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Evaluation of food chain transfer of the antibiotic oxytetracycline and human risk assessment
Chemosphere 93 (2013) 1009–1014
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Evaluation of food chain transfer of the antibiotic oxytetracycline and human risk assessment Maliwan Boonsaner a,⇑, Darryl W. Hawker b a b
Department of Environmental Science, Faculty of Science, Silpakorn University, Nakhon Pathom 73000, Thailand School of Environment, Griffith University, Nathan, Qld 4111, Australia
h i g h l i g h t s Shows for the first time that OTC is able to transfer from water to plant to fish. Characterizes bioconcentration of OTC by a floating aquatic plant (Wolffia globosa). Compares accumulation of OTC from water and watermeal by fish (Probarbus jullieni). Quantitative risk assessment for human consumption of contaminated fish undertaken.
a r t i c l e
i n f o
Article history: Received 6 January 2013 Received in revised form 16 May 2013 Accepted 25 May 2013 Available online 20 June 2013 Keywords: Food chain transfer Bioconcentration Biomagnification Oxytetracycline Watermeal (Wolffia globosa Hartog & Plas) Seven-striped carp (Probarbus jullieni)
a b s t r a c t There has been recent concern regarding the possibility of antibiotics entering the aquatic food chain and impacting human consumers. This work reports experimental results of the bioconcentration of the antibiotic oxytetracycline (OTC) by the Asian watermeal plant (Wolffia globosa Hartog & Plas) and bioaccumulation of OTC in watermeal and water by the seven-striped carp (Probarbus jullieni). They show, for the first time, the extent to which OTC is able to transfer from water to plant to fish and enter the food chain. The mean bioconcentration factor (dry weight basis) with watermeal was 1.28 103 L kg1. Separate experiments were undertaken to characterize accumulation of OTC by carp from water and watermeal. These showed the latter pathway to be dominant under the conditions employed. The bioconcentration and biomagnification factors for these processes were 1.75 L kg1 and 2 104 kg g1 respectively. Using an aqueous concentration range of 0.34–3.0 lg L1, hazard quotients for human consumption of contaminated fish of 1.3 102 to 1.15 101 were derived. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Antibiotics are biologically active compounds categorized as emerging environmental contaminants of concern (de la Torre et al., 2012). Widespread use and poor absorption following ingestion has resulted in a ubiquitous environmental presence of antibiotics. Despite this, little is known of their fate and behavior in the aquatic environment (Xu and Li, 2010). Concern has been expressed because of the potential for antibiotics to enter and move through food chains (Boxall and Ericson, 2012). Farré and Barceló (2012) have recently noted that farmed fish and shrimp are often produced in crowded facilities with little regulation of antibiotic use and that the discovery of chloramphenicol residues in shrimp has increased worldwide public awareness. In food chain transfer, a number of processes can occur that require explanation and delineation. In aquatic systems, plants and ⇑ Corresponding author. Tel.: +66 3 4219146; fax: +66 3 4273047. E-mail address: [email protected] (M. Boonsaner). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.05.070
other organisms can bioconcentrate chemicals such as antibiotics from water. Bioconcentration is hence defined as the accumulation of a chemical from the ambient environment of an organism via its respiratory and dermal surfaces. This process is quantified by the bioconcentration factor (BCF), the ratio of chemical concentration in an organism to that in the ambient environment. In contrast, bioaccumulation is accumulation from all sources, both ambient and dietary (Arnot and Gobas, 2006). Biomagnification may be described as the accumulation and transfer of chemicals via the food chain usually resulting in increased concentrations in organisms at higher trophic levels (European Commission, 2003). The biomagnification factor (BMF) is the ratio of a chemical’s concentration in an organism to that in its diet. Oxytetracycline (OTC) is a member of the tetracycline family of antibiotics, widely used for therapeutic purposes in humans as well as an antibiotic and growth promoter in animal farming (Holström et al., 2003). It is amongst the most commonly used antibiotics in fish farming in South East Asia (Holström et al., 2003). Previous studies have also demonstrated OTC bioaccumulation in
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various organisms including invertebrates and fish (Richardson et al., 2005). At present, there are growing concerns regarding the ecological/ environmental risks associated with OTC exposure. Given its antibacterial properties, development of antibiotic-resistant bacteria in humans, and the environment in general, has been cited as an obvious risk with use (Williams and Brooks, 2012). Antibiotics such as OTC have also been found to be very toxic to algae, the basis of aquatic food chains (Santos et al., 2010). Algal population changes could affect the structure of such chains. There is little evidence for acute toxic effects on fish at environmentally realistic concentrations (Crane et al., 2006). Given a likely entry route to aquatic ecosystems is via agricultural runoff at relatively low concentrations from veterinary use, chronic toxic effects are more likely (Williams and Brooks, 2012). The number of studies on human risk is relatively small (Kumar et al., 2010). Chronic exposure may result in gastrointestinal irritation and blood composition changes. Acute toxicity is manifested by hepatoxicity (WHO, 2012). Apart from direct therapeutic use, other routes of human exposure are via potable water and ingestion of contaminated food (e.g. fish and shrimp). Concentrations of antibiotics such as OTC in source waters for potable use are typically such that they are not a concern for human health (Boxall, 2004). However herbivorous animals such as fish can bioaccumulate antibiotics that in turn can be transferred to humans through the food chain (Kong et al., 2007). The extent to which this occurs is largely unknown. It has been speculated that this may produce tolerance to pathogens in animal consumers and humans (Boxall et al., 2006), but the direct risk to human health is uncharacterized. Many factors can affect the bioavailability of OTC in the aquatic food chain. Divalent cations (e.g. Ca2+ and Mg2+) in water can bind to OTC and form relatively stable complexes that reduce gastrointestinal absorption efficiency (Sassman and Lee, 2005). Environmental factors such as temperature, pH and light intensity can affect the degradation of OTC in water and sediment (Burhenne et al., 1997). Doi and Stoskopf (2000) found that the compound was more stable at low temperatures (4 °C) while Pouliquen et al. (2007) reported that first order degradation rates of OTC in water increase at least threefold with light exposure (1400 lux) compared to darkness and moreover were pH dependent. The accumulation of OTC at each trophic level of an aquatic food chain and ultimately the concentration in food for human intake are likely to be affected by these environmental factors, as well as the types of plant and animals involved. In this study, watermeal or swamp algae (Wolffia globosa Hartog & Plas.) was employed as the lowest trophic level, bioconcentrating OTC from water. Seven-striped carp (Probarbus jullieni) represented the second trophic level, bioaccumulating OTC from water itself and through consumption of contaminated watermeal. Watermeal is a free-floating plant without roots with a wide distribution in rivers across several continents including Asia and North America. The sevenstriped carp is a herbivorous fish, found in Thailand, Cambodia, Laos, Malaysia and Vietnam. The objectives of this study were to investigate the accumulation of OTC in watermeal and seven-striped carp as the first and second trophic levels in an aquatic food chain and evaluate
Water Seven-striped carp (Probarbus julleini)
Human Consumer
Watermeal (Wolffia globosa)
Fig. 1. Aquatic food chain for transfer of OTC to humans.
whether OTC can undergo food chain transfer. Finally, derived concentrations of OTC in seven-striped carp were employed to assess the risk involved in human consumption (EPA, 1989). The food chain considered is illustrated in Fig. 1. 2. Materials and methods 2.1. Reagents Oxytetracycline standard (as the hydrochloride) (>98.5% purity) for HPLC analysis was purchased from Dr. Ehrenstorfer GmbH, Augsburg, Germany. Commercial grade oxytetracycline hydrochloride used in experiments was obtained from Nova Medicine, Bangkok, Thailand and checked for impurities prior to use. Acetonitrile (Nanograde, Mallinckrodt, KY, USA), methanol (HPLC grade, Fisher, Pittsburgh, PA, USA), oxalic acid (AR, Ajax, Sydney, Australia), sodium EDTA (AR, Ajax, Sydney, Australia) and glass fiber filter papers (GF/C, Whatman, Maidstone, England) were used for extraction of the antibiotics. Solid phase extraction (SPE) tubes (Strata-X Polymeric Reversed Phase (200 mg/6 mL)) were obtained from Phenomenex (Torrance, CA, USA) and conditioned with 2 mL methanol followed by 2 mL of deionized water. Water used in accumulation experiments with fish was dechlorinated tap water [Ca2+ = 33.8 mg L1; Mg2+ = 8.8 mg L1]. 2.2. Analysis of OTC in water, plant and fish samples The concentrations of OTC in plant and fish samples (approximately 10 g) were determined by adding 25 mL of McIlvaine buffer-EDTA solution and followed the method described in Boonsaner and Hawker (2010). Samples were blended with an Ultra-Turrax homogenizer. Then, both sample types were placed in an ultrasonic bath for 2 min and filtered through a glass fiber filter. The filtrate was passed through a conditioned SPE cartridge and eluted with 4 mL of methanol. Resulting solutions containing OTC were analyzed by HPLC (Waters 600, Milford, MA, USA) with Photodiode Array Detection. The analytical column was a C18 column (HiQ Sil C18HS, 4.6 mm id 150 mm and 0.5 lm particle size) operated at room temperature and the chromatographic conditions were as follows: flow rate 1 mL min1; mobile phase 95% acetonitrile (v/v), 5% 0.01 M oxalic acid (v/v); injection volume 20 lL with detection at 333 nm. Water samples (100 mL) were passed through a conditioned SPE cartridge, eluted with 2 mL of methanol and analyzed by HPLC using the conditions described above. Method detection limits (MDL) for the analysis of OTC in plants and fish were 0.45 and 0.7 mg kg1 dry weight (dw) and 0.5 mg L1 for water. The mean recoveries from water, plant and fish samples were 97%, 70% and 66%, respectively. All concentrations reported herein account for these recoveries. 2.3. Bioconcentration of OTC by watermeal Watermeal was purchased from a local market in Nakhon Pathom province, central Thailand. Before commencing the experiments, it was determined to be OTC free. A preliminary investigation of OTC toxicity was undertaken by placing watermeal in 50, 80 and 100 mg L1 solutions. It was found that at concentrations of more than 50 mg L1 toxic effects occurred, evidenced by the watermeal turning brown, gelatinous and malodorous. Therefore, concentrations of 50 mg L1 and less were chosen for bioconcentration experiments. Experiments were conducted with three initial test OTC concentrations, nominally 10, 30 and 50 mg L1. For each concentration, 500 mL of test solution (pH 4.4–4.6) was added to each of 8 clear glass jars (800 mL capacity) followed by 20 g of watermeal. This
M. Boonsaner, D.W. Hawker / Chemosphere 93 (2013) 1009–1014
amount of watermeal covered the surface of the water in a jar. The daily illumination regime was 10 h light: 14 h darkness. The external sidewall of the jar was covered by aluminum foil to reduce degradation from illumination. All jars were placed in a room where temperature was 28 ± 2 °C. At days 0, 1, 3, 5, 9, 11, 13 and 15 of exposure, one jar of each OTC concentration was taken for analysis. Watermeal samples were filtered from water and separated for analysis. The filtered water was thoroughly stirred before collecting 100 mL samples for analysis. Duplicate samples were analyzed for plant tissue and water concentrations at all time periods. The bioconcentration factor (BCF) was obtained from the ratio of the test compound concentration in watermeal (CWATERMEAL) to the concentration found in the water (CW). A control experiment was performed by placing watermeal in clean sterilized deionized water and locating a jar in the same room. Following the 15-d experimental period, watermeal and water samples from the control jar were taken for OTC analysis. These showed no contamination and watermeal appeared normal. In order to assess the influence of environmental factors on aqueous OTC levels in bioconcentration experiments, a parallel investigation to determine the loss of OTC in water under the same conditions of the bioconcentration experiments was carried out, but without the addition of watermeal. OTC solutions (50 mg L1) in 250 mL bottles were prepared and covered with aluminum foil. Bottles were placed in the room together with jars of the bioconcentration experiment. Duplicate 100 mL samples of test solution were taken from each bottle at the same time that the samples were collected from the bioconcentration experiments. Numerical results were analyzed according to first order loss kinetics.
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daily (28 ± 2 °C), while fish samples were taken for OTC analysis every 2 weeks. For accumulation of OTC from food, twelve glass jars were each filled with 2 L of clean water and 7–8 fish (total mass approximately 20 g) placed in each jar. The fish were fed twice per day with the food described above, but contaminated with OTC (35 mg kg1 (dw)). Again, 2–3 g of contaminated food was added to each jar and 30 min allowed for eating. The leftover food was taken out, dried and weighed to determine the amount consumed. Fish were carefully transferred daily to OTC-free water. The jars were again placed in dim light and a temperature of 28 ± 2 °C. The experiment was conducted over a period of 15 d. Whole fish were sacrificed for OTC analysis on days 0, 1, 3, 5, 9, 11, 13 and 15 of exposure. Control experiments for accumulation from food were also conducted. The first involved one jar with 7–8 fish (total mass approximately 20 g) with clean water and uncontaminated food. Fish were transferred daily to new jars as for exposure experiments. The other comprised a jar containing water but no fish where contaminated food was added twice daily for 30 min. The results showed that after 15 d, no OTC was detected in control fish and there was none in water arising from desorption from food. Because fish were regularly transferred to clean water, there was likely to be little influence from desorption of OTC in faeces arising from incomplete dietary absorption. In the absence of regular water changes and in field situations however, this process may affect aqueous OTC concentrations and should not be neglected.
3. Results and discussion 2.4. Bioaccumulation of OTC by seven-striped carp
3.1. Bioconcentration of OTC by watermeal
Seven-striped carp (approximately 3–5 cm long and weighing 2–3 g each) were purchased from a local market in Nakhon Pathom province, Thailand. Before commencing the experiments, tissue OTC levels were determined to be below detection limits. Tap water used in the experiment was dechlorinated by aeration for 2 d prior to use. In this study, separate experiments were conducted viz. accumulation of OTC from water and accumulation of OTC from food (watermeal). During these experiments, fish were fed with food prepared from 300 g watermeal, 300 g dried ground fish, 100 g rice, 200 g rice bran, 50 g vegetable oil, 50 g starch and a small amount of water to assist mixing. This mixture was subsequently air dried and sieved to 2 mm size. A preliminary investigation of OTC toxicity found that there were no observable toxic effects on fish over the time frame of the current study at an aqueous concentration of 10 mg L1. As a result, a 10 mg L1 OTC solution was to investigate accumulation from water. For this, twelve glass jars were filled with 2 L of 10 mg L1 OTC solution and 7–8 fish (total mass approximately 20 g) placed in each jar. Due to relatively slow bioconcentration, the experiment was conducted over a period of 12 weeks. Fish were fed twice per day with the food described above. At each feeding period, 2–3 g of food was added and 30 min allowed for consumption. After this time period, the leftover food was taken out, dried and weighed to determine the mass of consumed food. Fish were carefully transferred daily to new jars containing 10 mg L1 OTC solution to ensure, as far as practicable, a constant exposure concentration. In addition, control experiments were conducted by using one jar with 15 fish in clean water and another jar with 10 mg L1 OTC solution without fish. All jars were placed in a room with dim light (8–10 lux) to imitate the natural conditions experienced by fish and to reduce the photodegradation of OTC. The room temperature was recorded
Regardless of initial ambient OTC concentrations, levels in watermeal increased rapidly within the first day of the experiments and then remained relatively stable until the end of the 15-d exposure (Fig. 2). Data on which this figure is based is found in Table S1 (Supplementary material). Similarly rapid uptake of OTC from aqueous solution has been observed with alfalfa (Medicago sativa L.) where equilibrium was attained after 4 h (Kong et al., 2007). OTC is susceptible to microbial transformation as well as abiotic processes such as hydrolysis and photolysis (Pouliquen et al., 2007; Ratasuk et al., 2012). The first order loss rate constant from the control experiment was 0.07 d1. In comparison, loss rate constants in water from bioconcentration experiments (0.12–0.15 d1) were larger. Although prima facie the difference is due to plant uptake it should be remembered that the watermeal covers the surface in bioconcentration experiments and phototransformation may be reduced here compared to the control. Aqueous OTC loss rate constants obtained in this study were comparable to those from other studies, e.g. 0.01–0.18 d1 (Doi and Stoskopf), and 0.01 d1 (Boonsaner and Hawker, 2012), bearing in mind the effect of variation in factors such as pH, temperature and light intensity. For compounds that are labile in aqueous systems such as OTC, Li et al. (2002) distinguish between a thermodynamic bioconcentration factor (the ratio of uptake and loss rate constants) and an actual bioconcentration factor (BCF) (the ratio of plant and water concentrations at each observation time). For this work, the relevant parameter is the actual BCF. The accumulated OTC concentrations in watermeal vary with the ambient levels of this antibiotic. As a result, BCF values are not a function of water concentrations. The mean of the coefficients of variation for BCFs with external aqueous concentrations for individual exposure times is 18.8%. Aqueous concentrations do however decrease with time, but watermeal concentrations are relatively constant, implying rela-
M. Boonsaner, D.W. Hawker / Chemosphere 93 (2013) 1009–1014
Aqueous Concentration (mg L -1)
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(a)
50
Nominal Initial Concentration 50 mg L-1 Nominal Initial Concentration 30 mg L-1
40
Nominal Initial Concentration 10 mg L-1
30 20 10 0 0
5
10
15
Watermeal Concentration (mg g-1)
Time (d)
(b)
30
20
10
0 0
5
10
15
Time (d) 10 8
BCF (L g -1)
3.2. Bioconcentration of OTC by seven-striped carp Concentrations in seven-striped carp tissue plateaued within the first week and were quite low (Table 1). As a result, BCF data expressed as mean (±standard error of the mean) over 12 weeks was 1.75 103 ± 2 104 L g1 (dw) or 1.75 ± 2 101 L kg1 (dw). The OTC concentration employed (10 mg L1) was selected to have no toxic effect on seven-striped carp based on results of preliminary bioassays. There is little other relevant toxicology data for this species. Ji et al. (2012) have reported that 50 mg L1 OTC did not affect the growth of juvenile and adult Japanese killifish (Oryzias latipes). OTC degradation measured in a control experiment without fish (k = 0.003 d1) was relatively low compared to that from the watermeal bioconcentration described above. This is likely the result of the dim illumination conditions employed, reducing photodegradation. Solutions were also renewed daily so that effectively, fish were exposed to a constant ambient OTC concentration. 3.3. Biomagnification of OTC by seven-striped carp
(c)
6 4 2 0 0
The magnitude of the BCFs for OTC here are similar to those observed for a plant possessing roots such as rice where BCF rose to >2 L g1 (dw) after a similar 15 d exposure (Boonsaner and Hawker, 2012). With rice, unionized organics are uptaken by roots largely by passive processes but active mechanisms are proposed for ionized species (Su and Zhu, 2007). Under the prevailing pH conditions (pH = 4.4–4.6) in the watermeal bioconcentation experiments, OTC exists largely in zwitterionic form. Previous research has shown OTC uptake into rice roots under these conditions is consistent with passive diffusion, controlled by diffusive transport through lipophilic membranes (Boonsaner and Hawker, 2012).
5
10
15
Time (d) Fig. 2. (a) Aqueous OTC concentrations ( 50 mg L1 nominal initial; j 30 mg L1 nominal initial; w 10 mg L1 nominal initial) with time in watermeal bioconcentration experiments. Shown in (b and c) are associated watermeal concentrations (mg g1) and BCF values (L g1) respectively.
tively slow or negligible loss from the plant in response to decreasing external concentrations. BCFs calculated from the ratio of CWATERMEAL/CW accordingly increase with time (Fig. 2). For the purposes of this work, the BCF value used in calculation of human health risk is the overall mean value from Fig. 2, i.e. 1.28 L g1 (dw) or 1.28 103 L kg1 (dw). Watermeal does not possess roots but similar plants have been shown to be useful for the removal of a range of aqueous organic contaminants by processes including surface sorption (Reinhold et al., 2010). In that work, it was concluded that equilibrium with the pharmaceutical fluoxetine was attained within 12–24 h after initial exposure. Close inspection of the results presented by Reinhold et al. (2010) show a relatively slow decrease in aqueous concentration after 24 h that was attributed to loss of antibiotic in the aqueous phase and uptake into the plant. Similar trends were observed in this current work with OTC as described above.
To characterize biomagnification of OTC from watermeal by seven-striped carp, 2–3 g of contaminated watermeal (35 mg kg1 (dw) OTC) was fed to the carp for two 30-min periods daily over 15 d. The level in contaminated food was considerably lower than the therapeutic dose for fish (50 mg kg1 for 2 weeks) (Choo, 1995) and thus no toxic effects were anticipated or observed. The cumulative amount of food consumption is shown in Table 2. Mean daily consumption was 2.3 g d1 from which can be calculated a mean daily OTC consumption of 0.080 mg d1. However, not all OTC consumed in food is actually absorbed by the fish. Measured OTC tissue concentrations are also shown in Table 2and increase relatively slowly with time. The mean of these values over the 15 d exposure period (0.0076 mg g1 (dw)) is used in conjunction with concentration in contaminated food to derive a biomagnification factor (BMF) of 2 104 kg g1. An unidentified peak was observed in chromatograms of fish tissue. Not present in controls, the peak increases in size with time and is attributed to a metabolite of OTC. Assuming the same response factor with the photodiode array detector as OTC itself, concentrations of OTC and metabolite are presented in Table 2. It is of interest that evidence of the OTC metabolite was not found in the bioconcentration of OTC directly from water by seven-striped carp, even when tissue concentrations from this process were greater. Previous work with common carp (Cyprinus carpio) has shown different pharmacokinetic behavior depending on route of administration however accumulation from ambient water and contaminated food were not compared (Grondel et al., 1987). A large fraction of compounds such as OTC absorbed from water by the gills is initially transported to the kidneys. In contrast, OTC from dietary exposure is first transported to the liver (Toutain et al., 2010). Hepatic cytochrome-P450 isozymes catalyze the oxidation of endogenous substrates such as OTC. They are known to exist in members of the carp family (Marionnet et al., 1998; Uno et al.,
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M. Boonsaner, D.W. Hawker / Chemosphere 93 (2013) 1009–1014 Table 1 Duplicate OTC concentrations and mean BCF values in seven-striped carp from bioconcentration over a 12-week exposure period to a 10 mg L1 aqueous concentration. Week
OTC concentration in seven-striped carp (mg g1 (dw))
Mean BCF (L g1 (dw))
0 1
0 0.013 0.007 0.009 0.005 0.021 0.017 0.012 0.018 0.010 0.016 0.012 0.005 0.016 0.008 0.014 0.011 0.031 0.019 0.023 0.017 0.023 0.020 0.012 0.020
0 0.001
2 3 4 5 6 7 8 9 10 11 12
C fish
1
¼ BMFðkg g1 Þ BCFðL kg Þ C w ðmg L1 Þ 4
¼ 2 10
kg g1 1:28 103 L kg
1
3:4 104 mg L1
¼ 8:70 105 mg g1
0.001
1
¼ 8:70 102 mg kg
0.002
ð2Þ
0.002
2
0.001 0.001 0.002 0.002 0.003 0.002 0.002 0.002
2012), but no details of metabolite identity for OTC currently exist. A first pass induction of hepatic enzymes could result in selective metabolism of OTC by this route of administration. As metabolite identity and properties are unknown however, it is not considered further in food chain transfer and risk calculations. 3.4. Assessment of human risk from consumption of fish containing OTC Comparison of the two pathways for bioaccumulation of OTC by seven-striped carp viz. from direct bioconcentration from water and from biomagnification of watermeal whose OTC burden is acquired by bioconcentration shows the latter to be dominant. For bioconcentration:
C fish
Concentrations of OTC in seven-striped carp from biomagnification are derived according to the methods described in the European Commission’s Technical Guidance Document on Risk Assessment (European Commission, 2003). Values of BMF and BCF employed are as derived above.
1
¼ BCFðL kg Þ C w ðmg L1 Þ ¼ 1:75 L kg
1 4
¼ 5:95 10
3:4 104 mg L1 mg kg
ð1Þ
1
Table 2 Cumulative amounts of food and OTC consumed together with the measured concentrations of OTC and metabolite in seven-striped carp from biomagnification. Day
Cumulative amount of food consumed (g)
Cumulative amount of OTC consumed (mg)a
OTC concentration in seven-striped carp (mg g1 (dw))
OTC + metabolite concentration in seven-striped carp (mg g1 (dw))
1 3 5 7 9 11 13 15
5.28 8.61 11.94 15.00 19.17 23.61 28.06 31.39
0.19 0.31 0.43 0.54 0.69 0.85 1.01 1.13
0.0053 0.0044 0.0096 0.0070 0.0072 0.0084 0.0090 0.0096
0.0058 0.0057 0.0092 0.0080 0.0112 0.0126 0.0140 0.0154
a Calculated from rate of food consumption using OTC concentration of 35 mg kg1.
1
The total body burden of OTC for fish (8.76 10 mg kg ) is taken as the sum of these concentrations. Kolpin et al. (2002) undertook a comprehensive reconnaissance of levels of pharmaceuticals in US streams and found a median level for OTC of 0.34 lg L1. Sampling sites were biased towards those susceptible to contamination such as sites downstream of urbanization or intensive livestock facilities. We employed the datum of Kolpin et al. in our calculations above, considering it to represent a conservative level of contamination that might be encountered in fish farms or surface water impacted by fish farms using antibiotics. However, there are many localized sites worldwide with higher concentrations. For example Liu et al. (2009) found levels of 3.0 lg L1 in the Nanming River, southwestern China. Using this concentration, the total body burden for OTC in fish calculated as above is 7.73 101 mg kg1. Using these values to represent a range for OTC levels in fish, the intake rate of OTC from fish by human consumers (mg kg1 d1) is determined using the following expression (EPA, 1989; Dunnivant and Anders, 2006): 1
Intake ðmg kg
1
d Þ¼
C fish a IR EF ED BW AT
ð3Þ
where the concentration in fish is expressed in mg kg1, a is the proportion of OTC absorbed by human from oral dosing (0.60) (WHO, 2012), IR the fish ingestion rate (0.054 kg d1) (Dunnivant and Anders, 2006), EF the exposure frequency (350 d y1) (Dunnivant and Anders, 2006), ED the duration of exposure (70 y) (Dunnivant and Anders, 2006), BW adult body weight (70 kg) and AT the time period over which exposure is averaged (70 y 365 d y1). This equation is modified from that usually employed by additionally including the absorption efficiency of OTC. On this basis, the intake rate of OTC ranges from 3.89 105 to 3.46 104 mg kg1 d1. Changes to parameters such as fish ingestion rate would modify this range. For example, if fish ingestion rate doubled, then OTC intake rate would also double. The hazard quotient is a metric describing risk posed by chemicals. It is used by many regulatory authorities, including the USEPA (Langdon et al., 2010). Here, it is derived from the ratio of the intake rate calculated above and an acceptable daily intake of OTC. The latter is 3 103 mg kg1 d1, although there is some uncertainty in this figure, and it may be somewhat higher (WHO, 2012). A ratio of 1.0 or above for the hazard quotient indicates a toxic effect is likely while conversely a ratio of <0.5 indicates a low risk. The resultant hazard quotient range derived here (1.3 102 to 1.15 101) suggests no direct detrimental effects from ingestion of contaminated fish where OTC has entered the aquatic food chain. Even with elevated concentrations in water, and increased ingestion rates, the hazard quotient is below the threshold value of 0.5. 4. Conclusion There is abundant evidence that antibiotics find their way into the environment from a variety of sources (Sarmah et al., 2006).
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However effects on aquatic ecosystems and consequent impacts on humans have not been well understood. Exploration of the mechanisms of uptake by plants and animals as well as the potential movement through food webs has been advocated (Boxall and Ericson, 2012). Behavior of OTC, a widely used antibiotic, in an aquatic food web has now been investigated and characterized. The compound is rapidly bioconcentrated from water by watermeal (W. globosa) (BCF = 1.28 103 L kg1). Fish (seven-striped carp (P. jullieni)) can accumulate OTC from contaminated watermeal (BMF = 2 104 kg g1). Fish OTC burdens from this pathway were over 100 times greater than those from direct bioconcentration by fish from water. A hazard quotient approach to risk assessment of human consumption of this fish suggested little likelihood of direct detrimental impact, even from sites with relatively high aqueous OTC levels of up to 3.0 lg L1. However, additional intake of OTC for therapeutic purposes can alter this scenario. Subtle effects such as increased antibiotic resistance of resident bacteria are also possible and should not be neglected. Acknowledgements This study was supported by the Thailand National Research Fund and the Silpakorn University Research and Development Institute. The authors would like to thank C. Teparak and N. Sanchan for their laboratory assistance and S. Jaipan for his plant preparations. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2013.05.070. References Arnot, J.A., Gobas, F.A.P.C., 2006. A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms. Environ. Rev. 14, 257–297. Boonsaner, M., Hawker, D.W., 2010. Accumulation of oxytetracycline and norfloxacin from saline soil by soybeans. Sci. Total Environ. 408, 1731–1737. Boonsaner, M., Hawker, D.W., 2012. Investigation of the mechanism of uptake and accumulation of zwitterionic tetracyclines by rice (Oryza sativa L.) Ecotoxicol. Environ. Saf. 78, 142–147. Boxall, A.B.A., 2004. The environmental side effects of medication. Embo. Rep. 5, 1110–1116. Boxall, A.B.A., Ericson, J.F., 2012. Environmental fate of human pharmaceuticals. Human Pharma. Environ., 63–83. Boxall, A.B., Johnson, P., Smith, E.J., Sinclair, C.J., Stutt, E., Levy, L.S., 2006. Uptake of veterinary medicines from soils into plants. J. Agric. Food Chem. 54, 2288–2297. Burhenne, J., Ludwig, M., Nikoloudis, P., Spiteller, M., 1997. Primary photoproducts and half-lives. Environ. Sci. Pollut. Res. Int. 4 (1), 10–15. Choo, P.S., 1995. Withdrawal time for oxytetracycline in Red Tilapia cultured in freshwater. Asian Fish. Sci. 8 (2), 169–176. Crane, M., Watts, C., Boucard, T., 2006. Chronic aquatic environmental risks from exposure to human pharmaceuticals. Sci. Total Environ. 367, 23–41. de la Torre, A., Iglesias, I., Carballo, M., Ramírez, P., Muñoz, M.J., 2012. An approach for mapping the vulnerability of European Union soils to antibiotic contamination. Sci. Total Environ. 414, 672–679. Doi, A.M., Stoskopf, M.K., 2000. The kinetics of oxytetracycline degradation in deionised water under varying temperature, pH, light, substrate, and organic matter. J. Aquat. Anim. Health 12, 246–253. Dunnivant, F.M., Anders, E., 2006. A Basic Introduction to Pollutant Fate and Transport. John Wiley and Sons, Inc., Hoboken, NJ, USA. EPA, 1989. Risk assessment, guidance for superfund volume 1 (Part A): human health evaluation manual. Office of Emergency and Remedial Response, USEPA Washington, DC 20450.
European Commission, 2003. Technical guidance document on risk assessment part 2. European Commission and European Chemicals Bureau, EUR 20418/EN/2, Luxembourg, Luxembourg. Farré, M., Barceló, D., 2012. Emerging organic contaminants and nanomaterials in food. In: Barceló, D. (Ed.), Emerging Organic Contaminants and Human Health, Handbook of Environmental Chemistry, vol. 20, pp. 1–46. Grondel, J.L., Nouws, J.F.M., deJong, M., Schutte, A.R., Driessens, F., 1987. Pharmocokinetics and tissue distribution of oxytetracycline in carp, Cyprinus carpio L., following different routes of administration. J. Fish Dis. 10, 153– 163. Holström, K., Gräslund, S., Wahlström, A., Poungshompoo, S., Bengtsson, B.-E., Kautsky, N., 2003. Antibiotic use in shrimp farming and implications for environmental impacts and human health. Int. J. Food Sci. Technol. 38, 255–266. Ji, K., Kim, S., Han, S., Seo, J., Lee, S., Park, Y., Choi, K., Kho, Y.-L., Kim, P.-G., Park, J., Choi, K., 2012. Risk assessment of chlortetracycline, oxytetracycline, sulfamethazole, sulfathiazole, and erythromycin in aquatic environment: are the current environmental concentrations safe? Ecotoxicology 21, 2031–2050. Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber, L.B., Buxton, H.T., 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999–2000: a national reconnaissance. Environ. Sci. Technol. 36 (6), 1202–1211. Kong, W.D., Zhu, Y.G., Liang, Y.C., Zhang, J., Smith, F.A., Yang, M., 2007. Uptake of oxytetracycline and its phytotoxicity to alfalfa (Medicago sativa L.). Environ. Pollut. 147, 187–193. Kumar, A., Chang, B., Xagoraraki, I., 2010. Human health risk assessment of pharmaceuticals in water: issues and challenges ahead. Int. J. Environ. Res. Publ. Health 7, 3929–3953. Langdon, K.A., Warne, M.St.J., Kookana, R.S., 2010. Aquatic hazard assessment for pharmaceuticals, personal care products and endocrine disrupting compounds from biosolids-amended land. Integr. Environ. Assess. Manage. 6 (4), 663– 676. Li, H., Sheng, G., Sheng, W., Xu, O., 2002. Uptake of trifluralin and lindane from water by ryegrass. Chemosphere 48, 335–341. Liu, H., Zhang, G., Liu, C.-Q., Li, L., Xiang, M., 2009. The occurrence of chloramphenicol and tetracyclines in municipal sewage and the Nanming River, Guiyang City, China. J. Environ. Monitor. 11, 1199–1205. Marionnet, D., Chambras, C., Taysse, T., Bosgireaud, C., Deschaux, P., 1998. Modulation of drug-metabolizing systems by bacterial endotoxin in carp liver and immune organs. Ecotoxicol. Environ. Saf. 41, 189–194. Pouliquen, H., Delépée, R., Larhantec-Verdier, M., Morvan, Le Bris, H., 2007. Comparative hydrolysis and photolysis of four antibacterial agents (oxytetracycline, oxolinic acid, flumequine and florfenicol) in deionised water, freshwater and seawater under abiotic conditions. Aquaculture 262 (1), 23– 28. Ratasuk, N., Boonsaner, M., Hawker, D.W., 2012. Effect of temperature, pH and illumination on abiotic degradation of oxytetracycline in sterilized swine manure. J. Environ. Sci. Health A 47, 1687–1694. Reinhold, D., Vishwanathan, S., Park, J.J., Oh, M., Saunders, F.M., 2010. Assessment of plant-driven removal of emerging organic pollutants by duckweed. Chemosphere 80, 687–692. Richardson, B.R., Lam, P.K.S., Martin, M., 2005. Emerging chemicals of concern: pharmaceuticals and personal care products (PPCPs) in Asia, with particular reference to Southern China. Mar. Pollut. Bull. 50, 913–920. Santos, L.H.M.L.M., Araujo, A.N., Fachini, A., Pena, A., Delerue-Matos, C., Montenegro, M.C.B.S.M., 2010. Ecotoxicological aspects related to the presence of pharmaceuticals in the aquatic environment. J. Hazard. Mater. 175, 45–95. Sarmah, A.K., Meyer, M.T., Boxall, A.B.A., 2006. A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 65, 725–729. Sassman, S.A., Lee, L.S., 2005. Sorption of three tetracyclines by several soils: assessing the role of pH and cation exchange. Environ. Sci. Technol. 39, 7452– 7459. Su, Y.-H., Zhu, Y.-G., 2007. Transport mechanisms for the uptake of organic compounds by rice (Oryza sativa) roots. Environ. Pollut. 148, 94–100. Toutain, P.-L., Ferran, A., Bousquet-Mélou, A., 2010. Species differences in pharmacokinetics and pharmacodynamics. In: Cunningham, F., Elliott, J., Lees, P. (Eds.), Comparative and Veterinary Pharmacology. Springer, Berlin, Heidelberg, pp. 19–48. Uno, T., Ishizuka, M., Itakura, T., 2012. Cytochrome P450 (CYP) in fish. Environ. Toxicol. Pharma. 34 (1), 1–13. WHO, 2012. Oxytetracyline (WHO food additives series 27). (accessed 08.01.12). Williams, E.S., Brooks, B.W., 2012. Health risk assessment of pharmaceuticals in the environment: existing practice, uncertainty and future directions. Human Pharma. Environ. 2, 167–224. Xu, X.-R., Li, X.-Y., 2010. Sorption and desorption of antibiotic tetracycline on marine sediment. Chemosphere 78, 430–436.
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Evaluation of food chain transfer of the antibiotic oxytetracycline and human risk assessment Maliwan Boonsaner a,⇑, Darryl W. Hawker b a b
Department of Environmental Science, Faculty of Science, Silpakorn University, Nakhon Pathom 73000, Thailand School of Environment, Griffith University, Nathan, Qld 4111, Australia
h i g h l i g h t s Shows for the first time that OTC is able to transfer from water to plant to fish. Characterizes bioconcentration of OTC by a floating aquatic plant (Wolffia globosa). Compares accumulation of OTC from water and watermeal by fish (Probarbus jullieni). Quantitative risk assessment for human consumption of contaminated fish undertaken.
a r t i c l e
i n f o
Article history: Received 6 January 2013 Received in revised form 16 May 2013 Accepted 25 May 2013 Available online 20 June 2013 Keywords: Food chain transfer Bioconcentration Biomagnification Oxytetracycline Watermeal (Wolffia globosa Hartog & Plas) Seven-striped carp (Probarbus jullieni)
a b s t r a c t There has been recent concern regarding the possibility of antibiotics entering the aquatic food chain and impacting human consumers. This work reports experimental results of the bioconcentration of the antibiotic oxytetracycline (OTC) by the Asian watermeal plant (Wolffia globosa Hartog & Plas) and bioaccumulation of OTC in watermeal and water by the seven-striped carp (Probarbus jullieni). They show, for the first time, the extent to which OTC is able to transfer from water to plant to fish and enter the food chain. The mean bioconcentration factor (dry weight basis) with watermeal was 1.28 103 L kg1. Separate experiments were undertaken to characterize accumulation of OTC by carp from water and watermeal. These showed the latter pathway to be dominant under the conditions employed. The bioconcentration and biomagnification factors for these processes were 1.75 L kg1 and 2 104 kg g1 respectively. Using an aqueous concentration range of 0.34–3.0 lg L1, hazard quotients for human consumption of contaminated fish of 1.3 102 to 1.15 101 were derived. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Antibiotics are biologically active compounds categorized as emerging environmental contaminants of concern (de la Torre et al., 2012). Widespread use and poor absorption following ingestion has resulted in a ubiquitous environmental presence of antibiotics. Despite this, little is known of their fate and behavior in the aquatic environment (Xu and Li, 2010). Concern has been expressed because of the potential for antibiotics to enter and move through food chains (Boxall and Ericson, 2012). Farré and Barceló (2012) have recently noted that farmed fish and shrimp are often produced in crowded facilities with little regulation of antibiotic use and that the discovery of chloramphenicol residues in shrimp has increased worldwide public awareness. In food chain transfer, a number of processes can occur that require explanation and delineation. In aquatic systems, plants and ⇑ Corresponding author. Tel.: +66 3 4219146; fax: +66 3 4273047. E-mail address: [email protected] (M. Boonsaner). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.05.070
other organisms can bioconcentrate chemicals such as antibiotics from water. Bioconcentration is hence defined as the accumulation of a chemical from the ambient environment of an organism via its respiratory and dermal surfaces. This process is quantified by the bioconcentration factor (BCF), the ratio of chemical concentration in an organism to that in the ambient environment. In contrast, bioaccumulation is accumulation from all sources, both ambient and dietary (Arnot and Gobas, 2006). Biomagnification may be described as the accumulation and transfer of chemicals via the food chain usually resulting in increased concentrations in organisms at higher trophic levels (European Commission, 2003). The biomagnification factor (BMF) is the ratio of a chemical’s concentration in an organism to that in its diet. Oxytetracycline (OTC) is a member of the tetracycline family of antibiotics, widely used for therapeutic purposes in humans as well as an antibiotic and growth promoter in animal farming (Holström et al., 2003). It is amongst the most commonly used antibiotics in fish farming in South East Asia (Holström et al., 2003). Previous studies have also demonstrated OTC bioaccumulation in
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various organisms including invertebrates and fish (Richardson et al., 2005). At present, there are growing concerns regarding the ecological/ environmental risks associated with OTC exposure. Given its antibacterial properties, development of antibiotic-resistant bacteria in humans, and the environment in general, has been cited as an obvious risk with use (Williams and Brooks, 2012). Antibiotics such as OTC have also been found to be very toxic to algae, the basis of aquatic food chains (Santos et al., 2010). Algal population changes could affect the structure of such chains. There is little evidence for acute toxic effects on fish at environmentally realistic concentrations (Crane et al., 2006). Given a likely entry route to aquatic ecosystems is via agricultural runoff at relatively low concentrations from veterinary use, chronic toxic effects are more likely (Williams and Brooks, 2012). The number of studies on human risk is relatively small (Kumar et al., 2010). Chronic exposure may result in gastrointestinal irritation and blood composition changes. Acute toxicity is manifested by hepatoxicity (WHO, 2012). Apart from direct therapeutic use, other routes of human exposure are via potable water and ingestion of contaminated food (e.g. fish and shrimp). Concentrations of antibiotics such as OTC in source waters for potable use are typically such that they are not a concern for human health (Boxall, 2004). However herbivorous animals such as fish can bioaccumulate antibiotics that in turn can be transferred to humans through the food chain (Kong et al., 2007). The extent to which this occurs is largely unknown. It has been speculated that this may produce tolerance to pathogens in animal consumers and humans (Boxall et al., 2006), but the direct risk to human health is uncharacterized. Many factors can affect the bioavailability of OTC in the aquatic food chain. Divalent cations (e.g. Ca2+ and Mg2+) in water can bind to OTC and form relatively stable complexes that reduce gastrointestinal absorption efficiency (Sassman and Lee, 2005). Environmental factors such as temperature, pH and light intensity can affect the degradation of OTC in water and sediment (Burhenne et al., 1997). Doi and Stoskopf (2000) found that the compound was more stable at low temperatures (4 °C) while Pouliquen et al. (2007) reported that first order degradation rates of OTC in water increase at least threefold with light exposure (1400 lux) compared to darkness and moreover were pH dependent. The accumulation of OTC at each trophic level of an aquatic food chain and ultimately the concentration in food for human intake are likely to be affected by these environmental factors, as well as the types of plant and animals involved. In this study, watermeal or swamp algae (Wolffia globosa Hartog & Plas.) was employed as the lowest trophic level, bioconcentrating OTC from water. Seven-striped carp (Probarbus jullieni) represented the second trophic level, bioaccumulating OTC from water itself and through consumption of contaminated watermeal. Watermeal is a free-floating plant without roots with a wide distribution in rivers across several continents including Asia and North America. The sevenstriped carp is a herbivorous fish, found in Thailand, Cambodia, Laos, Malaysia and Vietnam. The objectives of this study were to investigate the accumulation of OTC in watermeal and seven-striped carp as the first and second trophic levels in an aquatic food chain and evaluate
Water Seven-striped carp (Probarbus julleini)
Human Consumer
Watermeal (Wolffia globosa)
Fig. 1. Aquatic food chain for transfer of OTC to humans.
whether OTC can undergo food chain transfer. Finally, derived concentrations of OTC in seven-striped carp were employed to assess the risk involved in human consumption (EPA, 1989). The food chain considered is illustrated in Fig. 1. 2. Materials and methods 2.1. Reagents Oxytetracycline standard (as the hydrochloride) (>98.5% purity) for HPLC analysis was purchased from Dr. Ehrenstorfer GmbH, Augsburg, Germany. Commercial grade oxytetracycline hydrochloride used in experiments was obtained from Nova Medicine, Bangkok, Thailand and checked for impurities prior to use. Acetonitrile (Nanograde, Mallinckrodt, KY, USA), methanol (HPLC grade, Fisher, Pittsburgh, PA, USA), oxalic acid (AR, Ajax, Sydney, Australia), sodium EDTA (AR, Ajax, Sydney, Australia) and glass fiber filter papers (GF/C, Whatman, Maidstone, England) were used for extraction of the antibiotics. Solid phase extraction (SPE) tubes (Strata-X Polymeric Reversed Phase (200 mg/6 mL)) were obtained from Phenomenex (Torrance, CA, USA) and conditioned with 2 mL methanol followed by 2 mL of deionized water. Water used in accumulation experiments with fish was dechlorinated tap water [Ca2+ = 33.8 mg L1; Mg2+ = 8.8 mg L1]. 2.2. Analysis of OTC in water, plant and fish samples The concentrations of OTC in plant and fish samples (approximately 10 g) were determined by adding 25 mL of McIlvaine buffer-EDTA solution and followed the method described in Boonsaner and Hawker (2010). Samples were blended with an Ultra-Turrax homogenizer. Then, both sample types were placed in an ultrasonic bath for 2 min and filtered through a glass fiber filter. The filtrate was passed through a conditioned SPE cartridge and eluted with 4 mL of methanol. Resulting solutions containing OTC were analyzed by HPLC (Waters 600, Milford, MA, USA) with Photodiode Array Detection. The analytical column was a C18 column (HiQ Sil C18HS, 4.6 mm id 150 mm and 0.5 lm particle size) operated at room temperature and the chromatographic conditions were as follows: flow rate 1 mL min1; mobile phase 95% acetonitrile (v/v), 5% 0.01 M oxalic acid (v/v); injection volume 20 lL with detection at 333 nm. Water samples (100 mL) were passed through a conditioned SPE cartridge, eluted with 2 mL of methanol and analyzed by HPLC using the conditions described above. Method detection limits (MDL) for the analysis of OTC in plants and fish were 0.45 and 0.7 mg kg1 dry weight (dw) and 0.5 mg L1 for water. The mean recoveries from water, plant and fish samples were 97%, 70% and 66%, respectively. All concentrations reported herein account for these recoveries. 2.3. Bioconcentration of OTC by watermeal Watermeal was purchased from a local market in Nakhon Pathom province, central Thailand. Before commencing the experiments, it was determined to be OTC free. A preliminary investigation of OTC toxicity was undertaken by placing watermeal in 50, 80 and 100 mg L1 solutions. It was found that at concentrations of more than 50 mg L1 toxic effects occurred, evidenced by the watermeal turning brown, gelatinous and malodorous. Therefore, concentrations of 50 mg L1 and less were chosen for bioconcentration experiments. Experiments were conducted with three initial test OTC concentrations, nominally 10, 30 and 50 mg L1. For each concentration, 500 mL of test solution (pH 4.4–4.6) was added to each of 8 clear glass jars (800 mL capacity) followed by 20 g of watermeal. This
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amount of watermeal covered the surface of the water in a jar. The daily illumination regime was 10 h light: 14 h darkness. The external sidewall of the jar was covered by aluminum foil to reduce degradation from illumination. All jars were placed in a room where temperature was 28 ± 2 °C. At days 0, 1, 3, 5, 9, 11, 13 and 15 of exposure, one jar of each OTC concentration was taken for analysis. Watermeal samples were filtered from water and separated for analysis. The filtered water was thoroughly stirred before collecting 100 mL samples for analysis. Duplicate samples were analyzed for plant tissue and water concentrations at all time periods. The bioconcentration factor (BCF) was obtained from the ratio of the test compound concentration in watermeal (CWATERMEAL) to the concentration found in the water (CW). A control experiment was performed by placing watermeal in clean sterilized deionized water and locating a jar in the same room. Following the 15-d experimental period, watermeal and water samples from the control jar were taken for OTC analysis. These showed no contamination and watermeal appeared normal. In order to assess the influence of environmental factors on aqueous OTC levels in bioconcentration experiments, a parallel investigation to determine the loss of OTC in water under the same conditions of the bioconcentration experiments was carried out, but without the addition of watermeal. OTC solutions (50 mg L1) in 250 mL bottles were prepared and covered with aluminum foil. Bottles were placed in the room together with jars of the bioconcentration experiment. Duplicate 100 mL samples of test solution were taken from each bottle at the same time that the samples were collected from the bioconcentration experiments. Numerical results were analyzed according to first order loss kinetics.
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daily (28 ± 2 °C), while fish samples were taken for OTC analysis every 2 weeks. For accumulation of OTC from food, twelve glass jars were each filled with 2 L of clean water and 7–8 fish (total mass approximately 20 g) placed in each jar. The fish were fed twice per day with the food described above, but contaminated with OTC (35 mg kg1 (dw)). Again, 2–3 g of contaminated food was added to each jar and 30 min allowed for eating. The leftover food was taken out, dried and weighed to determine the amount consumed. Fish were carefully transferred daily to OTC-free water. The jars were again placed in dim light and a temperature of 28 ± 2 °C. The experiment was conducted over a period of 15 d. Whole fish were sacrificed for OTC analysis on days 0, 1, 3, 5, 9, 11, 13 and 15 of exposure. Control experiments for accumulation from food were also conducted. The first involved one jar with 7–8 fish (total mass approximately 20 g) with clean water and uncontaminated food. Fish were transferred daily to new jars as for exposure experiments. The other comprised a jar containing water but no fish where contaminated food was added twice daily for 30 min. The results showed that after 15 d, no OTC was detected in control fish and there was none in water arising from desorption from food. Because fish were regularly transferred to clean water, there was likely to be little influence from desorption of OTC in faeces arising from incomplete dietary absorption. In the absence of regular water changes and in field situations however, this process may affect aqueous OTC concentrations and should not be neglected.
3. Results and discussion 2.4. Bioaccumulation of OTC by seven-striped carp
3.1. Bioconcentration of OTC by watermeal
Seven-striped carp (approximately 3–5 cm long and weighing 2–3 g each) were purchased from a local market in Nakhon Pathom province, Thailand. Before commencing the experiments, tissue OTC levels were determined to be below detection limits. Tap water used in the experiment was dechlorinated by aeration for 2 d prior to use. In this study, separate experiments were conducted viz. accumulation of OTC from water and accumulation of OTC from food (watermeal). During these experiments, fish were fed with food prepared from 300 g watermeal, 300 g dried ground fish, 100 g rice, 200 g rice bran, 50 g vegetable oil, 50 g starch and a small amount of water to assist mixing. This mixture was subsequently air dried and sieved to 2 mm size. A preliminary investigation of OTC toxicity found that there were no observable toxic effects on fish over the time frame of the current study at an aqueous concentration of 10 mg L1. As a result, a 10 mg L1 OTC solution was to investigate accumulation from water. For this, twelve glass jars were filled with 2 L of 10 mg L1 OTC solution and 7–8 fish (total mass approximately 20 g) placed in each jar. Due to relatively slow bioconcentration, the experiment was conducted over a period of 12 weeks. Fish were fed twice per day with the food described above. At each feeding period, 2–3 g of food was added and 30 min allowed for consumption. After this time period, the leftover food was taken out, dried and weighed to determine the mass of consumed food. Fish were carefully transferred daily to new jars containing 10 mg L1 OTC solution to ensure, as far as practicable, a constant exposure concentration. In addition, control experiments were conducted by using one jar with 15 fish in clean water and another jar with 10 mg L1 OTC solution without fish. All jars were placed in a room with dim light (8–10 lux) to imitate the natural conditions experienced by fish and to reduce the photodegradation of OTC. The room temperature was recorded
Regardless of initial ambient OTC concentrations, levels in watermeal increased rapidly within the first day of the experiments and then remained relatively stable until the end of the 15-d exposure (Fig. 2). Data on which this figure is based is found in Table S1 (Supplementary material). Similarly rapid uptake of OTC from aqueous solution has been observed with alfalfa (Medicago sativa L.) where equilibrium was attained after 4 h (Kong et al., 2007). OTC is susceptible to microbial transformation as well as abiotic processes such as hydrolysis and photolysis (Pouliquen et al., 2007; Ratasuk et al., 2012). The first order loss rate constant from the control experiment was 0.07 d1. In comparison, loss rate constants in water from bioconcentration experiments (0.12–0.15 d1) were larger. Although prima facie the difference is due to plant uptake it should be remembered that the watermeal covers the surface in bioconcentration experiments and phototransformation may be reduced here compared to the control. Aqueous OTC loss rate constants obtained in this study were comparable to those from other studies, e.g. 0.01–0.18 d1 (Doi and Stoskopf), and 0.01 d1 (Boonsaner and Hawker, 2012), bearing in mind the effect of variation in factors such as pH, temperature and light intensity. For compounds that are labile in aqueous systems such as OTC, Li et al. (2002) distinguish between a thermodynamic bioconcentration factor (the ratio of uptake and loss rate constants) and an actual bioconcentration factor (BCF) (the ratio of plant and water concentrations at each observation time). For this work, the relevant parameter is the actual BCF. The accumulated OTC concentrations in watermeal vary with the ambient levels of this antibiotic. As a result, BCF values are not a function of water concentrations. The mean of the coefficients of variation for BCFs with external aqueous concentrations for individual exposure times is 18.8%. Aqueous concentrations do however decrease with time, but watermeal concentrations are relatively constant, implying rela-
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Aqueous Concentration (mg L -1)
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3.2. Bioconcentration of OTC by seven-striped carp Concentrations in seven-striped carp tissue plateaued within the first week and were quite low (Table 1). As a result, BCF data expressed as mean (±standard error of the mean) over 12 weeks was 1.75 103 ± 2 104 L g1 (dw) or 1.75 ± 2 101 L kg1 (dw). The OTC concentration employed (10 mg L1) was selected to have no toxic effect on seven-striped carp based on results of preliminary bioassays. There is little other relevant toxicology data for this species. Ji et al. (2012) have reported that 50 mg L1 OTC did not affect the growth of juvenile and adult Japanese killifish (Oryzias latipes). OTC degradation measured in a control experiment without fish (k = 0.003 d1) was relatively low compared to that from the watermeal bioconcentration described above. This is likely the result of the dim illumination conditions employed, reducing photodegradation. Solutions were also renewed daily so that effectively, fish were exposed to a constant ambient OTC concentration. 3.3. Biomagnification of OTC by seven-striped carp
(c)
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The magnitude of the BCFs for OTC here are similar to those observed for a plant possessing roots such as rice where BCF rose to >2 L g1 (dw) after a similar 15 d exposure (Boonsaner and Hawker, 2012). With rice, unionized organics are uptaken by roots largely by passive processes but active mechanisms are proposed for ionized species (Su and Zhu, 2007). Under the prevailing pH conditions (pH = 4.4–4.6) in the watermeal bioconcentation experiments, OTC exists largely in zwitterionic form. Previous research has shown OTC uptake into rice roots under these conditions is consistent with passive diffusion, controlled by diffusive transport through lipophilic membranes (Boonsaner and Hawker, 2012).
5
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Time (d) Fig. 2. (a) Aqueous OTC concentrations ( 50 mg L1 nominal initial; j 30 mg L1 nominal initial; w 10 mg L1 nominal initial) with time in watermeal bioconcentration experiments. Shown in (b and c) are associated watermeal concentrations (mg g1) and BCF values (L g1) respectively.
tively slow or negligible loss from the plant in response to decreasing external concentrations. BCFs calculated from the ratio of CWATERMEAL/CW accordingly increase with time (Fig. 2). For the purposes of this work, the BCF value used in calculation of human health risk is the overall mean value from Fig. 2, i.e. 1.28 L g1 (dw) or 1.28 103 L kg1 (dw). Watermeal does not possess roots but similar plants have been shown to be useful for the removal of a range of aqueous organic contaminants by processes including surface sorption (Reinhold et al., 2010). In that work, it was concluded that equilibrium with the pharmaceutical fluoxetine was attained within 12–24 h after initial exposure. Close inspection of the results presented by Reinhold et al. (2010) show a relatively slow decrease in aqueous concentration after 24 h that was attributed to loss of antibiotic in the aqueous phase and uptake into the plant. Similar trends were observed in this current work with OTC as described above.
To characterize biomagnification of OTC from watermeal by seven-striped carp, 2–3 g of contaminated watermeal (35 mg kg1 (dw) OTC) was fed to the carp for two 30-min periods daily over 15 d. The level in contaminated food was considerably lower than the therapeutic dose for fish (50 mg kg1 for 2 weeks) (Choo, 1995) and thus no toxic effects were anticipated or observed. The cumulative amount of food consumption is shown in Table 2. Mean daily consumption was 2.3 g d1 from which can be calculated a mean daily OTC consumption of 0.080 mg d1. However, not all OTC consumed in food is actually absorbed by the fish. Measured OTC tissue concentrations are also shown in Table 2and increase relatively slowly with time. The mean of these values over the 15 d exposure period (0.0076 mg g1 (dw)) is used in conjunction with concentration in contaminated food to derive a biomagnification factor (BMF) of 2 104 kg g1. An unidentified peak was observed in chromatograms of fish tissue. Not present in controls, the peak increases in size with time and is attributed to a metabolite of OTC. Assuming the same response factor with the photodiode array detector as OTC itself, concentrations of OTC and metabolite are presented in Table 2. It is of interest that evidence of the OTC metabolite was not found in the bioconcentration of OTC directly from water by seven-striped carp, even when tissue concentrations from this process were greater. Previous work with common carp (Cyprinus carpio) has shown different pharmacokinetic behavior depending on route of administration however accumulation from ambient water and contaminated food were not compared (Grondel et al., 1987). A large fraction of compounds such as OTC absorbed from water by the gills is initially transported to the kidneys. In contrast, OTC from dietary exposure is first transported to the liver (Toutain et al., 2010). Hepatic cytochrome-P450 isozymes catalyze the oxidation of endogenous substrates such as OTC. They are known to exist in members of the carp family (Marionnet et al., 1998; Uno et al.,
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M. Boonsaner, D.W. Hawker / Chemosphere 93 (2013) 1009–1014 Table 1 Duplicate OTC concentrations and mean BCF values in seven-striped carp from bioconcentration over a 12-week exposure period to a 10 mg L1 aqueous concentration. Week
OTC concentration in seven-striped carp (mg g1 (dw))
Mean BCF (L g1 (dw))
0 1
0 0.013 0.007 0.009 0.005 0.021 0.017 0.012 0.018 0.010 0.016 0.012 0.005 0.016 0.008 0.014 0.011 0.031 0.019 0.023 0.017 0.023 0.020 0.012 0.020
0 0.001
2 3 4 5 6 7 8 9 10 11 12
C fish
1
¼ BMFðkg g1 Þ BCFðL kg Þ C w ðmg L1 Þ 4
¼ 2 10
kg g1 1:28 103 L kg
1
3:4 104 mg L1
¼ 8:70 105 mg g1
0.001
1
¼ 8:70 102 mg kg
0.002
ð2Þ
0.002
2
0.001 0.001 0.002 0.002 0.003 0.002 0.002 0.002
2012), but no details of metabolite identity for OTC currently exist. A first pass induction of hepatic enzymes could result in selective metabolism of OTC by this route of administration. As metabolite identity and properties are unknown however, it is not considered further in food chain transfer and risk calculations. 3.4. Assessment of human risk from consumption of fish containing OTC Comparison of the two pathways for bioaccumulation of OTC by seven-striped carp viz. from direct bioconcentration from water and from biomagnification of watermeal whose OTC burden is acquired by bioconcentration shows the latter to be dominant. For bioconcentration:
C fish
Concentrations of OTC in seven-striped carp from biomagnification are derived according to the methods described in the European Commission’s Technical Guidance Document on Risk Assessment (European Commission, 2003). Values of BMF and BCF employed are as derived above.
1
¼ BCFðL kg Þ C w ðmg L1 Þ ¼ 1:75 L kg
1 4
¼ 5:95 10
3:4 104 mg L1 mg kg
ð1Þ
1
Table 2 Cumulative amounts of food and OTC consumed together with the measured concentrations of OTC and metabolite in seven-striped carp from biomagnification. Day
Cumulative amount of food consumed (g)
Cumulative amount of OTC consumed (mg)a
OTC concentration in seven-striped carp (mg g1 (dw))
OTC + metabolite concentration in seven-striped carp (mg g1 (dw))
1 3 5 7 9 11 13 15
5.28 8.61 11.94 15.00 19.17 23.61 28.06 31.39
0.19 0.31 0.43 0.54 0.69 0.85 1.01 1.13
0.0053 0.0044 0.0096 0.0070 0.0072 0.0084 0.0090 0.0096
0.0058 0.0057 0.0092 0.0080 0.0112 0.0126 0.0140 0.0154
a Calculated from rate of food consumption using OTC concentration of 35 mg kg1.
1
The total body burden of OTC for fish (8.76 10 mg kg ) is taken as the sum of these concentrations. Kolpin et al. (2002) undertook a comprehensive reconnaissance of levels of pharmaceuticals in US streams and found a median level for OTC of 0.34 lg L1. Sampling sites were biased towards those susceptible to contamination such as sites downstream of urbanization or intensive livestock facilities. We employed the datum of Kolpin et al. in our calculations above, considering it to represent a conservative level of contamination that might be encountered in fish farms or surface water impacted by fish farms using antibiotics. However, there are many localized sites worldwide with higher concentrations. For example Liu et al. (2009) found levels of 3.0 lg L1 in the Nanming River, southwestern China. Using this concentration, the total body burden for OTC in fish calculated as above is 7.73 101 mg kg1. Using these values to represent a range for OTC levels in fish, the intake rate of OTC from fish by human consumers (mg kg1 d1) is determined using the following expression (EPA, 1989; Dunnivant and Anders, 2006): 1
Intake ðmg kg
1
d Þ¼
C fish a IR EF ED BW AT
ð3Þ
where the concentration in fish is expressed in mg kg1, a is the proportion of OTC absorbed by human from oral dosing (0.60) (WHO, 2012), IR the fish ingestion rate (0.054 kg d1) (Dunnivant and Anders, 2006), EF the exposure frequency (350 d y1) (Dunnivant and Anders, 2006), ED the duration of exposure (70 y) (Dunnivant and Anders, 2006), BW adult body weight (70 kg) and AT the time period over which exposure is averaged (70 y 365 d y1). This equation is modified from that usually employed by additionally including the absorption efficiency of OTC. On this basis, the intake rate of OTC ranges from 3.89 105 to 3.46 104 mg kg1 d1. Changes to parameters such as fish ingestion rate would modify this range. For example, if fish ingestion rate doubled, then OTC intake rate would also double. The hazard quotient is a metric describing risk posed by chemicals. It is used by many regulatory authorities, including the USEPA (Langdon et al., 2010). Here, it is derived from the ratio of the intake rate calculated above and an acceptable daily intake of OTC. The latter is 3 103 mg kg1 d1, although there is some uncertainty in this figure, and it may be somewhat higher (WHO, 2012). A ratio of 1.0 or above for the hazard quotient indicates a toxic effect is likely while conversely a ratio of <0.5 indicates a low risk. The resultant hazard quotient range derived here (1.3 102 to 1.15 101) suggests no direct detrimental effects from ingestion of contaminated fish where OTC has entered the aquatic food chain. Even with elevated concentrations in water, and increased ingestion rates, the hazard quotient is below the threshold value of 0.5. 4. Conclusion There is abundant evidence that antibiotics find their way into the environment from a variety of sources (Sarmah et al., 2006).
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However effects on aquatic ecosystems and consequent impacts on humans have not been well understood. Exploration of the mechanisms of uptake by plants and animals as well as the potential movement through food webs has been advocated (Boxall and Ericson, 2012). Behavior of OTC, a widely used antibiotic, in an aquatic food web has now been investigated and characterized. The compound is rapidly bioconcentrated from water by watermeal (W. globosa) (BCF = 1.28 103 L kg1). Fish (seven-striped carp (P. jullieni)) can accumulate OTC from contaminated watermeal (BMF = 2 104 kg g1). Fish OTC burdens from this pathway were over 100 times greater than those from direct bioconcentration by fish from water. A hazard quotient approach to risk assessment of human consumption of this fish suggested little likelihood of direct detrimental impact, even from sites with relatively high aqueous OTC levels of up to 3.0 lg L1. However, additional intake of OTC for therapeutic purposes can alter this scenario. Subtle effects such as increased antibiotic resistance of resident bacteria are also possible and should not be neglected. Acknowledgements This study was supported by the Thailand National Research Fund and the Silpakorn University Research and Development Institute. The authors would like to thank C. Teparak and N. Sanchan for their laboratory assistance and S. Jaipan for his plant preparations. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2013.05.070. References Arnot, J.A., Gobas, F.A.P.C., 2006. A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms. Environ. Rev. 14, 257–297. Boonsaner, M., Hawker, D.W., 2010. Accumulation of oxytetracycline and norfloxacin from saline soil by soybeans. Sci. Total Environ. 408, 1731–1737. Boonsaner, M., Hawker, D.W., 2012. Investigation of the mechanism of uptake and accumulation of zwitterionic tetracyclines by rice (Oryza sativa L.) Ecotoxicol. Environ. Saf. 78, 142–147. Boxall, A.B.A., 2004. The environmental side effects of medication. Embo. Rep. 5, 1110–1116. Boxall, A.B.A., Ericson, J.F., 2012. Environmental fate of human pharmaceuticals. Human Pharma. Environ., 63–83. Boxall, A.B., Johnson, P., Smith, E.J., Sinclair, C.J., Stutt, E., Levy, L.S., 2006. Uptake of veterinary medicines from soils into plants. J. Agric. Food Chem. 54, 2288–2297. Burhenne, J., Ludwig, M., Nikoloudis, P., Spiteller, M., 1997. Primary photoproducts and half-lives. Environ. Sci. Pollut. Res. Int. 4 (1), 10–15. Choo, P.S., 1995. Withdrawal time for oxytetracycline in Red Tilapia cultured in freshwater. Asian Fish. Sci. 8 (2), 169–176. Crane, M., Watts, C., Boucard, T., 2006. Chronic aquatic environmental risks from exposure to human pharmaceuticals. Sci. Total Environ. 367, 23–41. de la Torre, A., Iglesias, I., Carballo, M., Ramírez, P., Muñoz, M.J., 2012. An approach for mapping the vulnerability of European Union soils to antibiotic contamination. Sci. Total Environ. 414, 672–679. Doi, A.M., Stoskopf, M.K., 2000. The kinetics of oxytetracycline degradation in deionised water under varying temperature, pH, light, substrate, and organic matter. J. Aquat. Anim. Health 12, 246–253. Dunnivant, F.M., Anders, E., 2006. A Basic Introduction to Pollutant Fate and Transport. John Wiley and Sons, Inc., Hoboken, NJ, USA. EPA, 1989. Risk assessment, guidance for superfund volume 1 (Part A): human health evaluation manual. Office of Emergency and Remedial Response, USEPA Washington, DC 20450.
European Commission, 2003. Technical guidance document on risk assessment part 2. European Commission and European Chemicals Bureau, EUR 20418/EN/2, Luxembourg, Luxembourg. Farré, M., Barceló, D., 2012. Emerging organic contaminants and nanomaterials in food. In: Barceló, D. (Ed.), Emerging Organic Contaminants and Human Health, Handbook of Environmental Chemistry, vol. 20, pp. 1–46. Grondel, J.L., Nouws, J.F.M., deJong, M., Schutte, A.R., Driessens, F., 1987. Pharmocokinetics and tissue distribution of oxytetracycline in carp, Cyprinus carpio L., following different routes of administration. J. Fish Dis. 10, 153– 163. Holström, K., Gräslund, S., Wahlström, A., Poungshompoo, S., Bengtsson, B.-E., Kautsky, N., 2003. Antibiotic use in shrimp farming and implications for environmental impacts and human health. Int. J. Food Sci. Technol. 38, 255–266. Ji, K., Kim, S., Han, S., Seo, J., Lee, S., Park, Y., Choi, K., Kho, Y.-L., Kim, P.-G., Park, J., Choi, K., 2012. Risk assessment of chlortetracycline, oxytetracycline, sulfamethazole, sulfathiazole, and erythromycin in aquatic environment: are the current environmental concentrations safe? Ecotoxicology 21, 2031–2050. Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber, L.B., Buxton, H.T., 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999–2000: a national reconnaissance. Environ. Sci. Technol. 36 (6), 1202–1211. Kong, W.D., Zhu, Y.G., Liang, Y.C., Zhang, J., Smith, F.A., Yang, M., 2007. Uptake of oxytetracycline and its phytotoxicity to alfalfa (Medicago sativa L.). Environ. Pollut. 147, 187–193. Kumar, A., Chang, B., Xagoraraki, I., 2010. Human health risk assessment of pharmaceuticals in water: issues and challenges ahead. Int. J. Environ. Res. Publ. Health 7, 3929–3953. Langdon, K.A., Warne, M.St.J., Kookana, R.S., 2010. Aquatic hazard assessment for pharmaceuticals, personal care products and endocrine disrupting compounds from biosolids-amended land. Integr. Environ. Assess. Manage. 6 (4), 663– 676. Li, H., Sheng, G., Sheng, W., Xu, O., 2002. Uptake of trifluralin and lindane from water by ryegrass. Chemosphere 48, 335–341. Liu, H., Zhang, G., Liu, C.-Q., Li, L., Xiang, M., 2009. The occurrence of chloramphenicol and tetracyclines in municipal sewage and the Nanming River, Guiyang City, China. J. Environ. Monitor. 11, 1199–1205. Marionnet, D., Chambras, C., Taysse, T., Bosgireaud, C., Deschaux, P., 1998. Modulation of drug-metabolizing systems by bacterial endotoxin in carp liver and immune organs. Ecotoxicol. Environ. Saf. 41, 189–194. Pouliquen, H., Delépée, R., Larhantec-Verdier, M., Morvan, Le Bris, H., 2007. Comparative hydrolysis and photolysis of four antibacterial agents (oxytetracycline, oxolinic acid, flumequine and florfenicol) in deionised water, freshwater and seawater under abiotic conditions. Aquaculture 262 (1), 23– 28. Ratasuk, N., Boonsaner, M., Hawker, D.W., 2012. Effect of temperature, pH and illumination on abiotic degradation of oxytetracycline in sterilized swine manure. J. Environ. Sci. Health A 47, 1687–1694. Reinhold, D., Vishwanathan, S., Park, J.J., Oh, M., Saunders, F.M., 2010. Assessment of plant-driven removal of emerging organic pollutants by duckweed. Chemosphere 80, 687–692. Richardson, B.R., Lam, P.K.S., Martin, M., 2005. Emerging chemicals of concern: pharmaceuticals and personal care products (PPCPs) in Asia, with particular reference to Southern China. Mar. Pollut. Bull. 50, 913–920. Santos, L.H.M.L.M., Araujo, A.N., Fachini, A., Pena, A., Delerue-Matos, C., Montenegro, M.C.B.S.M., 2010. Ecotoxicological aspects related to the presence of pharmaceuticals in the aquatic environment. J. Hazard. Mater. 175, 45–95. Sarmah, A.K., Meyer, M.T., Boxall, A.B.A., 2006. A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 65, 725–729. Sassman, S.A., Lee, L.S., 2005. Sorption of three tetracyclines by several soils: assessing the role of pH and cation exchange. Environ. Sci. Technol. 39, 7452– 7459. Su, Y.-H., Zhu, Y.-G., 2007. Transport mechanisms for the uptake of organic compounds by rice (Oryza sativa) roots. Environ. Pollut. 148, 94–100. Toutain, P.-L., Ferran, A., Bousquet-Mélou, A., 2010. Species differences in pharmacokinetics and pharmacodynamics. In: Cunningham, F., Elliott, J., Lees, P. (Eds.), Comparative and Veterinary Pharmacology. Springer, Berlin, Heidelberg, pp. 19–48. Uno, T., Ishizuka, M., Itakura, T., 2012. Cytochrome P450 (CYP) in fish. Environ. Toxicol. Pharma. 34 (1), 1–13. WHO, 2012. Oxytetracyline (WHO food additives series 27).