Progress of research on the toxicology of antibiotic pollution in aquatic organisms

Acta Ecologica Sinica 38 (2018) 36–41

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Progress of research on the toxicology of antibiotic pollution in aquatic organisms Lili Liu, Wei Wu, Jiayu Zhang, Peng Lv, Lei Xu, Yanchun Yan ⁎ Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China

a r t i c l e

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Article history: Received 18 July 2016 Received in revised form 30 November 2016 Accepted 9 January 2017 Keywords: Antibiotics Non-target organisms Aquatic vertebrate Developmental toxicity

a b s t r a c t Antibiotics are widely used to improve human and animal health and treat infections. Antibiotics are often used in livestock farms and fisheries to prevent diseases and promote growth. Recently, there has been increasing interest in the presence of antibiotics in aquatic environments. Low levels of antibiotic components are frequently detected in surface water, seawater, groundwater, and even drinking water. Antibiotics are consistently and continually discharged into the natural environment as parent molecules or metabolites, which are usually soluble and bioactive, and this results in a pseudo and persistent pollution. The effects of environmental antibiotic toxicity on non-target organisms, especially aquatic organisms, have become an increasing concern. Although antibiotics have been detected worldwide, their ecological and developmental effects have been poorly investigated, particularly in non-target organisms. This review describes the toxicity and underlying mechanism of antibiotic contamination in aquatic organisms, including the effects on vertebrate development. A considerable number of antibiotic effects on aquatic organisms have been investigated using acute toxicity assays, but only very little is known about the long-term effects. Aquatic photosynthetic autotrophs, such as Pseudokirchneriella subcapitata, Anabaena flos-aquae, and Lemna minor, were previously used for antibiotic toxicity tests because of low cost, simple operation, and high sensitivity. Certain antibiotics show a different degree of potency in algal toxicity tests on the basis of different test algae. Antibiotics inhibit protein synthesis, chloroplast development, and photosynthesis, ultimately leading to growth inhibition; some organisms exhibit growth stimulation at certain antibiotic concentrations. Daphnia magna and other aquatic invertebrates have also been used for checking the toxicity priority of antibiotics. When investigating the acute effect of antibiotics (e.g., growth inhibition), concentrations in standard laboratory organisms are usually about two or three orders of magnitude higher than the maximal concentrations in the aquatic environment, resulting in the underestimation of antibiotic hazards. Vertebrate organisms show a promising potential for chronic toxicity and potentially subtle effects of antibiotics, particularly on biochemical processes and molecular targets. The adverse developmental effects of macrolides, tetracyclines, sulfonamides, quinolones, and other antibiotic groups have been evaluated in aquatic vertebrates such as Danio rerio and Xenopus tropicalis. In acute toxicity tests, low levels of antibiotics have systematic teratogenic effects on fish. The effects of antibiotics on oxidative stress enzymes and cytochrome P450 have been investigated. Cytotoxicity, neurotoxicity, and genotoxicity have been observed for certain antibiotic amounts. However, there are no firm conclusions regarding the chronic toxicity of antibiotics at environmentally relevant levels because of the lack of long-term exposure studies. Herein, future perspectives and challenges of antibiotic toxicology were discussed. Researchers should pay more attention to the following points: chronic toxicity and potentially subtle effects of environmentally relevant antibiotics on vertebrates; effects of toxicity on biochemical processes and mode of action; combined toxicity of antibiotics and other antibiotics, metabolites, and heavy metals; and environmental factors such as temperature and pH. © 2018 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

1. Introduction Various antibiotics are widely and clinically used as over-thecounter (OTC) drugs with usually broad antimicrobial spectrum. Large amounts of antibiotics are also used to promote animal growth, inhibit ⁎ Corresponding author. E-mail address: [email protected] (Y. Yan).

https://doi.org/10.1016/j.chnaes.2018.01.006 1872-2032/© 2018 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

bacteria reproduction, prevent and cure diseases in aquaculture and livestock farm. The huge consumption of common antibiotics and the trait that most of the active antibiotics and their metabolites are water soluble lead to the pseudo and persistent pollution in aquatic environment and potential threat to ecosystem including human. The traditional view that antibiotics inhibit the bacteria growth and proliferation with no harm to the host is twisted now. Increasing evidence suggests that the parent antibiotics and metabolites discarded into surroundings

L. Liu et al. / Acta Ecologica Sinica 38 (2018) 36–41

are bioactive and persistent in low concentration (ng/L–μg/L) (Table 1), posting a potential hazard in food-chain system [12]. In 2013, the usage of 36 common antibiotics in China was 92,700 tons, among which 54,000 tons was excreted by human and animals, and an overwhelming majority entered into the surroundings [13]. Antibiotics are frequently detected in natural environment from different nations and regions, grabbing the attention to the toxicity in non-target organisms. Blue algae are sensitive to most antibiotics while daphnids and fish are more sensitive to macrolides [14]. An ecological risk assessment of over 226 antibiotics [15] suggests that 20% antibiotics are very toxic to algae (EC50 b 1 mg/L), while 16% were extremely toxic (EC50 b 0.1 mg/L) and 44% very toxic to daphnids. Meanwhile N50% antibiotics are predicted to be toxic (EC50 b 10 mg/L) and one third very toxic to fish. The research grabs attention to ecological risk of antibiotic pollutions on non-target organisms and suggests that sensitivity of different species to antibiotics varies. Certain antibiotics might show different toxicity between autotroph and heterotroph, or between lowly forms and higher lives. Therefore, based on test species and antibiotic structures, we reviewed the toxicology of common antibiotics on autotrophic hydrophytes (algae mainly), aquatic invertebrates and aquatic vertebrates. The three groups of organisms represent different nutrition and metabolism pathways, showing distinct different response to antibiotic pollution. 2. Toxicity of antibiotics on algae With low cost, high sensitivity and simplicity in manipulation, algae are one of the first applied to ecological risk assessment of antibiotics. 2.1. Ecotoxicity assessment and sensitivity rank analysis of antibiotics on algae Initially classical toxicity assessment tests of antibiotics based on algae are conducted with median effective concentration (EC50) or median lethal concentration (LC50) as main content. For example, Yang et al. [16] evaluated and ranked the growth inhibiting effects of 10 antibiotics such as roxithromycin and clarithromycin on Selenastrum capricornutum Pseudokirchneriella subcapitata based on the findings of 72 h-EC50, whose values varied from μg/L to mg/L. With longer exposure time of 7 days, the EC50 values of Lemna minor to erythrocin and tetracycline are 5.6 g/L and 4 g/L [17], separately, higher than that of microalgae (μg/L–mg/L). The variety of toxicity parameters in different test species

Table 1 Prominent aquatic antibiotics and their environmental concentrations. Major groups

Typical antibiotic

Environmental concentration

β-Lactams Aminoglycosides Macrolides

Lincomycin Gentamicin Erythromycin-H2O Clarithromycin Roxithromycin Tetracycline Oxytetracycline Chlortetracycline Sulfamethoxazole

Surface water 248.9 ng/L [1] Sewage plant export 1300 ng/L [2] Surface water 1700 ng/L [3] Surface water 260 ng/L [4] Surface water 560 ng/L [4] Underground water 3.8 ng/L [5] Surface water 340 ng/L1 [3] Surface water 690 ng/L [3] Surface water 1900 ng/L [3] Underground water 470 ng/L [4] Surface water 130 ng/L [3] Surface water 4660 ng/L [6] Underground water 160 ng/L [4] Surface water 460 ng/L [6] Sewage plant export 260 ng/L [7] Surface water 185 ng/L [8] Surface water 208 ng/L [9] Surface water 89 ng/L [9] Sewage plant export 210 ng/L [10] Livestock farm export 680 ng/L [11] Underground water 3 ng/L [5]

Tetracyclines

Sulfonamides

Sulfamethizole Sulfamethazine

Quinolones

Sulfadoxine Ciprofloxacin Norfloxacin Ofloxacin Enrofloxacin

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suggests the species diversity in toxic sensitivity to certain antibiotics. For example, the cyanobacterium Anabaena flosaquae and macrophyte Lemna minor exhibit higher sensibility to fluoroquinolones antibiotics than the freshwater microalga Desmodesmus subspicatus or the dicotyledonous macrophyte Myriophyllum spicatum [18]. 2.2. The toxicity of antibiotics on algae The toxic effects of antibiotics including tetracyclines [19], sulfonamides [20] and macrolides [17] and many other antibiotics on algae present mainly as adverse impact of growth and development, especially growth inhibition. For example, long-term exposure of sulfathiazole might induce growth inhibition on the macroalgae Ulva lactuca [21] and Lemna gibba [22], while tetracyclines, oxytetracycline and aureomycin would influence the cell growth in Microcystis aeruginosa [23]. An underlying mechanism for antibiotics to inhibit growth is the inducible production of abscisic acid [17]. Sulfonamides are reported to inhibit growth via influencing chlorophyll biosynthesis pathway [20]. Growth inhibiting is not the only effect of antibiotics on algae. Certain concentration of cefradine can stimulate the growth of Selenastrum capricornutum. Another example is the hormesis of cefalexin to the same algae, namely, promoting growth in low dosage and inhibition in high dosage [19]. Similarly, 0.001–0.1 μg/L erythrocin could stimulate the growth and photosynthesis while higher levels have the opposite effects to Microcystis flos-aquae [24]. Antibiotics affect algae photosynthesis via inhibiting chloroplast formation and protein biosynthesis [25] and damaging chlorophyll [26]. The decrease of chlorophyll weakens the capacity of photosynthesis and metabolism, resulting in inhibition of cell proliferation and growth. Hydrophyte containing chlorophyll is susceptive to even low levels of sulfonamide antibiotics. The EC50 of sulfamethoxazole to Synechococcus leopoliensis is reported as 0.0268 mg/L [20], far below the sulfamethoxazole concentrations in surroundings. Oxidative stress defense response is induced in algae when exposed to antibiotic pollution. Norfloxacin affects the antioxidative enzymes activity such as catalase (CAT) and glutathione S-transferase (GST) in a dosage-dependent manner [27]. Oxytetracycline (μg/L) is also reported to induce CAT and peroxidase activity changes significantly to activate enzymatic defense in aquatic plant [28]. Recently, Liu et al. [29] identified the candidate target proteins and inferred that the cellular biosynthesis process and the metabolism pathway were involved in the proteomic responses of cyanobacteria (Microcystis aeruginosa) exposed to amoxicillin. While this is the first study on the proteomic response of cyanobacteria to antibiotics, no more verification and further research on the key target proteins were conducted in this study. 2.3. Joint toxicity of antibiotics on algae Mixture of antibiotics might pose joint toxicity. Simple additive effects can be found in binary mixtures of a variety of antibiotics such as sulfonamides and other antibiotics or tylosin and macrolide antibiotics. Synergistic effects come from the binary mixtures of the same class or some combined drugs such as trimethoprim and sulfonamides [16]. Moreover, antagonistic effects are resulted from binary mixtures of tetracycline and 7-aminocephalosporanic acid, a main degradation product of beta-lactam antibiotic cephalosporin [19]. 3. Toxicity of antibiotics on aquatic invertebrates Aquatic invertebrate Daphnia magna is a popular species for the contaminant toxicity test. Related standard test guidelines such as Daphnia magna Acute Immobilisation Test (OECD 202) and Daphnia magna Reproduction Test (OECD 211) promote the application and interactive comparison analysis of data from different research groups. Also,

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Daphtoxkit F™ and other kits are commercially-available for researchers developing experiments rapidly. Nearly two decades ago, Migliore et al. [30] detected and ranked the toxicity of some primary agricultural antibiotics according to their lethal effect on nauplii of Artemia between 24 and 120 h (Table 2). The method was afterwards reformed by Wollenburger team in their research with Daphnia magna as test species [31], in which acute (EC50, mg/L) and chronic (EC50, mg/L) toxicity of antibiotics were separately determined (Table 2). The chronic toxicity was mainly based on reproduction test and the effect concentration was one magnitude lower than the acute toxic levels. The work drives the endpoints of toxicity from extreme toxic effect (lethality) into effective toxic effect (teratogenesis) covering acute and long-term exposure. Now, not only statistical analysis such as no observed effect concentration (NOEC) and EC50 [30], but also morphology observation including growth, hatch and pigmentation [31] and reproduction toxicity such as fecundity [32] are taken into consideration (Table 2). These parameters highlight the importance of the chronic toxicity test on environment relevant concentrations of antibiotics. The chronic toxicity of antibiotics on freshwater species is drawing public attention [33]. Molecular ecotoxicology researches are also a step forward, for example, ribosomal protein RpS3 mRNA abundance increases in Chironomus riparius with long-term sulfathiazole exposure [34], an evidence for DNA damage and immune response. The toxicity of antibiotics is determined by its own inner chemical construction and can be transformed by its surroundings to some degree. A typical example is that, the toxicity of sulfadiazine was 11 fold higher at pH 6.0 than at pH 8.5. The significantly high toxicity at the low pH might be caused by the high fraction of unionized sulfadiazine at acidic condition [35]. Parent compounds of chloramphenicol with high toxicity are transformed into less toxic metabolites after photooxidation treatment (Table 2), suggesting momentous effect of photooxidation on the toxicity of antibiotics [36]. 4. Toxicity of antibiotics on aquatic vertebrates Antibiotics were not considered to pose apparent ecological risk on fish at an earlier period. However, there is mounting evidence that antibiotics might be toxic to fish after acute or chronic exposure. Fish hepatocytes were more susceptible to drugs than hepatocytes from human and dog [37]. 4.1. The toxicity of macrolide antibiotics Low concentration of rapamycin (0–5 μmol/L) exposure affects zebrafish (Danio rerio) behavior [38]. Rapamycin induces zebrafish malformation such as yolk sac edema and uninflated swim bladder and Table 2 The toxicity research of antibiotics on aquatic invertebrate. Research field

Major research content

Prioritization of antibiotic toxicity

1. Toxicity assessment based on LC50: e.g., bacitracin N flumequine N lincomycin N Aminosidine N erythromycin [30]; 2. Toxicity assessment based on EC50: e.g., oxolinic acid N tiamulin N sulfadiazine N streptomycin N tylosin N oxytetracycline [31]. 1. Statistics analysis and morphology: e.g., LC50, EC50, NOEC, reproduction toxicity, teratogenesis, et al. [32]; 2. Molecular biomarker identification: e.g., RpS3 mRNA expression [34]. 1. Effect of pH on the toxicity of antibiotics: e.g., the sulfadiazine is with higher toxicity at lower pH [35]; 2. Effect of photooxidation on the toxicity of antibiotics: e.g., chloramphenicol toxicity decreases N30% after photooxidation process [36].

Effect characterization and mechanism of toxicity

Influence of surroundings on antibiotic toxicity

influences embryo spontaneous movement frequency and larval swimming movement speed. The activity of acetylcholine esterase (AChE) in brain, ethoxyresorufin-O-deethylase (EROD) and superoxide dismutase (SOD) in liver is changed significantly (P b 0.05) when Carassius auratus exposed to erythrocin [39] or roxithromycin [40]. AChE is a biomarker of neurotoxicity. SOD is a key enzyme in antioxidant system and EROD is the product of cytochrome P450 1A1 involved in xenobiotics metabolism. Though lacking verification and exploration of mechanisms, the enzyme activity studies [39,40] suggest the influence of macrolides on relevant system and necessity of toxicity research in non-target organism to antibiotics. 4.2. The toxicity of sulfonamide antibiotics Sulfonamides were once reported to be less toxic to the vertebrates than to microorganisms, algae and some plants [41]. Even though sulfathiazole and aureomycin (mg/L) might increase plasma 17-β estrogen level in medaka (Oryzias latipes), Ji et al. [42] insisted that the concentrations of environmental antibiotics were too low to pose risk to fish directly. Things changed over time. Aquatic antibiotic levels might be much higher than predicted due to the huge usage amount and inefficient elimination methods in sewage plants. Meanwhile, it's realized that ecological risk assessment should depend on more than single physiological or biochemical index as 17-β estrogen. And more complex external environmental condition and longer term exposure should be considered. Much evidence shows that weak concentrations of sulfonamide antibiotics are toxic to fish and result in developmental malformation [43], decreased locomotion ability, disordered organ function and oxidative stress [44]. Similar to macrolide antibiotics, sulfamethoxazole changes the EROD level in fish hepatocytes, suggesting that the involved signal pathway might be affected. 4.3. The toxicity of quinolone antibiotics Quinolone antibiotics and their metabolites often persist in the body for a long time, contributing to the chronic toxicity. The withdrawal time of enrofloxacin and its metabolite ciprofloxacin are predicted up to 22 days in Nile tilapia Oreochromis niloticus [45], adding to the risk of bioaccumulation toxicity. Quinolones induce the antioxidant defense system and detoxification pathway in fish. The activity of EROD, GST and SOD are changed and the antioxidant defense system is activated in male goldfish (Carassius auratus) [46] and zebrafish [47] exposed to norfloxacin. Enrofloxacin likewise changes the activity of oxidative stress enzymes significantly [48], meanwhile acts as a mechanism-based inhibitor inactivating cytochrome P450 isoform and is oxidatively deethylated into ciprofloxacin under the P450 system [49]. The metabolite ciprofloxacin could inactive the P450 enzymes too. Preliminary evidence suggests that quinolone antibiotics pose potential neurotoxicity to fish. Norfloxacin alone or combined with sulfamethoxazole may inhibit AChE activity in goldfish [46]. The activity of AChE in tra catfish (Pangasianodon hypophthalmus) is changed when exposed to enrofloxacin [48]. The DNA damage and increase of serum vitellogenin in norfloxacin treated goldfish imply the genetic toxicity and the potential estrogenic effects [46]. Molecular biomarker has been widespread applied in the field of toxicology of antibiotic contaminations, however, further research about toxic effect and molecular mechanism requires more complicated and sophisticated experimental methods. 4.4. The toxicity of tetracycline antibiotics More and more studies have been carried out on the toxicity of tetracycline antibiotics on aquatic vertebrates. The research is focused on toxicity priority and morphological depiction and statistical analysis

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toxic effect with growth, mortality and reproduction as major parameters. Recently, molecular ecotoxicology is coming into sight. However, toxicology research based on low concentrations and long-term exposure is still demanding more attention [50]. Toxicity priority research is conducted with different aquatic vertebrates. As reported, the susceptibility of Danio rerio is higher than that of Daphnia magna and lower than Carassius auratus [51]. The toxicity of tetracycline on fish is weaker than aureomycin [51], while its toxicity on Xenopus tropicalis embryo is stronger than chloramphenicol and erythrocin [52]. The toxic effect of tetracycline on Xenopus tropicalis embryo includes shortened body length, pericardial edema, enlarged proctodaeums and other malformations [52]. In zebrafish, tetracycline induces malformations such as yolk sac edema, uninflated swim bladder and dosage-dependent growth inhibition [53]. Environmental tetracycline induces histological changes in Gambusia holbrooki such as hepatocellular vacuolization and enlargement of the sinusoids, the former may be a sign of degenerative process because of antibiotics metabolic damage [54]. Antioxidative defense mechanisms are activated with different concentrations of tetracycline antibiotics. The degree of oxidative damage depends mainly on exposure time and concentrations of tetracyclines. Lipid peroxidation byproduct and severe oxidative damage are reported [55]. Low concentrations of tetracycline increase the activity of CAT in fish Gambusia holbrooki, meaning that tetracycline induces the production of hydrogen peroxide and activates antioxidative defense mechanism without damage or lipid peroxidation [54]. Oxytetracycline [56] and aureomycin [55] are also reported with pro-oxidative effect and changed antioxidant enzyme activity in fish. The lipid peroxidation resulting from antibiotics contamination further contributes to cell apoptosis, one of the main incentives to developmental toxicity. Gene expression analysis proves that the caspasedependent apoptosis pathway plays a vital role in tetracyclineinduced apoptosis to zebrafish embryo [53]. Besides development toxicity and teratogenesis, tetracycline antibiotics may also give rise to genotoxicity. DNA damage is reported when fish are exposed to oxytetracycline within limits [57]. 4.5. Joint toxicity and the effects of environmental conditions A growing concern is the joint toxicity of antibiotics with antibiotics or other contaminations such as antibiotic metabolites, heavy metals. For example, sulfadiazine and methoxybenzyl aminopyrimidine combined will lead to oxidative stress and hepatotoxicity in gilthead sea bream Sparus aurata L. [58]. Wang's team studied the joint toxicity of fluoroquinolone and tetracycline to zebrafish on morphology [59], histopathology [60], cytobiology [61], transcriptomics [62] and proteomics [60]. The findings suggest that fluoroquinolone combined with tetracycline lead to development toxicity, cardiotoxicity, immunotoxicity and disordered locomotion behavior (neurotoxicity) in antagonistic action. The temperature and other surrounding conditions alter the toxicity of antibiotics contaminations. Within certain limits, the toxicity of oxytetracycline to Euplotes crassus decreases along with temperature increment [63]. However, the toxicity of oxytetracycline with equi-toxic concentration of copper is in additive manner when temperature rises [63]. 5. Research prospect Amounts of antibiotic pollutions coming from wastewater in factories and hospitals, urine and feces, improper disposal of discarded antibiotics and sewage water lacking efficient treatment display inordinately persistent pollution. The risk assessment of the new class of pollutants is facing challenge due to the lack of specific legislation

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and efficient disposal method in sewage treatment. Thus, we propose the potential highlight and orientation in future research. 5.1. The chronic toxicity research of low concentration antibiotics Methodologically, the toxicity research of antibiotics is acute toxicity test with rate of growth, lethality and reproduction as endpoints. The experimental EC50 values (μg/L) are usually 100–1000 times higher than the environmental concentrations (ng/L), making it less likely that the environmental antibiotics pose significant toxic effects to life in aquatic environment. The values of LC50 and EC50 decrease while the exposure time is prolonged [64]. It is necessary in consequence to conduct chronic toxicity research of environmental levels of antibiotic contaminants on the basis of developmental toxicity, target organs toxicity, genotoxicity and toxic threshold value for the environmental risk management and establishment of laws and supervision regulations. 5.2. The molecular mechanism research of antibiotic toxicity to non-target organisms The toxicology research of antibiotic pollution currently focuses on morphological description and statistical analysis of non-target life including growth inhibition [32], teratogenesis [52,59], disordered locomotion behavior [44] and cytotoxicity [53,60]. As for the molecular mechanism of toxic effects, the researches of photosynthesis, antioxidant defense, oxidative damage and especially toxicity biomarker make much progress, though the fields of neurotoxicity and genotoxicity require more efforts (Table 3). However, the research in molecular ecotoxicology of antibiotics is facing difficulties, such as definition about damage degree, quantitative relations between biomarker expression level and pollutant concentration [65]. Besides the characterization of toxic effect, metabolic pathway of bioactive components in vivo, epigenetic alterations induced by contaminations [66] and other drug-related mechanism call for further research. 5.3. Joint toxicity research of antibiotics and other contaminations In the toxicity research of antibiotics, joint toxicity with other contaminations must be taken into consideration. First of all, growing evidence shows that joint toxicity of antibiotics act in synergistic effect, antagonistic effect and so on thus cannot be predicted by single antibiotic toxicity. All binary mixtures of ampicillin, amoxicillin, cephalothin,

Table 3 The common biomarkers in toxicology field of antibiotics. Classical biomarker

Abbreviation

Related pathway/mechanism

Abscisic acid [17] Chloroplast ATP synthase [29]

Plant growth Photosynthesis

Chlorophyll [26] Triosephosphate isomerase [29]

Abscisic acid Chloroplast ATP synthase Chlorophyll TPI

Phosphoglycerate kinase [29]

PGK

Catalase [28] Glutathione S-transferase [27] Superoxide dismutase [39,40] Aldehyde dehydrogenase 2 [58] 7-Ethoxy thiopheneoxazolonedeethylase [39,40] Cytochrome P450 [49] Ribosomal protein [34]

CAT GST SOD ALDH2 EROD

Acetylcholine esterase [39,40]

CYP450 Ribosomal protein AChE

Vitellogenin [46]

Vg

Photosynthesis Carbohydrate metabolism Carbohydrate metabolism Antioxidant defense Antioxidant defense Antioxidant defense Xenobiotics metabolism Xenobiotics metabolism

Xenobiotics metabolism DNA damage& Immunity response Neural signal transmission Estrogenic effect

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ciprofloxacin, gentamycin, and vancomycin are reported to exert synergistic effect to algae growth [67]. However, mixture exposure of trimethoprim, tylosin and jiemycin is well predicted with toxicity in concentration addition model [68]. Secondly, the toxicity of antibiotics can be altered by traditional pollutants such as heavy metals [69], for instance, alter. Thirdly, antibiotics are transformed into less active but not negligible metabolites after metabolic pathway in vivo [70]. A case in point is the metabolites of doramectin, which are less toxic than parent molecule, but still highly toxic to Daphnia (48 h-EC50 b 0.0011 mg/L) [71]. Particularly, after the electrochemical oxidation, the toxicity of ofloxacin increases in algal inhibition test [72]. 5.4. Effects of surrounding conditions on antibiotics toxicity The toxicity of antibiotic contaminations can be transformed by environmental factors such as ambient light [17], pH [35] and temperature [63]. Toxicity change of pollutants to aquatic organisms is also of great concern under the trend of global warming. The effects of surrounding conditions on antibiotics toxicity are in list of notable ecological issues. Acknowledgement This work was supported by the National Natural Science Foundation of China (31540067) and by the Basic Research Fund of CAAS (0042014006). We also thank Ruth Nahurira for the manuscript revision. References [1] D. Calamari, E. Zuccato, S. Castiglioni, R. Bagnati, R. Fanelli, Strategic survey of therapeutic drugs in the rivers Po and Lambro in northern Italy, Environ. Sci. Technol. 37 (7) (2003) 1241–1248. [2] D. Loffler, T.A. Ternes, Analytical method for the determination of the aminoglycoside gentamicin in hospital wastewater via liquid chromatography electrospraytandem mass spectrometry, J. Chromatogr. A 1000 (1) (2003) 583–588. [3] D.W. Kolpin, E.T. Furlong, M.T. Meyer, E.M. Thurman, S.D. Zaugg, L.B. Barber, H.T. Buxton, Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999−2000: a national reconnaissance, Environ. Sci. Technol. 36 (6) (2002) 1202–1211. [4] R. Hirsch, T. Ternes, K. Haberer, K.L. Kratz, Occurrence of antibiotics in the aquatic environment, Sci. Total Environ. 225 (1) (1999) 109–118. [5] L. Tong, P. Li, Y. Wang, K. Zhu, Analysis of veterinary antibiotic residues in swine wastewater and environmental water samples using optimized SPE-LC/MS/MS, Chemosphere 74 (7) (2009) 1090–1097. [6] R. Wei, F. Ge, S. Huang, M. Chen, R. Wang, Occurrence of veterinary antibiotics in animal wastewater and surface water around farms in Jiangsu Province, China, Chemosphere 82 (10) (2011) 1408–1414. [7] M.S. Kostich, A.L. Batt, J.M. Lazorchak, Concentrations of prioritized pharmaceuticals in effluents from 50 large wastewater treatment plants in the US and implications for risk estimation, Environ. Pollut. 184 (2014) 354–359. [8] Y. Bai, W. Meng, J. Xu, Y. Zhang, C. Guo, Occurrence, distribution and bioaccumulation of antibiotics in the Liao River Basin in China, Environ. Sci.: Processes Impacts 16 (13) (2014) 586. [9] W. Xu, G. Zhang, X. Li, S. Zou, P. Li, Z. Hu, J. Li, Occurrence and elimination of antibiotics at four sewage treatment plants in the Pearl River Delta (PRD), South China, Water Res. 41 (19) (2007) 4526–4534. [10] P. Guerra, M. Kim, A. Shah, M. Alaee, S.A. Smyth, Occurrence and fate of antibiotic, analgesic/anti-inflammatory, and antifungal compounds in five wastewater treatment processes, Sci. Total Environ. 473-474 (2014) 235–243. [11] M. Andrieu, A. Rico, T.M. Phu, D.T.T. Huong, N.T. Phuong, P.J. Van den Brink, Ecological risk assessment of the antibiotic enrofloxacin applied to Pangasius catfish farms in the Mekong Delta, Vietnam, Chemosphere 119 (2015) 407–414. [12] D. Huang, J. Hou, T. Kuo, H. Lai, Toxicity of the veterinary sulfonamide antibiotic sulfamonomethoxine to five aquatic organisms, Environ. Toxicol. Pharmacol. 38 (3) (2014) 874–880. [13] Q. Zhang, G. Ying, C. Pan, Y. Liu, J. Zhao, Comprehensive evaluation of antibiotics emission and fate in the river basins of china: source analysis, multimedia modeling, and linkage to bacterial resistance, Environ. Sci. Technol. 49 (11) (2015) 6772–6782. [14] A. Boxall, D.W. Kolpin, B. Halling-Sorensen, J. Tolls, Are veterinary medicines causing environmental risks? Environ. Sci. Technol. 37 (15) (2003) 286A–294A. [15] H. Sanderson, R.A. Brain, D.J. Johnson, C.J. Wilson, K.R. Solomon, Toxicity classification and evaluation of four pharmaceuticals classes: antibiotics, antineoplastics, cardiovascular, and sex hormones, Toxicology 203 (1) (2004) 27–40. [16] L. Yang, G. Ying, H. Su, J.L. Stauber, M.S. Adams, M.T. Binet, Growth-inhibiting effects of 12 antibacterial agents and their mixtures on the freshwater microalga Pseudokirchneriella subcapitata, Environ. Toxicol. Chem. 27 (5) (2008) 1201–1208.

[17] F. Pomati, A.G. Netting, D. Calamari, B.A. Neilan, Effects of erythromycin, tetracycline and ibuprofen on the growth of Synechocystis sp. and Lemna minor, Aquat. Toxicol. 67 (4) (2004) 387–396. [18] I. Ebert, J. Bachmann, U. Kühnen, A. Küster, C. Kussatz, D. Maletzki, C. Schlüter, Toxicity of the fluoroquinolone antibiotics enrofloxacin and ciprofloxacin to photoautotrophic aquatic organisms, Environ. Toxicol. Chem. 30 (12) (2011) 2786–2792. [19] L. Gao, L.J. Shi, T. Yuan, Growth inhibitive effect of typical antibiotics and their mixtures on Selenastrum capricornutum, J. Environ. Health 30 (6) (2013) 475–478. [20] B. Ferrari, R. Mons, B. Vollat, B. Fraysse, N. Paxeus, R. Lo Giudice, A. Pollio, J. Garric, 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) (2004) 1344–1354. [21] S. Leston, M. Nunes, I. Viegas, C. Nebot, A. Cepeda, M.Â. Pardal, F. Ramos, The influence of sulfathiazole on the macroalgae Ulva lactuca, Chemosphere 100 (2014) 105–110. [22] R.A. Brain, D.J. Johnson, S.M. Richards, H. Sanderson, P.K. Sibley, K.R. Solomon, Effects of 25 pharmaceutical compounds to Lemna gibba using a seven-day static-renewal test, Environ. Toxicol. Chem. 23 (2) (2004) 371. [23] A.H. Shang, J. Ye, D.H. Chen, X.X. Lu, H.D. Lu, Physiological effects of tetracycline antibiotic pollutants on non-target aquatic Microcystis aeruginosa, J. Environ. Sci. Health B 50 (1) (2015) 809–818. [24] J. Wan, P. Guo, X. Peng, K. Wen, Effect of erythromycin exposure on the growth, antioxidant system and photosynthesis of Microcystis flos-aquae, J. Hazard. Mater. 283 (2015) 778–786. [25] B. Halling-Sorensen, Algal toxicity of antibacterial agents used in intensive farming, Chemosphere 40 (7) (2000) 731–739. [26] C. Vannini, G. Domingo, M. Marsoni, F. De Mattia, M. Labra, S. Castiglioni, M. Bracale, Effects of a complex mixture of therapeutic drugs on unicellular algae Pseudokirchneriella subcapitata, Aquat. Toxicol. 101 (2) (2011) 459–465. [27] X.P. Nie, J.Y. Lu, X. Li, Y.F. Yang, Toxic effects of norfloxacin on the growth and the activity of antioxidase of Chlorella pyrenoidosa, Asian J. Ecotoxicol. 2 (3) (2007) 327–332. [28] E. Vilvert, V. Contardo-Jara, M. Esterhuizen-Londt, S. Pflugmacher, The effect of oxytetracycline on physiological and enzymatic defense responses in aquatic plant species Egeria densa, Azolla caroliniana, and Taxiphyllum barbieri, Toxicol. Environ. Chem. 99 (1) (2017) 104–116. [29] Y. Liu, S. Chen, J. Zhang, B. Gao, Growth, microcystin-production and proteomic responses of Microcystis aeruginosa under long-term exposure to amoxicillin, Water Res. 93 (2016) 141–152. [30] C.C.G.B. Luciana Migliore, Toxicity of several important agricultural antibiotics to Artemia, Water Res. 31 (7) (1997) 1801–1806. [31] L. Wollenberger, B. Halling-Sorensen, K.O. Kusk, Acute and chronic toxicity of veterinary antibiotics to Daphnia magna, Chemosphere 40 (7) (2000) 723–730. [32] A. Martins, L. Guimarães, L. Guilhermino, Chronic toxicity of the veterinary antibiotic florfenicol to Daphnia magna assessed at two temperatures, Environ. Toxicol. Pharmacol. 36 (3) (2013) 1022–1032. [33] N. Martins, R. Pereira, N. Abrantes, J. Pereira, F. Gonçalves, C.R. Marques, Ecotoxicological effects of ciprofloxacin on freshwater species: data integration and derivation of toxicity thresholds for risk assessment, Ecotoxicology 21 (4) (2012) 1167–1176. [34] K. Park, I. Kwak, Gene expression of ribosomal protein mRNA in Chironomus riparius: effects of endocrine disruptor chemicals and antibiotics, Comp. Biochem. Physiol. C: Pharmacol. Toxicol. 156 (2) (2012) 113–120. [35] G.G. Anskjær, C. Rendal, K.O. Kusk, Effect of pH on the toxicity and bioconcentration of sulfadiazine on Daphnia magna, Chemosphere 91 (8) (2013) 1183–1188. [36] B. Czech, I. Josko, P. Oleszczuk, Ecotoxicological evaluation of selected pharmaceuticals to Vibrio fischeri and Daphnia magna before and after photooxidation process, Ecotoxicol. Environ. Saf. 104 (2014) 247–253. [37] N. Laville, S. Ait-Aissa, E. Gomez, C. Casellas, J.M. Porcher, Effects of human pharmaceuticals on cytotoxicity, EROD activity and ROS production in fish hepatocytes, Toxicology 196 (1) (2004) 41–55. [38] J.F. Chen, X. Ma, Z.X. Gai, W.W. Li, Q.X. Dong, Acute rapamycin (RAP) exposure induce developmental, neurobehavioral and immunal toxicities in embryonic zebrafish, Environ. Chem. 33 (4) (2014) 556–561. [39] J. Liu, G. Lu, J. Ding, Z. Zhang, Y. Wang, Tissue distribution, bioconcentration, metabolism, and effects of erythromycin in crucian carp (Carassius auratus), Sci. Total Environ. 490 (2014) 914–920. [40] J. Liu, G. Lu, Y. Wang, Z. Yan, X. Yang, J. Ding, Z. Jiang, Bioconcentration, metabolism, and biomarker responses in freshwater fish Carassius auratus exposed to roxithromycin, Chemosphere 99 (2014) 102–108. [41] W. Baran, E. Adamek, J. Ziemiańska, A. Sobczak, Effects of the presence of sulfonamides in the environment and their influence on human health, J. Hazard. Mater. 196 (2011) 1–15. [42] K. Ji, K. Choi, S. Lee, S. Park, J.S. Khim, E. Jo, K. Choi, X. Zhang, J.P. Giesy, Effects of sulfathiazole, oxytetracycline and chlortetracycline on steroidogenesis in the human adrenocarcinoma (H295R) cell line and freshwater fish Oryzias latipes, J. Hazard. Mater. 182 (1–3) (2010) 494–502. [43] T. Lin, Y.Q. Chen, W. Chen, Toxic effect of sulfadiazine on the growth of zebrafish embryos in the water body, J. Saf. Environ. 14 (3) (2014) 324–327. [44] T. Lin, S. Yu, Y. Chen, W. Chen, Integrated biomarker responses in zebrafish exposed to sulfonamides, Environ. Toxicol. Pharmacol. 38 (2) (2014) 444–452. [45] W. Xu, X. Zhu, X. Wang, L. Deng, G. Zhang, Residues of enrofloxacin, furazolidone and their metabolites in Nile tilapia (Oreochromis niloticus), Aquaculture 254 (1–4) (2006) 1–8. [46] J. Liu, G. Lu, D. Wu, Z. Yan, A multi-biomarker assessment of single and combined effects of norfloxacin and sulfamethoxazole on male goldfish (Carassius auratus), Ecotoxicol. Environ. Saf. 102 (2014) 12–17.

L. Liu et al. / Acta Ecologica Sinica 38 (2018) 36–41 [47] M. Bartoskova, R. Dobsikova, V. Stancova, O. Pana, D. Zivna, L. Plhalova, J. Blahova, P. Marsalek, Norfloxacin—toxicity for zebrafish (Danio rerio) focused on oxidative stress parameters, Biomed. Res. Int. 2014 (2014) 1–6. [48] N. Wang, N. Noemie, N. Hien, T. Huynh, F. Silvestre, N. Phuong, S. Danyi, J. Widart, C. Douny, M. Scippo, P. Kestemont, D. Huong, Adverse effects of enrofloxacin when associated with environmental stress in Tra catfish (Pangasianodon hypophthalmus), Chemosphere 77 (11) (2009) 1577–1584. [49] E. Vaccaro, M. Giorgi, V. Longo, G. Mengozzi, P.G. Gervasi, Inhibition of cytochrome p450 enzymes by enrofloxacin in the sea bass (Dicentrarchus labrax), Aquat. Toxicol. 62 (1) (2003) 27–33. [50] J. Zhan, S.H. Wei, The distribution and eco-toxicology of tetracycfine in soil and water environment and its degradation: a review, Acta Ecol. Sin. 35 (9) (2015) 2820–2825. [51] H.Z. Wang, Y. Luo, W.Q. Xu, Q.X. Zhou, B.H. Tang, Y.Y. Wang, Ecotoxic effects of tetracycline and chlortetracycline on aquatic organisms, J. Agro-Environ. Sci. 27 (4) (2008) 1536–1539. [52] Z. Liu, Ecological Toxicity of Selected Antibiotics on the Embryo of Xenopus tropicalis, Shanghai Ocean University, 2012. [53] Q. Zhang, J. Cheng, Q. Xin, Effects of tetracycline on developmental toxicity and molecular responses in zebrafish (Danio rerio) embryos, Ecotoxicology 24 (4) (2015) 707–719. [54] B. Nunes, S.C. Antunes, R. Gomes, J.C. Campos, M.R. Braga, A.S. Ramos, A.T. Correia, Acute effects of tetracycline exposure in the freshwater fish Gambusia holbrooki: antioxidant effects, neurotoxicity and histological alterations, Arch. Environ. Contam. Toxicol. 68 (2) (2015) 371–381. [55] L. Dong, J. Gao, X. Xie, Q. Zhou, DNA damage and biochemical toxicity of antibiotics in soil on the earthworm Eisenia fetida, Chemosphere 89 (1) (2012) 44–51. [56] M.E. Yonar, The effect of lycopene on oxytetracycline-induced oxidative stress and immunosuppression in rainbow trout (Oncorhynchus mykiss, W.), Fish Shellfish Immunol. 32 (6) (2012) 994–1001. [57] M.M. Qu, L.W. Sun, J. Chen, Y.Q. Li, Y.G. Chen, Z.M. Kong, Toxicological characters of arsanilic acid and oxytetracycline, J. Agro-Environ. Sci. 23 (2) (2004) 240–242. [58] I. Varo, J.C. Navarro, G. Rigos, J. Del Ramo, J. Alvar Calduch-Giner, A. Hernandez, J. Pertusa, A. Torreblanca, Proteomic evaluation of potentiated sulfa treatment on gilthead sea bream (Sparus aurata L.) liver, Aquaculture 376-379 (2013) 36–44. [59] Y. Zhang, X. Wang, X. Yin, M. Shi, R.A. Dahlgren, H. Wang, Toxicity assessment of combined fluoroquinolone and tetracycline exposure in zebrafish, Environ. Toxicol. 31 (6) (2016) 736–750. [60] X. Yin, H. Wang, Y. Zhang, R.A. Dahlgren, H. Zhang, M. Shi, X.W. Ming Gao, Toxicological assessment of trace β-diketone antibiotic mixtures on zebrafish (Danio rerio) by proteomic analysis, PLoS One 9 (7) (2014), e102731.

41

[61] F. Li, H. Wang, J. Liu, J. Lin, A. Zeng, W. Ai, X. Wang, R.A. Dahlgren, H. Wang, Immunotoxicity of β-diketone antibiotic mixtures to zebrafish (Danio rerio) by transcriptome analysis, PLoS One 11 (4) (2016), e0152530. [62] H. Wang, X. Yin, F. Li, R.A. Dahlgren, Y. Zhang, H. Zhang, X. Wang, Chronic toxicological effects of β-diketone antibiotics on Zebrafish, Environ. Toxicol. 31 (11) (2016) 1357–1371. [63] A. Gomiero, A. Viarengo, Effects of elevated temperature on the toxicity of copper and oxytetracycline in the marine model, Euplotes crassus: a climate change perspective, Environ. Pollut. 194 (2014) 262–271. [64] A.J. Bartlett, V.K. Balakrishnan, J. Toito, L.R. Brown, Toxicity of four sulfonamide antibiotics to the freshwater amphipod, Environ. Toxicol. Chem. 32 (2013) 866–875. [65] L.X. Dong, X.J. Xie, Q.X. Zhou, P.P. Huang, Molecular ecotoxicology of antibiotics, an emerging type of environmental contaminants: a review, Chin. J. Ecol. 29 (10) (2010) 2042–2048. [66] J.L. Wilkinson, P.S. Hooda, J. Barker, S. Barton, J. Swinden, Ecotoxic pharmaceuticals, personal care products, and other emerging contaminants: a review of environmental, receptor-mediated, developmental, and epigenetic toxicity with discussion of proposed toxicity to humans, Crit. Rev. Environ. Sci. Technol. 46 (4) (2016) 336–381. [67] A. Magdaleno, M.E. Saenz, A.B. Juárez, J. Moretton, Effects of six antibiotics and their binary mixtures on growth of Pseudokirchneriella subcapitata, Ecotoxicol. Environ. Saf. 113 (2015) 72–78. [68] J. Guo, K. Selby, A.B.A. Boxall, Assessment of the risks of mixtures of major use veterinary antibiotics in European surface waters, Environ. Sci. Technol. 50 (15) (2016) 8282–8289. [69] Y. Zhang, X. Cai, X. Lang, X. Qiao, X. Li, J. Chen, Insights into aquatic toxicities of the antibiotics oxytetracycline and ciprofloxacin in the presence of metal: complexation versus mixture, Environ. Pollut. 166 (2012) 48–56. [70] M. Baumann, K. Weiss, D. Maletzki, W. Schüssler, D. Schudoma, W. Kopf, U. Kühnen, Aquatic toxicity of the macrolide antibiotic clarithromycin and its metabolites, Chemosphere 120 (2015) 192–198. [71] B. Halling-Sorensen, G. Sengelov, J. Tjornelund, Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria, including selected tetracycline-resistant bacteria, Arch. Environ. Contam. Toxicol. 42 (3) (2002) 263–271. [72] L. Zhu, B. Santiago-Schübel, H. Xiao, H. Hollert, S. Kueppers, Electrochemical oxidation of fluoroquinolone antibiotics: mechanism, residual antibacterial activity and toxicity change, Water Res. 102 (2016) 52–62.