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Sediment and salinity effects on the bioaccumulation of sulfamethoxazole in zebrafish (Danio rerio)
Chemosphere 180 (2017) 467e475
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Sediment and salinity effects on the bioaccumulation of sulfamethoxazole in zebrafish (Danio rerio) Y. Chen, J.L. Zhou*, L. Cheng, Y.Y. Zheng, J. Xu State Key Laboratory of Estuarine and Coastal Research, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Sulfamethoxazole bioaccumulation was quantified by direct concentration measurements. Sediment caused 13e28% reduction in sulfamethoxazole bioaccumulation. Salinity reduced sulfamethoxazole bioaccumulation to zebrafish. Equilibrium distribution of sulfamethoxazole was dominated by water. Sediment adsorption was most significant with small particles with large surface area.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 January 2017 Received in revised form 9 April 2017 Accepted 12 April 2017 Available online 14 April 2017
The dynamic distribution of a widely used antibiotic sulfamethoxazole between water, sediment and aquatic organisms (zebrafish) was studied in microcosms. Sulfamethoxazole concentrations in water were gradually reduced, while in sediment and zebrafish gradually increased, suggesting active adsorption and bioaccumulation processes occurring. The presence of sediment particles and their interactions with water reduced the bioaccumulation of sulfamethoxazole in zebrafish by 13e28%. The sediment of smaller particle size with more organic carbon content and higher surface area, adsorbed sulfamethoxazole more extensively and decreased its bioaccumulation most significantly. The effect became more severe with increasing salinity in water due to the salting out of sulfamethoxazole, resulting in 24e33% reduction in bioaccumulation. At equilibrium, the distribution of sulfamethoxazole in different phases was quantified, with most sulfamethoxazole being associated with water (97.3%), followed by sedimentary phase (2.7%) and finally zebrafish (0.05%). The findings provided important data for further research into antibiotics fate and bio-uptake in aquatic organisms, and subsequent ecotoxicity. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Sulfamethoxazole Bioaccumulation Sediment Salinity Zebrafish
1. Introduction Currently antibiotic residues in the environment have attracted worldwide attention due to their large-scale consumption, wide
* Corresponding author. E-mail address: [email protected] (J.L. Zhou). http://dx.doi.org/10.1016/j.chemosphere.2017.04.055 0045-6535/© 2017 Elsevier Ltd. All rights reserved.
occurrence, and potential risks for wildlife and human (Ahmed et al., 2015; Chen et al., 2015). In China alone, over 25,000 tons of antibiotics are produced every year (Yan et al., 2013), of which sulfonamides antibiotics are widely used in the animal feeding and human medicines (Zhang et al., 2015). Through different sources in particular sewage treatment works effluents (Michael et al., 2013) as hotspots, sulfonamides antibiotics will eventually be discharged into the natural aquatic environment, where their occurrence has
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been widely reported in water with concentrations as high as 211 mg/L (Zhao et al., 2015a). One of the main concerns with antibiotic contamination is that bacteria may develop antibiotic resistance gene (ARGs) from prolonged exposure to antibiotics, induced by plasmid horizontal transfer (Sengelov et al., 2003; Chen et al., 2017); and the migration and transformation of ARGs in the aquatic environment is potentially more harmful than the antibiotic residues (Ji et al., 2012). More attention should be given to sulfonamide antibiotics because of their widespread use, high excretion rate, high solubility and persistence in the environment n et al., 2011); for example it is reported that (García-Gala sulfonamide-resistant bacteria can remain stable in the aquatic environment for 5e10 years (Martinez et al., 2009). Antibiotics in general possess high migration ability, depending on their physicochemical properties such as water solubility, octanol/water partitioning coefficient (Kow), and acid dissociation constant (pKa) (Kay et al., 2005). It has been reported that sulfonamides have shown 100% detection and the highest concentrations in the water phase of Huangpu River (Chen and Zhou, 2014) and the Yangtze Estuary (Yan et al., 2013), China, where sulfamethoxazole is the dominant compound. It has been suggested that sulfonamides are highly stable in the aquatic environment (Yan et al., 2013), and sulfamethoxazole is difficult to be hydrolyzed and biodegraded (Benotti and Brownawell, 2009). Thus, sulfonamides residues have been predominantly detected in the water phase (Li et al., 2012; Na et al., 2013). In addition, aquatic sediment has been identified as an important medium to retain organic contaminants including relatively hydrophobic antibiotics (Zhou and Broodbank, 2014). Although Matongo et al. (2015) and Yang et al. (2015) reported relatively low concentrations of sulfamethoxazole in river sediments (<1 ng/g) from South Africa and Taiwan, respectively; significantly higher concentrations of sulfamethoxazole (up to 276 ng/g) were detected in southern Baltic Sea sediments by Siedlewicz et al. (2016). In addition, laboratory-based adsorption ndez et al. (2014) suggested strong experiments by Martínez-Herna affinity between sediment and sulfamethoxazole with only 4.9% of sorbed compound being desorbed. In addition, its adsorption may depend on the properties of contaminants and sediment e.g. particulate organic carbon content, and water properties such as temperature, pH, salinity and dissolved organic carbon (Hou et al., ndez et al., 2014). 2010; Xu and Li, 2010; Martínez-Herna In assessing the ecological risk of antibiotics, bioavailability is the key to determine bioaccumulation and adverse effects. Bioavailability is defined as the fraction of the unchanged chemical present in the organism (Alexander, 2000; Semple et al., 2004), and widely used in the environmental risk assessment of toxic chemicals (Wu and Zhu, 2016). Antibiotic contaminants may therefore bioaccumulate in aquatic organisms (Boonsaner and Hawker, 2013; Ding et al., 2015), for example, up to 1150 mg/kg of sulfonamides have been detected in aquatic organisms from field monitoring (Zhao et al., 2015a). Bayen et al. (2016) reported that sulfamethoxazole was bioavailable even to bivalves in mangrove habitats. There are chemical-based and biological-based approaches for assessing bioavailability, among which bioaccumulation experiments using animals is the most direct and relevant approach (Reichenberg and Mayer, 2006; Ortega-Calvo et al., 2015). The bioaccumulation of contaminants can be affected by the presence of suspended sediments and colloids in water (Gaillard et al., 2014). In addition, water salinity should be studied as a key parameter in estuarine environment where water pollution is particularly serious and can be highly influenced by tidal action (Zhao et al., 2015b). Zebrafish are widely used as the model organism for ecotoxicology experiments as they have many advantages e.g. small individual and easy breeding (Xia et al., 2016). In addition, as a
vertebrate organism, zebrafish (Danio rerio) possess significant physiological and morphological homology to human (Qiang et al., 2016). This study therefore aimed to determine the fate and bioaccumulation of sulfamethoxazole as a representative sulfonamides using zebrafish as the model organism, by hypothesizing that the compound will be bioavailable to zebrafish leading to bioaccumulation. Specifically the study examined the distribution of sulfamethoxazole between water, sediment and zebrafish phases in microcosm; the bioavailability and bioaccumulation of sulfamethoxazole to zebrafish; and the effects of sediment and salinity on sulfamethoxazole bioaccumulation. 2. Materials and methods 2.1. Chemicals, sediment and zebrafish Sulfamethoxazole standard was purchased from Dr. Ehrenstorfer (GmbH, Germany) and its physicochemical properties are shown in Supplementary Information Table A1. The compound sulfamethoxazole-d4 obtained from Dr. Ehrenstorfer (GmbH, Germany) was used as the internal standard to quantify sulfamethoxazole. All standard solutions were stored at 20 C. Sediment samples were collected in November 2014 from the east coast of Chongming Island in the Yangtze Estuary, China, as the sites did not show any antibiotic contamination from previous studies. The samples were freeze-dried for 72 h and homogenized, then successively sifted through 240-mesh (63 mm), 120-mesh (125 mm) and 60-mesh (250 mm) sieves, to obtain different sediment fractions (e.g. < 63 mm, 63e125 mm, and 125e250 mm). The specific surface area (SSA) of the sediments was 8.89 m2/g (<63 mm), 6.84 m2/g (63e125 mm), 6.60 m2/g (125e250 mm), and 6.78 m2/g (<250 mm), after measurement by a Quantchrome surface area analyzer using the BET method (Bowman et al., 2002). The organic carbon content of sediment samples was determined by high-temperature catalytic combustion following acidification twice by 5% sulfurous acid (Zhou and Broodbank, 2014). Wild-type zebrafish (Danio rerio, AB line) were maintained in several 40-L transparent plastic tanks filled with dechlorinated and carbon filtered tap water. The following conditions were monitored and maintained by the system: temperature 28 ± 1 C, dissolved oxygen >7 mg/L. Zebrafish were fed twice a day with dry flakes and brine shrimp (Artemia salina nauplii). A constant light/dark (14/ 10 h) cycle was used to meet its spawning condition. For this study, zebrafish of 3 months old, 2.8 ± 0.2 cm length, and 0.35 ± 0.08 g wet weight were used. Lipid content in zebrafish tissues based on wet weight was 4.76 ± 0.5%, which was measured in triplicate by the €lgyessy and Miha likova , 2015). All zebrafish gravimetric method (To were acclimatized to the laboratory conditions at least 14 days before use. For salinity experiments, zebrafish were acclimatized for a further 7 days in 10‰ and 20‰ salinity, during which no mortality was observed. 2.2. Fate and exposure microcosm experiments As shown in Fig. 1, four systems were designed in this study: (i) water, (ii) water-sediment, (iii) water-zebrafish, and (iv) watersediment-zebrafish, and each experiment was conducted in triplicate (i.e. 3 parallel tanks) to ensure statistical significance. The effects of sediment particle size (Fig. 1a) and salinity (Fig. 1b) on the bioaccumulation of sulfamethoxazole were investigated, respectively. In the water-sediment-zebrafish system, experiments were conducted in 8-L glass tanks containing 500 g (dry weight) of different sized sediments (<250 mm, < 63 mm, 63e125 mm, and 125e250 mm), respectively, which were mixed with 5 L of 10 mg/L sulfamethoxazole solution at a particular salinity (e.g. 0‰, 10‰ or
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Fig. 1. Experimental schematic for (a) the effect of sediment particle size and (b) the effect of salinity on the bioavailability and bioaccumulation of sulfamethoxazole (SMX) to zebrafish. 3 tanks per treatment, 40 zebrafish per tank.
20‰). Then 40 individuals of zebrafish were added to each tank to assess the antibiotic bioaccumulation ratio (BAR) in zebrafish: BAR ¼ Czebrafish/Cw
(1)
where BAR is defined as the ratio of sulfamethoxazole concentration in zebrafish (Czebrafish, ng/g dry weight) to sulfamethoxazole concentration in water (Cw, ng/mL). Without replacing water, the zebrafish were fed with the experimental diets without antibiotics contamination for 168 h. Water physicochemical parameters were checked daily to ensure a relatively stable water environment. Each chamber was equipped with a heater to maintain the water temperature at 28 ± 1 C, which was the optimal growth temperature for zebrafish. No mortality was observed during the experiments. Water, sediment, and zebrafish samples were collected at regular intervals and stored at 20 C. For zebrafish, 5 individuals were taken from each of the 3 parallel tanks, to obtain 3 aggregate samples. Then the sediment and zebrafish samples were freeze-dried for 72 h and grounded into powders. The wet and dry weight of each zebrafish was measured before and after freeze-drying, respectively. In the presence of sediment, sulfamethoxazole will interact with sediment at the same time when bioaccumulation is taking place. The binding strength of sediment-water interaction can be expressed with the distribution coefficient (KD): KD ¼ Cs/Cw
(2)
where Cs (ng/g dry weight) is sulfamethoxazole concentration in the sediment. 2.3. Antibiotic extraction and analysis in water, sediment and zebrafish Water samples (1 mL) were spiked with 20-ng of internal standard, before direct injection into high performance liquid chromatographetandem mass spectrometer (UHPLC-MS) for quantitative analysis. Sediment samples (3 g) were spiked with 20 ng of internal standard, and 9 mL of acetonitrile for extraction.
Each sample was stirred at 200 rpm for 20 min using a mechanical shaker before being sonicated in an ultrasonic bath for 15 min. The samples were centrifuged at 2500 rpm for 5 min to separate the solid and liquid phases. The liquid phase was collected, and the same extraction procedure was repeated twice to obtain a complete extraction of the target compound. When the extracts were evaporated to 0.5 mL under gentle N2 purging at 40 C, 5 mL of methanol was added, which was reduced to 0.5 mL. To the final extracts 0.5 mL of Milli-Q water with 0.1% formic acid was added before the chemical analysis. The processing of zebrafish samples was similar to the sediment samples, except an extra step of fat removal was introduced. The concentrated extracts were firstly reduced to 5 mL by evaporation under gentle N2 purging at 40 C, and stored at 20 C for 24 h. The lipids were removed via filtration. Each experimental group was replicated three times with two blank controls being used with each set of experiments. The final sample extracts were analyzed using a Waters Acquity™ UHPLCeMS/MS system with an HSS T3 column (2.1 mm 100 mm, 1.7 mm particle size), following methods by Zhou et al. (2012) and Chen and Zhou (2014). 2.4. Quality control and statistic analysis The antibiotic analysis was subject to quality assurance, by analyzing blanks and recovery samples and determining the limits of quantification (LOQs). The recovery of sulfamethoxazole in water, sediment and zebrafish samples was 96.3e101.1%, 82.2e88.1% and 68.8e78.9%, respectively. The LOQ values for sulfamethoxazole were estimated to be 0.34 ng/L in water, 0.11 ng/g (dry weight basis) in sediment, and 0.15 ng/g (dry weight basis) in zebrafish, respectively. All samples were analyzed in triplicate, and the relative standard deviation (RSD) values were less than 20%. All experimental data were expressed as the average ± standard error of mean. Homogeneity of variance and normality of all data set were first tested. As datasets of exposure tests were found to show equal variance, thus one-way analysis of variance (ANOVA) was applied to determine the statistical significance. Double tail Student's t-test was used to compare the difference between the exposure group and the control group. The value P < 0.05 was
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chosen as the threshold in both cases. All data were processed using Origin software (version 8.5). 3. Results and discussion 3.1. Sulfamethoxazole distribution between water, sediment and zebrafish in microcosm The results for the kinetics of sulfamethoxazole concentration changes in water, water-sediment, water-fish, and water-sedimentfish microcosms are shown in Fig. 2. In the water only control, sulfamethoxazole concentration decreased slightly from 9.98 to 9.70 mg/L (3%) over the 7 day period (Fig. 2a). It is apparent that this compound is stable during the experimental period, and suitable for adsorption and bioaccumulation experiments. The results agree with Carstens et al. (2013) who found there was still 92.1 ± 0.69% of sulfamethazine in pond water after 7 days. It has been reported that sulfonamides usually have high potential to resist degradation and hydrolysis in water (Lin and Gan, 2011). Yang et al. (2011) also reported that even in the presence of activated sludge, sulfonamide antibiotics were first removed by adsorption followed by biodegradation, and biodegradation was inhibited in the first 12 h due to competitive inhibition of xenobiotic oxidation by readily biodegradable substances. In comparison, the concentration of sulfamethoxazole in water decreased from 9.96 to 9.26 mg/L (7%) after 168 h in water-sediment microcosm, while sulfamethoxazole concentration in sediment (<250 mm) was increased from 0 to 2.55 ng/g (Fig. 2b). The results suggest significant interactions between sediment and water, similar to the behavior of other organic contaminants such as endocrine disrupting chemicals and pharmaceuticals (Bowman et al., 2002; Zhou and Broodbank, 2014). The results indicated that sediment played an important role in the distribution of sulfamethoxazole. Hou et al. (2010) reported high sorption of
sulfamethoxazole on both humic acids and inorganic mineral particles, with relatively low sorption being observed on sediment from Dianchi Lake, Yunan, China. In a study of sulfamethoxazole sorption by groundwater sediments from Madrid, Spain, Martínezndez et al. (2014) observed a high affinity between sediment Herna and sulfamethoxazole, evidenced by only 4.9% of the sorbed compound being released during desorption experiments. When zebrafish were introduced, sulfamethoxazole was found to accumulate rapidly in the body of zebrafish indicating that the compound was bioavailable (Fig. 2c). The concentration of sulfamethoxazole increased quickly in zebrafish, reaching the first peak level of 3.38 ng/g at 48 h of exposure. The bioaccumulated concentration then continued to rise slowly, reaching the highest concentration of 4.52 ng/g at 168 h. Meanwhile, sulfamethoxazole concentrations in water decreased from 10.01 to 9.62 mg/L i.e. 4%. Liu et al. (2014) also reported that roxithromycin was concentrated by freshwater fish (Carassius auratus) causing biological effects. When both sediment (<250 mm) and zebrafish were present in water (Fig. 2d), the concentration of sulfamethoxazole in water declined by 8% (from 9.98 to 9.21 mg/L) in the water-sedimentzebrafish microcosm, slightly faster than when only sediment was present (Fig. 2b) and significantly faster than when only zebrafish were present (Fig. 2c). The findings confirm that when more phases such as sediment were present in the microcosm, the amount of sulfamethoxazole removal from aqueous phase was enhanced. Sulfamethoxazole concentration in sediment was increasing with time, reaching the highest concentrations of 2.54 ng/g at 168 h. In addition, the concentration in zebrafish was also increased with time, reaching 3.42 ng/g at the end of experiment. The accumulation of sulfamethoxazole in zebrafish was therefore inhibited (24% reduction) in the presence of <250 mm sediment (Fig. 2c and d), confirmed by one-way ANOVA analysis with P < 0.01 compared to data without sediment. Compared with binary microcosms (water-fish, water-
Fig. 2. Kinetics of sulfamethoxazole concentration changes in (a) water, (b) water-sediment, (c) water-zebrafish, and (d) water-sediment-zebrafish microcosms. Sediment size is < 250 mm.
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sediment), the distribution of sulfamethoxazole became more complex in ternary microcosm due to competition between sediment adsorption and zebrafish bioaccumulation, leading to smaller body burden of antibiotic residues in zebrafish. 3.2. Effect of sediment on the bioaccumulation of sulfamethoxazole in zebrafish As shown in Fig. 3, the BAR values of sulfamethoxazole in waterzebrafish system increased with exposure time, reaching maximum values of 0.47 mL/g at 168 h. Similarly, BAR values in the presence of sediments also increased with time, however the equilibrium BAR value was reduced to 0.37 mL/g, a reduction of 21% due to competition of sediments for the antibiotic. One-way ANOVA Statistical analysis showed a significant difference between the two sets of BAR values, with P < 0.01. The BAR values of sulfamethoxazole are smaller than those observed by Hou et al. (2003), who reported higher BAR of 1.19 mL/g for sulfamethazine in the muscle of sturgeon after exposure to concentration of 100 mg/L. Such a difference could be due to differences in test species, fish biomass, antibiotics concentration and exposure time, all of which will affect the bioaccumulation potential. In the presence of sediment, the KD values of sulfamethoxazole at equilibrium were 0.28 and 0.26 mL/g in water-sediment microcosm and water-sediment-zebrafish microcosm, respectively as shown in Fig. 3. The results suggested significant sediment-water interaction which was unaffected by the presence of zebrafish. Previous study has reported that the KD values ranged from 2.23 to 6.05 mL/g for sulfamethoxazole in digested sludge (Carballa et al., 2008), which are 10 times higher than KD values in this study. Such difference would be caused by several factors: different types of solids with organic-rich sludge expected to have higher sorbing power than natural sediment, different concentrations of antibiotics being exposed, and different durations of solid-water interactions. These results implied that the KD values of antibiotics are most likely related to the hydrophobicity of the contaminants, physicochemical properties of sediment sorbents, and initial concentrations of the contaminants in water (Wegst-Uhrich et al., 2014). In addition, the hydrodynamics in the aquatic environment also play an important role due to kinetic controls and adsorptiondesorption cycles (Zhou and Broodbank, 2014).
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The distribution of contaminants between different environmental phases (water, air, sediment, and organisms) is determined by thermodynamic equilibria (Kummerer, 2010). In the present study, the mass balance of sulfamethoxazole distribution between water, sediment and zebrafish in the water-sediment-zebrafish microcosm is shown in Fig. 4. Most sulfamethoxazole tended to be associated with water at > 97% throughout the experiments, when sulfamethoxazole accumulation in sediments increased steadily while bioaccumulation began with a reduction followed by increases. At the equilibrium time of 168 h, the proportion of sulfamethoxazole in water, sediment and zebrafish was 97.3%, 2.7% and 0.05% of the total amount, respectively. The results confirmed that in ternary systems, the bioaccumulation of sulfamethoxazole was significantly reduced due to contaminant sorption to sediment. In addition, the bioaccumulation results agree with a previous study which reported zebrafish could accumulate 0.04% of erythromycin in a microcosm containing 500 mg/L of the contaminant in the presence of Elodea densa and sediment after 30 days (Wu et al., 2015). 3.3. Effect of sediment property on the bioaccumulation of sulfamethoxazole in zebrafish Since the bioaccumulation of sulfamethoxazole in the aquatic environment decreased after the sediment was introduced to the system, further experiments were performed to study the effects of the physicochemical properties of sediment on the distribution of sulfamethoxazole. Firstly, the effect of sediment particle size on the binding affinity of sulfamethoxazole in sediment and bioavailability of sulfamethoxazole in zebrafish was investigated. The calculated KD values of sulfamethoxazole at equilibrium were 0.36 mL/g (<63 mm), 0.31 mL/g (63e125 mm) and 0.26 mL/g (125e250 mm), as shown in Supplementary Information Fig. A1a. The results suggest that with increasing sediment particle size, the KD values gradually decreased, with the reduction being statistically significant (P < 0.05, One-way ANOVA). The SSA of the sediment fractions also decreased with increasing particle size from 8.89 m2/g for the <63 mm fraction, to 6.84 m2/g for the 63e125 mm fraction, and 6.60 m2/g for the 125e250 mm fraction. The results (Fig. 5) indicated that KD increased with SSA (r2 ¼ 0.79, P < 0.05).
Fig. 3. KD and bioaccumulation ratio (BAR) values in water-sediment and water-sediment-zebrafish microcosms. Sediment size <250 mm.
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Fig. 4. Mass balance distribution of sulfamethoxazole in water-sediment-zebrafish microcosm during exposure. Sediment size <250 mm.
Fig. 5. Relationship between sediment/water partition coefficient (KD) and zebrafish/water BAR of sulfamethoxazole and sediment specific surface area, in water-sedimentzebrafish microcosm.
The results are consistent with a similar study by Bowman et al. (2002) who showed the KD for endocrine disrupting chemicals decreased with sediment particle size and increased with sediment SSA. It is widely recognized that sediment sorption of endocrine disrupting chemicals and antibiotic contaminants is highly affected by the organic matter content in sediments (Bowman et al., 2002; Zhou and Broodbank, 2014). Similarly it is reported that the dominant sorption matrix for sulfamethoxazole is the organic matter (Hou et al., 2010; Sangster et al., 2015). These results implied that the KD values of antibiotics are most likely related to the hydrophobicity of the contaminants, physicochemical properties of sediment and initial concentrations of the contaminants in water (Wegst-Uhrich et al., 2014). The KD results for sulfamethoxazole exhibited a positive relationship with fraction organic carbon (foc)
in sediments. To compensate for the different foc values in different sediment fractions, organic carbon normalized distribution coefficient (Koc) values were calculated: Koc ¼ KD/foc
(3)
As shown in Fig. A1b, the Koc values of sulfamethoxazole increased with exposure time, ranged from 8.9 to 19.3 L/kg-oc at 168 h, which agreed with the range of Koc values from 9 to 1565 L/ kg-oc for sulfamethoxazole partition between sediment and water in the Huangpu River, Shanghai, China (Chen and Zhou, 2014). The adsorption rates of sediment increased with decreasing particle size, as the smaller particle size of sediment contained higher organic carbon with reported extraordinarily strong sediment adsorption affinity for organic contaminants (Xu and Li, 2010).
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Fig. 6. Effect of salinity on sulfamethoxazole proportion in water-zebrafish, and water-sediment-zebrafish systems at equilibrium. Sediment size <250 mm.
As shown in Fig. A1c, the BAR values of sulfamethoxazole increased with exposure time, reaching maximum values of 0.34e0.41 mL/g at 168 h in water-sediment-zebrafish microcosm. In comparison to water only control (Fig. 2c), BAR values in the presence of sediments were reduced by 15e28%, due to competition of sediments for the contaminant hence causing a reduction in bioaccumulation. Of the different sediment size fractions, the <63 mm fraction caused the most significant reduction (28%) in bioavailability; the results are consistent with this fraction possessing the highest KD values. In addition, there is a significant negative relation between BAR and sediment SSA (Fig. 5), suggesting the importance of sediment interfacial properties. In general, the bioavailability of these chemicals decreases with increasing sediment foc content (Duong et al., 2009; Reid et al., 2000). Furthermore, bottom sediments act as a sink by reducing the bioaccumulation potential and hence toxicity to aquatic organisms. In this study, the movement of sulfamethoxazole from the aqueous phase to sediment phase most likely reduced its bioavailability to zebrafish.
3.4. Effect of salinity on the bioavailability of sulfamethoxazole in zebrafish Estuaries are the interface between land and ocean, and act as a filter of river-derived contaminants. As a result, major biogeochemical processes are occurring there in association with salinity changes. It has been reported that salinity could change the density, ionic strength and dissolved oxygen in water (Shabala and Munns, 2012). Furthermore, the differences in such properties could affect the distribution of contaminants between the water phase and solid phase, and influence the growth of aquatic organisms (Bartley et al., 2013). Thus the effects of salinity on the binding affinity of sulfamethoxazole in sediment and bioaccumulation of sulfamethoxazole in zebrafish was investigated. The calculated sulfamethoxazole proportion in sediment during 168-h exposure is shown in Fig. A2a. Sulfamethoxazole concentration in sediment was increasing with time and exhibited a positive relationship with salinity. It is well known that the aqueous solubility of many organic contaminants decreases with increasing salt concentration as a result of salting out effect. Thus, the sorption efficiency of organic contaminants in the sorbent may be increased. Previous
studies have reported that under the condition of high salinity, endocrine disrupting chemicals and antibiotics are more likely to adsorb to sediment (Shi et al., 2014). It is observed that concentrations of sulfamethoxazole in 10‰ and 20‰ salinity waters showed higher sorption on sediment which agreed with previous studies. Furthermore, Boeuf and Payan (2001) reported that the salinity of water not only affected the osmotic regulation and metabolism of aquatic animals, but also activated or inhibited the activity of digestive enzymes in their body which affected the animals’ ability for the absorption and digestion of food. In both the binary water-zebrafish microcosm and the ternary watersediment-zebrafish microcosms, the extent of sulfamethoxazole bioaccumulation in zebrafish during the exposure decreased with increasing salinity. The results demonstrated that high salinity inhibited zebrafish to bioaccumulate sulfamethoxazole whether sediment was absent or present (Fig. A2b), although the inhibition became more significant when sediment was present. This is more clearly shown in Fig. 6 as equilibrium distribution. In addition, a comparison of bioaccumulation data between the binary and ternary microcosms showed that sulfamethoxazole proportion in zebrafish was approximately 0.06%, which was substantially reduced by sediment presence. In addition, there was a negative relation between sulfamethoxazole proportion in zebrafish and salinity. These findings demonstrated for the first time the importance of water salinity on the antibiotic bioaccumulation and bioavailability in zebrafish, raising awareness about potential impacts of emerging contaminants in estuarine systems on fish health.
4. Conclusions The bioaccumulation of sulfamethoxazole in zebrafish was determined by direct concentration measurement in microcosms. The results confirmed that sediment adsorbed antibiotic sulfamethoxazole from water. The adsorption capacity of sulfamethoxazole in different sized sediment was compared, with the highest adsorption being associated with the small size (<63 mm) fraction, due to its high surface area and organic carbon content. Sulfamethoxazole sorption on sediments exhibited a positive relationship with salinity, consistent with salting out effect. As a result, more sulfamethoxazole was found to become adsorbed on
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sediment in high salinity water, leading to reduced bioavailability and small body burden of antibiotic residues in zebrafish. The equilibrium data further confirmed that the distribution of the antibiotic in water, sediment and zebrafish accounted for 97.3%, 2.7% and 0.05% of the dosing quantity, respectively. These results provide important insights into the transport and biological fate of a widely used antibiotic in microcosm, which form the basis for further research on antibiotic residues in aquatic environments. Acknowledgements This work was funded by the State Key Laboratory of Estuarine and Coastal Research (2016RCDW02). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2017.04.055. References Ahmed, M.B., Zhou, J.L., Ngo, H.H., Guo, W., 2015. Adsorptive removal of antibiotics from water and wastewater: progress and challenges. Sci. Total Environ. 532, 112e126. Alexander, M., 2000. Aging, bioavailability, and overestimation of risk from environmental pollutants. Environ. Sci. Technol. 34 (20), 4259e4265. Bartley, M.L., Boeing, W.J., Corcoran, A.A., Holguin, F.O., Schaub, T., 2013. Effects of salinity on growth and lipid accumulation of biofuel microalga Nannochloropsis salina and invading organisms. Biomass Bioenergy 54, 83e88. Bayen, S., Estrada, E.S., Juhel, G., Kit, L.W., Kelly, B.C., 2016. Pharmaceutically active compounds and endocrine disrupting chemicals in water, sediments and mollusks in mangrove ecosystems from Singapore. Mar. Pollut. Bull. 109 (2), 716e722. Benotti, M.J., Brownawell, B.J., 2009. Microbial degradation of pharmaceuticals in estuarine and coastal seawater. Environ. Pollut. 157 (3), 994e1002. Boeuf, G., Payan, P., 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. C 130 (4), 411e423. Boonsaner, M., Hawker, D.W., 2013. Evaluation of food chain transfer of the antibiotic oxytetracycline and human risk assessment. Chemosphere 93 (6), 1009e1014. Bowman, J.C., Zhou, J.L., Readman, J.W., 2002. Sediment-water interactions of natural oestrogens under estuarine conditions. Mar. Chem. 77 (4), 263e276. Carballa, M., Fink, G., Omil, F., Lema, J.M., Ternes, T., 2008. Determination of the solid-water distribution coefficient (Kd) for pharmaceuticals, estrogens and musk fragrances in digested sludge. Water Res. 42 (1e2), 287e295. Carstens, K.L., Gross, A.D., Moorman, T.B., Coats, J.R., 2013. Sorption and photodegradation processes govern distribution and fate of sulfamethazine in freshwater-sediment microcosms. Environ. Sci. Technol. 47 (19), 10877e10883. Chen, C.Q., Zheng, L., Zhou, J.L., Zhao, H., 2017. Persistence and risk of antibiotic residues and antibiotic resistance genes in major mariculture sites in Southeast China. Sci. Total Environ. 580, 1175e1184. Chen, H., Liu, S., Xu, X.-R., Liu, S.-S., Zhou, G.-J., Sun, K.-Y., Zhao, J.-L., Ying, G.-G., 2015. Antibiotics in typical marine aquaculture farms surrounding Hailing Island, South China: occurrence, bioaccumulation and human dietary exposure. Mar. Pollut. Bull. 90 (1e2), 181e187. Chen, K., Zhou, J.L., 2014. Occurrence and behavior of antibiotics in water and sediments from the Huangpu River, Shanghai, China. Chemosphere 95, 604e612. Ding, J.N., Lu, G.H., Liu, J.C., Zhang, Z.H., 2015. Evaluation of the potential for trophic transfer of roxithromycin along an experimental food chain. Environ. Sci. Pollut. Res. 22 (14), 10592e10600. Duong, C.N., Schlenk, D., Chang, N.I., Kim, S.D., 2009. The effect of particle size on the bioavailability of estrogenic chemicals from sediments. Chemosphere 76 (3), 395e401. Gaillard, J., Banas, D., Thomas, M., Fournier, A., Feidt, C., 2014. Bioavailability and bioaccumulation of sediment-bound polychlorinated biphenyls to carp. Environ. Toxicol. Chem. 33 (6), 1324e1330. , D., 2011. Occurrence of sulfonamide García-Gal an, M.J., Díaz-Cruz, M.S., Barcelo residues along the Ebro river basin: removal in wastewater treatment plants and environmental impact assessment. Environ. Int. 37, 462e473. Hou, J., Pan, B., Niu, X., Chen, J., Xing, B., 2010. Sulfamethoxazole sorption by sediment fractions in comparison to pyrene and bisphenol A. Environ. Pollut. 158, 2826e2832. Hou, X.L., Shen, J.Z., Zhang, S.X., Jiang, H.Y., Coats, J.R., 2003. Bioconcentration and elimination of sulfamethazine and its main metabolite in sturgeon (Acipenser schrenkii). J. Agric. Food Chem. 51, 7725e7729. Ji, X., Shen, Q., Liu, F., Ma, J., Xu, G., Wang, Y., Wu, M., 2012. Antibiotic resistance gene abundances associated with antibiotics and heavy metals in animal manures
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Reid, B.J., Jones, K.C., Semple, K.T., 2000. Bioavailability of persistent organic pollutants in soils and sediments - a perspective on mechanisms, consequences and assessment. Environ. Pollut. 108 (1), 103e112. Sangster, J.L., Oke, H., Zhang, Y., Bartelt-Hunt, S.L., 2015. The effect of particle size on sorption of estrogens, androgens and progestagens in aquatic sediment. J. Hazard. Mater. 299, 112e121. Semple, K.T., Doick, K.J., Jones, K.C., Burauel, P., Craven, A., Harms, H., 2004. Defining bioavailability and bioaccessibility of contaminated soil and sediment is complicated. Environ. Sci. Technol. 38 (12), 228Ae231A. Sengelov, G., Agerso, Y., Halling-Sorensen, B., Baloda, S.B., Andersen, J.S., Jensen, L.B., 2003. Bacterial antibiotic resistance levels in Danish farmland as a result of treatment with pig manure slurry. Environ. Int. 28, 587e595. Shabala, S., Munns, R., 2012. Salinity stress: physiological constraints and adaptive mechanisms. In: Shabala, Sergey (Ed.), Plant Stress Physiology,. Centre for Agriculture and Biosciences International, pp. 59e93. Shi, X., Zhou, J.L., Zhao, H., Hou, L., Yang, Y., 2014. Application of passive sampling in assessing the occurrence and risk of antibiotics and endocrine disrupting chemicals in the Yangtze Estuary, China. Chemosphere 111, 344e351. ska, A., Sikora, K., Stepnowski, P., Pazdro, K., Siedlewicz, G., Borecka, M., Białk-Bielin 2016. Determination of antibiotic residues in southern Baltic Sea sediments using tandem solid-phase extraction and liquid chromatography coupled with tandem mass spectrometry. Oceanologia 58, 221e234. €lgyessy, P., Miha likov To a, Z., 2015. Rapid determination of total lipids in fish samples employing extraction/partitioning with acetone/ethyl acetate solvent mixture and gravimetric quantification. Food Control 60, 44e49. Wegst-Uhrich, S.R., Navarro, D.A.G., Zimmerman, L., Aga, D.S., 2014. 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Sediment and salinity effects on the bioaccumulation of sulfamethoxazole in zebrafish (Danio rerio) Y. Chen, J.L. Zhou*, L. Cheng, Y.Y. Zheng, J. Xu State Key Laboratory of Estuarine and Coastal Research, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Sulfamethoxazole bioaccumulation was quantified by direct concentration measurements. Sediment caused 13e28% reduction in sulfamethoxazole bioaccumulation. Salinity reduced sulfamethoxazole bioaccumulation to zebrafish. Equilibrium distribution of sulfamethoxazole was dominated by water. Sediment adsorption was most significant with small particles with large surface area.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 January 2017 Received in revised form 9 April 2017 Accepted 12 April 2017 Available online 14 April 2017
The dynamic distribution of a widely used antibiotic sulfamethoxazole between water, sediment and aquatic organisms (zebrafish) was studied in microcosms. Sulfamethoxazole concentrations in water were gradually reduced, while in sediment and zebrafish gradually increased, suggesting active adsorption and bioaccumulation processes occurring. The presence of sediment particles and their interactions with water reduced the bioaccumulation of sulfamethoxazole in zebrafish by 13e28%. The sediment of smaller particle size with more organic carbon content and higher surface area, adsorbed sulfamethoxazole more extensively and decreased its bioaccumulation most significantly. The effect became more severe with increasing salinity in water due to the salting out of sulfamethoxazole, resulting in 24e33% reduction in bioaccumulation. At equilibrium, the distribution of sulfamethoxazole in different phases was quantified, with most sulfamethoxazole being associated with water (97.3%), followed by sedimentary phase (2.7%) and finally zebrafish (0.05%). The findings provided important data for further research into antibiotics fate and bio-uptake in aquatic organisms, and subsequent ecotoxicity. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Sulfamethoxazole Bioaccumulation Sediment Salinity Zebrafish
1. Introduction Currently antibiotic residues in the environment have attracted worldwide attention due to their large-scale consumption, wide
* Corresponding author. E-mail address: [email protected] (J.L. Zhou). http://dx.doi.org/10.1016/j.chemosphere.2017.04.055 0045-6535/© 2017 Elsevier Ltd. All rights reserved.
occurrence, and potential risks for wildlife and human (Ahmed et al., 2015; Chen et al., 2015). In China alone, over 25,000 tons of antibiotics are produced every year (Yan et al., 2013), of which sulfonamides antibiotics are widely used in the animal feeding and human medicines (Zhang et al., 2015). Through different sources in particular sewage treatment works effluents (Michael et al., 2013) as hotspots, sulfonamides antibiotics will eventually be discharged into the natural aquatic environment, where their occurrence has
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been widely reported in water with concentrations as high as 211 mg/L (Zhao et al., 2015a). One of the main concerns with antibiotic contamination is that bacteria may develop antibiotic resistance gene (ARGs) from prolonged exposure to antibiotics, induced by plasmid horizontal transfer (Sengelov et al., 2003; Chen et al., 2017); and the migration and transformation of ARGs in the aquatic environment is potentially more harmful than the antibiotic residues (Ji et al., 2012). More attention should be given to sulfonamide antibiotics because of their widespread use, high excretion rate, high solubility and persistence in the environment n et al., 2011); for example it is reported that (García-Gala sulfonamide-resistant bacteria can remain stable in the aquatic environment for 5e10 years (Martinez et al., 2009). Antibiotics in general possess high migration ability, depending on their physicochemical properties such as water solubility, octanol/water partitioning coefficient (Kow), and acid dissociation constant (pKa) (Kay et al., 2005). It has been reported that sulfonamides have shown 100% detection and the highest concentrations in the water phase of Huangpu River (Chen and Zhou, 2014) and the Yangtze Estuary (Yan et al., 2013), China, where sulfamethoxazole is the dominant compound. It has been suggested that sulfonamides are highly stable in the aquatic environment (Yan et al., 2013), and sulfamethoxazole is difficult to be hydrolyzed and biodegraded (Benotti and Brownawell, 2009). Thus, sulfonamides residues have been predominantly detected in the water phase (Li et al., 2012; Na et al., 2013). In addition, aquatic sediment has been identified as an important medium to retain organic contaminants including relatively hydrophobic antibiotics (Zhou and Broodbank, 2014). Although Matongo et al. (2015) and Yang et al. (2015) reported relatively low concentrations of sulfamethoxazole in river sediments (<1 ng/g) from South Africa and Taiwan, respectively; significantly higher concentrations of sulfamethoxazole (up to 276 ng/g) were detected in southern Baltic Sea sediments by Siedlewicz et al. (2016). In addition, laboratory-based adsorption ndez et al. (2014) suggested strong experiments by Martínez-Herna affinity between sediment and sulfamethoxazole with only 4.9% of sorbed compound being desorbed. In addition, its adsorption may depend on the properties of contaminants and sediment e.g. particulate organic carbon content, and water properties such as temperature, pH, salinity and dissolved organic carbon (Hou et al., ndez et al., 2014). 2010; Xu and Li, 2010; Martínez-Herna In assessing the ecological risk of antibiotics, bioavailability is the key to determine bioaccumulation and adverse effects. Bioavailability is defined as the fraction of the unchanged chemical present in the organism (Alexander, 2000; Semple et al., 2004), and widely used in the environmental risk assessment of toxic chemicals (Wu and Zhu, 2016). Antibiotic contaminants may therefore bioaccumulate in aquatic organisms (Boonsaner and Hawker, 2013; Ding et al., 2015), for example, up to 1150 mg/kg of sulfonamides have been detected in aquatic organisms from field monitoring (Zhao et al., 2015a). Bayen et al. (2016) reported that sulfamethoxazole was bioavailable even to bivalves in mangrove habitats. There are chemical-based and biological-based approaches for assessing bioavailability, among which bioaccumulation experiments using animals is the most direct and relevant approach (Reichenberg and Mayer, 2006; Ortega-Calvo et al., 2015). The bioaccumulation of contaminants can be affected by the presence of suspended sediments and colloids in water (Gaillard et al., 2014). In addition, water salinity should be studied as a key parameter in estuarine environment where water pollution is particularly serious and can be highly influenced by tidal action (Zhao et al., 2015b). Zebrafish are widely used as the model organism for ecotoxicology experiments as they have many advantages e.g. small individual and easy breeding (Xia et al., 2016). In addition, as a
vertebrate organism, zebrafish (Danio rerio) possess significant physiological and morphological homology to human (Qiang et al., 2016). This study therefore aimed to determine the fate and bioaccumulation of sulfamethoxazole as a representative sulfonamides using zebrafish as the model organism, by hypothesizing that the compound will be bioavailable to zebrafish leading to bioaccumulation. Specifically the study examined the distribution of sulfamethoxazole between water, sediment and zebrafish phases in microcosm; the bioavailability and bioaccumulation of sulfamethoxazole to zebrafish; and the effects of sediment and salinity on sulfamethoxazole bioaccumulation. 2. Materials and methods 2.1. Chemicals, sediment and zebrafish Sulfamethoxazole standard was purchased from Dr. Ehrenstorfer (GmbH, Germany) and its physicochemical properties are shown in Supplementary Information Table A1. The compound sulfamethoxazole-d4 obtained from Dr. Ehrenstorfer (GmbH, Germany) was used as the internal standard to quantify sulfamethoxazole. All standard solutions were stored at 20 C. Sediment samples were collected in November 2014 from the east coast of Chongming Island in the Yangtze Estuary, China, as the sites did not show any antibiotic contamination from previous studies. The samples were freeze-dried for 72 h and homogenized, then successively sifted through 240-mesh (63 mm), 120-mesh (125 mm) and 60-mesh (250 mm) sieves, to obtain different sediment fractions (e.g. < 63 mm, 63e125 mm, and 125e250 mm). The specific surface area (SSA) of the sediments was 8.89 m2/g (<63 mm), 6.84 m2/g (63e125 mm), 6.60 m2/g (125e250 mm), and 6.78 m2/g (<250 mm), after measurement by a Quantchrome surface area analyzer using the BET method (Bowman et al., 2002). The organic carbon content of sediment samples was determined by high-temperature catalytic combustion following acidification twice by 5% sulfurous acid (Zhou and Broodbank, 2014). Wild-type zebrafish (Danio rerio, AB line) were maintained in several 40-L transparent plastic tanks filled with dechlorinated and carbon filtered tap water. The following conditions were monitored and maintained by the system: temperature 28 ± 1 C, dissolved oxygen >7 mg/L. Zebrafish were fed twice a day with dry flakes and brine shrimp (Artemia salina nauplii). A constant light/dark (14/ 10 h) cycle was used to meet its spawning condition. For this study, zebrafish of 3 months old, 2.8 ± 0.2 cm length, and 0.35 ± 0.08 g wet weight were used. Lipid content in zebrafish tissues based on wet weight was 4.76 ± 0.5%, which was measured in triplicate by the €lgyessy and Miha likova , 2015). All zebrafish gravimetric method (To were acclimatized to the laboratory conditions at least 14 days before use. For salinity experiments, zebrafish were acclimatized for a further 7 days in 10‰ and 20‰ salinity, during which no mortality was observed. 2.2. Fate and exposure microcosm experiments As shown in Fig. 1, four systems were designed in this study: (i) water, (ii) water-sediment, (iii) water-zebrafish, and (iv) watersediment-zebrafish, and each experiment was conducted in triplicate (i.e. 3 parallel tanks) to ensure statistical significance. The effects of sediment particle size (Fig. 1a) and salinity (Fig. 1b) on the bioaccumulation of sulfamethoxazole were investigated, respectively. In the water-sediment-zebrafish system, experiments were conducted in 8-L glass tanks containing 500 g (dry weight) of different sized sediments (<250 mm, < 63 mm, 63e125 mm, and 125e250 mm), respectively, which were mixed with 5 L of 10 mg/L sulfamethoxazole solution at a particular salinity (e.g. 0‰, 10‰ or
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Fig. 1. Experimental schematic for (a) the effect of sediment particle size and (b) the effect of salinity on the bioavailability and bioaccumulation of sulfamethoxazole (SMX) to zebrafish. 3 tanks per treatment, 40 zebrafish per tank.
20‰). Then 40 individuals of zebrafish were added to each tank to assess the antibiotic bioaccumulation ratio (BAR) in zebrafish: BAR ¼ Czebrafish/Cw
(1)
where BAR is defined as the ratio of sulfamethoxazole concentration in zebrafish (Czebrafish, ng/g dry weight) to sulfamethoxazole concentration in water (Cw, ng/mL). Without replacing water, the zebrafish were fed with the experimental diets without antibiotics contamination for 168 h. Water physicochemical parameters were checked daily to ensure a relatively stable water environment. Each chamber was equipped with a heater to maintain the water temperature at 28 ± 1 C, which was the optimal growth temperature for zebrafish. No mortality was observed during the experiments. Water, sediment, and zebrafish samples were collected at regular intervals and stored at 20 C. For zebrafish, 5 individuals were taken from each of the 3 parallel tanks, to obtain 3 aggregate samples. Then the sediment and zebrafish samples were freeze-dried for 72 h and grounded into powders. The wet and dry weight of each zebrafish was measured before and after freeze-drying, respectively. In the presence of sediment, sulfamethoxazole will interact with sediment at the same time when bioaccumulation is taking place. The binding strength of sediment-water interaction can be expressed with the distribution coefficient (KD): KD ¼ Cs/Cw
(2)
where Cs (ng/g dry weight) is sulfamethoxazole concentration in the sediment. 2.3. Antibiotic extraction and analysis in water, sediment and zebrafish Water samples (1 mL) were spiked with 20-ng of internal standard, before direct injection into high performance liquid chromatographetandem mass spectrometer (UHPLC-MS) for quantitative analysis. Sediment samples (3 g) were spiked with 20 ng of internal standard, and 9 mL of acetonitrile for extraction.
Each sample was stirred at 200 rpm for 20 min using a mechanical shaker before being sonicated in an ultrasonic bath for 15 min. The samples were centrifuged at 2500 rpm for 5 min to separate the solid and liquid phases. The liquid phase was collected, and the same extraction procedure was repeated twice to obtain a complete extraction of the target compound. When the extracts were evaporated to 0.5 mL under gentle N2 purging at 40 C, 5 mL of methanol was added, which was reduced to 0.5 mL. To the final extracts 0.5 mL of Milli-Q water with 0.1% formic acid was added before the chemical analysis. The processing of zebrafish samples was similar to the sediment samples, except an extra step of fat removal was introduced. The concentrated extracts were firstly reduced to 5 mL by evaporation under gentle N2 purging at 40 C, and stored at 20 C for 24 h. The lipids were removed via filtration. Each experimental group was replicated three times with two blank controls being used with each set of experiments. The final sample extracts were analyzed using a Waters Acquity™ UHPLCeMS/MS system with an HSS T3 column (2.1 mm 100 mm, 1.7 mm particle size), following methods by Zhou et al. (2012) and Chen and Zhou (2014). 2.4. Quality control and statistic analysis The antibiotic analysis was subject to quality assurance, by analyzing blanks and recovery samples and determining the limits of quantification (LOQs). The recovery of sulfamethoxazole in water, sediment and zebrafish samples was 96.3e101.1%, 82.2e88.1% and 68.8e78.9%, respectively. The LOQ values for sulfamethoxazole were estimated to be 0.34 ng/L in water, 0.11 ng/g (dry weight basis) in sediment, and 0.15 ng/g (dry weight basis) in zebrafish, respectively. All samples were analyzed in triplicate, and the relative standard deviation (RSD) values were less than 20%. All experimental data were expressed as the average ± standard error of mean. Homogeneity of variance and normality of all data set were first tested. As datasets of exposure tests were found to show equal variance, thus one-way analysis of variance (ANOVA) was applied to determine the statistical significance. Double tail Student's t-test was used to compare the difference between the exposure group and the control group. The value P < 0.05 was
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chosen as the threshold in both cases. All data were processed using Origin software (version 8.5). 3. Results and discussion 3.1. Sulfamethoxazole distribution between water, sediment and zebrafish in microcosm The results for the kinetics of sulfamethoxazole concentration changes in water, water-sediment, water-fish, and water-sedimentfish microcosms are shown in Fig. 2. In the water only control, sulfamethoxazole concentration decreased slightly from 9.98 to 9.70 mg/L (3%) over the 7 day period (Fig. 2a). It is apparent that this compound is stable during the experimental period, and suitable for adsorption and bioaccumulation experiments. The results agree with Carstens et al. (2013) who found there was still 92.1 ± 0.69% of sulfamethazine in pond water after 7 days. It has been reported that sulfonamides usually have high potential to resist degradation and hydrolysis in water (Lin and Gan, 2011). Yang et al. (2011) also reported that even in the presence of activated sludge, sulfonamide antibiotics were first removed by adsorption followed by biodegradation, and biodegradation was inhibited in the first 12 h due to competitive inhibition of xenobiotic oxidation by readily biodegradable substances. In comparison, the concentration of sulfamethoxazole in water decreased from 9.96 to 9.26 mg/L (7%) after 168 h in water-sediment microcosm, while sulfamethoxazole concentration in sediment (<250 mm) was increased from 0 to 2.55 ng/g (Fig. 2b). The results suggest significant interactions between sediment and water, similar to the behavior of other organic contaminants such as endocrine disrupting chemicals and pharmaceuticals (Bowman et al., 2002; Zhou and Broodbank, 2014). The results indicated that sediment played an important role in the distribution of sulfamethoxazole. Hou et al. (2010) reported high sorption of
sulfamethoxazole on both humic acids and inorganic mineral particles, with relatively low sorption being observed on sediment from Dianchi Lake, Yunan, China. In a study of sulfamethoxazole sorption by groundwater sediments from Madrid, Spain, Martínezndez et al. (2014) observed a high affinity between sediment Herna and sulfamethoxazole, evidenced by only 4.9% of the sorbed compound being released during desorption experiments. When zebrafish were introduced, sulfamethoxazole was found to accumulate rapidly in the body of zebrafish indicating that the compound was bioavailable (Fig. 2c). The concentration of sulfamethoxazole increased quickly in zebrafish, reaching the first peak level of 3.38 ng/g at 48 h of exposure. The bioaccumulated concentration then continued to rise slowly, reaching the highest concentration of 4.52 ng/g at 168 h. Meanwhile, sulfamethoxazole concentrations in water decreased from 10.01 to 9.62 mg/L i.e. 4%. Liu et al. (2014) also reported that roxithromycin was concentrated by freshwater fish (Carassius auratus) causing biological effects. When both sediment (<250 mm) and zebrafish were present in water (Fig. 2d), the concentration of sulfamethoxazole in water declined by 8% (from 9.98 to 9.21 mg/L) in the water-sedimentzebrafish microcosm, slightly faster than when only sediment was present (Fig. 2b) and significantly faster than when only zebrafish were present (Fig. 2c). The findings confirm that when more phases such as sediment were present in the microcosm, the amount of sulfamethoxazole removal from aqueous phase was enhanced. Sulfamethoxazole concentration in sediment was increasing with time, reaching the highest concentrations of 2.54 ng/g at 168 h. In addition, the concentration in zebrafish was also increased with time, reaching 3.42 ng/g at the end of experiment. The accumulation of sulfamethoxazole in zebrafish was therefore inhibited (24% reduction) in the presence of <250 mm sediment (Fig. 2c and d), confirmed by one-way ANOVA analysis with P < 0.01 compared to data without sediment. Compared with binary microcosms (water-fish, water-
Fig. 2. Kinetics of sulfamethoxazole concentration changes in (a) water, (b) water-sediment, (c) water-zebrafish, and (d) water-sediment-zebrafish microcosms. Sediment size is < 250 mm.
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sediment), the distribution of sulfamethoxazole became more complex in ternary microcosm due to competition between sediment adsorption and zebrafish bioaccumulation, leading to smaller body burden of antibiotic residues in zebrafish. 3.2. Effect of sediment on the bioaccumulation of sulfamethoxazole in zebrafish As shown in Fig. 3, the BAR values of sulfamethoxazole in waterzebrafish system increased with exposure time, reaching maximum values of 0.47 mL/g at 168 h. Similarly, BAR values in the presence of sediments also increased with time, however the equilibrium BAR value was reduced to 0.37 mL/g, a reduction of 21% due to competition of sediments for the antibiotic. One-way ANOVA Statistical analysis showed a significant difference between the two sets of BAR values, with P < 0.01. The BAR values of sulfamethoxazole are smaller than those observed by Hou et al. (2003), who reported higher BAR of 1.19 mL/g for sulfamethazine in the muscle of sturgeon after exposure to concentration of 100 mg/L. Such a difference could be due to differences in test species, fish biomass, antibiotics concentration and exposure time, all of which will affect the bioaccumulation potential. In the presence of sediment, the KD values of sulfamethoxazole at equilibrium were 0.28 and 0.26 mL/g in water-sediment microcosm and water-sediment-zebrafish microcosm, respectively as shown in Fig. 3. The results suggested significant sediment-water interaction which was unaffected by the presence of zebrafish. Previous study has reported that the KD values ranged from 2.23 to 6.05 mL/g for sulfamethoxazole in digested sludge (Carballa et al., 2008), which are 10 times higher than KD values in this study. Such difference would be caused by several factors: different types of solids with organic-rich sludge expected to have higher sorbing power than natural sediment, different concentrations of antibiotics being exposed, and different durations of solid-water interactions. These results implied that the KD values of antibiotics are most likely related to the hydrophobicity of the contaminants, physicochemical properties of sediment sorbents, and initial concentrations of the contaminants in water (Wegst-Uhrich et al., 2014). In addition, the hydrodynamics in the aquatic environment also play an important role due to kinetic controls and adsorptiondesorption cycles (Zhou and Broodbank, 2014).
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The distribution of contaminants between different environmental phases (water, air, sediment, and organisms) is determined by thermodynamic equilibria (Kummerer, 2010). In the present study, the mass balance of sulfamethoxazole distribution between water, sediment and zebrafish in the water-sediment-zebrafish microcosm is shown in Fig. 4. Most sulfamethoxazole tended to be associated with water at > 97% throughout the experiments, when sulfamethoxazole accumulation in sediments increased steadily while bioaccumulation began with a reduction followed by increases. At the equilibrium time of 168 h, the proportion of sulfamethoxazole in water, sediment and zebrafish was 97.3%, 2.7% and 0.05% of the total amount, respectively. The results confirmed that in ternary systems, the bioaccumulation of sulfamethoxazole was significantly reduced due to contaminant sorption to sediment. In addition, the bioaccumulation results agree with a previous study which reported zebrafish could accumulate 0.04% of erythromycin in a microcosm containing 500 mg/L of the contaminant in the presence of Elodea densa and sediment after 30 days (Wu et al., 2015). 3.3. Effect of sediment property on the bioaccumulation of sulfamethoxazole in zebrafish Since the bioaccumulation of sulfamethoxazole in the aquatic environment decreased after the sediment was introduced to the system, further experiments were performed to study the effects of the physicochemical properties of sediment on the distribution of sulfamethoxazole. Firstly, the effect of sediment particle size on the binding affinity of sulfamethoxazole in sediment and bioavailability of sulfamethoxazole in zebrafish was investigated. The calculated KD values of sulfamethoxazole at equilibrium were 0.36 mL/g (<63 mm), 0.31 mL/g (63e125 mm) and 0.26 mL/g (125e250 mm), as shown in Supplementary Information Fig. A1a. The results suggest that with increasing sediment particle size, the KD values gradually decreased, with the reduction being statistically significant (P < 0.05, One-way ANOVA). The SSA of the sediment fractions also decreased with increasing particle size from 8.89 m2/g for the <63 mm fraction, to 6.84 m2/g for the 63e125 mm fraction, and 6.60 m2/g for the 125e250 mm fraction. The results (Fig. 5) indicated that KD increased with SSA (r2 ¼ 0.79, P < 0.05).
Fig. 3. KD and bioaccumulation ratio (BAR) values in water-sediment and water-sediment-zebrafish microcosms. Sediment size <250 mm.
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Fig. 4. Mass balance distribution of sulfamethoxazole in water-sediment-zebrafish microcosm during exposure. Sediment size <250 mm.
Fig. 5. Relationship between sediment/water partition coefficient (KD) and zebrafish/water BAR of sulfamethoxazole and sediment specific surface area, in water-sedimentzebrafish microcosm.
The results are consistent with a similar study by Bowman et al. (2002) who showed the KD for endocrine disrupting chemicals decreased with sediment particle size and increased with sediment SSA. It is widely recognized that sediment sorption of endocrine disrupting chemicals and antibiotic contaminants is highly affected by the organic matter content in sediments (Bowman et al., 2002; Zhou and Broodbank, 2014). Similarly it is reported that the dominant sorption matrix for sulfamethoxazole is the organic matter (Hou et al., 2010; Sangster et al., 2015). These results implied that the KD values of antibiotics are most likely related to the hydrophobicity of the contaminants, physicochemical properties of sediment and initial concentrations of the contaminants in water (Wegst-Uhrich et al., 2014). The KD results for sulfamethoxazole exhibited a positive relationship with fraction organic carbon (foc)
in sediments. To compensate for the different foc values in different sediment fractions, organic carbon normalized distribution coefficient (Koc) values were calculated: Koc ¼ KD/foc
(3)
As shown in Fig. A1b, the Koc values of sulfamethoxazole increased with exposure time, ranged from 8.9 to 19.3 L/kg-oc at 168 h, which agreed with the range of Koc values from 9 to 1565 L/ kg-oc for sulfamethoxazole partition between sediment and water in the Huangpu River, Shanghai, China (Chen and Zhou, 2014). The adsorption rates of sediment increased with decreasing particle size, as the smaller particle size of sediment contained higher organic carbon with reported extraordinarily strong sediment adsorption affinity for organic contaminants (Xu and Li, 2010).
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Fig. 6. Effect of salinity on sulfamethoxazole proportion in water-zebrafish, and water-sediment-zebrafish systems at equilibrium. Sediment size <250 mm.
As shown in Fig. A1c, the BAR values of sulfamethoxazole increased with exposure time, reaching maximum values of 0.34e0.41 mL/g at 168 h in water-sediment-zebrafish microcosm. In comparison to water only control (Fig. 2c), BAR values in the presence of sediments were reduced by 15e28%, due to competition of sediments for the contaminant hence causing a reduction in bioaccumulation. Of the different sediment size fractions, the <63 mm fraction caused the most significant reduction (28%) in bioavailability; the results are consistent with this fraction possessing the highest KD values. In addition, there is a significant negative relation between BAR and sediment SSA (Fig. 5), suggesting the importance of sediment interfacial properties. In general, the bioavailability of these chemicals decreases with increasing sediment foc content (Duong et al., 2009; Reid et al., 2000). Furthermore, bottom sediments act as a sink by reducing the bioaccumulation potential and hence toxicity to aquatic organisms. In this study, the movement of sulfamethoxazole from the aqueous phase to sediment phase most likely reduced its bioavailability to zebrafish.
3.4. Effect of salinity on the bioavailability of sulfamethoxazole in zebrafish Estuaries are the interface between land and ocean, and act as a filter of river-derived contaminants. As a result, major biogeochemical processes are occurring there in association with salinity changes. It has been reported that salinity could change the density, ionic strength and dissolved oxygen in water (Shabala and Munns, 2012). Furthermore, the differences in such properties could affect the distribution of contaminants between the water phase and solid phase, and influence the growth of aquatic organisms (Bartley et al., 2013). Thus the effects of salinity on the binding affinity of sulfamethoxazole in sediment and bioaccumulation of sulfamethoxazole in zebrafish was investigated. The calculated sulfamethoxazole proportion in sediment during 168-h exposure is shown in Fig. A2a. Sulfamethoxazole concentration in sediment was increasing with time and exhibited a positive relationship with salinity. It is well known that the aqueous solubility of many organic contaminants decreases with increasing salt concentration as a result of salting out effect. Thus, the sorption efficiency of organic contaminants in the sorbent may be increased. Previous
studies have reported that under the condition of high salinity, endocrine disrupting chemicals and antibiotics are more likely to adsorb to sediment (Shi et al., 2014). It is observed that concentrations of sulfamethoxazole in 10‰ and 20‰ salinity waters showed higher sorption on sediment which agreed with previous studies. Furthermore, Boeuf and Payan (2001) reported that the salinity of water not only affected the osmotic regulation and metabolism of aquatic animals, but also activated or inhibited the activity of digestive enzymes in their body which affected the animals’ ability for the absorption and digestion of food. In both the binary water-zebrafish microcosm and the ternary watersediment-zebrafish microcosms, the extent of sulfamethoxazole bioaccumulation in zebrafish during the exposure decreased with increasing salinity. The results demonstrated that high salinity inhibited zebrafish to bioaccumulate sulfamethoxazole whether sediment was absent or present (Fig. A2b), although the inhibition became more significant when sediment was present. This is more clearly shown in Fig. 6 as equilibrium distribution. In addition, a comparison of bioaccumulation data between the binary and ternary microcosms showed that sulfamethoxazole proportion in zebrafish was approximately 0.06%, which was substantially reduced by sediment presence. In addition, there was a negative relation between sulfamethoxazole proportion in zebrafish and salinity. These findings demonstrated for the first time the importance of water salinity on the antibiotic bioaccumulation and bioavailability in zebrafish, raising awareness about potential impacts of emerging contaminants in estuarine systems on fish health.
4. Conclusions The bioaccumulation of sulfamethoxazole in zebrafish was determined by direct concentration measurement in microcosms. The results confirmed that sediment adsorbed antibiotic sulfamethoxazole from water. The adsorption capacity of sulfamethoxazole in different sized sediment was compared, with the highest adsorption being associated with the small size (<63 mm) fraction, due to its high surface area and organic carbon content. Sulfamethoxazole sorption on sediments exhibited a positive relationship with salinity, consistent with salting out effect. As a result, more sulfamethoxazole was found to become adsorbed on
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sediment in high salinity water, leading to reduced bioavailability and small body burden of antibiotic residues in zebrafish. The equilibrium data further confirmed that the distribution of the antibiotic in water, sediment and zebrafish accounted for 97.3%, 2.7% and 0.05% of the dosing quantity, respectively. These results provide important insights into the transport and biological fate of a widely used antibiotic in microcosm, which form the basis for further research on antibiotic residues in aquatic environments. Acknowledgements This work was funded by the State Key Laboratory of Estuarine and Coastal Research (2016RCDW02). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2017.04.055. References Ahmed, M.B., Zhou, J.L., Ngo, H.H., Guo, W., 2015. Adsorptive removal of antibiotics from water and wastewater: progress and challenges. Sci. Total Environ. 532, 112e126. Alexander, M., 2000. Aging, bioavailability, and overestimation of risk from environmental pollutants. Environ. Sci. Technol. 34 (20), 4259e4265. Bartley, M.L., Boeing, W.J., Corcoran, A.A., Holguin, F.O., Schaub, T., 2013. Effects of salinity on growth and lipid accumulation of biofuel microalga Nannochloropsis salina and invading organisms. Biomass Bioenergy 54, 83e88. Bayen, S., Estrada, E.S., Juhel, G., Kit, L.W., Kelly, B.C., 2016. Pharmaceutically active compounds and endocrine disrupting chemicals in water, sediments and mollusks in mangrove ecosystems from Singapore. Mar. Pollut. Bull. 109 (2), 716e722. Benotti, M.J., Brownawell, B.J., 2009. Microbial degradation of pharmaceuticals in estuarine and coastal seawater. Environ. Pollut. 157 (3), 994e1002. Boeuf, G., Payan, P., 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. C 130 (4), 411e423. Boonsaner, M., Hawker, D.W., 2013. Evaluation of food chain transfer of the antibiotic oxytetracycline and human risk assessment. Chemosphere 93 (6), 1009e1014. Bowman, J.C., Zhou, J.L., Readman, J.W., 2002. Sediment-water interactions of natural oestrogens under estuarine conditions. Mar. Chem. 77 (4), 263e276. Carballa, M., Fink, G., Omil, F., Lema, J.M., Ternes, T., 2008. Determination of the solid-water distribution coefficient (Kd) for pharmaceuticals, estrogens and musk fragrances in digested sludge. Water Res. 42 (1e2), 287e295. Carstens, K.L., Gross, A.D., Moorman, T.B., Coats, J.R., 2013. Sorption and photodegradation processes govern distribution and fate of sulfamethazine in freshwater-sediment microcosms. Environ. Sci. Technol. 47 (19), 10877e10883. Chen, C.Q., Zheng, L., Zhou, J.L., Zhao, H., 2017. Persistence and risk of antibiotic residues and antibiotic resistance genes in major mariculture sites in Southeast China. Sci. Total Environ. 580, 1175e1184. Chen, H., Liu, S., Xu, X.-R., Liu, S.-S., Zhou, G.-J., Sun, K.-Y., Zhao, J.-L., Ying, G.-G., 2015. Antibiotics in typical marine aquaculture farms surrounding Hailing Island, South China: occurrence, bioaccumulation and human dietary exposure. Mar. Pollut. Bull. 90 (1e2), 181e187. Chen, K., Zhou, J.L., 2014. Occurrence and behavior of antibiotics in water and sediments from the Huangpu River, Shanghai, China. Chemosphere 95, 604e612. Ding, J.N., Lu, G.H., Liu, J.C., Zhang, Z.H., 2015. Evaluation of the potential for trophic transfer of roxithromycin along an experimental food chain. Environ. Sci. Pollut. Res. 22 (14), 10592e10600. Duong, C.N., Schlenk, D., Chang, N.I., Kim, S.D., 2009. The effect of particle size on the bioavailability of estrogenic chemicals from sediments. Chemosphere 76 (3), 395e401. Gaillard, J., Banas, D., Thomas, M., Fournier, A., Feidt, C., 2014. Bioavailability and bioaccumulation of sediment-bound polychlorinated biphenyls to carp. Environ. Toxicol. Chem. 33 (6), 1324e1330. , D., 2011. Occurrence of sulfonamide García-Gal an, M.J., Díaz-Cruz, M.S., Barcelo residues along the Ebro river basin: removal in wastewater treatment plants and environmental impact assessment. Environ. Int. 37, 462e473. Hou, J., Pan, B., Niu, X., Chen, J., Xing, B., 2010. Sulfamethoxazole sorption by sediment fractions in comparison to pyrene and bisphenol A. Environ. Pollut. 158, 2826e2832. Hou, X.L., Shen, J.Z., Zhang, S.X., Jiang, H.Y., Coats, J.R., 2003. Bioconcentration and elimination of sulfamethazine and its main metabolite in sturgeon (Acipenser schrenkii). J. Agric. Food Chem. 51, 7725e7729. Ji, X., Shen, Q., Liu, F., Ma, J., Xu, G., Wang, Y., Wu, M., 2012. Antibiotic resistance gene abundances associated with antibiotics and heavy metals in animal manures
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