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Uptake and depuration of eight fluoroquinolones (FQs) in common carp (Cyprinus carpio)
Ecotoxicology and Environmental Safety 180 (2019) 202–207
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Uptake and depuration of eight fluoroquinolones (FQs) in common carp (Cyprinus carpio)
T
Mo Chen, Hongxia Zhao∗, Yan Wang, Tadiyose Girma Bekele, Wanyu Liu, Jingwen Chen Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Linggong Road 2, Dalian, 116024, China
A R T I C LE I N FO
A B S T R A C T
Keywords: FQs Common carp Bioconcentration Tissue distribution
Fluoroquinolones (FQs) are extensively used in humans and animals, which have aroused wide attention due to the emergence of FQ resistant bacteria and frequent detection in water, sediment and organism. However, little information is available about the bioconcentration and tissue distribution of FQs in fish. In the present study, we investigated the uptake and depuration of eight FQs (balofloxacin (BAL), enoxacin (ENO), enrofloxacin (ENR), fleroxacin (FLE), lomefloxacin (LOM), moxifloxacin (MOX), ofloxacin (OFL), sparfloxacin (SPA)) in common carp under controlled laboratory conditions. The results showed that all target FQs could accumulate in fish tissues, and had a similar tendency over time during the whole uptake and depuration periods. The uptake rate constant (k1), depuration rate constant (k2) and half-lives (t1/2) were in the ranges of 0.007–3.599 L/(kg·d), 0.051–0.283 d−1 and 2.4–10.7 d, respectively. The ranges of bioconcentration factors (BCFs) were 0.24–39.55 L/ kg, 0.21–24.97 L/kg and 0.04–1.07 L/kg in liver, kidney and muscle, respectively. BCFs of eight FQs decreased in the order: MOX > ENR > ENO ≈ BAL ≈ FLE ≈ OFL ≈ LOM ≈ SPA, which may be correlated with the substituents at positions 7 and 8 of the basic quinolone nucleus and the metabolic capacity. Besides, BCFs were relative with pH-adjusted distribution coefficient (log D), suggesting that molecular status of ionizable compounds strongly influenced the bioconcentration processes. The present study provides important insights for understanding the bioconcentration and tissues distribution of FQs.
1. Introduction Fluoroquinolones (FQs), are a kind of synthetic antimicrobial drugs, which can be active against gram-positive bacteria, gram-negative bacteria and mycoplasma by inhibiting their DNA gyrase (Chen et al., 2010). Due to their broad-spectrum characteristic, FQs are widely used in most types of animal husbandry and aquaculture to prevent and treat diseases (Tufa et al., 2015; Van et al., 2014). So far, a large number of FQs are produced and used around world every year (Ferech et al., 2006; FDA, 2012; FDA, 2014; Zhang et al., 2015). Especially in China, the largest producer and user of antibiotics in the world (Zhu et al., 2013; Hvistendahl, 2012), consumed about 27300 tons of FQs in 2013 alone, which accounted for 17% of the total antibiotics usage (Zhang et al., 2015). It has been proven that FQs are incompletely absorbed and only partially metabolized in humans and animals, and approximately 70% of these are ultimately excreted as parent compounds (Kümmerer, 2009). As a consequence, FQs have been frequently detected in water (Du et al., 2017; Ma et al., 2015; Zou et al., 2011), soil (Wei et al., 2016; Chen et al., 2015) and biota samples (Liu et al., 2017; Chen et al.,
∗
2015). Nowadays, residues of FQs in edible animals have aroused wide concern (Kang et al., 2018; Song et al., 2017; Barani and Fallah, 2015). These residues may accumulate in human bodies through food chains, which could contribute to the emergence of antibiotic resistance bacterial (Xiong et al., 2015). Furthermore, FQs also had a risk of irreversible nerve damage announced by the Food and Drug Adminstration (FDA) (Marchant, 2018). To protect public health and ensure safety of animal origin food, European Union (EU), and the Codex Alimentarius Commission have set maximum residue limits (MRLs) of FQs in edible tissues (European Commission, 2010; Codex Alimentarius Commission, 2015). FQs were easy to enter aquatic biotas due to continuous discharge into water environment (Minh et al., 2009; Gulkowska et al., 2007; Glassmeyer et al., 2005). It has been proven that FQs were bioaccumulative in fish tissues with bioaccumulation factor (BAF) up to 39801 L/kg by some field studies (Liu et al., 2018; Xie et al., 2017). At present, several controlled laboratory studies have been operated to investigate bioconcentration of pharmaceuticals in aquatic animals. For instance, Liu et al. (2014) investigate the uptake of roxithromycin
Corresponding author. E-mail address: [email protected] (H. Zhao).
https://doi.org/10.1016/j.ecoenv.2019.04.075 Received 25 January 2019; Received in revised form 2 April 2019; Accepted 25 April 2019 Available online 13 May 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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chromatography-tandem mass spectrometry (UPLC-MS/MS) system (Waters, MA, USA). All detailed instrument conditions were included in the SI.
(ROX) in crucian carp (Carassius auratus) and results showed that liver exhibited the highest concentration followed by bile, gills and muscle tissues. Ding et al. (2016) observed the influence of pH on bioconcentration of ROX and propranolol (PRP) in daphnia magna, and found that bioconcentration factors (BCFs) increased with increasing pH levels. However, reports on the uptake and depuration kinetics of FQs in aquatic organisms are still limited, and this information is essential for ecological risk assessment. Common carp (Cyprinus carpio) is commonly used as bioindicator species due to its wide distribution, easy availability, sensitivity to xenobiotic exposure and laboratory conditions adaptability (SaucedoVence et al., 2017). Therefore, the objectives of this study were to investigate: (1) the concentration levels of eight FQs in common carp liver, kidney and muscle tissues under controlled laboratory conditions; (2) the bioconcentration kinetics, including uptake/depuration rate constans (k1/k2), half-lives (t1/2) and BCF values; (3) tissue distribution. The results of the present study provided basic data for antibiotic risk assessments in fish.
2.4. Quality assurance and quality control The limits of detection (LODs) and the limits of quantification (LOQs) were defined as three and ten times the ratios of signal to noise (n = 6). The LODs and LOQs were in the range of 0.033–0.137 μg/L and 0.110–0.457 μg/L, respectively. The method recovery and reproducibility were determined by analyses of six replicates of target compoundfree fish tissues spiked with standards. The recoveries were 45.0%–92.1%, and relative standard deviations (RSD) were 12.1%–19.6%. 2.5. Data analysis The bioconcentration kinetic parameters for each FQ in liver, kidney and muscle were calculated based on a mass balance model (Mackay and Fraser, 2000). In this model, the uptake and depuration process can be described as eq. (1):
2. Materials and methods 2.1. Chemicals
dCB = k1 CW − k2 CB dt
Balofloxacin (BAL), enoxacin (ENO), enrofloxacin (ENR), fleroxacin (FLE), lomefloxacin (LOM), moxifloxacin (MOX), ofloxacin (OFL), sparfloxacin (SPA) were purchased from Dalian Meilun Biological Technology Co., Ltd (Dalian, China). Their structures were shown in Fig. S1 in Supporting Information (SI). Ciprofloxacin-D8 (CIP-D8) was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). All of their purities were > 98%. All the chemicals used were analytical or HPLC grade. A Millipore Milli-Q system was used to purify water for all experiments.
(1)
where CB was the FQ concentration in each fish tissue (ng/g, ww), CW was the chemical concentration in water phase (ng/L), k1 was the chemical uptake rate constant (L/(kg·d)), k2 was depuration rate constant (d−1), t was the exposure time (d). If elimination from the respiratory and loss due to metabolism and egestion were ignored, then k1 was calculated by iteratively fitting a nonlinear regression to the eq. (2):
CB = [(k1/ k2) CW ] (1 − e−k2 t )
(2)
k2 was obtained by fitting a nonlinear regression to the eq. (3):
2.2. Uptake and depuration experiments
CB = Ae−k2 t
In this study, common carp (Cyprinus carpio) was selected as test species, and experiments were conducted in a semi-static aquarium system with continuous aeration. Juvenile common carp (7.8 ± 0.7 cm, 4.3 ± 0.9 g) were purchased from a local aquarium in Dalian. All fish were acclimated in aerated tap water at least one week prior to use, fed with commercial pellets daily, and excess food was siphoned from aquariums after feeding. Water temperature, dissolved oxygen, and pH were 20.9 ± 1.4 °C, 95.8 ± 2.9% and 7.4 ± 0.1, respectively. The uptake experiment was conducted in glass tanks containing 90 L of water. Fish was exposed to constant concentration (10 μg/L for each analyte) in replicate aquaria, and a non-spiked tank served as control. The concentration of exposure group was similar to that investigated by Zou et al. (2011), who reported the concentrations of FQs were in the range of not detected to 12.29 μg/L in the Bohai Bay. To confirm the aqueous stability of analytes, water samples were collected from each tank during the exposure period. After 28-d exposure, tanks were refilled and replaced with equal volumes of tap water. Then fish were exposed to the semi-static clean water for 20 days. Duplicate samples (each sample with 3 fish) were randomly selected and euthanized instantly during exposure (i.e., days 2, 4, 6, 9, 12, 16, 20, 24, and 28) and depuration (i.e., days 32, 36, 40, 44, and 48) periods. After measuring the weight and length of each individual fish, their tissues (including liver, kidney and muscle) were removed, weighed separately. Samples were stored in fridges at −20 °C until analysis.
(3)
At the steady state, BCF could be derived using the eq. (4):
BCF =
k1 k2
(4)
And the half-lives (t1/2) of chemicals in biological samples were calculated according to the eq. (5):
t1/2 =
ln2 k2
(5)
Statistical analysis was performed using Origin 8.5 (Origin Lab Corporation) and Simca 13.0 (Umetri AB & Erisoft AB). An analysis of variance method (ANOVA) was applied to examine significant differences. A p-value of < 0.05 was used as statistically significant. 3. Results and discussions 3.1. Fish mortality and morphological indices No adverse behavior was observed in the both control and treatment groups during the experiment period. Mortalities of each group are shown in Table S2. Morphological indices (MI) were proposed as “exposure indices” to environmental pollutants, indicating the overall effect of contaminants on individual fish (Li et al., 2011). Therefore, in this study, one typical MI, hepatosomatic index (HSI) was determined. HSI was calculated by the ratio of liver weight to the whole body weight, and shown in Table S2.
2.3. Sample analysis Treatment procedure was followed the method employed in our previous study with some modifications (Zhao et al., 2018), and shown in the SI. The samples were analyzed by an ultra-performance liquid
3.2. Uptake and depuration of eight FQs in common carp The concentrations of target FQs in exposure water are shown in 203
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Fig. 1. Uptake and depuration of target FQs in common carp liver, kidney and muscle exposed to constant concentration of 10 μg/L.
and 0.04–1.07 L/kg in liver, kidney and muscle, respectively. We noted that muscle had smallest BCF values compared to liver and kidney, which was relative with its low uptake rates and high depuration rates. For example, the k1 values in liver and kidney were 2.45–16.22 and 2.73–9.51 times higher than those in muscle, respectively, and k2 values in muscle of MOX were 1.23 and 1.15 times higher compared to those in kidney and liver, respectively. As a result, BCF values in liver and kidney were 2.14–36.09 and 1.85–22.41 times higher compared with those in muscle, respectively. And this phenomenon might be correlative with phospholipid content in each tissue. FQs are a kind of ionogenic organic chemicals. Phospholipids are zwitterionic with a positively charged choline group and a negatively charged phosphate group. FQs may interact with phospholipids via electrostatic forces, besides the hydrophobic effect (Escher et al., 2000; Armitage et al., 2012). And a previous study has proven that liver and kidney exhibit higher phospholipid content than muscle (Schmitt, 2008). Consequently, tissue rich in phospholipids (e.g., liver, kidney) could be the primary phase for accumulation of FQs. Apart from that, we found the BCF values of FQs in common carp liver in the present study were lower than those reported previously for grass carp and tilapia, respectively (Chen et al., 2018; Zhao et al., 2015). This variance in BCF values of FQs from different species may be explained by the interspecies variations in biological uptake and depuration. BCF values of eight FQs decreased in the order: MOX > ENR > ENO ≈ BAL ≈ FLE ≈ OFL ≈ LOM ≈ SPA. Most FQs have the similar bioconcentration potentials except for MOX and ENR, which might be correlated with their chemical structures and metabolic capacity. As we all know, FQs are named based on C6 fluorine substituent of quinolone structure, and mainly distinguished by the substituents in position C7 and C8. The characteristic substituents of MOX are C7 diazabicyclo and C8 methoxy group. C7 diazabicyclo might increase lipophilicity and C8 methoxy group might enhance cell penetration, which has been reported by Van Doorslaer et al. (2015) and Kocsis et al. (2016). Under the effects of above two substituents, MOX had the biggest BCF value, which was similar to that in mussel, with BCF up to 70 L/kg (Gilroy Ève et al., 2014). Although BAL has a methoxy group in position C8, it still exhibited low BCF value. And a previous study has proven that an optimal combination of C7 and C8 substituents may be important for the activity against bacteria (Malik and Drlica, 2006). Therefore, we speculated that bioconcentration potentials of FQs might also be influenced by the interaction of substituents in the position of C7 and C8. Besides, ENR also had high BCF
Table S3. The bioconcentration profiles in common carp over 48 days of exposure to constant concentration (10 μg/L) are presented at Fig. 1. All target FQs were consistently detected in liver, kidney and muscle samples. Their concentrations showed a similar tendency over time. During the whole exposure periods, FQ concentrations increased with time, and rapid uptake was observed at initial time. For example, the growth rate of MOX was 14.10 ng/(g·d) in the first four days. The concentrations of ENR and MOX increased all the time, up to 63.28 ng/ g (ww) and 295.12 ng/g (ww), respectively, while the other six FQs leveled off on day 28. According to testing guidelines published by OECD (OECD, 1996), the times to 95% of steady state (i.e., t95 = 3.0/k2) in the uptake periods were in the ranges of 10.6 d (for SPA in liver) and 58.8 d (for ENR in muscle) (Table S4). Thus, we speculated that ENR and MOX appeared to pseudo-steady state after 28 d exposure. This phenomenon, which has also been observed in the uptake of ROX in Daphnia magna (Ding et al., 2016), might be attributed to the mask of further uptake by growth dilution or the compensation by depuration (Maes et al., 2014). During the depuration periods, the concentrations of target compounds decreased with a high speed at initial stage, and then with a smooth slow speed until under detection limits. We also noted that SPA displayed strong clearance ability, with concentration dropped down nearly 90% between days 28 and 32. On the contrary, 96.4% of ENR could still be detected in the fish tissues after 96 h of elimination. The uptake/depuration rate constant (k1/k2) and half-lives (t1/2) of the target chemicals were calculated, and provided in Table 1. The k1 values ranged from 0.038 to 3.599 L/(kg·d) in liver, 0.037–2.197 L/ (kg·d) in kidney, and 0.007–0.209 L/(kg·d) in muscle. The maximum k1 was 3.599 L/(kg·d), found in liver for MOX, the minimum k1 was 0.007 L/(kg·d), discovered in muscle for OFL. The k2 values of eight FQs were in the range of 0.065–0.283 d−1, 0.058–0.270 d−1 and 0.051–0.223 d−1 in liver, kidney and muscle, respectively, with corresponding half-lives of 2.4–10.7 d, 2.6–12.0 d and 3.1–13.6 d, respectively. The maximum and minimum k2 were 0.283 d−1 and 0.051 d−1, found in liver for SPA and muscle for ENR, respectively. The k1 and k2 values of ENR in the liver were similar to that reported by Liu et al. (2014) observed for ROX in crucian carp.
3.3. Bioconcentration patterns The BCF values of eight FQs in different tissues are shown in Table 1. The ranges of BCFs were 0.24–39.55 L/kg, 0.21–24.97 L/kg 204
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Table 1 Kinetic parameters of eight FQs in common carp at concentration of 10 ng/mL. Compounds
Tissues
k1(L/(kg·d))
R2
k2(d−1)
BAL
Liver Kidney Muscle Liver Kidney Muscle Liver Kidney Muscle Liver Kidney Muscle Liver Kidney Muscle Liver Kidney Muscle Liver Kidney Muscle Liver Kidney Muscle
0.077 0.122 0.022 0.115 0.094 0.018 0.189 0.149 0.040 0.114 0.202 0.020 0.038 0.096 0.011 3.599 2.197 0.209 0.068 0.037 0.007 0.067 0.058 0.012
0.97 0.84 0.88 0.98 0.88 0.86 0.91 0.94 0.78 0.95 0.88 0.87 0.99 0.96 0.98 0.89 0.82 0.86 0.85 0.93 0.91 0.95 0.90 0.90
0.085 0.142 0.148 0.112 0.138 0.137 0.065 0.058 0.051 0.133 0.178 0.210 0.124 0.173 0.223 0.091 0.088 0.196 0.094 0.120 0.176 0.283 0.270 0.159
ENO
ENR
FLE
LOM
MOX
OFL
SPA
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.010 0.033 0.005 0.014 0.026 0.005 0.051 0.028 0.014 0.018 0.114 0.005 0.002 0.011 0.001 0.808 0.647 0.053 0.018 0.007 0.002 0.009 0.011 0.002
values in fish tissues, which might be relative with its metabolic capacity. Metabolic reactions often added the eOH group(s) to ENR (Morales-Gutiérrez et al., 2015; Junza et al., 2014). This type of transformation increased the H-bond donor strength of the molecule (Endo et al., 2011). Thereby ENR might be likely to remain in tissues rather than to be excreted, which made it show high BCF values. In summary, the bioconcentration potentials of FQs might be influenced by the optimal combination of C7 and C8 substituents of quinolone ring and metabolic capacity. The bioconcentration potential is an important criterion to evaluate food safety and ecosystem risk. Octanol-water partition coefficient (Kow) has been proven to be a good predictor of bioconcentration for organic chemicals (Dai et al., 2013; Lai et al., 2002). The BCF values of FQs in liver and muscle increased with log Kow, however there wasn't significant correlation (r = 0.78–0.79, p > 0.05, Fig. S2). It wasn't surprising to find this, because FQs were a kind of ionizable compounds, which can exhibit various ionization states at environmental pH values. Due ionic form can hardly diffuse across biomembrane, bioconcentration of ionizable compounds was relative with the fraction of neutral molecules (fn) and the pH-adjusted distribution coefficient (log D) (Trapp, 2009; Wu et al., 2013). Therefore, to explore the role of log D in bioconcentration process of FQs, we evaluated the relationship between BCFs and their respective log D values (Table S5) at pH 7.4 (Tanoue et al., 2015). A significant positive correlation (r = 0.89–0.90, p < 0.05) was observed between BCFs and log D values (Fig. S2). These results suggested that the bioconcentration of ionizable compounds was strongly correlated with the fraction of neutral molecules and log D could better reflect bioconcentration potential, as noted in several previous studies (Liu et al., 2017; Zhao et al., 2017; Ding et al., 2016).
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.021 0.029 0.026 0.015 0.020 0.020 0.012 0.015 0.007 0.006 0.019 0.024 0.017 0.027 0.038 0.024 0.017 0.048 0.037 0.018 0.035 0.063 0.065 0.054
R2
t1/2(d)
BCF(L/kg)
0.75 0.82 0.87 0.92 0.90 0.90 0.86 0.73 0.92 0.99 0.95 0.94 0.91 0.89 0.87 0.73 0.83 0.76 0.52 0.89 0.83 0.79 0.76 0.60
8.2 4.9 4.7 6.2 5.0 5.1 10.7 12.0 13.6 5.2 3.9 3.3 5.6 4.0 3.1 7.6 7.9 3.5 7.4 5.8 3.9 2.4 2.6 4.4
0.91 0.86 0.15 1.03 0.68 0.13 2.91 2.57 0.78 0.86 1.13 0.10 0.31 0.55 0.05 39.55 24.97 1.07 0.72 0.31 0.04 0.24 0.21 0.08
Fig. 2. Tissue distribution of eight FQs in common carp after 28 days exposure in term of concentration.
concentrations (Fig. 3). The principal component (PC) 1 and 2 accounted for 71.3% and 28.7%, respectively. Points which were closed to each other in score scatter plot had the similar distribution. So for the target FQs, there were several types of distribution patterns, which might be relative with the characteristic substituents at positions 7 and 8 of the basic quinolone nucleus. There were two subsets (I, II), with the meaning of BAL and FLE, LOM and SPA might have the same distribution in common carp tissues, respectively. In general, FQs concentrations in biological samples decreased in the order: liver > kidney > muscle, which may be attributed to the predominance of quinolone nucleus. In a word, the difference of FQs in the proportion among liver, kidney and muscle might be relative with the substituents in positions C7 and C8 of quinolone ring, and the same trend in the concentrations of three tissues might be correlative with predominance of quinolone ring. Some previous studies have focused on tissue distribution of pharmaceuticals in fish. Zhao et al. (2016) discovered the highest concentration of sulfamethazine in marine medaka bile, followed by liver, gill and muscle. It was observed that the concentrations of methocarbamol and temazepam in bluegill tissues increased in the
3.4. Tissue distribution To understand the tissue distribution, the concentration percentages of FQs in various tissues, including liver, kidney and muscle were calculated after 28 d of exposure, as shown in Fig. 2. Compared to the other antibiotics, MOX was the easiest to concentrate in liver, accounting for 70.99%, followed by ENR, ENO, SPA, LOM, BAL, FLE and OFL. OFL most likely enriched in kidney and muscle, accounting for 51.93% and 6.67%, respectively. And principle component analysis (PCA) was performed to investigate tissue distribution in terms of 205
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Chen, H., Liu, S., Xu, X.R., et al., 2015b. Antibiotics in typical marine aquaculture farms surrounding Hailing Island, South China: Occurrence, bioaccumulation and human dietary exposure. Mar. Pollut. Bull. 90, 181–187. Chen, H., Liu, S., Xu, X.R., et al., 2018. Tissue distribution, bioaccumulation characteristics and health risk of antibiotics in cultured fish from a typical aquaculture area. J. Hazard Mater. 343 (5), 140–148. Codex Alimentarius Commission, 2015. Maximum Residue Limits (MRLs) and Risk Management Recommendations (RMRs) for Residues of Veterinary Drugs in Foods. 2015. Updated as at the 38th Session of the Codex Alimentarius Commission. Codex Alimentarius Commission. http://www.codexalimentarius.org/standards/ veterinary-drugs-mrls/en/. Dai, Z., Xia, X., Guo, J., et al., 2013. Bioaccumulation and uptake routes of perfluoroalkyl acids in Daphnia magna. Chemosphere 90, 1589–1596. Ding, J.N., Lu, G.H., Liu, J.C., et al., 2016. Uptake, depuration, and bioconcentration of two pharmaceuticals, roxithromycin and propranolol, in Daphnia magna. Ecotoxicol. Environ. Saf. 126, 85–93. Du, J., Zhao, H.X., Liu, S.S., et al., 2017. Antibiotics in the coastal water of the South Yellow Sea in China: Occurrence, distribution and ecological risks. Sci. Total Environ. 595, 521–527. Endo, S., Escher, B.I., Goss, K.U., 2011. Capacities of membrane lipids to accumulate neutral organic chemicals. Environ. Sci. Technol. 45, 5912–5921. Escher, B.I., Schwarzenbach, R.P., Westall, J.C., 2000. Evaluation of liposome-water partitioning of organic acids and bases. 1. Development of a sorption model. Environ. Sci. Technol. 34, 3954–3961. European Commission, 2010. Commission regulation 37/2010 pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off. J. Eur. Communities 8 (6), 1–72. FDA, 2012. Drug use review. In: Department of Health and Human Services, Public Health Service, Food and Drug Administration, Center for Drug Evaluation and Research. Office of Surveillance and Epidemiology Available at: http://www.fda.gov/ downloads/Drugs/DrugSafety/InformationbyDrugClass/UCM319435.pdf. FDA, 2014. Summary report on antimicrobials sold or distributed for use in food-producing animals. In: Food and Drug Administration. Department of Health and Human Services, USA Available at: http://www.fda.gov/downloads/forindustry/userfees/ animaldruguserfeeactadufa/ucm416983.pdf. Ferech, M., Coenen, S., Malhotra-Kumar, S., et al., 2006. European surveillance of antimicrobial consumption (ESAC): Outpatient antibiotic use in Europe. J. Antimicrob. Chemother. 58 (2), 401–407. Gilroy Ève, A.M., Klinck Joel, S., Campbell, S.D., et al., 2014. Toxicity and bioconcentration of the pharmaceuticals moxifloxacin, rosuvastatin, and drospirenone to the unionid mussel Lampsilis siliquoidea. Sci. Total Environ. 487, 537–544. Glassmeyer, S.T., Furlong, E.T., Kolpin, D.W., et al., 2005. Transport of chemical and microbial compounds from known wastewater discharge: Potential for use as indicators of human fecal contamination. Environ. Sci. Technol. 39, 5157–5169. Gulkowska, A., He, Y.H., So, M.K., et al., 2007. The occurrence of selected antibiotics in Hong Kong coastal water. Mar. Pollut. Bull. 54 (8), 1287–1293. Hvistendahl, M., 2012. China takes aim at rampant antibiotic resistance. Science 336 (6083) 795-795. Junza, A., Barbosa, S., Codony, M.R., et al., 2014. Identification of metabolites and thermal transformation products of quinolones in raw cow's milk by liquid chromatography coupled to high-resolution mass spectrometry. J. Agric. Food Chem. 62 (8), 2008–2021. Kang, H.S., Lee, S.B., Shin, D., et al., 2018. Occurrence of veterinary drug residues in farmed fishery products in South Korea. Food Control 85, 57–65. Kocsis, B., Domokos, J., Szabo, D., 2016. Chemical structure and pharmacokinetics of novel quinolone agents represented by avarofloxacin, delafloxacin, finafloxacin, zabofloxacin and nemonoxacin. Ann. Clin. Microbiol. Antimicrob. 15, 34. Kümmerer, K., 2009. Antibiotics in the aquatic environment-a review-part I. Chemosphere 75 (4), 417–434. Lai, K.M., Scrimshaw, M.D., Lester, J.N., 2002. Prediction of the bioaccumulation factors and body burden of natural and synthetic estrogens in aquatic organisms in the river systems. Sci. Total Environ. 289, 159–168. Li, Z.H., Velisek, J., Zlabek, V., et al., 2011. Chronic toxicity of verapamil on juvenile rainbow trout (Oncorhynchus mykiss): Effects on morphological indices, hematological parameters and antioxidant responses. J. Hazard Mater. 185, 870–880. Liu, J.C., Lu, G.H., Wang, Y.H., et al., 2014. Bioconcentration, metabolism, and biomarker responses in freshwater fish Carassius auratus exposed to roxithromycin. Chemosphere 99, 102–108. Liu, S.S., Zhao, H.X., Hans-Joachim, L., et al., 2017. Antibiotics pollution in marine food webs in Laizhou Bay, North China: Trophodynamics and human exposure implication. Environ. Sci. Technol. 51 (4), 2392–2400. Liu, S.S., Bekele, T.G., Zhao, H.X., et al., 2018. Bioaccumulation and tissue distribution of antibiotics in wild marine fish from Laizhou Bay, North China. Sci. Total Environ. 631–632, 1398–1405. Ma, Y.P., Li, M., Wu, M.M., et al., 2015. Occurrences and regional distributions of 20 antibiotics in water bodies during groundwater recharge. Sci. Total Environ. S. 518–519, 498–506. Mackay, D., Fraser, A., 2000. Bioaccumulation of persistent organic chemicals: mechanisms and models. Environ. Pollut. 110, 375–391. Maes, H.M., Maletz, S.X., Ratte, H.T., et al., 2014. Uptake, elimination, and biotransformation of 17α-ethinylestradiol by the freshwater alga Desmodesmus subspicatus. Environ. Sci. Technol. 48 (20), 12354–12361. Malik, M., Drlica, K., 2006. Moxifloxacin lethality against mycobacterium tuberculosis in the presence and absence of chloramphenicol. Antimicrob. Agents Chemother. 50, 2842–2844. Marchant, J., 2018. When antibiotics turn toxic. Nature 555 (7697), 431–433.
Fig. 3. Two-dimensional principal component plots for tissue distribution of target FQs.
following order: muscle < brain < liver (Zhao et al., 2017). And the tissue distributions in our study were in accordance with the results of above research (Zhao et al., 2016, 2017). 4. Conclusion In summary, the present study investigated the uptake and depuration of eight FQs in common carp tissues under controlled experimental conditions. The results indicated that the tissue distribution of FQs was strongly influenced by phospholipids content, and the bioconcentration potential might be correlative with the interaction of substituents in the position of C7 and C8 of quinolone ring and the metabolic capacity. In addition, a positive correlation has also been observed between BCFs of target FQs and log D. Overall, the results of this study provide basic data for antibiotic risk assessment. However, the biotransformation of FQs in common carp needs to be investigated further. Acknowledgment This research was financially supported by the National Natural Science Foundation of China (21677023). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.04.075. References Armitage, J.M., Arnot, J.A., Wania, F., 2012. Potential role of phospholipids in determining the internal tissue distribution of perfluoroalkyl acids in biota. Environ. Sci. Technol. 46, 12285–12286. Barani, A., Fallah, A.A., 2015. Occurrence of tetracyclines, sulfonamides, fluoroquinolones and florfenicol in farmed rainbow trout in Iran. Food Agric. Immunol. 26 (3), 420–429. Chen, L., Zhang, X., Xu, Y., et al., 2010. Determination of fluoroquinolone antibiotics in environmental water samples based on magnetic molecularly imprinted polymer extraction followed by liquid chromatography-tandem mass spectrometry. Anal. Chim. Acta 662 (1), 31–38. Chen, G.L., Li, M., Liu, X., 2015a. Fluoroquinolone antibacterial agent contaminants in soil/groundwater: A literature review of sources, fate, and occurrence. Water Air Soil Pollut. 226 (12), 418.
206
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M. Chen, et al.
manure-amended soils in Eastern China. Chemosphere 144, 2377–2383. Wu, X., Ernst, F., Conkle, J.L., 2013. Comparative uptake and translocation of pharmaceutical and personal care products (PPCPs) by common vegetables. Environ. Int. 60, 15–22. Xie, Z.G., Lu, G.H., Yan, Z.H., et al., 2017. Bioaccumulation and trophic transfer of pharmaceuticals in food webs from a large freshwater lake. Environ. Pollut. 222, 356–366. Xiong, W.G., Sun, Y.G., Zhang, T., et al., 2015. Antibiotics, antibiotic resistance genes, and bacterial community composition in fresh water aquaculture environment in China. Microb. Ecol. 70 (2), 425–432. Zhang, Q.Q., Ying, G.G., Pan, C.G., et al., 2015. 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), 6772–6782. Zhao, J.L., Liu, Y.S., Liu, W.R., et al., 2015. Tissue-specific bioaccumulation of human and veterinary antibiotics in bile, plasma, liver and muscle tissues of wild fish from a highly urbanized region. Environ. Pollut. 198, 15–24. Zhao, S.H., Wang, X.H., Li, Y.Y., et al., 2016. Bioconcentration, metabolism, and biomarker responses in marine medaka (Oryzias melastigma) exposed to sulfamethazine. Aquat. Toxicol. 181, 29–36. Zhao, J.L., Furlong, E.T., Schoenfuss, H.L., et al., 2017. Uptake and disposition of select pharmaceuticals by bluegill exposed at constant concentrations in a flow-through aquatic exposure system. Environ. Sci. Technol. 51 (8), 4434–4444. Zhao, H.X., Quan, W.N., Bekele, T.G., et al., 2018. Effect of copper on the accumulation and elimination kinetics of fluoroquinolones in the zebrafish (Danio rerio). Ecotoxicol. Environ. Saf. 156, 135–140. Zhu, Y.G., Johnson, T.A., Su, J.Q., et al., 2013. Diverse and abundant antibiotic resistance genes in Chinese swine farms. Proc. Natl. Acad. Sci. USA 110 (9), 3435–3440. Zou, S.C., Xu, W.H., Zhang, R.J., et al., 2011. Occurrence and distribution of antibiotics in coastal water of the Bohai Bay, China: Impacts of river discharge and aquaculture activities. Environ. Pollut. 159, 2913–2920.
Minh, T.B., Leung, H.W., Loi, I.H., et al., 2009. Antibiotics in the Hong Kong metropolitan area: Ubiquitous distribution and fate in Victoria Harboue. Mar. Pollut. Bull. 58 (7), 1052–1062. Morales-Gutiérrez, F.J., Barbosa, J., Barrón, D., 2015. Metabolic study of enrofloxacin and metabolic profile modifications in broiler chicken tissues after drug administration. Food Chem. 172, 30–39. OECD, 1996. Bioconcentration: Flow-Through Fish Test. OECD Guidelines for the Testing Chemicals No. 305E. Organization for Economic Cooperation and Development, Paris, France, pp. 23. Saucedo-Vence, K., Elizalde-Velazquea, A., Dublan-Garcia, O., et al., 2017. Toxicological hazard induces by sucralose to environmentally relevant concentrations in common carp (Cyprinus carpio). Sci. Total Environ. 575, 347–357. Schmitt, W., 2008. General approach for the calculation of tissue to plasma partition coefficients. Toxicol. In Vitro 22, 457–467. Song, C., Zhang, C., Kamira, B., et al., 2017. Occurrence and human dietary assessment of fluoroquinolones antibiotics in cultured fish around Tai Lake, China. Environ. Toxicol. Chem. 36 (11), 2899–2905. Tanoue, R., Nomiyama, K., Nakamura, H., et al., 2015. Uptake and tissue distribution of pharmaceuticals and personal care products in wild fish from treated-wastewaterimpacted streams. Environ. Sci. Technol. 49, 11649–11658. Trapp, S., 2009. Bioaccumulation of polar and ionizable compounds in plants. In: In: Devillers, J. (Ed.), Ecotoxicology Modeling, vol. 2. Springer, New York, pp. 299–353. Tufa, R.A., Pinacho, D.G., Pascual, N., et al., 2015. Development and validation of an enzyme linked immunosorbent assay for fluoroquinolones in animal feeds. Food Control 57, 195–201. Van Doorslaer, X., Haylamicheal, I.D., Dewulf, J., et al., 2015. Heterogeneous photocatalysis of moxifloxacin in water: Chemical transformation and ecotoxicity. Chemosphere 119, S75–S80. Van, D.X., Dewulf, J., Van, L.H., et al., 2014. Fluoroquinolone antibiotics: An emerging class of environmental micropollutants. Sci. Total Environ. 500–501, 250–269. Wei, R.C., Ge, F., Zhang, L.L., et al., 2016. Occurrence of 13 veterinary drugs in animal
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Uptake and depuration of eight fluoroquinolones (FQs) in common carp (Cyprinus carpio)
T
Mo Chen, Hongxia Zhao∗, Yan Wang, Tadiyose Girma Bekele, Wanyu Liu, Jingwen Chen Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Linggong Road 2, Dalian, 116024, China
A R T I C LE I N FO
A B S T R A C T
Keywords: FQs Common carp Bioconcentration Tissue distribution
Fluoroquinolones (FQs) are extensively used in humans and animals, which have aroused wide attention due to the emergence of FQ resistant bacteria and frequent detection in water, sediment and organism. However, little information is available about the bioconcentration and tissue distribution of FQs in fish. In the present study, we investigated the uptake and depuration of eight FQs (balofloxacin (BAL), enoxacin (ENO), enrofloxacin (ENR), fleroxacin (FLE), lomefloxacin (LOM), moxifloxacin (MOX), ofloxacin (OFL), sparfloxacin (SPA)) in common carp under controlled laboratory conditions. The results showed that all target FQs could accumulate in fish tissues, and had a similar tendency over time during the whole uptake and depuration periods. The uptake rate constant (k1), depuration rate constant (k2) and half-lives (t1/2) were in the ranges of 0.007–3.599 L/(kg·d), 0.051–0.283 d−1 and 2.4–10.7 d, respectively. The ranges of bioconcentration factors (BCFs) were 0.24–39.55 L/ kg, 0.21–24.97 L/kg and 0.04–1.07 L/kg in liver, kidney and muscle, respectively. BCFs of eight FQs decreased in the order: MOX > ENR > ENO ≈ BAL ≈ FLE ≈ OFL ≈ LOM ≈ SPA, which may be correlated with the substituents at positions 7 and 8 of the basic quinolone nucleus and the metabolic capacity. Besides, BCFs were relative with pH-adjusted distribution coefficient (log D), suggesting that molecular status of ionizable compounds strongly influenced the bioconcentration processes. The present study provides important insights for understanding the bioconcentration and tissues distribution of FQs.
1. Introduction Fluoroquinolones (FQs), are a kind of synthetic antimicrobial drugs, which can be active against gram-positive bacteria, gram-negative bacteria and mycoplasma by inhibiting their DNA gyrase (Chen et al., 2010). Due to their broad-spectrum characteristic, FQs are widely used in most types of animal husbandry and aquaculture to prevent and treat diseases (Tufa et al., 2015; Van et al., 2014). So far, a large number of FQs are produced and used around world every year (Ferech et al., 2006; FDA, 2012; FDA, 2014; Zhang et al., 2015). Especially in China, the largest producer and user of antibiotics in the world (Zhu et al., 2013; Hvistendahl, 2012), consumed about 27300 tons of FQs in 2013 alone, which accounted for 17% of the total antibiotics usage (Zhang et al., 2015). It has been proven that FQs are incompletely absorbed and only partially metabolized in humans and animals, and approximately 70% of these are ultimately excreted as parent compounds (Kümmerer, 2009). As a consequence, FQs have been frequently detected in water (Du et al., 2017; Ma et al., 2015; Zou et al., 2011), soil (Wei et al., 2016; Chen et al., 2015) and biota samples (Liu et al., 2017; Chen et al.,
∗
2015). Nowadays, residues of FQs in edible animals have aroused wide concern (Kang et al., 2018; Song et al., 2017; Barani and Fallah, 2015). These residues may accumulate in human bodies through food chains, which could contribute to the emergence of antibiotic resistance bacterial (Xiong et al., 2015). Furthermore, FQs also had a risk of irreversible nerve damage announced by the Food and Drug Adminstration (FDA) (Marchant, 2018). To protect public health and ensure safety of animal origin food, European Union (EU), and the Codex Alimentarius Commission have set maximum residue limits (MRLs) of FQs in edible tissues (European Commission, 2010; Codex Alimentarius Commission, 2015). FQs were easy to enter aquatic biotas due to continuous discharge into water environment (Minh et al., 2009; Gulkowska et al., 2007; Glassmeyer et al., 2005). It has been proven that FQs were bioaccumulative in fish tissues with bioaccumulation factor (BAF) up to 39801 L/kg by some field studies (Liu et al., 2018; Xie et al., 2017). At present, several controlled laboratory studies have been operated to investigate bioconcentration of pharmaceuticals in aquatic animals. For instance, Liu et al. (2014) investigate the uptake of roxithromycin
Corresponding author. E-mail address: [email protected] (H. Zhao).
https://doi.org/10.1016/j.ecoenv.2019.04.075 Received 25 January 2019; Received in revised form 2 April 2019; Accepted 25 April 2019 Available online 13 May 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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chromatography-tandem mass spectrometry (UPLC-MS/MS) system (Waters, MA, USA). All detailed instrument conditions were included in the SI.
(ROX) in crucian carp (Carassius auratus) and results showed that liver exhibited the highest concentration followed by bile, gills and muscle tissues. Ding et al. (2016) observed the influence of pH on bioconcentration of ROX and propranolol (PRP) in daphnia magna, and found that bioconcentration factors (BCFs) increased with increasing pH levels. However, reports on the uptake and depuration kinetics of FQs in aquatic organisms are still limited, and this information is essential for ecological risk assessment. Common carp (Cyprinus carpio) is commonly used as bioindicator species due to its wide distribution, easy availability, sensitivity to xenobiotic exposure and laboratory conditions adaptability (SaucedoVence et al., 2017). Therefore, the objectives of this study were to investigate: (1) the concentration levels of eight FQs in common carp liver, kidney and muscle tissues under controlled laboratory conditions; (2) the bioconcentration kinetics, including uptake/depuration rate constans (k1/k2), half-lives (t1/2) and BCF values; (3) tissue distribution. The results of the present study provided basic data for antibiotic risk assessments in fish.
2.4. Quality assurance and quality control The limits of detection (LODs) and the limits of quantification (LOQs) were defined as three and ten times the ratios of signal to noise (n = 6). The LODs and LOQs were in the range of 0.033–0.137 μg/L and 0.110–0.457 μg/L, respectively. The method recovery and reproducibility were determined by analyses of six replicates of target compoundfree fish tissues spiked with standards. The recoveries were 45.0%–92.1%, and relative standard deviations (RSD) were 12.1%–19.6%. 2.5. Data analysis The bioconcentration kinetic parameters for each FQ in liver, kidney and muscle were calculated based on a mass balance model (Mackay and Fraser, 2000). In this model, the uptake and depuration process can be described as eq. (1):
2. Materials and methods 2.1. Chemicals
dCB = k1 CW − k2 CB dt
Balofloxacin (BAL), enoxacin (ENO), enrofloxacin (ENR), fleroxacin (FLE), lomefloxacin (LOM), moxifloxacin (MOX), ofloxacin (OFL), sparfloxacin (SPA) were purchased from Dalian Meilun Biological Technology Co., Ltd (Dalian, China). Their structures were shown in Fig. S1 in Supporting Information (SI). Ciprofloxacin-D8 (CIP-D8) was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). All of their purities were > 98%. All the chemicals used were analytical or HPLC grade. A Millipore Milli-Q system was used to purify water for all experiments.
(1)
where CB was the FQ concentration in each fish tissue (ng/g, ww), CW was the chemical concentration in water phase (ng/L), k1 was the chemical uptake rate constant (L/(kg·d)), k2 was depuration rate constant (d−1), t was the exposure time (d). If elimination from the respiratory and loss due to metabolism and egestion were ignored, then k1 was calculated by iteratively fitting a nonlinear regression to the eq. (2):
CB = [(k1/ k2) CW ] (1 − e−k2 t )
(2)
k2 was obtained by fitting a nonlinear regression to the eq. (3):
2.2. Uptake and depuration experiments
CB = Ae−k2 t
In this study, common carp (Cyprinus carpio) was selected as test species, and experiments were conducted in a semi-static aquarium system with continuous aeration. Juvenile common carp (7.8 ± 0.7 cm, 4.3 ± 0.9 g) were purchased from a local aquarium in Dalian. All fish were acclimated in aerated tap water at least one week prior to use, fed with commercial pellets daily, and excess food was siphoned from aquariums after feeding. Water temperature, dissolved oxygen, and pH were 20.9 ± 1.4 °C, 95.8 ± 2.9% and 7.4 ± 0.1, respectively. The uptake experiment was conducted in glass tanks containing 90 L of water. Fish was exposed to constant concentration (10 μg/L for each analyte) in replicate aquaria, and a non-spiked tank served as control. The concentration of exposure group was similar to that investigated by Zou et al. (2011), who reported the concentrations of FQs were in the range of not detected to 12.29 μg/L in the Bohai Bay. To confirm the aqueous stability of analytes, water samples were collected from each tank during the exposure period. After 28-d exposure, tanks were refilled and replaced with equal volumes of tap water. Then fish were exposed to the semi-static clean water for 20 days. Duplicate samples (each sample with 3 fish) were randomly selected and euthanized instantly during exposure (i.e., days 2, 4, 6, 9, 12, 16, 20, 24, and 28) and depuration (i.e., days 32, 36, 40, 44, and 48) periods. After measuring the weight and length of each individual fish, their tissues (including liver, kidney and muscle) were removed, weighed separately. Samples were stored in fridges at −20 °C until analysis.
(3)
At the steady state, BCF could be derived using the eq. (4):
BCF =
k1 k2
(4)
And the half-lives (t1/2) of chemicals in biological samples were calculated according to the eq. (5):
t1/2 =
ln2 k2
(5)
Statistical analysis was performed using Origin 8.5 (Origin Lab Corporation) and Simca 13.0 (Umetri AB & Erisoft AB). An analysis of variance method (ANOVA) was applied to examine significant differences. A p-value of < 0.05 was used as statistically significant. 3. Results and discussions 3.1. Fish mortality and morphological indices No adverse behavior was observed in the both control and treatment groups during the experiment period. Mortalities of each group are shown in Table S2. Morphological indices (MI) were proposed as “exposure indices” to environmental pollutants, indicating the overall effect of contaminants on individual fish (Li et al., 2011). Therefore, in this study, one typical MI, hepatosomatic index (HSI) was determined. HSI was calculated by the ratio of liver weight to the whole body weight, and shown in Table S2.
2.3. Sample analysis Treatment procedure was followed the method employed in our previous study with some modifications (Zhao et al., 2018), and shown in the SI. The samples were analyzed by an ultra-performance liquid
3.2. Uptake and depuration of eight FQs in common carp The concentrations of target FQs in exposure water are shown in 203
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Fig. 1. Uptake and depuration of target FQs in common carp liver, kidney and muscle exposed to constant concentration of 10 μg/L.
and 0.04–1.07 L/kg in liver, kidney and muscle, respectively. We noted that muscle had smallest BCF values compared to liver and kidney, which was relative with its low uptake rates and high depuration rates. For example, the k1 values in liver and kidney were 2.45–16.22 and 2.73–9.51 times higher than those in muscle, respectively, and k2 values in muscle of MOX were 1.23 and 1.15 times higher compared to those in kidney and liver, respectively. As a result, BCF values in liver and kidney were 2.14–36.09 and 1.85–22.41 times higher compared with those in muscle, respectively. And this phenomenon might be correlative with phospholipid content in each tissue. FQs are a kind of ionogenic organic chemicals. Phospholipids are zwitterionic with a positively charged choline group and a negatively charged phosphate group. FQs may interact with phospholipids via electrostatic forces, besides the hydrophobic effect (Escher et al., 2000; Armitage et al., 2012). And a previous study has proven that liver and kidney exhibit higher phospholipid content than muscle (Schmitt, 2008). Consequently, tissue rich in phospholipids (e.g., liver, kidney) could be the primary phase for accumulation of FQs. Apart from that, we found the BCF values of FQs in common carp liver in the present study were lower than those reported previously for grass carp and tilapia, respectively (Chen et al., 2018; Zhao et al., 2015). This variance in BCF values of FQs from different species may be explained by the interspecies variations in biological uptake and depuration. BCF values of eight FQs decreased in the order: MOX > ENR > ENO ≈ BAL ≈ FLE ≈ OFL ≈ LOM ≈ SPA. Most FQs have the similar bioconcentration potentials except for MOX and ENR, which might be correlated with their chemical structures and metabolic capacity. As we all know, FQs are named based on C6 fluorine substituent of quinolone structure, and mainly distinguished by the substituents in position C7 and C8. The characteristic substituents of MOX are C7 diazabicyclo and C8 methoxy group. C7 diazabicyclo might increase lipophilicity and C8 methoxy group might enhance cell penetration, which has been reported by Van Doorslaer et al. (2015) and Kocsis et al. (2016). Under the effects of above two substituents, MOX had the biggest BCF value, which was similar to that in mussel, with BCF up to 70 L/kg (Gilroy Ève et al., 2014). Although BAL has a methoxy group in position C8, it still exhibited low BCF value. And a previous study has proven that an optimal combination of C7 and C8 substituents may be important for the activity against bacteria (Malik and Drlica, 2006). Therefore, we speculated that bioconcentration potentials of FQs might also be influenced by the interaction of substituents in the position of C7 and C8. Besides, ENR also had high BCF
Table S3. The bioconcentration profiles in common carp over 48 days of exposure to constant concentration (10 μg/L) are presented at Fig. 1. All target FQs were consistently detected in liver, kidney and muscle samples. Their concentrations showed a similar tendency over time. During the whole exposure periods, FQ concentrations increased with time, and rapid uptake was observed at initial time. For example, the growth rate of MOX was 14.10 ng/(g·d) in the first four days. The concentrations of ENR and MOX increased all the time, up to 63.28 ng/ g (ww) and 295.12 ng/g (ww), respectively, while the other six FQs leveled off on day 28. According to testing guidelines published by OECD (OECD, 1996), the times to 95% of steady state (i.e., t95 = 3.0/k2) in the uptake periods were in the ranges of 10.6 d (for SPA in liver) and 58.8 d (for ENR in muscle) (Table S4). Thus, we speculated that ENR and MOX appeared to pseudo-steady state after 28 d exposure. This phenomenon, which has also been observed in the uptake of ROX in Daphnia magna (Ding et al., 2016), might be attributed to the mask of further uptake by growth dilution or the compensation by depuration (Maes et al., 2014). During the depuration periods, the concentrations of target compounds decreased with a high speed at initial stage, and then with a smooth slow speed until under detection limits. We also noted that SPA displayed strong clearance ability, with concentration dropped down nearly 90% between days 28 and 32. On the contrary, 96.4% of ENR could still be detected in the fish tissues after 96 h of elimination. The uptake/depuration rate constant (k1/k2) and half-lives (t1/2) of the target chemicals were calculated, and provided in Table 1. The k1 values ranged from 0.038 to 3.599 L/(kg·d) in liver, 0.037–2.197 L/ (kg·d) in kidney, and 0.007–0.209 L/(kg·d) in muscle. The maximum k1 was 3.599 L/(kg·d), found in liver for MOX, the minimum k1 was 0.007 L/(kg·d), discovered in muscle for OFL. The k2 values of eight FQs were in the range of 0.065–0.283 d−1, 0.058–0.270 d−1 and 0.051–0.223 d−1 in liver, kidney and muscle, respectively, with corresponding half-lives of 2.4–10.7 d, 2.6–12.0 d and 3.1–13.6 d, respectively. The maximum and minimum k2 were 0.283 d−1 and 0.051 d−1, found in liver for SPA and muscle for ENR, respectively. The k1 and k2 values of ENR in the liver were similar to that reported by Liu et al. (2014) observed for ROX in crucian carp.
3.3. Bioconcentration patterns The BCF values of eight FQs in different tissues are shown in Table 1. The ranges of BCFs were 0.24–39.55 L/kg, 0.21–24.97 L/kg 204
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Table 1 Kinetic parameters of eight FQs in common carp at concentration of 10 ng/mL. Compounds
Tissues
k1(L/(kg·d))
R2
k2(d−1)
BAL
Liver Kidney Muscle Liver Kidney Muscle Liver Kidney Muscle Liver Kidney Muscle Liver Kidney Muscle Liver Kidney Muscle Liver Kidney Muscle Liver Kidney Muscle
0.077 0.122 0.022 0.115 0.094 0.018 0.189 0.149 0.040 0.114 0.202 0.020 0.038 0.096 0.011 3.599 2.197 0.209 0.068 0.037 0.007 0.067 0.058 0.012
0.97 0.84 0.88 0.98 0.88 0.86 0.91 0.94 0.78 0.95 0.88 0.87 0.99 0.96 0.98 0.89 0.82 0.86 0.85 0.93 0.91 0.95 0.90 0.90
0.085 0.142 0.148 0.112 0.138 0.137 0.065 0.058 0.051 0.133 0.178 0.210 0.124 0.173 0.223 0.091 0.088 0.196 0.094 0.120 0.176 0.283 0.270 0.159
ENO
ENR
FLE
LOM
MOX
OFL
SPA
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.010 0.033 0.005 0.014 0.026 0.005 0.051 0.028 0.014 0.018 0.114 0.005 0.002 0.011 0.001 0.808 0.647 0.053 0.018 0.007 0.002 0.009 0.011 0.002
values in fish tissues, which might be relative with its metabolic capacity. Metabolic reactions often added the eOH group(s) to ENR (Morales-Gutiérrez et al., 2015; Junza et al., 2014). This type of transformation increased the H-bond donor strength of the molecule (Endo et al., 2011). Thereby ENR might be likely to remain in tissues rather than to be excreted, which made it show high BCF values. In summary, the bioconcentration potentials of FQs might be influenced by the optimal combination of C7 and C8 substituents of quinolone ring and metabolic capacity. The bioconcentration potential is an important criterion to evaluate food safety and ecosystem risk. Octanol-water partition coefficient (Kow) has been proven to be a good predictor of bioconcentration for organic chemicals (Dai et al., 2013; Lai et al., 2002). The BCF values of FQs in liver and muscle increased with log Kow, however there wasn't significant correlation (r = 0.78–0.79, p > 0.05, Fig. S2). It wasn't surprising to find this, because FQs were a kind of ionizable compounds, which can exhibit various ionization states at environmental pH values. Due ionic form can hardly diffuse across biomembrane, bioconcentration of ionizable compounds was relative with the fraction of neutral molecules (fn) and the pH-adjusted distribution coefficient (log D) (Trapp, 2009; Wu et al., 2013). Therefore, to explore the role of log D in bioconcentration process of FQs, we evaluated the relationship between BCFs and their respective log D values (Table S5) at pH 7.4 (Tanoue et al., 2015). A significant positive correlation (r = 0.89–0.90, p < 0.05) was observed between BCFs and log D values (Fig. S2). These results suggested that the bioconcentration of ionizable compounds was strongly correlated with the fraction of neutral molecules and log D could better reflect bioconcentration potential, as noted in several previous studies (Liu et al., 2017; Zhao et al., 2017; Ding et al., 2016).
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.021 0.029 0.026 0.015 0.020 0.020 0.012 0.015 0.007 0.006 0.019 0.024 0.017 0.027 0.038 0.024 0.017 0.048 0.037 0.018 0.035 0.063 0.065 0.054
R2
t1/2(d)
BCF(L/kg)
0.75 0.82 0.87 0.92 0.90 0.90 0.86 0.73 0.92 0.99 0.95 0.94 0.91 0.89 0.87 0.73 0.83 0.76 0.52 0.89 0.83 0.79 0.76 0.60
8.2 4.9 4.7 6.2 5.0 5.1 10.7 12.0 13.6 5.2 3.9 3.3 5.6 4.0 3.1 7.6 7.9 3.5 7.4 5.8 3.9 2.4 2.6 4.4
0.91 0.86 0.15 1.03 0.68 0.13 2.91 2.57 0.78 0.86 1.13 0.10 0.31 0.55 0.05 39.55 24.97 1.07 0.72 0.31 0.04 0.24 0.21 0.08
Fig. 2. Tissue distribution of eight FQs in common carp after 28 days exposure in term of concentration.
concentrations (Fig. 3). The principal component (PC) 1 and 2 accounted for 71.3% and 28.7%, respectively. Points which were closed to each other in score scatter plot had the similar distribution. So for the target FQs, there were several types of distribution patterns, which might be relative with the characteristic substituents at positions 7 and 8 of the basic quinolone nucleus. There were two subsets (I, II), with the meaning of BAL and FLE, LOM and SPA might have the same distribution in common carp tissues, respectively. In general, FQs concentrations in biological samples decreased in the order: liver > kidney > muscle, which may be attributed to the predominance of quinolone nucleus. In a word, the difference of FQs in the proportion among liver, kidney and muscle might be relative with the substituents in positions C7 and C8 of quinolone ring, and the same trend in the concentrations of three tissues might be correlative with predominance of quinolone ring. Some previous studies have focused on tissue distribution of pharmaceuticals in fish. Zhao et al. (2016) discovered the highest concentration of sulfamethazine in marine medaka bile, followed by liver, gill and muscle. It was observed that the concentrations of methocarbamol and temazepam in bluegill tissues increased in the
3.4. Tissue distribution To understand the tissue distribution, the concentration percentages of FQs in various tissues, including liver, kidney and muscle were calculated after 28 d of exposure, as shown in Fig. 2. Compared to the other antibiotics, MOX was the easiest to concentrate in liver, accounting for 70.99%, followed by ENR, ENO, SPA, LOM, BAL, FLE and OFL. OFL most likely enriched in kidney and muscle, accounting for 51.93% and 6.67%, respectively. And principle component analysis (PCA) was performed to investigate tissue distribution in terms of 205
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Chen, H., Liu, S., Xu, X.R., et al., 2015b. Antibiotics in typical marine aquaculture farms surrounding Hailing Island, South China: Occurrence, bioaccumulation and human dietary exposure. Mar. Pollut. Bull. 90, 181–187. Chen, H., Liu, S., Xu, X.R., et al., 2018. Tissue distribution, bioaccumulation characteristics and health risk of antibiotics in cultured fish from a typical aquaculture area. J. Hazard Mater. 343 (5), 140–148. Codex Alimentarius Commission, 2015. Maximum Residue Limits (MRLs) and Risk Management Recommendations (RMRs) for Residues of Veterinary Drugs in Foods. 2015. Updated as at the 38th Session of the Codex Alimentarius Commission. Codex Alimentarius Commission. http://www.codexalimentarius.org/standards/ veterinary-drugs-mrls/en/. Dai, Z., Xia, X., Guo, J., et al., 2013. Bioaccumulation and uptake routes of perfluoroalkyl acids in Daphnia magna. Chemosphere 90, 1589–1596. Ding, J.N., Lu, G.H., Liu, J.C., et al., 2016. Uptake, depuration, and bioconcentration of two pharmaceuticals, roxithromycin and propranolol, in Daphnia magna. Ecotoxicol. Environ. Saf. 126, 85–93. Du, J., Zhao, H.X., Liu, S.S., et al., 2017. Antibiotics in the coastal water of the South Yellow Sea in China: Occurrence, distribution and ecological risks. Sci. Total Environ. 595, 521–527. Endo, S., Escher, B.I., Goss, K.U., 2011. Capacities of membrane lipids to accumulate neutral organic chemicals. Environ. Sci. Technol. 45, 5912–5921. Escher, B.I., Schwarzenbach, R.P., Westall, J.C., 2000. Evaluation of liposome-water partitioning of organic acids and bases. 1. Development of a sorption model. Environ. Sci. Technol. 34, 3954–3961. European Commission, 2010. Commission regulation 37/2010 pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off. J. Eur. Communities 8 (6), 1–72. FDA, 2012. Drug use review. In: Department of Health and Human Services, Public Health Service, Food and Drug Administration, Center for Drug Evaluation and Research. Office of Surveillance and Epidemiology Available at: http://www.fda.gov/ downloads/Drugs/DrugSafety/InformationbyDrugClass/UCM319435.pdf. FDA, 2014. Summary report on antimicrobials sold or distributed for use in food-producing animals. In: Food and Drug Administration. Department of Health and Human Services, USA Available at: http://www.fda.gov/downloads/forindustry/userfees/ animaldruguserfeeactadufa/ucm416983.pdf. Ferech, M., Coenen, S., Malhotra-Kumar, S., et al., 2006. European surveillance of antimicrobial consumption (ESAC): Outpatient antibiotic use in Europe. J. Antimicrob. Chemother. 58 (2), 401–407. Gilroy Ève, A.M., Klinck Joel, S., Campbell, S.D., et al., 2014. Toxicity and bioconcentration of the pharmaceuticals moxifloxacin, rosuvastatin, and drospirenone to the unionid mussel Lampsilis siliquoidea. Sci. Total Environ. 487, 537–544. Glassmeyer, S.T., Furlong, E.T., Kolpin, D.W., et al., 2005. Transport of chemical and microbial compounds from known wastewater discharge: Potential for use as indicators of human fecal contamination. Environ. Sci. Technol. 39, 5157–5169. Gulkowska, A., He, Y.H., So, M.K., et al., 2007. The occurrence of selected antibiotics in Hong Kong coastal water. Mar. Pollut. Bull. 54 (8), 1287–1293. Hvistendahl, M., 2012. China takes aim at rampant antibiotic resistance. Science 336 (6083) 795-795. Junza, A., Barbosa, S., Codony, M.R., et al., 2014. Identification of metabolites and thermal transformation products of quinolones in raw cow's milk by liquid chromatography coupled to high-resolution mass spectrometry. J. Agric. Food Chem. 62 (8), 2008–2021. Kang, H.S., Lee, S.B., Shin, D., et al., 2018. Occurrence of veterinary drug residues in farmed fishery products in South Korea. Food Control 85, 57–65. Kocsis, B., Domokos, J., Szabo, D., 2016. Chemical structure and pharmacokinetics of novel quinolone agents represented by avarofloxacin, delafloxacin, finafloxacin, zabofloxacin and nemonoxacin. Ann. Clin. Microbiol. Antimicrob. 15, 34. Kümmerer, K., 2009. Antibiotics in the aquatic environment-a review-part I. Chemosphere 75 (4), 417–434. Lai, K.M., Scrimshaw, M.D., Lester, J.N., 2002. Prediction of the bioaccumulation factors and body burden of natural and synthetic estrogens in aquatic organisms in the river systems. Sci. Total Environ. 289, 159–168. Li, Z.H., Velisek, J., Zlabek, V., et al., 2011. Chronic toxicity of verapamil on juvenile rainbow trout (Oncorhynchus mykiss): Effects on morphological indices, hematological parameters and antioxidant responses. J. Hazard Mater. 185, 870–880. Liu, J.C., Lu, G.H., Wang, Y.H., et al., 2014. Bioconcentration, metabolism, and biomarker responses in freshwater fish Carassius auratus exposed to roxithromycin. Chemosphere 99, 102–108. Liu, S.S., Zhao, H.X., Hans-Joachim, L., et al., 2017. Antibiotics pollution in marine food webs in Laizhou Bay, North China: Trophodynamics and human exposure implication. Environ. Sci. Technol. 51 (4), 2392–2400. Liu, S.S., Bekele, T.G., Zhao, H.X., et al., 2018. Bioaccumulation and tissue distribution of antibiotics in wild marine fish from Laizhou Bay, North China. Sci. Total Environ. 631–632, 1398–1405. Ma, Y.P., Li, M., Wu, M.M., et al., 2015. Occurrences and regional distributions of 20 antibiotics in water bodies during groundwater recharge. Sci. Total Environ. S. 518–519, 498–506. Mackay, D., Fraser, A., 2000. Bioaccumulation of persistent organic chemicals: mechanisms and models. Environ. Pollut. 110, 375–391. Maes, H.M., Maletz, S.X., Ratte, H.T., et al., 2014. Uptake, elimination, and biotransformation of 17α-ethinylestradiol by the freshwater alga Desmodesmus subspicatus. Environ. Sci. Technol. 48 (20), 12354–12361. Malik, M., Drlica, K., 2006. Moxifloxacin lethality against mycobacterium tuberculosis in the presence and absence of chloramphenicol. Antimicrob. Agents Chemother. 50, 2842–2844. Marchant, J., 2018. When antibiotics turn toxic. Nature 555 (7697), 431–433.
Fig. 3. Two-dimensional principal component plots for tissue distribution of target FQs.
following order: muscle < brain < liver (Zhao et al., 2017). And the tissue distributions in our study were in accordance with the results of above research (Zhao et al., 2016, 2017). 4. Conclusion In summary, the present study investigated the uptake and depuration of eight FQs in common carp tissues under controlled experimental conditions. The results indicated that the tissue distribution of FQs was strongly influenced by phospholipids content, and the bioconcentration potential might be correlative with the interaction of substituents in the position of C7 and C8 of quinolone ring and the metabolic capacity. In addition, a positive correlation has also been observed between BCFs of target FQs and log D. Overall, the results of this study provide basic data for antibiotic risk assessment. However, the biotransformation of FQs in common carp needs to be investigated further. Acknowledgment This research was financially supported by the National Natural Science Foundation of China (21677023). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.04.075. References Armitage, J.M., Arnot, J.A., Wania, F., 2012. Potential role of phospholipids in determining the internal tissue distribution of perfluoroalkyl acids in biota. Environ. Sci. Technol. 46, 12285–12286. Barani, A., Fallah, A.A., 2015. Occurrence of tetracyclines, sulfonamides, fluoroquinolones and florfenicol in farmed rainbow trout in Iran. Food Agric. Immunol. 26 (3), 420–429. Chen, L., Zhang, X., Xu, Y., et al., 2010. Determination of fluoroquinolone antibiotics in environmental water samples based on magnetic molecularly imprinted polymer extraction followed by liquid chromatography-tandem mass spectrometry. Anal. Chim. Acta 662 (1), 31–38. Chen, G.L., Li, M., Liu, X., 2015a. Fluoroquinolone antibacterial agent contaminants in soil/groundwater: A literature review of sources, fate, and occurrence. Water Air Soil Pollut. 226 (12), 418.
206
Ecotoxicology and Environmental Safety 180 (2019) 202–207
M. Chen, et al.
manure-amended soils in Eastern China. Chemosphere 144, 2377–2383. Wu, X., Ernst, F., Conkle, J.L., 2013. Comparative uptake and translocation of pharmaceutical and personal care products (PPCPs) by common vegetables. Environ. Int. 60, 15–22. Xie, Z.G., Lu, G.H., Yan, Z.H., et al., 2017. Bioaccumulation and trophic transfer of pharmaceuticals in food webs from a large freshwater lake. Environ. Pollut. 222, 356–366. Xiong, W.G., Sun, Y.G., Zhang, T., et al., 2015. Antibiotics, antibiotic resistance genes, and bacterial community composition in fresh water aquaculture environment in China. Microb. Ecol. 70 (2), 425–432. Zhang, Q.Q., Ying, G.G., Pan, C.G., et al., 2015. 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), 6772–6782. Zhao, J.L., Liu, Y.S., Liu, W.R., et al., 2015. Tissue-specific bioaccumulation of human and veterinary antibiotics in bile, plasma, liver and muscle tissues of wild fish from a highly urbanized region. Environ. Pollut. 198, 15–24. Zhao, S.H., Wang, X.H., Li, Y.Y., et al., 2016. Bioconcentration, metabolism, and biomarker responses in marine medaka (Oryzias melastigma) exposed to sulfamethazine. Aquat. Toxicol. 181, 29–36. Zhao, J.L., Furlong, E.T., Schoenfuss, H.L., et al., 2017. Uptake and disposition of select pharmaceuticals by bluegill exposed at constant concentrations in a flow-through aquatic exposure system. Environ. Sci. Technol. 51 (8), 4434–4444. Zhao, H.X., Quan, W.N., Bekele, T.G., et al., 2018. Effect of copper on the accumulation and elimination kinetics of fluoroquinolones in the zebrafish (Danio rerio). Ecotoxicol. Environ. Saf. 156, 135–140. Zhu, Y.G., Johnson, T.A., Su, J.Q., et al., 2013. Diverse and abundant antibiotic resistance genes in Chinese swine farms. Proc. Natl. Acad. Sci. USA 110 (9), 3435–3440. Zou, S.C., Xu, W.H., Zhang, R.J., et al., 2011. Occurrence and distribution of antibiotics in coastal water of the Bohai Bay, China: Impacts of river discharge and aquaculture activities. Environ. Pollut. 159, 2913–2920.
Minh, T.B., Leung, H.W., Loi, I.H., et al., 2009. Antibiotics in the Hong Kong metropolitan area: Ubiquitous distribution and fate in Victoria Harboue. Mar. Pollut. Bull. 58 (7), 1052–1062. Morales-Gutiérrez, F.J., Barbosa, J., Barrón, D., 2015. Metabolic study of enrofloxacin and metabolic profile modifications in broiler chicken tissues after drug administration. Food Chem. 172, 30–39. OECD, 1996. Bioconcentration: Flow-Through Fish Test. OECD Guidelines for the Testing Chemicals No. 305E. Organization for Economic Cooperation and Development, Paris, France, pp. 23. Saucedo-Vence, K., Elizalde-Velazquea, A., Dublan-Garcia, O., et al., 2017. Toxicological hazard induces by sucralose to environmentally relevant concentrations in common carp (Cyprinus carpio). Sci. Total Environ. 575, 347–357. Schmitt, W., 2008. General approach for the calculation of tissue to plasma partition coefficients. Toxicol. In Vitro 22, 457–467. Song, C., Zhang, C., Kamira, B., et al., 2017. Occurrence and human dietary assessment of fluoroquinolones antibiotics in cultured fish around Tai Lake, China. Environ. Toxicol. Chem. 36 (11), 2899–2905. Tanoue, R., Nomiyama, K., Nakamura, H., et al., 2015. Uptake and tissue distribution of pharmaceuticals and personal care products in wild fish from treated-wastewaterimpacted streams. Environ. Sci. Technol. 49, 11649–11658. Trapp, S., 2009. Bioaccumulation of polar and ionizable compounds in plants. In: In: Devillers, J. (Ed.), Ecotoxicology Modeling, vol. 2. Springer, New York, pp. 299–353. Tufa, R.A., Pinacho, D.G., Pascual, N., et al., 2015. Development and validation of an enzyme linked immunosorbent assay for fluoroquinolones in animal feeds. Food Control 57, 195–201. Van Doorslaer, X., Haylamicheal, I.D., Dewulf, J., et al., 2015. Heterogeneous photocatalysis of moxifloxacin in water: Chemical transformation and ecotoxicity. Chemosphere 119, S75–S80. Van, D.X., Dewulf, J., Van, L.H., et al., 2014. Fluoroquinolone antibiotics: An emerging class of environmental micropollutants. Sci. Total Environ. 500–501, 250–269. Wei, R.C., Ge, F., Zhang, L.L., et al., 2016. Occurrence of 13 veterinary drugs in animal
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