ

SMX

STZ Trimethoprim

Macrolides

TMP

CLA ERY

Nitrosomonas europeaea Activated sludge bacteria Ammonium Oxidizing Bacteria Activated sludge bacteria ANAMMOX bacteria Activated sludge bacteria Ammonium Oxidizing Bacteria Ammonium Oxidizing Bacteria Activated sludge bacteria Nitrifying bacteria Nitrifying bacteria

Penicillins Tetracyclines

AMP TCN

Anaerobic bacteria Nitrosomonas europeaea Nitrifying bacteria Activated sludge bacteria Ammonium Oxidizing Bacteria

TCN hydrochloride

ANAMMOX bacteria

CTC

Nitrosomonas europeaea Activated sludge bacteria Anaerobic bacteria

OTC

DXC

Nitrosomonas europeaea Nitrifying bacteria Activated sludge bacteria ANAMMOX bacteria Anaerobic bacteria

Nitroimidazoles

MNZ

Activated sludge bacteria

Lincosamides

LIN

Nitrifying bacteria of activated sludge (UN a = 4.95) Nitrifying bacteria of activated sludge (UN a = 6.81)

a

UN – mg NH4-N/gSSV/h.

Halling-Sørensen (2001)

566

P. Välitalo et al. / International Journal of Hygiene and Environmental Health 220 (2017) 558–569

Table 5 Comparison of lowest effective concentrations reported for antibiotics for cyanobacteria, green algae, marine and wastewater bacteria. Red color indicates effect concentrations below 0.05 mg/L, and dark green indicates high effect concentrations (>100 mg/L). The values are selected from the tables presented in this mini-review.

4. Summary and future challenges The heat map (Table 5) shows that cyanobacteria and ammonium oxidizing bacteria are the most sensitive micro-organisms to antibiotics. Proteobacteria come across as least sensitive, emphasizing the need to replace the acute toxicity tests with chronic tests. However, challenges related to the development of antibiotic resistance can make the application of chronic tests more problematic than acute tests. The use of other species of bacteria or other endpoints could also provide more sensitive tools. Brandt et al. (2015) reviewed possible alternatives for the classic V.fischeri inhibition test. Those included respiration and enzyme activity assays, functional diversity assays, phospholipid fatty acid analysis (PLFA) fingerprinting, and several others. The few studies carried out with an activated sludge process show a good example of how more complex microbial communities might respond to antibiotics (Katipoglu-Yazan et al., 2013, 2015). Moreover, recent developments in the field of microbial DNA and RNA analysis offer good tools for toxicological assessments. Similar approaches could be adopted with natural microbial communities where toxicity studies are lacking. A few studies have investigated the effects of antibiotics on marine biofilms and cyanobacterial biofilms (Johansson et al., 2014a,b; Tan et al., 2016). So far, most of the studies on antibiotics have focused on single-substance type approaches, and the potential mechanisms of inhibition were described for the single substances. However, it is important to bear in mind that antibiotics never appear in environmental samples on their own but rather as very complex mixtures with hundreds or even thousands of chemicals Therefore, investigation of the mixture effect can provide more relevant data on inhibiting processes (Roose-Amsaleg and Laverman, 2016). There are some studies available on mixture effects (Backhaus et al., 2000; Yang et al., 2008; Ghosh et al., 2009; González-Pleiter et al., 2013; Magdaleno et al., 2015). González-Pleiter et al. (2013) concluded that certain specific combinations of antibiotics may pose a potential ecological risk for aquatic ecosystems at environ-

mentally relevant concentrations. Also, Ghosh et al. (2009) showed that for CLA, ENR, SMX, TCN, and TMP, the ammonium oxidation inhibition of each single compound is lower than 15% in concentration of 0.05 mg/L, whereas the mixture of compounds in the same concentration gave a 25% inhibition effect. Magdaleno et al. (2015) showed that mixture effects were more significant than single substance effects for green algae by analyzing the toxicity of several binary mixtures of antibiotics. The binary mixtures showed synergistic effects at concentrations below the EC10 of each individual antibiotic. In addition to mixture effect studies, some studies have focused on the metabolites of toxic antibiotics (López-Serna et al., 2012; Baumann et al., 2015). For example, up to 60% of the consumed clarithromycin is excreted metabolized and only 40% remain as the parent compound, indicating that a significant share of the antibiotics enter the environment transformed (Baumann et al., 2015). Baumann et al. (2015) showed that a major metabolite of clarithromycin, 14-hydroxy(R)-clarithromycin, was toxic to aquatic photoautotrophs and that the effect concentrations for the metabolite were on a similar level compared to the parent compound. And even if the metabolites are extracted as inactive compounds, they may be transferred back to the bioactive parent compounds in the environment. Other antibiotics may also have pharmacologically active metabolites that can be toxic to non-target organisms, so therefore including metabolites in ecotoxicological assessments should be considered. More research is definitely needed on the mixture effects and metabolite toxicity of antibiotics to non-target micro-organisms to provide more comprehensive knowledge on how antibiotics affect non-target micro-organisms in the environment. Furthermore, there are still big knowledge gaps in ecotoxicological data on antibiotics. As seen from the present mini-review, there is no ecotoxicological data available for all of the antibiotics that were listed as being of high ecotoxicological concern. Some antibiotics (e.g., tetracycline and oxytetracycline) have received more attention than others have. For compounds such as lincomicin, doxycycline, and sulfamethazine, only one or two studies exist.

P. Välitalo et al. / International Journal of Hygiene and Environmental Health 220 (2017) 558–569

Even azithromycin, which was detected at relatively high concentrations in effluents samples (1.22 ␮g/L) compared to other antibiotics (Biroˇsová et al., 2014), has received minimal attention. It can be concluded based on this mini-review that in general cyanobacteria and ammonium oxidizing bacteria seem the most sensitive micro-organisms to antibiotics. Regarding proteobacteria, chronic tests should be included in the testing because acute tests are not always appropriate due to the short exposure times. However, the possibility of rapid development of antibiotic resistance should be taken into account in chronic testing. Also the application of other species of bacteria and endpoints should be considered, not forgetting mixture effect studies and bacterial community studies. Though the concerns related to antibiotics in the environment have grown and more ecotoxicological studies on antibiotics have been conducted in the past decade, there are still big knowledge gaps related to antibiotics. Data on the toxicological impacts of many potentially harmful antibiotics are still lacking.

Acknowledgments The authors would like to thank the Maj ja Tor Nessling Foundation and Maa- ja vesitekniikan tuki ry. for funding this research.

References Aguirre-Martínez, G.V., Owuor, M.A., Garrido-Pérez, C., Salamanca, M.J., Del Valls, T.A., Martín-Díaz, M.L., 2015. Are standard tests sensitive enough to evaluate effects of human pharmaceuticals in aquatic biota? Facing changes in research approaches when performing risk assessment of drugs. Chemosphere 120, 75–85. Alygizakis, N.A., Gago-Ferrero, P., Borova, V.L., Pavlidou, A., Hatzianestis, I., Thomaidis, N.S., 2016. Occurrence and spatial distribution of 158 pharmaceuticals, drugs of abuse and related metabolites in offshore seawater. Sci. Total Environ. 541, 1097–1105. Ando, T., Nagase, H., Eguchi, K., Hirooka, T., Nakamura, T., Miyamoto, K., Hirata, K., 2007. A novel method using cyanobacteria for ecotoxicity test of veterinary antimicrobial agents? Environ. Toxicol. Chem. 26 (4), 601–606. Andreozzi, R., Caprio, V., Ciniglia, C., de Champdoré, M., Lo Giudice, R., Marotta, R., Zuccato, E., 2004. Antibiotics in the environment: occurrence in Italian STPs fate, and preliminary assessment on algal toxicity of amoxicillin. Environ. Sci. Technol. 38 (24), 6832–6838. Ashby, M.J., Neale, J.E., Knott, S.J., Critchley, I.A., 1994. Effect of antibiotics on non-growing planktonic cells and biofilms of Escherichia coli. J. Antimicrob. Chemother. 33 (3), 443–452. Backhaus, T., Grimme, L.H., 1999. The toxicity of antibiotic agents to the luminescent bacterium Vibrio fischeri. Chemosphere 38 (14), 3291–3301. Backhaus, T., Scholze, M., Grimme, L.H., 2000. The single substance and mixture toxicity of quinolones to the bioluminescent bacterium Vibrio fischeri. Aquat. Toxicol. 49 (1), 49–61. Baquero, F., Martínez, J.L., Cantón, R., 2008. Antibiotics and antibiotic resistance in water environments. Curr. Opin. Biotechnol. 19 (3), 260–265. Barnes, K.K., Kolpin, D.W., Furlong, E.T., Zaugg, S.D., Meyer, M.T., Barber, L.B., 2008. A national reconnaissance of pharmaceuticals and other organic wastewater contaminants in the United States—I) Groundwater groundwater. Sci. Total Environ. 402 (2), 192–200. Bartelt-Hunt, S.L., Snow, D.D., Damon, T., Shockley, J., Hoagland, K., 2009. The occurrence of illicit and therapeutic pharmaceuticals in wastewater effluent and surface waters in Nebraska? Environ. Pollut. 157 (3), 786–791. Batt, A.L., Kim, S., Aga, D.S., 2007. Comparison of the occurrence of antibiotics in four full-scale wastewater treatment plants with varying designs and operations. Chemosphere 68 (3), 428–435. Baumann, M., Weiss, K., Maletzki, D., Schüssler, W., Schudoma, D., Kopf, W., Kühnen, U., 2015. Aquatic toxicity of the macrolide antibiotic clarithromycin and its metabolites. Chemosphere 120, 192–198. Braschi, I., Blasioli, S., Fellet, C., Lorenzini, R., Garelli, A., Pori, M., Giacomini, D., 2013. Persistence and degradation of new ␤-lactam antibiotics in the soil and water environment. Chemosphere 93 (1), 152–159. Berman-Frank, I., Lundgren, P., Falkowski, P., 2003. Nitrogen fixation and photosynthetic oxygen evolution in cyanobacteria. Res. Microbiol. 154 (3), 157–164. ´ Białk-Bielinska, A., Stolte, S., Arning, J., Uebers, U., Böschen, A., Stepnowski, P., Matzke, M., 2011. Ecotoxicity evaluation of selected sulfonamides. Chemosphere 85 (6), 928–933. ˇ Biroˇsová, L., Mackulak, T., Bodík, I., Ryba, J., Skubák, J., Grabic, R., 2014. Pilot study of seasonal occurrence and distribution of antibiotics and drug resistant bacteria in wastewater treatment plants in Slovakia. Sci. Total Environ. 15 (490), 440–444.

567

Bradel, B.G., Preil, W., Jeske, H., 2000. Remission of the free-branching pattern of euphorbia pulcherrima by tetracycline treatment. J. Phytopathol. 148 (11–12), 587–590. Brandt, K.K., Amézquita, A., Backhaus, T., Boxall, A., Coors, A., Heberer, T., Lawrence, J.R., Lazorchak, J., Schönfeld, J., Snape, J.R., Zhu, Y.-G., Topp, E., 2015. Ecotoxicological assessment of antibiotics: a call for improved consideration of microorganisms. Environ. Int. 85, 189–205. Brausch, J.M., Connors, K.A., Brooks, B.W., Rand, G.M., 2012. Human pharmaceuticals in the aquatic environment: a review of recent toxicological studies and considerations for toxicity testing. Reviews of Environmental Contamination and Toxicology, vol. 218. Springer, US, pp. 1–99. Brown, K.D., Kulis, J., Thomson, B., Chapman, T.H., Mawhinney, D.B., 2006. Occurrence of antibiotics in hospital residential, and dairy effluent, municipal wastewater, and the Rio Grande in New Mexico. Sci. Total Environ. 366 (2), 772–783. Calamari, D., Zuccato, E., Castiglioni, S., Bagnati, R., Fanelli, R., 2003. Strategic survey of therapeutic drugs in the rivers Po and Lambro in northern Italy. Environ. Sci. Technol. 37, 1241–1248. Campos, J.L., Garrido, J.M., Méndez, R., Lema, J.M., 2001. Effect of two broad-spectrum antibiotics on activity and stability of continuous nitrifying system. Appl. Biochem. Biotechnol. 95 (1), 1–10. Carucci, A., Cappai, G., Piredda, M., 2006. Biodegradability and toxicity of pharmaceuticals in biological wastewater treatment plants? J. Environ. Sci. Health A: Tox. Hazard. Subst. Environ. Eng. 41 (9), 1831–1842. Christensen, A.M., Ingerslev, F., Baun, A., 2006. Ecotoxicity of mixtures of antibiotics used in aquacultures. Environ. Toxicol. Chem. 25 (8), 2208–2215. De Liguoro, M., Di Leva, V., Dalla Bona, M., Merlanti, R., Caporale, G., Radaelli, G., 2012. Sublethal effects of trimethoprim on four freshwater organisms. Ecotoxicol. Environ. Saf. 82, 114–121. Dinh, Q.T., Alliot, F., Moreau-Guigon, E., Eurin, J., Chevreuil, M., Labadie, P., 2011. Measurement of trace levels of antibiotics in river water using on-line enrichment and triple-quadrupole LC–MS/MS? Talanta 85 (3), 1238–1245. Dong, H., Yuan, X., Wang, W., Qiang, Z., 2016. Occurrence and removal of antibiotics in ecological and conventional wastewater treatment processes: a field study. J. Environ. Manage. 178, 11–19. Dorival-García, N., Zafra-Gómez, A., Navalón, A., González-López, J., Hontoria, E., Vílchez, J.L., 2013. Removal and degradation characteristics of quinolone antibiotics in laboratory-scale activated sludge reactors under aerobic, nitrifying and anoxic conditions. J. Environ. Manage. 120, 75–83. Dosler, S., Karaaslan, E., 2014. Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides. Peptides 62, 32–37. Ebert, I., Bachmann, J., Kühnen, U., Küster, A., Kussatz, C., Maletzki, D., Schlüter, C., 2011. Toxicity of the fluoroquinolone antibiotics enrofloxacin and ciprofloxacin to photoautotrophic aquatic organisms? Environ. Toxicol. Chem. 30 (12), 2786–2792. Eguchi, K., Nagase, H., Ozawa, M., Endoh, Y.S., Goto, K., Hirata, K., et al., 2004. Evaluation of antimicrobial agents for veterinary use in the ecotoxicity test using microalgae. Chemosphere 57 (11), 1733–1738. Fernandez, I., Mosquera-Corral, A., Campos, J.L., Mendez, R., 2009. Operation of an anammox SBR in the presence of two broad-spectrum antibiotics? Process Biochem. 44 (4), 494–498. ¯ Ferrari, B., Mons, R., Vollat, B., Fraysse, B., Paxeaus, N., Giudice, R.L., et al., 2004. Environmental risk assessment of six human pharmaceuticals: are the current environmental risk assessment procedures sufficient for the protection of the aquatic environment? Environ. Toxicol. Chem. 23 (5), 1344–1354. Flemming, H.C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S.A., Kjelleberg, S., 2016. Biofilms: an emergent form of bacterial life? Nat. Rev. Microbiol. 14 (9), 563–575. Froehner, K., Backhaus, T., Grimme, L.H., 2000. Bioassays with Vibrio fischeri for the assessment of delayed toxicity. Chemosphere 40 (8), 821–828. Gao, L., Shi, Y., Li, W., Niu, H., Liu, J., Cai, Y., 2012. Occurrence of antibiotics in eight sewage treatment plants in Beijing, China. Chemosphere 86 (6), 665–671. García-Galán, M.J., Díaz-Cruz, M.S., Barceló, D., 2012. Occurrence and fate of sulfonamide antibiotics in surface waters: climatic effects on their presence in the Mediterranean region and aquatic ecosystem vulnerability. In: The Llobregat. Springer, Berlin, Heidelberg, pp. 167–192. Ghosh, G.C., Okuda, T., Yamashita, N., Tanaka, H., 2009. Occurrence and elimination of antibiotics at four sewage treatment plants in Japan and their effects on bacterial ammonia oxidation. Water Sci. Technol. 59 (4), 779–786. Giang, C.N.D., Sebesvari, Z., Renaud, F., Rosendahl, I., Minh, Q.H., Amelung, W., 2015. Occurrence and dissipation of the antibiotics sulfamethoxazole, sulfadiazine, trimethoprim, and enrofloxacin in the mekong delta, vietnam. PLoS One 10 (7), e0131855. González-Pleiter, M., Gonzalo, S., Rodea-Palomares, I., Leganés, F., Rosal, R., Boltes, K., et al., 2013. Toxicity of five antibiotics and their mixtures towards photosynthetic aquatic organisms: implications for environmental risk assessment. Water Res. 47 (6), 2050–2064. ´ M., Ginebreda, A., Barceló, D., 2010. Removal of pharmaceuticals Gros, M., Petrovic, during wastewater treatment and environmental risk assessment using hazard indexes? Environ. Int. 36 (1), 15–26. Guo, J., Selby, K., Boxall, A.B., 2016. Effects of antibiotics on the growth and physiology of chlorophytes cyanobacteria, and a diatom. Arch. Environ. Contam. Toxicol. 71 (4), 589–602.

568

P. Välitalo et al. / International Journal of Hygiene and Environmental Health 220 (2017) 558–569

Hagenbuch, I.M., Pinckney, J.L., 2012. Toxic effect of the combined antibiotics ciprofloxacin, lincomycin, and tylosin on two species of marine diatoms. Water Res. 46 (16), 5028–5036. Halling-Sørensen, B., Lützhøft, H.C.H., Andersen, H.R., Ingerslev, F., 2000. Environmental risk assessment of antibiotics: comparison of mecillinam, trimethoprim and ciprofloxacin. J. Antimicrob. Chemother. 46 (suppl. 1), 53–58. Halling-Sørensen, B., Sengeløv, G., Ingerslev, F., Jensen, L.B., 2003. Reduced antimicrobial potencies of oxytetracycline, tylosin, sulfadiazin, streptomycin, ciprofloxacin, and olaquindox due to environmental processes. Arch. Environ. Contam. Toxicol. 44 (1), 7–16. Halling-Sørensen, B., 2000. Algal toxicity of antibacterial agents used in intensive farming. Chemosphere 40 (7), 731–739. Halling-Sørensen, B., 2001. Inhibition of aerobic growth and nitrification of bacteria in sewage sludge by antibacterial agents? Arch. Environ. Contam. Toxicol. 40 (4), 451–460. Hernando, M.D., De Vettori, S., Bueno, M.M., Fernández-Alba, A.R., 2007. Toxicity evaluation with Vibrio fischeri test of organic chemicals used in aquaculture. Chemosphere 68 (4), 724–730. Isidori, M., Lavorgna, M., Nardelli, A., Pascarella, L., Parrella, A., 2005. Toxic and genotoxic evaluation of six antibiotics on non-target organisms. Sci. Total Environ. 346 (1), 87–98. Ji, J.Y., Xing, Y.J., Ma, Z.T., Cai, J., Zheng, P., Lu, H.F., 2013. Toxicity assessment of anaerobic digestion intermediates and antibiotics in pharmaceutical wastewater by luminescent bacterium. J. Hazard. Mater. 246, 319–323. Johansson, C.H., Janmar, L., Backhaus, T., 2014a. Triclosan causes toxic effects to algae in marine biofilms, but does not inhibit the metabolic activity of marine biofilm bacteria. Mar. Pollut. Bull. 84 (1), 208–212. Johansson, C.H., Janmar, L., Backhaus, T., 2014b. Toxicity of ciprofloxacin and sulfamethoxazole to marine periphytic algae and bacteria. Aquat. Toxicol. 156, 248–258. Kümmerer, K., Al-Ahmad, A., Mersch-Sundermann, V., 2000. Biodegradability of some antibiotics, elimination of the genotoxicity and affection of wastewater bacteria in a simple test. Chemosphere 40 (7), 701–710. Kümmerer, K., Alexy, R., Hüttig, J., Schöll, A., 2004. Standardized tests fail to assess the effects of antibiotics on environmental bacteria. Water Res. 38 (8), 2111–2116. Kümmerer, K., 2009. Antibiotics in the aquatic environment—a review—part I. Chemosphere 75, 417–434. Kasai, K., Kanno, T., Endo, Y., Wakasa, K., Tozawa, Y., 2004. Guanosine tetra-and pentaphosphate synthase activity in chloroplasts of a higher plant: association with 70S ribosomes and inhibition by tetracycline. Nucleic Acids Res. 32 (19), 5732–5741. Kasprzyk-Hordern, B., Dinsdale, R.M., Guwy, A.J., 2008a. The occurrence of pharmaceuticals, personal care products, endocrine disruptors and illicit drugs in surface water in South Wales, UK. Water Res. 42, 3498–3518. Kasprzyk-Hordern, B., Dinsdale, R.M., Guwy, A.J., 2008b. Multiresidue methods for the analysis of pharmaceuticals, personal care products and illicit drugs in surface water and wastewater by solid-phase extraction and ultra performance liquid chromatography–electrospray tandem mass spectrometry. Anal. Bioanal. Chem. 391, 1293–1308. Kasprzyk-Hordern, B., Dinsdale, R.M., Guwy, A.J., 2009. The removal of pharmaceuticals personal care products, endocrine disruptors and illicit drugs during wastewater treatment and its impact on the quality of receiving waters. Water Res. 43, 363–380. Katipoglu-Yazan, T., Pala-Ozkok, I., Ubay-Cokgor, E., Orhon, D., 2013. Acute impact of erythromycin and tetracycline on the kinetics of nitrification and organic carbon removal in mixed microbial culture. Bioresour. Technol. 144, 410–419. Katipoglu-Yazan, T., Merlin, C., Pons, M.N., Ubay-Cokgor, E., Orhon, D., 2015. Chronic impact of tetracycline on nitrification kinetics and the activity of enriched nitrifying microbial culture. Water Res. 72, 227–238. Kelly 2nd, R.T., Henriques, I.D., Love, N.G., 2004. Chemical inhibition of nitrification in activated sludge. Biotechnol. Bioeng. 85 (6), 683–694. Kersters, K., Vos, P., Gillis, M., Swings, J., Vandamme, P., Stackebrandt, E., 2006. Introduction to the Proteobacteria. In: Balows, A., Dworkin, M., Hardes, W., Schleifer, K.H., Trüper, H.G. (Eds.), The Prokaryotes. An Electronic Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, and Applications. Springer-Verlag KG, Berlin, Germany. Kim, Y., Choi, K., Jung, J., Park, S., Kim, P.G., Park, J., 2007. Aquatic toxicity of acetaminophen carbamazepine, cimetidine, diltiazem and six major sulfonamides, and their potential ecological risks in Korea. Environ. Int. 33 (3), 370–375. Kimosop, S.J., Getenga, Z.M., Orata, F., Okello, V.A., Cheruiyot, J.K., 2016. Residue levels and discharge loads of antibiotics in wastewater treatment plants (WWTPs), hospital lagoons, and rivers within Lake Victoria Basin, Kenya. Environ. Monit. Assess. 188 (9), 532. Kolar, B., Arnuˇs, L., Jeretin, B., Gutmaher, A., Drobne, D., Durjava, M.K., 2014. The toxic effect of oxytetracycline and trimethoprim in the aquatic environment. Chemosphere 115, 75–80. ´ M., Barceló, D., 2012. Occurrence and distribution of López-Serna, R., Petrovic, multi-class pharmaceuticals and their active metabolites and transformation products in the Ebro River basin (NE Spain). Sci. Total Environ. 440, 280–289. Lützhøft, H.C.H., Halling-Sørensen, B., Jørgensen, S.E., 1999. Algal toxicity of antibacterial agents applied in Danish fish farming? Arch. Environ. Contam. Toxicol. 36 (1), 1–6.

Lee, Y.J., Lee, S.E., Lee, D.S., Kim, Y.H., 2008. Risk assessment of human antibiotics in Korean aquatic environment. Environ. Toxicol. Pharmacol. 26 (2), 216–221. Leung, H.W., Minh, T.B., Murphy, M.B., Lam, J.C., So, M.K., Martin, M., et al., 2012. Distribution, fate and risk assessment of antibiotics in sewage treatment plants in Hong Kong, South China. Environ. Int. 42, 1–9. Liu, B.Y., Nie, X.P., Liu, W.Q., Snoeijs, P., Guan, C., Tsui, M.T., 2011. Toxic effects of erythromycin, ciprofloxacin and sulfamethoxazole on photosynthetic apparatus in Selenastrum capricornutum. Ecotoxicol. Environ. Saf. 74 (4), 1027–1035. Liu, Y., Zhang, J., Gao, B., Feng, S., 2014. Combined effects of two antibiotic contaminants on Microcystis aeruginosa. J. Hazard. Mater. 279, 148–155. Lotti, T., Cordola, M., Kleerebezem, R., Caffaz, S., Lubello, C., Van Loosdrecht, M.C.M., 2012. Inhibition effect of swine wastewater heavy metals and antibiotics on anammox activity. Water Sci. Technol. 66 (7), 1519–1526. Louvet, J.N., Heluin, Y., Attik, G., Dumas, D., Potier, O., Pons, M.N., 2010. Assessment of erythromycin toxicity on activated sludge via batch experiments and microscopic techniques (epifluorescence and CLSM)? Process Biochem. 45 (11), 1787–1794. Luo, Y., Guo, W., Ngo, H.H., Nghiem, L.D., Hai, F.I., Zhang, J., et al., 2014. A review on the occurrence of micropollutants in the aquatic environment and their fate and removal during wastewater treatment. Sci. Total Environ. 473, 619–641. Magdaleno, A., Saenz, M.E., Juárez, A.B., Moretton, J., 2015. Effects of six antibiotics and their binary mixtures on growth of Pseudokirchneriella subcapitata. Ecotoxicol. Environ. Saf. 113, 72–78. Martinez, J.L., 2009. Environmental pollution by antibiotics and by antibiotic resistance determinants. Environ. Pollut. 157, 2893–2902. Martins, N., Pereira, R., Abrantes, N., Pereira, J., Gonc¸alves, F., Marques, C.R., 2012. Ecotoxicological effects of ciprofloxacin on freshwater species: data integration and derivation of toxicity thresholds for risk assessment. Ecotoxicology 21 (4), 1167–1176. Marx, C., Mühlbauer, V., Krebs, P., Kuehn, V., 2015. Environmental risk assessment of antibiotics including synergistic and antagonistic combination effects. Sci. Total Environ. 524, 269–279. Matsumoto, H., Takechi, K., Sato, H., Takio, S., Takano, H., 2012. Treatment with antibiotics that interfere with peptidoglycan biosynthesis inhibits chloroplast division in the desmid Closterium. PLoS One 7 (7), e40734. Mitchell, S.M., Ullman, J.L., Teel, A.L., Watts, R.J., 2014. pH and temperature effects on the hydrolysis of three ␤-lactam antibiotics: ampicillin, cefalotin and cefoxitin. Sci. Total Environ. 466, 547–555. Mitsui, A., Kumazawa, S., Takahashi, A., Ikemoto, H., Cao, S., Arai, T., 1986. Strategy by which nitrogen-fixing unicellular cyanobacteria grow photoautotrophically. Nature 323, 720–722. Moullan, N., Mouchiroud, L., Wang, X., Ryu, D., Williams, E.G., Mottis, A., et al., 2015. Tetracyclines disturb mitochondrial function across eukaryotic models: a call for caution in biomedical research. Cell Rep. 10 (10), 1681–1691. Mulo, P., Pursiheimo, S., Hou, C.X., Tyystjärvi, T., Aro, E.M., 2003. Multiple effects of antibiotics on chloroplast and nuclear gene expression. Funct. Plant Biol. 30 (11), 1097–1103. Na, G., Fang, X., Cai, Y., Ge, L., Zong, H., Yuan, X., et al., 2013. Occurrence, distribution, and bioaccumulation of antibiotics in coastal environment of Dalian, China. Mar. Pollut. Bull. 69 (1), 233–237. Ngumba, E., Gachanja, A., Tuhkanen, T.1, 2016. Occurrence of selected antibiotics and antiretroviral drugs in Nairobi River Basin, Kenya. Sci. Total Environ. 539, 206–213. Olson, M.E., Ceri, H., Morck, D.W., Buret, A.G., Read, R.R., 2002. Biofilm bacteria: formation and comparative susceptibility to antibiotics. Can. J. Vet. Res. 66 (2), 86. Pailler, J.Y., Krein, A., Pfister, L., Hoffmann, L., Guignard, C., 2009. Solid phase extraction coupled to liquid chromatography–tandem mass spectrometry analysis of sulfonamides, tetracyclines, analgesics and hormones in surface water and wastewater in Luxembourg. Sci. Total Environ. 407 (16), 4736–4743. Pan, X., Zhang, D., Chen, X., Mu, G., Li, L., Bao, A., 2009. Effects of levofloxacin hydrochlordie on photosystem II activity and heterogeneity of Synechocystis sp. Chemosphere 77 (3), 413–418. Park, S., Choi, K., 2008. Hazard assessment of commonly used agricultural antibiotics on aquatic ecosystems. Ecotoxicology 17 (6), 526–538. Qin, M., Lin, Z., Wang, D., Long, X., Zheng, M., Qiu, Y., 2016. What are the differences between aerobic and anaerobic toxic effects of sulfonamides on Escherichia coli? Environ. Toxicol. Pharmacol. 41, 251–258. Robinson, A.A., Belden, J.B., Lydy, M.J., 2005. Toxicity of fluoroquinolone antibiotics to aquatic organisms? Environ. Toxicol. Chem. 24 (2), 423–430. Rodríguez-Gil, J.L., Catalá, M., Alonso, S.G., Maroto, R.R., Valcárcel, Y., Segura, Y., et al., 2010. Heterogeneous photo-Fenton treatment for the reduction of pharmaceutical contamination in Madrid rivers and ecotoxicological evaluation by a miniaturized fern spores bioassay. Chemosphere 80 (4), 381–388. Ronquillo, M.G., Hernandez, J.C.A., 2017. Antibiotic and synthetic growth promoters in animal diets: review of impact and analytical methods. Food Control 72, 255–267. Roose-Amsaleg, C., Laverman, A.M., 2016. Do antibiotics have environmental side-effects? Impact of synthetic antibiotics on biogeochemical processes. Environ. Sci. Pollut. Res. 23 (5), 4000–4012. Santos, L.H., Araújo, A.N., Fachini, A., Pena, A., Delerue-Matos, C., Montenegro, M.C.B.S.M., 2010. Ecotoxicological aspects related to the presence of pharmaceuticals in the aquatic environment. J. Hazard. Mater. 175 (1), 45–95.

P. Välitalo et al. / International Journal of Hygiene and Environmental Health 220 (2017) 558–569 Sanz, J.L., Rodriguez, N., Amils, Á.R., 1996. The action of antibiotics on the anaerobic digestion process. Appl. Microbiol. Biotechnol. 46 (5–6), 587–592. Sarmah, A.K., Meyer, M.T., Boxall, A.B., 2006. A global perspective on the use sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 65 (5), 725–759. Schmidt, S., Winter, J., Gallert, C., 2012. Long-term effects of antibiotics on the elimination of chemical oxygen demand, nitrification, and viable bacteria in laboratory-scale wastewater treatment plants. Arch. Environ. Contam. Toxicol. 63 (3), 354–364. Seoane, M., Rioboo, C., Herrero, C., Cid, Á., 2014. Toxicity induced by three antibiotics commonly used in aquaculture on the marine microalga Tetraselmis suecica (Kylin) Butch. Mar. Environ. Res. 101, 1–7. Stewart, P.S., Costerton, J.W., 2001. Antibiotic resistance of bacteria in biofilms. Lancet 358 (9276), 135–138. Stewart, P.S., Franklin, M.J., Williamson, K.S., Folsom, J.P., Boegli, L., James, G.A., 2015. Contribution of stress responses to antibiotic tolerance in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 59 (7), 3838–3847. Tan, L.R., Xia, P.F., Sun, X.F., Guo, N., Song, C., Li, Q., Wang, S.G., 2016. Ecological insights into low-level antibiotics interfered biofilms of Synechococcus elongatus? RSC Adv. 6 (81), 78132–78135. Ternes, T.A., 1998. Occurrence of drugs in German sewage treatment plants and rivers. Water Res. 32 (11), 3245–3260. Thomulka, K.W., McGee, D.J., Lange, J.H., 1993. Detection of biohazardous materials in water by measuring bioluminescence reduction with the marine organism Vibrio harveyi. J. Environ. Sci. Health Part A 28 (9), 2153–2166. Tobajas, M., Verdugo, V., Polo, A.M., Rodriguez, J.J., Mohedano, A.F., 2016. Assessment of toxicity and biodegradability on activated sludge of priority and emerging pollutants. Environ. Technol. 37 (6), 713–721. van der Grinten, E., Pikkemaat, M.G., van den Brandhof, E.-J., Stroomberg, G.J., Kraak, M.H., 2010. Comparing the sensitivity of algal, cyanobacterial and bacterial bioassays to different groups of antibiotics. Chemosphere 80 (1), 1–6. Wang, X., Ryu, D., Houtkooper, R.H., Auwerx, J., 2015. Antibiotic use and abuse: a threat to mitochondria and chloroplasts with impact on research health, and environment. Bioessays 37 (10), 1045–1053.

569

Wang, L., Qiang, Z., Li, Y., Ben, W., 2016. An insight into the removal of fluoroquinolones in activated sludge process: sorption and biodegradation characteristics. J. Environ. Sci., http://dx.doi.org/10.1016/j.jes.2016.10.006, in press. Watkinson, A.J., Murby, E.J., Costanzo, S.D., 2007. Removal of antibiotics in conventional and advanced wastewater treatment: implications for environmental discharge and wastewater recycling. Water Res. 41 (18), 4164–4176. Watkinson, A.J., Murby, E.J., Kolpin, D.W., Costanzo, S.D., 2009. The occurrence of antibiotics in an urban watershed: from wastewater to drinking water. Sci. Total Environ. 407, 2711–2723. Wei, R., Ge, F., Huang, S., Chen, M., Wang, R., 2011. Occurrence of veterinary antibiotics in animal wastewater and surface water around farms in Jiangsu Province, China. Chemosphere 82 (10), 1408–1414. Wei, R., Ge, F., Chen, M., Wang, R., 2012. Occurrence of ciprofloxacin enrofloxacin, and florfenicol in animal wastewater and water resources. J. Environ. Qual. 41 (5), 1481–1486. Wille, K., Noppe, H., Verheyden, K., Bussche, J.V., De Wulf, E., Van Caeter, P., et al., 2010. Validation and application of an LC–MS/MS method for the simultaneous quantification of 13 pharmaceuticals in seawater. Anal. Bioanal. Chem. 397 (5), 1797–1808. Wollenberger, L., Halling-Sørensen, B., Kusk, K.O., 2000. Acute and chronic toxicity of veterinary antibiotics to Daphnia magna. Chemosphere 40 (7), 723–730. Yang, S., Carlson, K., 2003. Evolution of antibiotic occurrence in a river through pristine: urban and agricultural landscapes. Water Res. 37, 4645–4656. Yang, L.H., Ying, G.G., Su, H.C., Stauber, J.L., Adams, M.S., Binet, M.T., 2008. Growth-inhibiting effects of 12 antibacterial agents and their mixtures on the freshwater microalga pseudokirchneriella subcapitata. Environ. Toxicol. Chem. 27 (5), 1201–1208. Yasser, E.N., Adli, A., 2015. Toxicity of single and mixtures of antibiotics to cyanobacteria. J. Environ. Anal. Toxicol. 5 (3), 1. Zuccato, E., Castiglioni, S., Bagnati, R., Melis, M., Fanelli, R., 2010. Source, occurrence and fate of antibiotics in the Italian aquatic environment. J. Hazard. Mater. 179 (1), 1042–1048.