- Email: [email protected]
The toxic effects of three active pharmaceutical ingredients (APIs) with different efficacy to Vibrio fischeri
Emerging Contaminants 5 (2019) 297e302
Contents lists available at ScienceDirect
Emerging Contaminants journal homepage: http://www.keaipublishing.com/en/journals/ emerging-contaminants/
The toxic effects of three active pharmaceutical ingredients (APIs) with different efficacy to Vibrio fischeri Yuying Dong*, Zheng Fang, Yan Xu, Qiying Wang, Xuejun Zou Department of Environmental Science and Engineering, Dalian Minzu University, Dalian, 116600, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2019 Received in revised form 21 August 2019 Accepted 29 August 2019
The environmental residues of active pharmaceutical ingredients (APIs) are associated with environmental risks and health problems, the influence to environment have become a matter of public concern. In this paper, the individual and joint toxicities to vibrio fischeri of three APIs, including ibuprofen, azithromycin, and triclosan were investigated. The EC50 endpoint values of the above tested APIs were 36.5 105 mol L1, 30.26 105 mol L1, and 0.0155 105 mol L1 respectively. It was indicated that the endpoint toxicities to vibrio fischeri for different mixtures are higher than those of the individual toxins. The joint toxicities of the multiple systems of three pharmaceuticals were evaluated by the additive index, toxicity unit, and mixed toxicity index methods. The consistent evaluation results were obtained. It was observed that antagonistic effects in binary and ternary systems were appeared different antagonistic strengths. It was deduced that different functional groups of the studied pharmaceuticals could affect the physiological and biochemical reaction processes of organisms. The obtained data of the acute toxicities of pharmaceuticals to vibrio fischeri can facilitate the evaluation of the environmental risks associated with emerging pollutants. Copyright © 2019, KeAi Communications Co., Ltd. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Ibuprofen Azithromycin Triclosan Joint toxic effects
1. Introduction With the detection of trace organic pollutants related to active pharmaceutical ingredients (APIs) in different water sources, the influences to ecological environment and human survival have been widely concerned [1]. When the environment passes through the self-purification ability cannot completely remove the new pollutants remaining in the environment, such as antibiotics' active ingredients, there may also be an ecological phenomenon in which a large number of drug-resistant strains in the environment increase, and the drug-resistant bacteria are increased, the risk of other biological infections will increase. Ibuprofen is a typical representative of a new generation of non-steroidal anti-inflammatory analgesic pharmaceuticals for phenylpropionic acid. It is the only recommendation of the World Health Organization and the US Food and Drug Administration (FDA) because of its low toxicity, good efficacy and small side effects have been widely used in the world [2,3]. Azithromycin is a semi-synthetic fifteen-membered
* Corresponding author. E-mail address: [email protected] (Y. Dong). Peer review under responsibility of KeAi Communications Co., Ltd.
cyclic lactone antibiotic, which is used in the treatment of human and animal infections because of its low incidence of adverse reactions and good sexual acceptability. It has better curative effect than general antibiotics [4e6]. Triclosan (TCS) is a commonly used high-efficiency antibacterial agent, commonly known as triclosan cream in pharmaceuticals, it has the ability to kill and inhibit the sterilization and preservatives of pharmaceuticals and personal care products [7]. The release of active ingredients in these common medicines can cause different degrees of threat to human health and the ecological environment. At the same time, such pollutants are ubiquitous on a global scale, have high stability in the environment, and are difficult to degrade. It is easy to be enriched in the ecosystem [8,9], and the harm to various organisms including humans in the environment can't be ignored. Common pharmaceutical products represented by antibiotics often ubiquitous in the form of a mixture, the mixture of pharmaceuticals such as antibiotics in the environment always shows different toxic effects from a single compound [10]. The selected three common pharmaceuticals are used as antipyretic, analgesic, anti-infective and sterilization pharmaceuticals have good curative effects, and the three pharmaceutical mixture impacts to environment research were few. As of 2006, the annual use of triclosan has
https://doi.org/10.1016/j.emcon.2019.08.004 2405-6650/Copyright © 2019, KeAi Communications Co., Ltd. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
298
Y. Dong et al. / Emerging Contaminants 5 (2019) 297e302
exceeded 360t, and the use of ibuprofen and azithromycin are widespread worldwide. The toxic effects study of ibuprofen and triclosan on the yellow catfish were observed that single and mixed exposures ibuprofen and triclosan to yellow catfish were different [11]. Li analyzed the toxicity of ibuprofen and triclosan on selenastrum capricornutum in the study of toxic effects of triclosan, ibuprofen and cadmium chloride to selenastrum capricornutum, and the results showed that triclosan is a highly toxic substance to the algae, and ibuprofen also has a great influence on the catalase and alondialdehyde in the antioxidant enzyme system of selenastrum capricornutum. The joint toxicity of cadmium chloride and triclosan are antagonistic to selenastrum capricornutum, and the joint toxicity of cadmium and ibuprofen have synergistic effects to selenastrum capricornutum [12]. The study on the joint toxicity of ibuprofen, azithromycin and triclosan in the environment is still relatively rare. Therefore, it is urgent to understand the ecotoxic effects of these three common pharmaceutical mixtures in the environment. Vibrio fischeri are sensitive and fast, easy to operate, and have a wide range of applications. They are used as test organisms to study the single and joint effects of ibuprofen, azithromycin and triclosan. The mixture toxicity data of the selected APIs in the paper can provide basic data on the environmental and biological impacts of new pollutants.
IR ¼
phase was used. The above operations were carried out under aseptic conditions and cultured at 20 C [9]. Due to the luminous intensity of the vibrio fischeri could not be controlled, take a certain amount of shake flask liquid in 3% NaCl solution, to control the test luminous intensity range in 300e900. 2.3.2. Pre-test of toxicity The concentration gradient of the selected reagents was tested, and the luminescence inhibition rate of the compound to be tested on the vibrio fischeri at 15 min was observed, and the suitable concentration range of the toxicity test was determined. 2.3.3. Determination of single toxicity EC50 At least 13 different concentrations of each pharmaceutical were test in triplicates. 2 mL 3% NaCl solution was used as a blank control. The spacing of the concentrations tested was adjusted to the different steepness values of the individual concentrationeresponse curves so that the complete effect range between 1 and at least 95% effect is described. As for data processing, the concentrations and the corresponding luminescence inhibition rate (IR) were showed a good linear relationship around EC50. The ratio of the luminescence intensity of the sample solution to the luminescence intensity of the blank control was calculated by the following equation.
Control luminescence intensity Experimental sample luminous intensity 100% Control luminescence intensity
(1)
2. Materials and methods 2.1. Reagents Ibuprofen sustained release capsules and azithromycin dispersible tablets were purchased from Changchun Overseas Pharmaceutical Group, and Northeast Pharmaceutical Group Shenyang First Pharmaceutical, and were prepared using the corrected active ingredient content. The reagents such as triclosan and sodium chloride are analytical grade. Lyophilized powder was purchased from Wuxi Keliying Environmental Protection. 2.2. Equipments The Microtox test instrument, Model toxicity analyzer DXY-2 was made by the Institute of Soil Science, Academia Sciences, Nanjing P. R. China, THZ-28 thermostatic oscillator made by Environmental Protection Instrument Factory, Taicang, Jiangsu, High atmospheric pressure bacterium-destroy boiler, Thermostatic magnetic stirrer. 2.3. Methods of test 2.3.1. Cultivation of vibrio fischeri Under sterile conditions, 1 mL of sterilized 3% NaCl solution was added to the lyophilized powder preparation of vibrio fischeri, placed at room temperature for 2 min, vibrio fischeri is recovery. The revived vibrio fischeri are transferred to the slant medium with the sterilized inoculation rod immediately, cultured for 24 h (T1), transferred T1 to the second generation (T2) cultured for 24 h, then transferred to T3 cultured for 12 h. At last, T3 was transferred to a 250 mL shake flask containing 100 mL of the culture solution, and shake it at thermostatic oscillator until the logarithmic growth
2.3.4. Determination of joint toxicity EC50 According to the experimental results of the single toxicity test, the effect-dose curve of the toxic effect was drawn to obtain the single toxicity EC50 of the three pharmaceuticals to vibrio fischeri, and the test substances ibuprofen, azithromycin and triclosan were measured according to single toxicity. The EC40, EC45, EC50, EC55, EC60 and other toxic ratios were prepared in a binary and ternary mixed system. The concentrations of each group were set to 3 parallels with 3% NaCl as the blank control, ensuring that the standard deviation of the three parallel experiments was less than 10%. 2.3.5. Assessment method The types of joint toxicity of pollutants to organisms under multi-component systems include independent, synergistic, additive and antagonistic effects. In this experiment, additive index method (AI), toxic unit method (TU), mixing three evaluation methods of Toxicity Index Method (MTI) quantitatively discriminate and analyze the types of action of three pharmaceuticals’ binary and ternary mixed systems. The combined toxicity type evaluation criteria are shown in Table 1. 2.4. Quality control procedures Acetone, ether, tetrahydrofuran (THF), ethanol were selected. The four kinds of co-solvents are analytically pure. The corresponding series concentration gradient was prepared with 3% NaCl solution. Then determine the toxicity of four co-solvents to vibrio fischeri, the 3% NaCl as a blank control. The standard error of the effect of acetone as co-solvent used in the experiment on the biotoxicity of the vibrio fischeri is determined to be no more than 5%.
Y. Dong et al. / Emerging Contaminants 5 (2019) 297e302
299
Table 1 Joint toxicity types and standards of different evaluating methods. Interaction type
Synergistic
Simple additive
Independent
Antagonism
Partial additive
TU AI MIT
M<1 AI >1 MIT >1
M¼1 AI ¼ 1 MIT ¼ 1
M ¼ M0
M > M0 AI < 0 MIT <0
M0>M > 1
MIT ¼ 0
M is the unit of toxicity of the mixture equal to the sum of the toxicity units of the components, M ¼ TUmix ¼ toxicity in the mixture), the Joint toxicity types can be evaluated according to the M value and M0.
The limiting concentration of co-solvent addition as the actual operation allows.
Table 3 EC50 values when three kinds of pharmaceuticals act alone. Compounds
3. Results and discussion
(a)
3.1. Influence of co-solvents to vibrio fischeri in acute toxicity test Since some pharmaceuticals are hardly soluble in water, the addition of a co-solvent will affect the toxicity analysis of the compound. The organic solvent is toxic and can produce synergistic, additive or antagonistic effects with tested compounds. Therefore, determine the specific requirements of the influence to co-solvent and the limit of dosages, can provide guarantee for the uncertain factors brought by the actual analysis work. The effects of four common organic co-solvents including acetone, ether, tetrahydrofuran (THF) and ethanol to vibrio fischeri were studied. The concentration ranges corresponding to the different inhibitory effects of four common organic solvents on the luminescence intensity of vibrio fischeri were studied and compared. As is clear from Table 2, when the inhibition rate of the luminescent activity of the vibrio fischeri was less than 15%, the concentration level corresponding to the specific inhibition rate was different. If the corresponding concentration value under the same inhibition rate is larger, it indicates that the effect of the solvent on the toxic effect of the vibrio fischeri is smaller. Through the study on the effects of the biotoxicity of commonly used co-solvents, the specific requirements for the selection and dosage of co-solvents were made. Combined with the properties of ibuprofen are easily soluble in ethanol and azithromycin are easily soluble in acetone, ethanol and acetone was chosen as a co-solvent in the following experiments. The amount of co-solvent does not exceed the minimum limit of EC5. 3.2. Single toxicity test analysis The three pharmaceuticals showed different degrees of inhibition on vibrio fischeri (Table 3), and their half-effect concentration (EC50) to vibrio fischeri are 36.5 105, 30.26 105 and 0.0155 105 mol L1, its toxicity is: triclosan > azithromycin > ibuprofen, that is triclosan has the strongest toxic effect. Fig. 1 (a, b and c) shows the molecular structure of three pharmaceutical products in the order of ibuprofen, azithromycin, and triclosan respectively. As can be seen from the structure diagram (Fig. 2), the substituents of the three pharmaceutical products are eOH and -Cl. Zhai selected 17 kinds of substituted benzene compounds to study the acute toxicity of vibrio fischeri and obtained the order of the
0 < MIT<1 P TUi, if M0 ¼ M/maxTUi (maxTUi represents the maximum
Ibuprofen Azithromycin(b) Triclosan(c)
Linear equation
R2
EC50( 105)(mol$L1)
y ¼ 0.0067x -0.0099 y ¼ 0.0054x - 0.7239 y ¼ 8.5927x þ 0.1724
0.9615 0.945 0.9157
36.5 30.26 0.0155
toxic contribution of the substituents to vibrio fischeri was: eNO2>Cl > -CH3>-NH2>-OH (Zhai, 2003). Therefore, it is inferred that the difference in the inhibitory effect of three different pharmaceuticals on vibrio fischeri is caused by the different effects of different substituents. Further research on the molecular interaction mechanism is needed to get quantitative explanation. 3.3. Joint toxicity evaluation of binary mixed system Comprehensive analysis of the combined toxicity of binary mixed system on vibrio fischeri (Table 4), the EC50 value of the binary toxicity of the three pharmaceuticals is significantly greater than the EC50 of the single toxicity when mixed in the same toxicity ratio, indicating that the toxicity of the mixed system is lower than that of the single toxicity. As for the binary mixtures of azithromycin þ ibuprofen, triclosan þ ibuprofen, triclosan þ azithromycin, the M values are greater than M0, AI and MTI are less than zero, so the role of these three mixed systems types are characterized by antagonism. Since the closer the AI is to zero, the type tends to antagonism, and the closer the MTI is to zero, the role the type of action tends to be independent. Therefore, according to the difference of AI and MTI of the three binary systems, it can be judged. The difference in antagonism is determined by the intensity of the response of the vibrio fischeri to the binary mixed system of three pharmaceutical products: triclosan þ azithromycin > triclosan þ ibuprofen > azithromycin þ ibuprofen. 3.4. Joint toxicity evaluation of ternary mixed system Due to the joint toxicity of binary mixed system is antagonistic, it is suspected that the interaction between the three pharmaceuticals can lead to a decrease in the combined toxicity of the system, multiple binary antagonistic systems are accumulated, and the enhanced interaction between the molecules can lead to the continuation of the multi-component system. The experimental results show that the combined toxicity evaluation parameters and action types of the ternary mixed system are shown in Table 5. The toxicity EC50 of ternary mixed system with equal toxicity ratio is
Table 2 Effect of organic solvents on the luminescence intensity of vibrio fischeri. Compounds
Linear equation
R2
EC5 ( 105)(mol$L1)
EC10 ( 105)(mol$L1)
EC15 ( 105)(mol$L1)
Acetone Ether THF Ethanol
y%¼-8.459 þ 555.342x y%¼-18.054 þ 4.588 103x y%¼-8.018 þ 3.973 103x y%¼-11.288 þ 225.791x
0.997 0.995 0.999 0.998
24.24 5.025 3.277 72.14
33.24 6.115 4.535 94.28
42.24 7.204 5.794 116.4
300
Y. Dong et al. / Emerging Contaminants 5 (2019) 297e302
Fig. 1. Three kinds of pharmaceutical single toxicity test (Ibuprofen(a), Azithromycin(b), Triclosan(c)).
Fig. 2. Three kinds of pharmaceutical structure.
Table 4 EC50 values and joint toxicity evaluation parameters and action types of binary mixtures. Binary mixed system
EC50( 105) mol$L1
TUi
M
M0
AI
MTI
Action type
Azithromycinþ Ibuprofen Triclosan þ Ibuprofen Triclosan þ Azithromycin
47.66
1.122 1.292 1.426 2.633 3.089 1.668
2.414
1.868
0.665
0.409
Antagonistic
4.059
1.541
3.059
2.238
Antagonistic
4.756
1.540
13.04
2.612
Antagonistic
52.59 50.47
Y. Dong et al. / Emerging Contaminants 5 (2019) 297e302
301
Table 5 EC50 values and joint toxicity evaluation parameters and action types of ternary mixtures. Ternary Mixture
EC50( 105) mol$L1
TUi
M
M0
AI
MTI
Action type
Ibuprofen þ Azithromycin þ Triclosan
54.99
1.490 1.211 2.831
5.532
1.954
0.850
1.534
Antagonistic
significantly greater than the EC50 values of binary and single toxic effects, indicating that the ternary mixed system are less toxic. The M of the ternary mixed system is greater than M0, the AI is less than zero, and the MTI is less than zero, the type of joint toxicity was obtained as an antagonistic effect, which was consistent with the results of previous studies. Researchers found that tetracycline antibiotics and chloramphenicol antibiotics also showed antagonistic effects on the combined acute toxicity of vibrio fischeri [13] 3.5. Analysis on the luminous mechanism of vibrio fischeri Comprehensive analysis of the combined toxicity of binary and ternary mixed systems is an antagonistic effect. Therefore, it is speculated that the active ingredients of the three pharmaceuticals can interact and lead to a decrease in joint toxicity, and the interaction between the various active ingredients leads to the antagonism of the multi-component system. Our experimental results are also consistent with the hypothesis. From the research on the combined toxicity of cineine hydrochloride with chlortetracycline, salinomycin and flavomycin to vibrio fischeri, it could be drawn that molecular hybrid systems with strong antagonistic effects play a leading role in multiple mixtures [14]. In the research of ternary chronic joint toxicity of antibiotics and quorum to vibrio fischeri, proved the antagonism of the two-component system is the root cause of the antagonism of the ternary system and the pollutants are mixed in various ways in the environment, their interaction may lead to weakening of toxicity [15]. The preliminary combined toxicity analysis can also be carried out by the principle of luminescent luminescence in the combined effect of present, The luminescence mechanism of vibrio fischeri is basically the presence of bacterial luciferase (LE), reduced flavin mononucleotide (FMNH2), oxygen (O2), participation of long-chain fatty aldehydes (RCHO) [16e18]. The luminescence reaction equation is as follows:
FMNH2 þ O2 þ RCHO/FMN þ PR COOH þ H2 O þ Light
(2)
During this reaction, the conversion between the oxidized form (FMN) and the reduced form (FMNH2) of the flavin mononucleotide as an important coenzyme plays a role in the transfer of hydrogen throughout the reaction. In the experiment, the three common pharmaceutical molecules contain hydroxyl groups (-OH), which easily combine with flavin mononucleotides to form hydrogen bonds, which hinders the hydrogen transfer between FMNH2 in oxidation and reduction in the luminescence reaction, and interferes with the normal luminescence reaction process. At the same time, since both azithromycin and ibuprofen contain N elements in the molecule, N element as a nutrient factor can promote the luminescence reaction process to a certain extent [19,20], reducing other inhibitory effect of the compound on the luminescence intensity. In addition, we have proposed a method for predicting the toxicity of pharmaceuticals' mode of action. We defined the compound itself or its metabolites which can interact with biological macromolecules’ certain structures as reactive. For example, an electron withdrawing group (electrophilic group) forms a covalent bond with a biological target (nucleophilic group), especially a biological macromolecule such as a nucleophilic group in a polypeptide, a protein, and a nucleic acid (amino group eNH2),
hydroxyl eOH and thiol eSH. We defined a hydrophobic noncovalent interaction between the pharmaceuticals and the cell membrane that reversibly alters the structure and function of the cell membrane, thereby toxic to the organism or the interaction between the pharmaceutical and the biomacromolecule may be through physical rather than chemical reactions as narcosis. According to the data of acute toxicity LC50 concentration and chronic toxicity NOCE concentration of the three pharmaceuticals, and calculated by our formula, the following conclusions were obtained: ibuprofen and azithromycin demonstrated to exhibit reactive, triclosan demonstrated to exhibit narcosis, the conclusions were verified our experimental inference the type of this action is antagonizes. If we want to analyze the causes of antagonism more specifically, we need to further explore the mechanism of the combined action of testing pollutants and the physiological and biochemical reactions of vibrio fischeri. 4. Conclusion Three selected APIs could inhibit the luminescent bacteria to varying degrees, and triclosan had the strongest single toxicity. The toxicity of the binary mixed system was lower than that of the single toxicity, and showed antagonistic effects. The mixed system of triclosan and azithromycin had the strongest antagonistic effect. The toxicity of the ternary mixed system was weaker than that of binary mixed systems. The obtained toxicity data of three pharmaceuticals to vibrio fischeri could be helpful to deduce the toxic effects on other aquatic organisms. Co-solvent screening among acetone, aether, ethanol, tetrahydrofuran on vibrio fischeri toxicity test was carried and different effective concentration (EC) values were obtained. Acetone and ethanol was selected as the most suitable solvent of chemical compounds with low water-solubility according to its good characters, less toxicity effect to vibrio fischeri and properties of pharmaceuticals. The successful application of acetone used as co-solvent provides a good example and a good idea to improve traditional acute toxicity test of vibrio fischeri. Besides, The mode of action of three pharmaceuticals were calculated obtained: ibuprofen and azithromycin demonstrated to exhibit reactive, triclosan demonstrated to exhibit narcosis, the conclusions was verified our experimental inference the type of this action is antagonizes. Our study filled the knowledge gap of the joint effect of pharmaceuticals and could provide a basis for assessing the potential risks that different pharmaceuticals mixture might bring to the aquatic ecosystem. Acknowledgement This research was financially supported by Liaoning Science Technology Project Foundation (20180550107) and Basic Scientific Research Funds of DLNU (2019). References [1] H. Jin, C. Wang, J.Q. Shi, L. Chen, J. Hazard Mater. 279 (279) (2014) 156e162. https://doi.org/10.1016/j.jhazmat.2014.06.068. [2] M. Li, J. Sun, Y. Hai, L. Wang, A.L. Xiao, H.M. Xu, S.L. Zhang, M. Ni, J.Y. Wang, G. Gan, X.X. Du, Chin. J. Pharmacovigilance 7 (03) (2010) 151e153. https://doi. org/10.3969/j.issn.1672-8629.2010.03.007.
302
Y. Dong et al. / Emerging Contaminants 5 (2019) 297e302
[3] L. Ferrando-Climent, N. Collado, G. Buttiglieri, M. Gros, I. Rodriguez-Roda, , Sci. Total Environ. 438 (3) (2012) 404e413. S. Rodriguez-Mozaz, D. Barcelo https://doi.org/10.1016/j.scitotenv.2012.08.073. [4] Z. Miao, Comparison of effect on azithromycin and clindamycin in the treatment of acute tonsillitis, Clin. Med. Res. Pract. 1 (24) (2016) 52e53. https://doi. org/10.3969/j.issn.2096-1413.2016.24.025. [5] D.E. Koch, A. Bhandari, L. Close, R.P. Hunter, J. Chromatogr. A 1074 (1) (2005) 17e22. https://doi.org/10.1016/j.chroma.2005.03.052. [6] T. Edward, J. Renaud, M.W. Sumarah, S. Lyne, Sci. Total Environ. 562 (2016) 136e144. https://doi.org/10.1016/j.scitotenv.2016.03.210. [7] J. Chen, R.J. Qu, X.X. Pan, Z.Y. Wang, Water Res. 103 (2016) 215e223. https:// doi.org/10.1016/j.watres.2016.07.041. [8] X.N. Feng, Z. Yang, J. Sun, N. Wang, L. Yang, X.H. Wang, Y.H. Zhao, J. Ecotoxicol. 12 (3) (2017) 687e694. https://doi.org/10.7524/AJE.1673-5897.20161122001. [9] W.Q. Gao, Y.Y. Dong, H.Q. Wang, X.J. Zou, Hubei Agric. Sci. 56 (16) (2017) 3074e3077. https://doi.org/10.14088/j.cnki.issn0439-8114.2017.16.018. [10] S.X. Fang, D.L. Wang, L.H. Zhu, T.T. Shi, M.N. Qin, Z.F. Lin, J. Ecotoxicol. 10 (02) (2015) 69e75. https://doi.org/10.7524/AJE.1673-5897.20141102001. [11] X.Y. Wu, Toxic Effects of Ibuprofen and Triclosan on P450 Enzymes and Antioxidantive System Og the Yellow Catfish, Jinan University, 2013, pp. 37e38. http://kns.cnki.net/KCMS/detail/detail.aspx?dbcode¼CMFD& dbname¼CMFD201401&filename¼1013027646.nh&v¼MjM5MTZyV00xRnJD VVJMT2ZaT1J2Rnkza1ViN0pWRjI2SGJPNkdkZklxWkViUElSOGVYMUx1eFlTN 0RoMVQzcVQ¼. [12] Y.G. Li, Toxic Effects of Triclosan, Ibuprofen and Cadmium Chloride to Selenastrum Capricornutum, Jinan University, 2013, pp. 62e65. http://kns.cnki. net/KCMS/detail/detail.aspx?dbcode¼CMFD&dbname¼CMFD201501&
[13] [14]
[15] [16] [17] [18] [19] [20]
filename¼1013027640.nh&uid¼WEEvREdxOWJmbC9oM1NjYkZCbDdrdTVi ZlNLV1hVVnJxa3MwU2ZCY1Y2cGM¼$R1yZ0H6jyaa0en3RxVUd8df-oHi7XM MDo7mtKT6mSmEvTuk11l2gFA!!&v¼MjY2OTN5M2tXN3JPVkYyNkhiTzZH ZGZJcjVFYlBJUjhlWDFMdXhZUzdEaDFUM3FUcldNMUZyQ1VSTE9mWk9Sdk Y¼. Y.P. Cong, Z.F. Lin, T. Wang, X.M. Zou, H.M. Ge, D.Q. Yin, Environ. Chem. (07) (2013) 1348e1352. https://doi.org/10.7524/j.issn.0254-6108.2013.07.031. H. Ren, J.R. Wang, X.J. Chen, J.G. Liu, P. Lou, Feed Process. Test. 47 (21) (2011) 49e52. http://kns.cnki.net/KCMS/detail/detail.aspx?dbcode¼CJFQ&dbname¼ CJFD2011&filename¼ZGXM201121014&uid¼WEEvREdxOWJmbC9oM1NjY kZCbDdrdTViZlNLV1hVVnJxa3MwU2ZCY1Y2cGM¼$R1yZ0H6jyaa0en3RxVUd 8df-oHi7XMMDo7mtKT6mSmEvTuk11l2gFA!!&v¼MjU0MDNMdXhZUzdEaD FUM3FUcldNMUZyQ1VSTE9mWk9SdkZ5M25VNzdOUHlyVFk3RzRIOURPcm 85RVlJUjhlWDE¼. Z.S. Fang, Z.F. Yao, T. Wang, Z.F. Lin, S.P. Zuo, Asian J. Ecotoxicol. 11 (2) (2016) 369e373. https://doi.org/10.7524/AJE.1673-5897.20151102001. L. Tong, L.L. Yao, H. Liu, Y.X. Wang, J. Ecotoxicol. 11 (2) (2016) 27e36. https:// doi.org/10.7524/AJE.1673-5897.20151127002. S.H.A. Hassan, S. Eun Oh, B: Biol. 101 (1) (2010) 16e21. https://doi.org/10. 1016/j.jphotobiol.2010.06.006. Y.Y. Dong, J. Wang, L. Ding, Y.Y. Liu, Procedia Environ. Sci. 18 (2013) 143e148. https://doi.org/10.1016/j.proenv.2013.04.019. D.Y. Tian, Z.F. Lin, X.H. Zhou, D.Q. Yin, Toxicol. Appl. Pharmacol. 272 (2) (2013) 551e558. https://doi.org/10.1016/j.taap.2013.06.015. L.M. Su, X.J. Zhang, X. Yuan, Y.H. Zhao, D.M. Zhang, W.C. Qin, J. Hazard Mater. (2012) 241e242. https://doi.org/10.1016/j.jhazmat.2012.09.065.
Contents lists available at ScienceDirect
Emerging Contaminants journal homepage: http://www.keaipublishing.com/en/journals/ emerging-contaminants/
The toxic effects of three active pharmaceutical ingredients (APIs) with different efficacy to Vibrio fischeri Yuying Dong*, Zheng Fang, Yan Xu, Qiying Wang, Xuejun Zou Department of Environmental Science and Engineering, Dalian Minzu University, Dalian, 116600, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2019 Received in revised form 21 August 2019 Accepted 29 August 2019
The environmental residues of active pharmaceutical ingredients (APIs) are associated with environmental risks and health problems, the influence to environment have become a matter of public concern. In this paper, the individual and joint toxicities to vibrio fischeri of three APIs, including ibuprofen, azithromycin, and triclosan were investigated. The EC50 endpoint values of the above tested APIs were 36.5 105 mol L1, 30.26 105 mol L1, and 0.0155 105 mol L1 respectively. It was indicated that the endpoint toxicities to vibrio fischeri for different mixtures are higher than those of the individual toxins. The joint toxicities of the multiple systems of three pharmaceuticals were evaluated by the additive index, toxicity unit, and mixed toxicity index methods. The consistent evaluation results were obtained. It was observed that antagonistic effects in binary and ternary systems were appeared different antagonistic strengths. It was deduced that different functional groups of the studied pharmaceuticals could affect the physiological and biochemical reaction processes of organisms. The obtained data of the acute toxicities of pharmaceuticals to vibrio fischeri can facilitate the evaluation of the environmental risks associated with emerging pollutants. Copyright © 2019, KeAi Communications Co., Ltd. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Ibuprofen Azithromycin Triclosan Joint toxic effects
1. Introduction With the detection of trace organic pollutants related to active pharmaceutical ingredients (APIs) in different water sources, the influences to ecological environment and human survival have been widely concerned [1]. When the environment passes through the self-purification ability cannot completely remove the new pollutants remaining in the environment, such as antibiotics' active ingredients, there may also be an ecological phenomenon in which a large number of drug-resistant strains in the environment increase, and the drug-resistant bacteria are increased, the risk of other biological infections will increase. Ibuprofen is a typical representative of a new generation of non-steroidal anti-inflammatory analgesic pharmaceuticals for phenylpropionic acid. It is the only recommendation of the World Health Organization and the US Food and Drug Administration (FDA) because of its low toxicity, good efficacy and small side effects have been widely used in the world [2,3]. Azithromycin is a semi-synthetic fifteen-membered
* Corresponding author. E-mail address: [email protected] (Y. Dong). Peer review under responsibility of KeAi Communications Co., Ltd.
cyclic lactone antibiotic, which is used in the treatment of human and animal infections because of its low incidence of adverse reactions and good sexual acceptability. It has better curative effect than general antibiotics [4e6]. Triclosan (TCS) is a commonly used high-efficiency antibacterial agent, commonly known as triclosan cream in pharmaceuticals, it has the ability to kill and inhibit the sterilization and preservatives of pharmaceuticals and personal care products [7]. The release of active ingredients in these common medicines can cause different degrees of threat to human health and the ecological environment. At the same time, such pollutants are ubiquitous on a global scale, have high stability in the environment, and are difficult to degrade. It is easy to be enriched in the ecosystem [8,9], and the harm to various organisms including humans in the environment can't be ignored. Common pharmaceutical products represented by antibiotics often ubiquitous in the form of a mixture, the mixture of pharmaceuticals such as antibiotics in the environment always shows different toxic effects from a single compound [10]. The selected three common pharmaceuticals are used as antipyretic, analgesic, anti-infective and sterilization pharmaceuticals have good curative effects, and the three pharmaceutical mixture impacts to environment research were few. As of 2006, the annual use of triclosan has
https://doi.org/10.1016/j.emcon.2019.08.004 2405-6650/Copyright © 2019, KeAi Communications Co., Ltd. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
298
Y. Dong et al. / Emerging Contaminants 5 (2019) 297e302
exceeded 360t, and the use of ibuprofen and azithromycin are widespread worldwide. The toxic effects study of ibuprofen and triclosan on the yellow catfish were observed that single and mixed exposures ibuprofen and triclosan to yellow catfish were different [11]. Li analyzed the toxicity of ibuprofen and triclosan on selenastrum capricornutum in the study of toxic effects of triclosan, ibuprofen and cadmium chloride to selenastrum capricornutum, and the results showed that triclosan is a highly toxic substance to the algae, and ibuprofen also has a great influence on the catalase and alondialdehyde in the antioxidant enzyme system of selenastrum capricornutum. The joint toxicity of cadmium chloride and triclosan are antagonistic to selenastrum capricornutum, and the joint toxicity of cadmium and ibuprofen have synergistic effects to selenastrum capricornutum [12]. The study on the joint toxicity of ibuprofen, azithromycin and triclosan in the environment is still relatively rare. Therefore, it is urgent to understand the ecotoxic effects of these three common pharmaceutical mixtures in the environment. Vibrio fischeri are sensitive and fast, easy to operate, and have a wide range of applications. They are used as test organisms to study the single and joint effects of ibuprofen, azithromycin and triclosan. The mixture toxicity data of the selected APIs in the paper can provide basic data on the environmental and biological impacts of new pollutants.
IR ¼
phase was used. The above operations were carried out under aseptic conditions and cultured at 20 C [9]. Due to the luminous intensity of the vibrio fischeri could not be controlled, take a certain amount of shake flask liquid in 3% NaCl solution, to control the test luminous intensity range in 300e900. 2.3.2. Pre-test of toxicity The concentration gradient of the selected reagents was tested, and the luminescence inhibition rate of the compound to be tested on the vibrio fischeri at 15 min was observed, and the suitable concentration range of the toxicity test was determined. 2.3.3. Determination of single toxicity EC50 At least 13 different concentrations of each pharmaceutical were test in triplicates. 2 mL 3% NaCl solution was used as a blank control. The spacing of the concentrations tested was adjusted to the different steepness values of the individual concentrationeresponse curves so that the complete effect range between 1 and at least 95% effect is described. As for data processing, the concentrations and the corresponding luminescence inhibition rate (IR) were showed a good linear relationship around EC50. The ratio of the luminescence intensity of the sample solution to the luminescence intensity of the blank control was calculated by the following equation.
Control luminescence intensity Experimental sample luminous intensity 100% Control luminescence intensity
(1)
2. Materials and methods 2.1. Reagents Ibuprofen sustained release capsules and azithromycin dispersible tablets were purchased from Changchun Overseas Pharmaceutical Group, and Northeast Pharmaceutical Group Shenyang First Pharmaceutical, and were prepared using the corrected active ingredient content. The reagents such as triclosan and sodium chloride are analytical grade. Lyophilized powder was purchased from Wuxi Keliying Environmental Protection. 2.2. Equipments The Microtox test instrument, Model toxicity analyzer DXY-2 was made by the Institute of Soil Science, Academia Sciences, Nanjing P. R. China, THZ-28 thermostatic oscillator made by Environmental Protection Instrument Factory, Taicang, Jiangsu, High atmospheric pressure bacterium-destroy boiler, Thermostatic magnetic stirrer. 2.3. Methods of test 2.3.1. Cultivation of vibrio fischeri Under sterile conditions, 1 mL of sterilized 3% NaCl solution was added to the lyophilized powder preparation of vibrio fischeri, placed at room temperature for 2 min, vibrio fischeri is recovery. The revived vibrio fischeri are transferred to the slant medium with the sterilized inoculation rod immediately, cultured for 24 h (T1), transferred T1 to the second generation (T2) cultured for 24 h, then transferred to T3 cultured for 12 h. At last, T3 was transferred to a 250 mL shake flask containing 100 mL of the culture solution, and shake it at thermostatic oscillator until the logarithmic growth
2.3.4. Determination of joint toxicity EC50 According to the experimental results of the single toxicity test, the effect-dose curve of the toxic effect was drawn to obtain the single toxicity EC50 of the three pharmaceuticals to vibrio fischeri, and the test substances ibuprofen, azithromycin and triclosan were measured according to single toxicity. The EC40, EC45, EC50, EC55, EC60 and other toxic ratios were prepared in a binary and ternary mixed system. The concentrations of each group were set to 3 parallels with 3% NaCl as the blank control, ensuring that the standard deviation of the three parallel experiments was less than 10%. 2.3.5. Assessment method The types of joint toxicity of pollutants to organisms under multi-component systems include independent, synergistic, additive and antagonistic effects. In this experiment, additive index method (AI), toxic unit method (TU), mixing three evaluation methods of Toxicity Index Method (MTI) quantitatively discriminate and analyze the types of action of three pharmaceuticals’ binary and ternary mixed systems. The combined toxicity type evaluation criteria are shown in Table 1. 2.4. Quality control procedures Acetone, ether, tetrahydrofuran (THF), ethanol were selected. The four kinds of co-solvents are analytically pure. The corresponding series concentration gradient was prepared with 3% NaCl solution. Then determine the toxicity of four co-solvents to vibrio fischeri, the 3% NaCl as a blank control. The standard error of the effect of acetone as co-solvent used in the experiment on the biotoxicity of the vibrio fischeri is determined to be no more than 5%.
Y. Dong et al. / Emerging Contaminants 5 (2019) 297e302
299
Table 1 Joint toxicity types and standards of different evaluating methods. Interaction type
Synergistic
Simple additive
Independent
Antagonism
Partial additive
TU AI MIT
M<1 AI >1 MIT >1
M¼1 AI ¼ 1 MIT ¼ 1
M ¼ M0
M > M0 AI < 0 MIT <0
M0>M > 1
MIT ¼ 0
M is the unit of toxicity of the mixture equal to the sum of the toxicity units of the components, M ¼ TUmix ¼ toxicity in the mixture), the Joint toxicity types can be evaluated according to the M value and M0.
The limiting concentration of co-solvent addition as the actual operation allows.
Table 3 EC50 values when three kinds of pharmaceuticals act alone. Compounds
3. Results and discussion
(a)
3.1. Influence of co-solvents to vibrio fischeri in acute toxicity test Since some pharmaceuticals are hardly soluble in water, the addition of a co-solvent will affect the toxicity analysis of the compound. The organic solvent is toxic and can produce synergistic, additive or antagonistic effects with tested compounds. Therefore, determine the specific requirements of the influence to co-solvent and the limit of dosages, can provide guarantee for the uncertain factors brought by the actual analysis work. The effects of four common organic co-solvents including acetone, ether, tetrahydrofuran (THF) and ethanol to vibrio fischeri were studied. The concentration ranges corresponding to the different inhibitory effects of four common organic solvents on the luminescence intensity of vibrio fischeri were studied and compared. As is clear from Table 2, when the inhibition rate of the luminescent activity of the vibrio fischeri was less than 15%, the concentration level corresponding to the specific inhibition rate was different. If the corresponding concentration value under the same inhibition rate is larger, it indicates that the effect of the solvent on the toxic effect of the vibrio fischeri is smaller. Through the study on the effects of the biotoxicity of commonly used co-solvents, the specific requirements for the selection and dosage of co-solvents were made. Combined with the properties of ibuprofen are easily soluble in ethanol and azithromycin are easily soluble in acetone, ethanol and acetone was chosen as a co-solvent in the following experiments. The amount of co-solvent does not exceed the minimum limit of EC5. 3.2. Single toxicity test analysis The three pharmaceuticals showed different degrees of inhibition on vibrio fischeri (Table 3), and their half-effect concentration (EC50) to vibrio fischeri are 36.5 105, 30.26 105 and 0.0155 105 mol L1, its toxicity is: triclosan > azithromycin > ibuprofen, that is triclosan has the strongest toxic effect. Fig. 1 (a, b and c) shows the molecular structure of three pharmaceutical products in the order of ibuprofen, azithromycin, and triclosan respectively. As can be seen from the structure diagram (Fig. 2), the substituents of the three pharmaceutical products are eOH and -Cl. Zhai selected 17 kinds of substituted benzene compounds to study the acute toxicity of vibrio fischeri and obtained the order of the
0 < MIT<1 P TUi, if M0 ¼ M/maxTUi (maxTUi represents the maximum
Ibuprofen Azithromycin(b) Triclosan(c)
Linear equation
R2
EC50( 105)(mol$L1)
y ¼ 0.0067x -0.0099 y ¼ 0.0054x - 0.7239 y ¼ 8.5927x þ 0.1724
0.9615 0.945 0.9157
36.5 30.26 0.0155
toxic contribution of the substituents to vibrio fischeri was: eNO2>Cl > -CH3>-NH2>-OH (Zhai, 2003). Therefore, it is inferred that the difference in the inhibitory effect of three different pharmaceuticals on vibrio fischeri is caused by the different effects of different substituents. Further research on the molecular interaction mechanism is needed to get quantitative explanation. 3.3. Joint toxicity evaluation of binary mixed system Comprehensive analysis of the combined toxicity of binary mixed system on vibrio fischeri (Table 4), the EC50 value of the binary toxicity of the three pharmaceuticals is significantly greater than the EC50 of the single toxicity when mixed in the same toxicity ratio, indicating that the toxicity of the mixed system is lower than that of the single toxicity. As for the binary mixtures of azithromycin þ ibuprofen, triclosan þ ibuprofen, triclosan þ azithromycin, the M values are greater than M0, AI and MTI are less than zero, so the role of these three mixed systems types are characterized by antagonism. Since the closer the AI is to zero, the type tends to antagonism, and the closer the MTI is to zero, the role the type of action tends to be independent. Therefore, according to the difference of AI and MTI of the three binary systems, it can be judged. The difference in antagonism is determined by the intensity of the response of the vibrio fischeri to the binary mixed system of three pharmaceutical products: triclosan þ azithromycin > triclosan þ ibuprofen > azithromycin þ ibuprofen. 3.4. Joint toxicity evaluation of ternary mixed system Due to the joint toxicity of binary mixed system is antagonistic, it is suspected that the interaction between the three pharmaceuticals can lead to a decrease in the combined toxicity of the system, multiple binary antagonistic systems are accumulated, and the enhanced interaction between the molecules can lead to the continuation of the multi-component system. The experimental results show that the combined toxicity evaluation parameters and action types of the ternary mixed system are shown in Table 5. The toxicity EC50 of ternary mixed system with equal toxicity ratio is
Table 2 Effect of organic solvents on the luminescence intensity of vibrio fischeri. Compounds
Linear equation
R2
EC5 ( 105)(mol$L1)
EC10 ( 105)(mol$L1)
EC15 ( 105)(mol$L1)
Acetone Ether THF Ethanol
y%¼-8.459 þ 555.342x y%¼-18.054 þ 4.588 103x y%¼-8.018 þ 3.973 103x y%¼-11.288 þ 225.791x
0.997 0.995 0.999 0.998
24.24 5.025 3.277 72.14
33.24 6.115 4.535 94.28
42.24 7.204 5.794 116.4
300
Y. Dong et al. / Emerging Contaminants 5 (2019) 297e302
Fig. 1. Three kinds of pharmaceutical single toxicity test (Ibuprofen(a), Azithromycin(b), Triclosan(c)).
Fig. 2. Three kinds of pharmaceutical structure.
Table 4 EC50 values and joint toxicity evaluation parameters and action types of binary mixtures. Binary mixed system
EC50( 105) mol$L1
TUi
M
M0
AI
MTI
Action type
Azithromycinþ Ibuprofen Triclosan þ Ibuprofen Triclosan þ Azithromycin
47.66
1.122 1.292 1.426 2.633 3.089 1.668
2.414
1.868
0.665
0.409
Antagonistic
4.059
1.541
3.059
2.238
Antagonistic
4.756
1.540
13.04
2.612
Antagonistic
52.59 50.47
Y. Dong et al. / Emerging Contaminants 5 (2019) 297e302
301
Table 5 EC50 values and joint toxicity evaluation parameters and action types of ternary mixtures. Ternary Mixture
EC50( 105) mol$L1
TUi
M
M0
AI
MTI
Action type
Ibuprofen þ Azithromycin þ Triclosan
54.99
1.490 1.211 2.831
5.532
1.954
0.850
1.534
Antagonistic
significantly greater than the EC50 values of binary and single toxic effects, indicating that the ternary mixed system are less toxic. The M of the ternary mixed system is greater than M0, the AI is less than zero, and the MTI is less than zero, the type of joint toxicity was obtained as an antagonistic effect, which was consistent with the results of previous studies. Researchers found that tetracycline antibiotics and chloramphenicol antibiotics also showed antagonistic effects on the combined acute toxicity of vibrio fischeri [13] 3.5. Analysis on the luminous mechanism of vibrio fischeri Comprehensive analysis of the combined toxicity of binary and ternary mixed systems is an antagonistic effect. Therefore, it is speculated that the active ingredients of the three pharmaceuticals can interact and lead to a decrease in joint toxicity, and the interaction between the various active ingredients leads to the antagonism of the multi-component system. Our experimental results are also consistent with the hypothesis. From the research on the combined toxicity of cineine hydrochloride with chlortetracycline, salinomycin and flavomycin to vibrio fischeri, it could be drawn that molecular hybrid systems with strong antagonistic effects play a leading role in multiple mixtures [14]. In the research of ternary chronic joint toxicity of antibiotics and quorum to vibrio fischeri, proved the antagonism of the two-component system is the root cause of the antagonism of the ternary system and the pollutants are mixed in various ways in the environment, their interaction may lead to weakening of toxicity [15]. The preliminary combined toxicity analysis can also be carried out by the principle of luminescent luminescence in the combined effect of present, The luminescence mechanism of vibrio fischeri is basically the presence of bacterial luciferase (LE), reduced flavin mononucleotide (FMNH2), oxygen (O2), participation of long-chain fatty aldehydes (RCHO) [16e18]. The luminescence reaction equation is as follows:
FMNH2 þ O2 þ RCHO/FMN þ PR COOH þ H2 O þ Light
(2)
During this reaction, the conversion between the oxidized form (FMN) and the reduced form (FMNH2) of the flavin mononucleotide as an important coenzyme plays a role in the transfer of hydrogen throughout the reaction. In the experiment, the three common pharmaceutical molecules contain hydroxyl groups (-OH), which easily combine with flavin mononucleotides to form hydrogen bonds, which hinders the hydrogen transfer between FMNH2 in oxidation and reduction in the luminescence reaction, and interferes with the normal luminescence reaction process. At the same time, since both azithromycin and ibuprofen contain N elements in the molecule, N element as a nutrient factor can promote the luminescence reaction process to a certain extent [19,20], reducing other inhibitory effect of the compound on the luminescence intensity. In addition, we have proposed a method for predicting the toxicity of pharmaceuticals' mode of action. We defined the compound itself or its metabolites which can interact with biological macromolecules’ certain structures as reactive. For example, an electron withdrawing group (electrophilic group) forms a covalent bond with a biological target (nucleophilic group), especially a biological macromolecule such as a nucleophilic group in a polypeptide, a protein, and a nucleic acid (amino group eNH2),
hydroxyl eOH and thiol eSH. We defined a hydrophobic noncovalent interaction between the pharmaceuticals and the cell membrane that reversibly alters the structure and function of the cell membrane, thereby toxic to the organism or the interaction between the pharmaceutical and the biomacromolecule may be through physical rather than chemical reactions as narcosis. According to the data of acute toxicity LC50 concentration and chronic toxicity NOCE concentration of the three pharmaceuticals, and calculated by our formula, the following conclusions were obtained: ibuprofen and azithromycin demonstrated to exhibit reactive, triclosan demonstrated to exhibit narcosis, the conclusions were verified our experimental inference the type of this action is antagonizes. If we want to analyze the causes of antagonism more specifically, we need to further explore the mechanism of the combined action of testing pollutants and the physiological and biochemical reactions of vibrio fischeri. 4. Conclusion Three selected APIs could inhibit the luminescent bacteria to varying degrees, and triclosan had the strongest single toxicity. The toxicity of the binary mixed system was lower than that of the single toxicity, and showed antagonistic effects. The mixed system of triclosan and azithromycin had the strongest antagonistic effect. The toxicity of the ternary mixed system was weaker than that of binary mixed systems. The obtained toxicity data of three pharmaceuticals to vibrio fischeri could be helpful to deduce the toxic effects on other aquatic organisms. Co-solvent screening among acetone, aether, ethanol, tetrahydrofuran on vibrio fischeri toxicity test was carried and different effective concentration (EC) values were obtained. Acetone and ethanol was selected as the most suitable solvent of chemical compounds with low water-solubility according to its good characters, less toxicity effect to vibrio fischeri and properties of pharmaceuticals. The successful application of acetone used as co-solvent provides a good example and a good idea to improve traditional acute toxicity test of vibrio fischeri. Besides, The mode of action of three pharmaceuticals were calculated obtained: ibuprofen and azithromycin demonstrated to exhibit reactive, triclosan demonstrated to exhibit narcosis, the conclusions was verified our experimental inference the type of this action is antagonizes. Our study filled the knowledge gap of the joint effect of pharmaceuticals and could provide a basis for assessing the potential risks that different pharmaceuticals mixture might bring to the aquatic ecosystem. Acknowledgement This research was financially supported by Liaoning Science Technology Project Foundation (20180550107) and Basic Scientific Research Funds of DLNU (2019). References [1] H. Jin, C. Wang, J.Q. Shi, L. Chen, J. Hazard Mater. 279 (279) (2014) 156e162. https://doi.org/10.1016/j.jhazmat.2014.06.068. [2] M. Li, J. Sun, Y. Hai, L. Wang, A.L. Xiao, H.M. Xu, S.L. Zhang, M. Ni, J.Y. Wang, G. Gan, X.X. Du, Chin. J. Pharmacovigilance 7 (03) (2010) 151e153. https://doi. org/10.3969/j.issn.1672-8629.2010.03.007.
302
Y. Dong et al. / Emerging Contaminants 5 (2019) 297e302
[3] L. Ferrando-Climent, N. Collado, G. Buttiglieri, M. Gros, I. Rodriguez-Roda, , Sci. Total Environ. 438 (3) (2012) 404e413. S. Rodriguez-Mozaz, D. Barcelo https://doi.org/10.1016/j.scitotenv.2012.08.073. [4] Z. Miao, Comparison of effect on azithromycin and clindamycin in the treatment of acute tonsillitis, Clin. Med. Res. Pract. 1 (24) (2016) 52e53. https://doi. org/10.3969/j.issn.2096-1413.2016.24.025. [5] D.E. Koch, A. Bhandari, L. Close, R.P. Hunter, J. Chromatogr. A 1074 (1) (2005) 17e22. https://doi.org/10.1016/j.chroma.2005.03.052. [6] T. Edward, J. Renaud, M.W. Sumarah, S. Lyne, Sci. Total Environ. 562 (2016) 136e144. https://doi.org/10.1016/j.scitotenv.2016.03.210. [7] J. Chen, R.J. Qu, X.X. Pan, Z.Y. Wang, Water Res. 103 (2016) 215e223. https:// doi.org/10.1016/j.watres.2016.07.041. [8] X.N. Feng, Z. Yang, J. Sun, N. Wang, L. Yang, X.H. Wang, Y.H. Zhao, J. Ecotoxicol. 12 (3) (2017) 687e694. https://doi.org/10.7524/AJE.1673-5897.20161122001. [9] W.Q. Gao, Y.Y. Dong, H.Q. Wang, X.J. Zou, Hubei Agric. Sci. 56 (16) (2017) 3074e3077. https://doi.org/10.14088/j.cnki.issn0439-8114.2017.16.018. [10] S.X. Fang, D.L. Wang, L.H. Zhu, T.T. Shi, M.N. Qin, Z.F. Lin, J. Ecotoxicol. 10 (02) (2015) 69e75. https://doi.org/10.7524/AJE.1673-5897.20141102001. [11] X.Y. Wu, Toxic Effects of Ibuprofen and Triclosan on P450 Enzymes and Antioxidantive System Og the Yellow Catfish, Jinan University, 2013, pp. 37e38. http://kns.cnki.net/KCMS/detail/detail.aspx?dbcode¼CMFD& dbname¼CMFD201401&filename¼1013027646.nh&v¼MjM5MTZyV00xRnJD VVJMT2ZaT1J2Rnkza1ViN0pWRjI2SGJPNkdkZklxWkViUElSOGVYMUx1eFlTN 0RoMVQzcVQ¼. [12] Y.G. Li, Toxic Effects of Triclosan, Ibuprofen and Cadmium Chloride to Selenastrum Capricornutum, Jinan University, 2013, pp. 62e65. http://kns.cnki. net/KCMS/detail/detail.aspx?dbcode¼CMFD&dbname¼CMFD201501&
[13] [14]
[15] [16] [17] [18] [19] [20]
filename¼1013027640.nh&uid¼WEEvREdxOWJmbC9oM1NjYkZCbDdrdTVi ZlNLV1hVVnJxa3MwU2ZCY1Y2cGM¼$R1yZ0H6jyaa0en3RxVUd8df-oHi7XM MDo7mtKT6mSmEvTuk11l2gFA!!&v¼MjY2OTN5M2tXN3JPVkYyNkhiTzZH ZGZJcjVFYlBJUjhlWDFMdXhZUzdEaDFUM3FUcldNMUZyQ1VSTE9mWk9Sdk Y¼. Y.P. Cong, Z.F. Lin, T. Wang, X.M. Zou, H.M. Ge, D.Q. Yin, Environ. Chem. (07) (2013) 1348e1352. https://doi.org/10.7524/j.issn.0254-6108.2013.07.031. H. Ren, J.R. Wang, X.J. Chen, J.G. Liu, P. Lou, Feed Process. Test. 47 (21) (2011) 49e52. http://kns.cnki.net/KCMS/detail/detail.aspx?dbcode¼CJFQ&dbname¼ CJFD2011&filename¼ZGXM201121014&uid¼WEEvREdxOWJmbC9oM1NjY kZCbDdrdTViZlNLV1hVVnJxa3MwU2ZCY1Y2cGM¼$R1yZ0H6jyaa0en3RxVUd 8df-oHi7XMMDo7mtKT6mSmEvTuk11l2gFA!!&v¼MjU0MDNMdXhZUzdEaD FUM3FUcldNMUZyQ1VSTE9mWk9SdkZ5M25VNzdOUHlyVFk3RzRIOURPcm 85RVlJUjhlWDE¼. Z.S. Fang, Z.F. Yao, T. Wang, Z.F. Lin, S.P. Zuo, Asian J. Ecotoxicol. 11 (2) (2016) 369e373. https://doi.org/10.7524/AJE.1673-5897.20151102001. L. Tong, L.L. Yao, H. Liu, Y.X. Wang, J. Ecotoxicol. 11 (2) (2016) 27e36. https:// doi.org/10.7524/AJE.1673-5897.20151127002. S.H.A. Hassan, S. Eun Oh, B: Biol. 101 (1) (2010) 16e21. https://doi.org/10. 1016/j.jphotobiol.2010.06.006. Y.Y. Dong, J. Wang, L. Ding, Y.Y. Liu, Procedia Environ. Sci. 18 (2013) 143e148. https://doi.org/10.1016/j.proenv.2013.04.019. D.Y. Tian, Z.F. Lin, X.H. Zhou, D.Q. Yin, Toxicol. Appl. Pharmacol. 272 (2) (2013) 551e558. https://doi.org/10.1016/j.taap.2013.06.015. L.M. Su, X.J. Zhang, X. Yuan, Y.H. Zhao, D.M. Zhang, W.C. Qin, J. Hazard Mater. (2012) 241e242. https://doi.org/10.1016/j.jhazmat.2012.09.065.