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Acute effects of triclosan, diclofenac and carbamazepine on feeding performance of Japanese medaka fish (Oryzias latipes)
Chemosphere 80 (2010) 1095–1100
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Acute effects of triclosan, diclofenac and carbamazepine on feeding performance of Japanese medaka fish (Oryzias latipes) Mohamed Nassef a, Shuhei Matsumoto a, Masanori Seki b, Fatma Khalil a, Ik Joon Kang c, Yohei Shimasaki a, Yuji Oshima a,*, Tsuneo Honjo a a b c
Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan Chemicals Evaluation and Research Institute (CERI), Hita Laboratory, 3-822 Ishiimachi, Hita-shi, Oita 877-0061, Japan Aquatic Biomonitoring and Environmental Laboratory, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan
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Article history: Received 17 February 2010 Received in revised form 24 April 2010 Accepted 27 April 2010 Available online 26 May 2010 Keywords: Triclosan Diclofenac Carbamazepine Fish behavior
a b s t r a c t The toxicity of three pharmaceutical and personal care products (PPCPs) – carbamazepine (CBMZ), diclofenac (DCF), and triclosan (TCS) – was examined by measuring their effects on feeding behavior and swimming speed of adult Japanese medaka fish (Oryzias latipes). Medaka were exposed to 6.15 mg L 1 CBMZ, 1.0 mg L 1 DCF, 0.17 mg L 1 TCS, or no PPCP (control) for 9 d. Fish behaviors were monitored during days 5–9 of the exposure period. Feeding behavior (time to eat midge larvae, TE) and swimming speed (SS) of individual exposed and control fish were tracked in two dimensions, using an automated system with a digital charge-coupled device camera. As a result, feeding behavior was affected by exposure to CBMZ and DCF, while SS was altered by exposure to CBMZ and TCS. Thus, TCS, DCF and CBMZ appear to affect fish behaviors through different mechanisms. Overall, the results suggest that behavioral changes may provide a sensitive indicator for assessing the toxicity of PPCPs to aquatic organisms. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Pharmaceuticals and personal care products (PPCPs) have recently been identified as potentially toxic chemicals in aquatic environments (Laura Martín-Díaz et al., 2009). Triclosan (TCS), diclofenac (DCF), and carbamazepine (CBMZ) are among the most frequently detected PPCPs worldwide (Kolpin et al., 2002; Ferrari et al., 2003). TCS is widely used as an antibacterial agent in many personal care products such as soaps, detergents, toothpastes, disinfectants, cosmetics and pharmaceuticals (Ying and Kookana, 2007), and its widespread use has been shown to pose a potential risk to aquatic organisms (Dussault et al., 2008). DCF is an antiinflammatory drug with large production volumes. CBMZ is an antiepileptic drug, and its environmental persistence raises concerns about potential effects on non-target organisms (Zaremba et al., 2006). The maximum environmentally detected concentrations of these PPCPs in waste water treatment plant (WWTP) effluents were 0.269 mg L 1 TCS in Spain (Mezcua et al., 2004), 0.006 mg L 1 CBMZ and 0.002 mg L 1 DCF in Germany (Ternes, 1998). Assessing the human risk posed by PPCPs in water is a high priority; however, the impacts of these chemicals on aquatic
* Corresponding author. Tel.: +81 92 642 2905; fax: +81 92 642 2908. E-mail address: [email protected] (Y. Oshima). 0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2010.04.073
organisms and communities are also important (Bendz et al., 2005; Dove, 2006). Our previous studies evaluated the acute toxicity of TCS, DCF and CBMZ to adult and embryos of medaka fish, and concluded that TCS and DCF may pose ecological risks to aquatic organisms (Nassef et al., 2009, 2010). Although acute lethality tests are useful for generating guidelines to protect against physiological death (i.e., mortality) of aquatic animals, these tests ignore ‘‘ecological death,” i.e., the inability to function in an ecological context when normal behaviors are altered. Such effects may occur at much lower toxicant exposures, even if animals are not overtly harmed by a contaminant. Indeed, environmental impacts in natural ecosystems often occur at concentrations well below those causing significant mortality (Jensen and Bro-Rasmussen, 1992; Cabrera et al., 1998; Norris et al., 1999; Gaworecki and Klaine, 2008). A better understanding of the toxicological effects of contaminants can be achieved by examining behavioral changes and determining how they relate to effects at other levels (Scott and Sloman, 2004). Behavioral alterations can provide estimates of endpoints for sublethal toxicity, and serve as a tool for environmental risk assessment and analysis of toxicological impact (Andrew et al., 2004). Appropriate feeding is essential for growth and reproduction of most aquatic animals, including fish (Volkoff and Wyatt, 2009). The ability to capture prey could be impacted by prolonged exposure to low concentrations of contaminants, including PPCPs (De Lange
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et al., 2006; Kristen and Stephen, 2008). Swimming performance is closely related to food capture (Zeng et al., 2009), and is considered to be a primary determinant of survival in many species of fish and other aquatic animals (Jones and Hill, 1974; Taylor and McPhail, 1986). For example, De Lange et al. (2006) observed reduced locomotion in Gammarus pulex exposed to CBMZ (1–1000 ng L–1 for 1.5 h) and speculated that the reduction may interfere with feeding behavior. Japanese medaka have been used in previous studies of basic fish biology and behavior, as well as toxicology, and the species has been proposed by the Organization for Economic Cooperation and Development for use as the standard fish for toxicology tests (OECD, 1999). Our previous research showed that exposure of medaka to sublethal concentrations of 17b-estradiol impaired sexual behavior and decreased reproductive success (Oshima et al., 2003). Significant changes in swimming speed were observed in medaka exposed to cyanide or aldicarb (Kang et al., 2009). Although previous studies used medaka to examine the accumulation and toxicity of anthropogenic chemicals, there is limited information on behavioral effects. Even though the neurotoxicity of PPCPs has been characterized in non-target species (Gao and Chuang, 1992; Gokcimen et al., 2007), the effects on behavior of aquatic organisms have not been well studied. The goal of the present study was to investigate the effects of sublethal concentrations of two pharmaceuticals (CBMZ and DCF) and one personal care product (TCS) on the behavior of Japanese medaka (Oryzias latipes). Based on ecological relevance, feeding behavior and swimming performance of medaka were chosen as indicators of TCS, DCF and CBMZ toxicity to aquatic animals. 2. Materials and methods 2.1. Test chemicals TCS (>98.0% purity), DCF (>98.0% purity), CBMZ (>97.0% purity) and dimethyl sulfoxide (DMSO, >99.0% purity) were obtained from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). Stock solutions (1.7 mg mL–1 TCS, 1.0 mg mL–1 DCF, and 6.15 mg mL–1 CBMZ) were prepared by dissolving pure chemicals in DMSO, and stored prior to use. Treatment solutions were prepared by mixing appropriate amounts of stock solutions with 1 L of artificial seawater (0.01% salinity). PPCP concentrations in treatment solutions (0.17 mg L–1 TCS, 1.0 mg L–1 DCF, and 6.15 mg L–1 CBMZ) were determined as 10% of the calculated 96 h LC50 for adult medaka, based on our previous results (Nassef et al., 2009). Final DMSO concentrations were <0.1% (v/v) in the treatment solutions. 2.2. Test organisms Fifty-four medaka (O. latipes, orange-red strain), 3 months old, 0.3 ± 0.03 g average weight, and 3.0 ± 0.34 cm average length, were collected from brood stock maintained in the Laboratory of Marine and Environmental Science at Kyushu University, Fukuoka, Japan. The test fish were acclimated to laboratory conditions for 1 week prior to testing. The selected fish were held in a 56 L glass aquarium (60 cm long 30 cm wide 36 cm high) equipped with an aerating filter system, in artificial seawater at 0.01% salinity and 25 ± 1 °C. The exposure media were prepared from dechlorinated tap water as artificial seawater (salinity was adjusted at 0.01%) to retain constant water quality. The fish were kept under a 16:8 h light: dark regime and fed with Artemia nauplii (<24 h after hatching) twice a day, in the morning and evening. Half the water was replaced daily. Frozen midge larvae (Chironomus sp., ML), obtained from a pet shop, were used as fish bait during feeding experiments.
2.3. Experimental design On the first day of exposure, nine fish were randomly assigned to each of three treatment groups: 0.17 mg L–1 TCS, 1.0 mg L–1 DCF, or 6.15 mg L–1 CBMZ. Each treatment group was paired with a control group of nine fish. Each treatment and control group was held in a 2.5 L, clear glass test chamber (18 cm long 12 cm wide 12 cm high). Fish in treatment groups were exposed to TCS, DCF or CBMZ for 9 d under semi-static conditions and the fish behavior was monitored for 5 d started on day 5 from exposure until the day 9, the end of exposure time. The fish were fed 9 ML per tank daily during the first 2 d to condition them to that food. Feeding and swimming behaviors were monitored on days 5–9 of exposure. The fish were not fed for 2 d before and during the behavior monitoring, to ensure that they were hungry. Fish mortality was checked throughout the experimental period. On day 5 of exposure, fish from each treatment and control group were transferred individually to a 2 L test box (18 cm long, 12 cm wide, and 12 cm high), containing the respective concentration of the same PPCP for treated fish, or no PPCP for control fish. Black acrylic boards were attached to the walls of the test box to prevent reflections of fish and lamps. Black screens were arranged around the test boxes, so that the presence of an experimenter would not disturb the behavior of the fish. Fluorescent lamps were used as the sole light source. A flat LED panel was fixed below the bottom of the test box, and a white acrylic board, 2 mm thick, was placed on the bottom to provide uniform illumination. An incomplete vertical glass partition was placed in the test box, leaving a narrow space for introducing the fish (Fig. 1). Prior to placing the fish in the test box, 1 ML was placed in the bottom left corner. All experiments were run at 25 ± 1 °C. 2.4. Behavioral measurements The behaviors of individual exposed and control fish in the test boxes were videotaped using a computerized tracking method. A digital video camera (CCD, model GE60, Library, Japan) was installed in the center of the test box, 30 cm above the water surface. The fish was placed in the test box, and the recording process started when the fish passed the vertical partition and moved toward the ML. Behavioral data were collected for 10 min in each trial. If the fish did not move toward the larva within 5 min of starting the recording, the glass partition was removed. The behavior of each fish was recorded at the same time daily on days 5–9 of the exposure period. Time to eat the midge larva (TE) and swimming speed (SS) were calculated for each trial by analyzing the instantaneous video images recorded at 0.033 s intervals (= dte). The two-dimensional (2D) coordinates in the horizontal plane were determined using motion analyzer software (Move-Tr 32/2D; Library, Tokyo, Japan). When the fish did not successfully eat the ML, the TE value was set at 600 s. 2.5. Statistical analysis Statistical significance of differences in the means of SS and medians of TE between treatment and control groups were analyzed using Mann–Whitney U-tests (Stat View J 5.0, Abacus Concepts, Berkeley, CA, USA). Significant difference was determined at p 6 0.05. The box-and-whisker plot displays a statistical summary of a variable: median, quartiles, range and possibly extreme values (outliers). An outlier value is defined as a value that is smaller than the lower quartile (25 percentile) minus 1.5 times the interquartile range, or larger than the upper quartile (75 percentile) plus 1.5 times the interquartile range.
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Entrance Start point for recording behavior
Swimming pathway
Incomplete vertical glass partition Midge larva Medaka fish Test box filled with PPCP in artificial seawater Fig. 1. Schematic diagram of experimental setup for behavior monitoring.
swimming speed (SS) on days 6 and 8 (Fig. 2). Conversely, exposure to DCF increased TE in exposed fish compared to controls (Fig. 3A); on days 8 and 9, most of the medaka did not eat the larvae at all and the number of fish did not eat ML was four and five fish,
3. Results Exposure to TCS had no significant effect on time to eat the midge larva (TE) by medaka, but did significantly decrease mean
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Exposure time (day) Fig. 2. (TCS) (A) Changes in time to eat midge larvae (TE) of medaka unexposed (Cont.) or exposed (Expo.) to 0.17 mg L–1 TCS. Box-whisker plots represent percentiles (bottom of box = 25th, horizontal line inside of box = 50th, top of box = 75th, top whisker = 90th. Open circle represents lower outlier value). An outlier value is defined as a value that is smaller than the lower quartile (25 percentile) minus 1.5 times the interquartile range, or larger than the upper quartile (75 percentile) plus 1.5 times the interquartile range. (B) Mean swimming speed (SS; ±SD, n = 9) of medaka unexposed (Cont.) or exposed (Expo.) to 0.17 mg L–1 TCS. p 6 0.05.
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Exposure time (day) Fig. 3. (DCF) (A) Changes in time to eat midge larvae (TE) of medaka unexposed (Cont.) or exposed (Expo.) to 1.0 mg L–1 DCF. Box-whisker plots represent percentiles (bottom of box = 25th, horizontal line inside of box = 50th, top of box = 75th, top whisker = 90th. Open circles represent upper outlier values). An outlier value is defined as a value that is smaller than the lower quartile (25 percentile) minus 1.5 times the interquartile range, or larger than the upper quartile (75 percentile) plus 1.5 times the interquartile range. p 6 0.05. (B) Mean swimming speed (SS; ±SD, n = 9) of medaka unexposed (Cont.) or exposed (Expo.) to 1.0 mg L–1 DCF.
respectively. However, exposure to DCF had no significant effect on SS (Fig. 3B). Similarly to DCF, exposure to CMBZ increased TE (Fig. 4A). However, CMBZ caused a significant decrease in SS on days 8 and 9 (Fig. 4B), which was similar to the effect of TCS. Thus, CBMZ had deleterious effects on both feeding behavior and swimming speed of medaka.
4. Discussion Results of the present study clearly showed that feeding behavior of medaka was affected by exposure to DCF and CBMZ at concentrations of 1.0 and 6.15 mg L–1, respectively, but not by exposure to TCS at 0.17 mg L–1. On the other hand, swimming speed was affected by exposure to TCS and CBMZ, but not by exposure to DCF. Thus, the mechanisms of the behavioral effects appear to be different for each chemical. The observed effects of exposure to TCS, DCF and CBMZ on SS of medaka are similar to effects on SS of other species of fish and aquatic invertebrates reported by previous studies. For example, Alteration on fish behavior, erratic swimming, was reported in rainbow trout (Oncorhynchus mykiss) exposed to 0.071 mg L–1 of TCS for 61 d (Orvos et al., 2002) and in zebrafish (Danio rerio) exposed to TCS at concentrations of 0.5 and 0.4 mg L–1 (Oliveira et al., 2009). Swimming activity of the amphipod Gammarus pulex
exposed to 10 ng L–1 CBMZ for 1.5 h decreased to around 45% of total time compared to 65% in controls (De Lange et al., 2006). Altogether, these observations suggest that the deleterious effects of exposure to TCS and CBMZ on swimming behavior of aquatic organisms might decrease survival rates and ultimately impact populations. Our observation that exposure to DCF did not significantly affect SS in medaka demonstrates that deleterious effects on swimming behavior of aquatic organisms are not consistent among PPCPs, probably due to chemical-specific mechanisms of action. For example, the decrease in SS caused by exposure to CBMZ may have resulted from a change in acetylcholine (ACh) level. A positive relationship between swimming speed and ACh level was previously reported in fish exposed to chemicals (Kavitha and Rao, 2007). A toxic dose of CBMZ inhibited the synthesis and release of extracellular ACh in the brains of Wistar rats (Mizuno et al., 2000). CBMZ inhibits Na+ channel activity (McLean and Macdonald, 1986; Mattson, 1997), which regulates the ACh release (Westerink et al., 1989). No studies were found in literature to evaluate the mechanism of swimming ability in fish exposed to TCS. Further studies with aquatic organisms are needed to investigate the relationships among CBMZ and TCS toxicity, ACh and swimming activity. Our observations of the effects of exposure to TCS, DCF and CBMZ on feeding behavior are in agreement with results of some
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Exposure time (day) Fig. 4. (CBMZ) (A) Changes in time to eat midge larvae (TE) of medaka unexposed (Cont.) or exposed (Expo.) to 6.15 mg L–1 CBMZ. Box-whisker plots represent percentiles (bottom of box = 25th, horizontal line inside of box = 50th, top of box = 75th top whisker = 90th. Open circle represents lower outlier value). An outlier value is defined as a value that is smaller than the lower quartile (25 percentile) minus 1.5 times the interquartile range, or larger than the upper quartile (75 percentile) plus 1.5 times the interquartile range. p 6 0.05; p 6 0.01. (B) Mean swimming speed (SS; ±SD, n = 9) of medaka unexposed (Cont.) or exposed (Expo.) to 6.15 mg L–1 CBMZ.p 6 0.01.
previous studies of aquatic organisms. Quinn et al. (2008) demonstrated that exposure to 50 mg L–1 CBMZ for 96 h significantly reduces feeding activity in Hydra attenuata, and that exposure to 10 mg L–1 ibuprofen for 96 h significantly reduces the time for prey ingestion. However, our results are in contrast to those of Richards et al. (2004), who reported that food intake increases in Wistar rats exposed to 2.5 mg kg–1 DCF for 10 d, resulting from effects on stress and pro-inflammatory activation. The difference in observed effects of DCF on feeding behavior of medaka and Wistar rats may be due to species or dose differences. Nevertheless, decreased feeding by fish in natural environments, due to long-term exposure to DCF and/or CBMZ at subchronic levels, may have far-reaching effects on growth, reproduction, and population success. Effects of exposure to PPCPs on swimming speed and feeding behavior may be interrelated, at least in some cases (Schmidt et al., 2004). The decreased feeding rate of medaka exposed to CBMZ could certainly be explained by the simultaneous reduction in swimming speed. De Lange et al. (2006) similarly speculated that reduced locomotion of Gammarus pulex exposed to CBMZ or ibuprofen may interfere with feeding behavior. In contrast to
CBMZ, exposure to DCF or TCS did not have consistent effects on SS and TE in medaka: DCF decreased feeding behavior without significantly affecting swimming speed; TCS decreased swimming speed, but did not affect feeding behavior. These results suggest that TCS, DCF and CBMZ may affect different behaviors through different neuron-dependent factors, such as appetite. As for swimming speed, differential effects of TCS, DCF and CBMZ on feeding behavior suggest different mechanisms of action. For example, inhibition of feeding behavior by CBMZ and DCF may be related to serotonin (5-HT). Serotonin functions as a neurotransmitter and regulates a wide range of behaviors, including feeding activity (Barton et al., 2002; Fent et al., 2006). Dailey et al. (1998) suggested that toxic effects of CBMZ on feeding behavior of Sprague–Dawley rats may be related to the inhibition activity of serotonin. DCF (10–300 lM) inhibited serotonin-induced reproducible levels in pig ureter (Mastrangelo et al., 2000). The relationships between DCF and CBMZ toxicity, serotonin, and feeding behavior in aquatic organisms are worthy of future research. The PPCP concentrations used in the present study were 10% of the 96 h LC50 values calculated for medaka in our previous study
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(Nassef et al., 2009). Our present results clearly showed that these lower concentrations of TCS, DCF and CBMZ have significant effects on swimming and feeding behaviors of O. latipes, suggesting that behavior is a more sensitive indicator of toxicity than mortality. Previous research demonstrated that a variety of contaminants disrupt normal fish behavior after exposures much less severe than those causing significant mortality (Hernando et al., 2006). Thus, behavioral indicators are useful for assessing sublethal impacts of pollutants. Furthermore, the present study demonstrated that objective characterization of behavioral data using computational methods can provide real-time, on-line tools for monitoring TCS, DCF or CBMZ toxicity. The chronic exposure of aquatic organisms to a mixture of PPCPs in nature may result in greater ecological risk than exposure to individual chemicals at comparable concentrations (Schwaiger et al., 2004). Thus, mixtures of PPCP residues may cause adverse effects on aquatic ecosystems, if WWTP effluents are discharged without dilution or without the use of appropriate removal technologies (Hernando et al., 2006). In summary, exposure of medaka to CBMZ, DCF, or TCS had deleterious effects on feeding behavior and swimming speed. In addition, behavioral changes may provide a sensitive indicator for assessing the toxicity of TCS, DCF and CBMZ to aquatic organisms. Additional research on mixtures of TCS, DCF and CBMZ at low concentrations is needed to further our understanding of how these chemicals affect aquatic species in contaminated environments. References Andrew, S.K., James, D.S., Geoffrey, T.G., Timothy, C.A.M., Colin, H., 2004. A videobased movement analysis system to quantify behavioral stress responses of fish. Water Res. 38, 3993–4001. Barton, B.A., Morgan, J.D., Vijayan, M.M., 2002. Physiological and condition-related indicators of environmental stress in fish. In: Adams, S.M. (Ed.), Biological Indicators of Aquatic Ecosystem Stress. American Fisheries Society, Bethesda, MD, pp. 111–148. Bendz, D., Paxéus, N.A., Ginn, T.R., Loge, F.J., 2005. Occurrence and fate of pharmaceutically active compounds in the environment, a case study: Höje River in Sweden. J. Hazard. Mater. 122 (3), 1995–2204. Cabrera, C., Ortega, E., Lorenzo, M.L., López, M.D.C., 1998. Cadmium contamination of vegetable crops, farmlands, and irrigation waters. Rev. Environ. Contam. Toxicol. 154, 55–81. Dailey, J.W., Reith, M.E., Steidley, K.R., Milbrandt, J.C., Jobe, P.C., 1998. Carbamazepine-induced release of serotonin from rat hippocampus in vitro. Epilepsia 39 (10), 1054–1063. De Lange, H.J., Noordoven, W., Murkc, A.J., Lürling, M., Peeters, E.T.H.M., 2006. Behavioural responses of Gammarus pulex (Crustacea, Amphipoda) to low concentrations of pharmaceuticals. Aquat. Toxicol. 78, 209–216. Dove, A., 2006. Drugs down the drain. Nat. Med. 12, 376–377. Dussault, E.B., Balakrishnan, V.K., Sverko, E., Solomon, K.R., Sibley, P.K., 2008. Toxicity of human pharmaceuticals and personal care products to benthic invertebrates. Environ. Toxicol. Chem. 27, 425–432. Fent, K., Weston, A.A., Caminada, D., 2006. Ecotoxicology of human pharmaceuticals. Aquat. Toxicol. 76, 122–159. Ferrari, B., Paxéus, N., Giudice, R.L., Pollio, A., Garrica, J., 2003. Ecotoxicological impact of pharmaceuticals found in treated wastewaters: study of carbamazepine, clofibric acid, and diclofenac. Ecotoxicol. Environ. Saf. 55, 359–370. Gao, X.M., Chuang, D.M., 1992. Carbamazepine-induced neurotoxicity and its prevention by NMDA in cultured cerebellar granule cells. Neurosci. Lett. 135, 159–162. Gaworecki, K.M., Klaine, S.J., 2008. Behavioral and biochemical responses of hybrid striped bass during and after fluoxetine exposure. Aquat. Toxicol. 88, 207–213. Gokcimen, A., Rag˘betli, M.Ç., Basß, O., Tunc, A.T., Aslan, H., Yazici, A.C., Kaplan, S., 2007. Effect of prenatal exposure to an anti-inflammatory drug on neuron number in cornu ammonis and dentate gyrus of the rat hippocampus: a stereological study. Brain Res. 1127, 185–192. Hernando, M.D., Mezcua, M., Fernandez-Alba, A.R., Barcelo, D., 2006. Environmental risk assessment of pharmaceutical residues in wastewater effluents, surface waters and sediments. Talanta 69, 334–342. Jensen, A., Bro-Rasmussen, F., 1992. Environmental cadmium in Europe. Rev. Environ. Contam. Toxicol. 125, 101–181. Jones, K.H., Hill, S.A., 1974. The toxicology absorption and pharmacokinetics of amoxicillin. Adv. Clin. Pharmacol. 7, 20–27. Kang, I.J., Moroishi, J., Nakamura, A., Nagafuchi, K., Kim, S.G., Oshima, Y., 2009. Biological monitoring for detection of toxic chemicals in water by the
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Effects of pharmaceutical mixtures in aquatic microcosms. Environ. Toxicol. Chem. 23, 1035–1042. Schmidt, K., Steinberg, C.E.W., Pflugmacher, S., Staaks, G.B.O., 2004. Xenobiotic substances such as PCB mixtures (Aroclor 1254) and TBT can influence swimming behavior and biotransformation activity (GST) of carp (Cyprinus carpio). Environ. Toxicol. 19, 460–470. Schwaiger, J., Ferling, H., Mallow, U., Wintermayr, H., Negele, R.D., 2004. Toxic effects of the non-steroidal anti-inflammatory drug diclofenac. Part I: histopathological alterations and bioaccumulation in rainbow trout. Aquat. Toxicol. 68, 141–150. Scott, G.R., Sloman, K.A., 2004. The effects of environmental pollutants on complex fish behaviour: integrating behavioural and physiological indicators of toxicity. Aquat. Toxicol. 68, 369–392. Taylor, E.B., McPhail, J.D., 1986. Prolong and burst swimming in anadromous and freshwater threespine stickleback, Gasterosteus aculeatus. Can. J. Zool. 64, 416– 420. Ternes, T.A., 1998. Occurrence of drugs in German sewage treatment plants and rivers. Water Res. 32 (11), 3245–3260. Volkoff, H., Wyatt, J.L., 2009. Apelin in goldfish (Carassius auratus): cloning, distribution and role in appetite regulation. Peptides 30, 1434–1440. Westerink, B.H., Damsma, G., deVries, J.B., 1989. Effect of ouabain applied by intrastriatal microdialysis on the in vivo release of dopamine, acetylcholine, and amino acids in the brain of conscious rats. J. Neurochem. 52, 705–712. Ying, G.G., Kookana, R.S., 2007. Triclosan in wastewaters and biosolids from Australian wastewater treatment plants. Environ. Int. 33, 199–205. Zaremba, P.D., Bialek, M., Blaszczyk, B., Cioczek, P., Czuczwar, S.J., 2006. Nonepilepsy uses of antiepileptic drugs. Pharmacol. Rep. 58, 1–12. Zeng, L., Cao, Z., Fu, S., Peng, J., Wang, Y., 2009. Effect of temperature on swimming performance in juvenile southern catfish (Silurus meridionalis). Comp. Biochem. Physiol., A. 153, 125–130.
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Acute effects of triclosan, diclofenac and carbamazepine on feeding performance of Japanese medaka fish (Oryzias latipes) Mohamed Nassef a, Shuhei Matsumoto a, Masanori Seki b, Fatma Khalil a, Ik Joon Kang c, Yohei Shimasaki a, Yuji Oshima a,*, Tsuneo Honjo a a b c
Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan Chemicals Evaluation and Research Institute (CERI), Hita Laboratory, 3-822 Ishiimachi, Hita-shi, Oita 877-0061, Japan Aquatic Biomonitoring and Environmental Laboratory, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan
a r t i c l e
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Article history: Received 17 February 2010 Received in revised form 24 April 2010 Accepted 27 April 2010 Available online 26 May 2010 Keywords: Triclosan Diclofenac Carbamazepine Fish behavior
a b s t r a c t The toxicity of three pharmaceutical and personal care products (PPCPs) – carbamazepine (CBMZ), diclofenac (DCF), and triclosan (TCS) – was examined by measuring their effects on feeding behavior and swimming speed of adult Japanese medaka fish (Oryzias latipes). Medaka were exposed to 6.15 mg L 1 CBMZ, 1.0 mg L 1 DCF, 0.17 mg L 1 TCS, or no PPCP (control) for 9 d. Fish behaviors were monitored during days 5–9 of the exposure period. Feeding behavior (time to eat midge larvae, TE) and swimming speed (SS) of individual exposed and control fish were tracked in two dimensions, using an automated system with a digital charge-coupled device camera. As a result, feeding behavior was affected by exposure to CBMZ and DCF, while SS was altered by exposure to CBMZ and TCS. Thus, TCS, DCF and CBMZ appear to affect fish behaviors through different mechanisms. Overall, the results suggest that behavioral changes may provide a sensitive indicator for assessing the toxicity of PPCPs to aquatic organisms. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Pharmaceuticals and personal care products (PPCPs) have recently been identified as potentially toxic chemicals in aquatic environments (Laura Martín-Díaz et al., 2009). Triclosan (TCS), diclofenac (DCF), and carbamazepine (CBMZ) are among the most frequently detected PPCPs worldwide (Kolpin et al., 2002; Ferrari et al., 2003). TCS is widely used as an antibacterial agent in many personal care products such as soaps, detergents, toothpastes, disinfectants, cosmetics and pharmaceuticals (Ying and Kookana, 2007), and its widespread use has been shown to pose a potential risk to aquatic organisms (Dussault et al., 2008). DCF is an antiinflammatory drug with large production volumes. CBMZ is an antiepileptic drug, and its environmental persistence raises concerns about potential effects on non-target organisms (Zaremba et al., 2006). The maximum environmentally detected concentrations of these PPCPs in waste water treatment plant (WWTP) effluents were 0.269 mg L 1 TCS in Spain (Mezcua et al., 2004), 0.006 mg L 1 CBMZ and 0.002 mg L 1 DCF in Germany (Ternes, 1998). Assessing the human risk posed by PPCPs in water is a high priority; however, the impacts of these chemicals on aquatic
* Corresponding author. Tel.: +81 92 642 2905; fax: +81 92 642 2908. E-mail address: [email protected] (Y. Oshima). 0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2010.04.073
organisms and communities are also important (Bendz et al., 2005; Dove, 2006). Our previous studies evaluated the acute toxicity of TCS, DCF and CBMZ to adult and embryos of medaka fish, and concluded that TCS and DCF may pose ecological risks to aquatic organisms (Nassef et al., 2009, 2010). Although acute lethality tests are useful for generating guidelines to protect against physiological death (i.e., mortality) of aquatic animals, these tests ignore ‘‘ecological death,” i.e., the inability to function in an ecological context when normal behaviors are altered. Such effects may occur at much lower toxicant exposures, even if animals are not overtly harmed by a contaminant. Indeed, environmental impacts in natural ecosystems often occur at concentrations well below those causing significant mortality (Jensen and Bro-Rasmussen, 1992; Cabrera et al., 1998; Norris et al., 1999; Gaworecki and Klaine, 2008). A better understanding of the toxicological effects of contaminants can be achieved by examining behavioral changes and determining how they relate to effects at other levels (Scott and Sloman, 2004). Behavioral alterations can provide estimates of endpoints for sublethal toxicity, and serve as a tool for environmental risk assessment and analysis of toxicological impact (Andrew et al., 2004). Appropriate feeding is essential for growth and reproduction of most aquatic animals, including fish (Volkoff and Wyatt, 2009). The ability to capture prey could be impacted by prolonged exposure to low concentrations of contaminants, including PPCPs (De Lange
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et al., 2006; Kristen and Stephen, 2008). Swimming performance is closely related to food capture (Zeng et al., 2009), and is considered to be a primary determinant of survival in many species of fish and other aquatic animals (Jones and Hill, 1974; Taylor and McPhail, 1986). For example, De Lange et al. (2006) observed reduced locomotion in Gammarus pulex exposed to CBMZ (1–1000 ng L–1 for 1.5 h) and speculated that the reduction may interfere with feeding behavior. Japanese medaka have been used in previous studies of basic fish biology and behavior, as well as toxicology, and the species has been proposed by the Organization for Economic Cooperation and Development for use as the standard fish for toxicology tests (OECD, 1999). Our previous research showed that exposure of medaka to sublethal concentrations of 17b-estradiol impaired sexual behavior and decreased reproductive success (Oshima et al., 2003). Significant changes in swimming speed were observed in medaka exposed to cyanide or aldicarb (Kang et al., 2009). Although previous studies used medaka to examine the accumulation and toxicity of anthropogenic chemicals, there is limited information on behavioral effects. Even though the neurotoxicity of PPCPs has been characterized in non-target species (Gao and Chuang, 1992; Gokcimen et al., 2007), the effects on behavior of aquatic organisms have not been well studied. The goal of the present study was to investigate the effects of sublethal concentrations of two pharmaceuticals (CBMZ and DCF) and one personal care product (TCS) on the behavior of Japanese medaka (Oryzias latipes). Based on ecological relevance, feeding behavior and swimming performance of medaka were chosen as indicators of TCS, DCF and CBMZ toxicity to aquatic animals. 2. Materials and methods 2.1. Test chemicals TCS (>98.0% purity), DCF (>98.0% purity), CBMZ (>97.0% purity) and dimethyl sulfoxide (DMSO, >99.0% purity) were obtained from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). Stock solutions (1.7 mg mL–1 TCS, 1.0 mg mL–1 DCF, and 6.15 mg mL–1 CBMZ) were prepared by dissolving pure chemicals in DMSO, and stored prior to use. Treatment solutions were prepared by mixing appropriate amounts of stock solutions with 1 L of artificial seawater (0.01% salinity). PPCP concentrations in treatment solutions (0.17 mg L–1 TCS, 1.0 mg L–1 DCF, and 6.15 mg L–1 CBMZ) were determined as 10% of the calculated 96 h LC50 for adult medaka, based on our previous results (Nassef et al., 2009). Final DMSO concentrations were <0.1% (v/v) in the treatment solutions. 2.2. Test organisms Fifty-four medaka (O. latipes, orange-red strain), 3 months old, 0.3 ± 0.03 g average weight, and 3.0 ± 0.34 cm average length, were collected from brood stock maintained in the Laboratory of Marine and Environmental Science at Kyushu University, Fukuoka, Japan. The test fish were acclimated to laboratory conditions for 1 week prior to testing. The selected fish were held in a 56 L glass aquarium (60 cm long 30 cm wide 36 cm high) equipped with an aerating filter system, in artificial seawater at 0.01% salinity and 25 ± 1 °C. The exposure media were prepared from dechlorinated tap water as artificial seawater (salinity was adjusted at 0.01%) to retain constant water quality. The fish were kept under a 16:8 h light: dark regime and fed with Artemia nauplii (<24 h after hatching) twice a day, in the morning and evening. Half the water was replaced daily. Frozen midge larvae (Chironomus sp., ML), obtained from a pet shop, were used as fish bait during feeding experiments.
2.3. Experimental design On the first day of exposure, nine fish were randomly assigned to each of three treatment groups: 0.17 mg L–1 TCS, 1.0 mg L–1 DCF, or 6.15 mg L–1 CBMZ. Each treatment group was paired with a control group of nine fish. Each treatment and control group was held in a 2.5 L, clear glass test chamber (18 cm long 12 cm wide 12 cm high). Fish in treatment groups were exposed to TCS, DCF or CBMZ for 9 d under semi-static conditions and the fish behavior was monitored for 5 d started on day 5 from exposure until the day 9, the end of exposure time. The fish were fed 9 ML per tank daily during the first 2 d to condition them to that food. Feeding and swimming behaviors were monitored on days 5–9 of exposure. The fish were not fed for 2 d before and during the behavior monitoring, to ensure that they were hungry. Fish mortality was checked throughout the experimental period. On day 5 of exposure, fish from each treatment and control group were transferred individually to a 2 L test box (18 cm long, 12 cm wide, and 12 cm high), containing the respective concentration of the same PPCP for treated fish, or no PPCP for control fish. Black acrylic boards were attached to the walls of the test box to prevent reflections of fish and lamps. Black screens were arranged around the test boxes, so that the presence of an experimenter would not disturb the behavior of the fish. Fluorescent lamps were used as the sole light source. A flat LED panel was fixed below the bottom of the test box, and a white acrylic board, 2 mm thick, was placed on the bottom to provide uniform illumination. An incomplete vertical glass partition was placed in the test box, leaving a narrow space for introducing the fish (Fig. 1). Prior to placing the fish in the test box, 1 ML was placed in the bottom left corner. All experiments were run at 25 ± 1 °C. 2.4. Behavioral measurements The behaviors of individual exposed and control fish in the test boxes were videotaped using a computerized tracking method. A digital video camera (CCD, model GE60, Library, Japan) was installed in the center of the test box, 30 cm above the water surface. The fish was placed in the test box, and the recording process started when the fish passed the vertical partition and moved toward the ML. Behavioral data were collected for 10 min in each trial. If the fish did not move toward the larva within 5 min of starting the recording, the glass partition was removed. The behavior of each fish was recorded at the same time daily on days 5–9 of the exposure period. Time to eat the midge larva (TE) and swimming speed (SS) were calculated for each trial by analyzing the instantaneous video images recorded at 0.033 s intervals (= dte). The two-dimensional (2D) coordinates in the horizontal plane were determined using motion analyzer software (Move-Tr 32/2D; Library, Tokyo, Japan). When the fish did not successfully eat the ML, the TE value was set at 600 s. 2.5. Statistical analysis Statistical significance of differences in the means of SS and medians of TE between treatment and control groups were analyzed using Mann–Whitney U-tests (Stat View J 5.0, Abacus Concepts, Berkeley, CA, USA). Significant difference was determined at p 6 0.05. The box-and-whisker plot displays a statistical summary of a variable: median, quartiles, range and possibly extreme values (outliers). An outlier value is defined as a value that is smaller than the lower quartile (25 percentile) minus 1.5 times the interquartile range, or larger than the upper quartile (75 percentile) plus 1.5 times the interquartile range.
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Entrance Start point for recording behavior
Swimming pathway
Incomplete vertical glass partition Midge larva Medaka fish Test box filled with PPCP in artificial seawater Fig. 1. Schematic diagram of experimental setup for behavior monitoring.
swimming speed (SS) on days 6 and 8 (Fig. 2). Conversely, exposure to DCF increased TE in exposed fish compared to controls (Fig. 3A); on days 8 and 9, most of the medaka did not eat the larvae at all and the number of fish did not eat ML was four and five fish,
3. Results Exposure to TCS had no significant effect on time to eat the midge larva (TE) by medaka, but did significantly decrease mean
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Exposure time (day) Fig. 2. (TCS) (A) Changes in time to eat midge larvae (TE) of medaka unexposed (Cont.) or exposed (Expo.) to 0.17 mg L–1 TCS. Box-whisker plots represent percentiles (bottom of box = 25th, horizontal line inside of box = 50th, top of box = 75th, top whisker = 90th. Open circle represents lower outlier value). An outlier value is defined as a value that is smaller than the lower quartile (25 percentile) minus 1.5 times the interquartile range, or larger than the upper quartile (75 percentile) plus 1.5 times the interquartile range. (B) Mean swimming speed (SS; ±SD, n = 9) of medaka unexposed (Cont.) or exposed (Expo.) to 0.17 mg L–1 TCS. p 6 0.05.
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Exposure time (day) Fig. 3. (DCF) (A) Changes in time to eat midge larvae (TE) of medaka unexposed (Cont.) or exposed (Expo.) to 1.0 mg L–1 DCF. Box-whisker plots represent percentiles (bottom of box = 25th, horizontal line inside of box = 50th, top of box = 75th, top whisker = 90th. Open circles represent upper outlier values). An outlier value is defined as a value that is smaller than the lower quartile (25 percentile) minus 1.5 times the interquartile range, or larger than the upper quartile (75 percentile) plus 1.5 times the interquartile range. p 6 0.05. (B) Mean swimming speed (SS; ±SD, n = 9) of medaka unexposed (Cont.) or exposed (Expo.) to 1.0 mg L–1 DCF.
respectively. However, exposure to DCF had no significant effect on SS (Fig. 3B). Similarly to DCF, exposure to CMBZ increased TE (Fig. 4A). However, CMBZ caused a significant decrease in SS on days 8 and 9 (Fig. 4B), which was similar to the effect of TCS. Thus, CBMZ had deleterious effects on both feeding behavior and swimming speed of medaka.
4. Discussion Results of the present study clearly showed that feeding behavior of medaka was affected by exposure to DCF and CBMZ at concentrations of 1.0 and 6.15 mg L–1, respectively, but not by exposure to TCS at 0.17 mg L–1. On the other hand, swimming speed was affected by exposure to TCS and CBMZ, but not by exposure to DCF. Thus, the mechanisms of the behavioral effects appear to be different for each chemical. The observed effects of exposure to TCS, DCF and CBMZ on SS of medaka are similar to effects on SS of other species of fish and aquatic invertebrates reported by previous studies. For example, Alteration on fish behavior, erratic swimming, was reported in rainbow trout (Oncorhynchus mykiss) exposed to 0.071 mg L–1 of TCS for 61 d (Orvos et al., 2002) and in zebrafish (Danio rerio) exposed to TCS at concentrations of 0.5 and 0.4 mg L–1 (Oliveira et al., 2009). Swimming activity of the amphipod Gammarus pulex
exposed to 10 ng L–1 CBMZ for 1.5 h decreased to around 45% of total time compared to 65% in controls (De Lange et al., 2006). Altogether, these observations suggest that the deleterious effects of exposure to TCS and CBMZ on swimming behavior of aquatic organisms might decrease survival rates and ultimately impact populations. Our observation that exposure to DCF did not significantly affect SS in medaka demonstrates that deleterious effects on swimming behavior of aquatic organisms are not consistent among PPCPs, probably due to chemical-specific mechanisms of action. For example, the decrease in SS caused by exposure to CBMZ may have resulted from a change in acetylcholine (ACh) level. A positive relationship between swimming speed and ACh level was previously reported in fish exposed to chemicals (Kavitha and Rao, 2007). A toxic dose of CBMZ inhibited the synthesis and release of extracellular ACh in the brains of Wistar rats (Mizuno et al., 2000). CBMZ inhibits Na+ channel activity (McLean and Macdonald, 1986; Mattson, 1997), which regulates the ACh release (Westerink et al., 1989). No studies were found in literature to evaluate the mechanism of swimming ability in fish exposed to TCS. Further studies with aquatic organisms are needed to investigate the relationships among CBMZ and TCS toxicity, ACh and swimming activity. Our observations of the effects of exposure to TCS, DCF and CBMZ on feeding behavior are in agreement with results of some
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Exposure time (day) Fig. 4. (CBMZ) (A) Changes in time to eat midge larvae (TE) of medaka unexposed (Cont.) or exposed (Expo.) to 6.15 mg L–1 CBMZ. Box-whisker plots represent percentiles (bottom of box = 25th, horizontal line inside of box = 50th, top of box = 75th top whisker = 90th. Open circle represents lower outlier value). An outlier value is defined as a value that is smaller than the lower quartile (25 percentile) minus 1.5 times the interquartile range, or larger than the upper quartile (75 percentile) plus 1.5 times the interquartile range. p 6 0.05; p 6 0.01. (B) Mean swimming speed (SS; ±SD, n = 9) of medaka unexposed (Cont.) or exposed (Expo.) to 6.15 mg L–1 CBMZ.p 6 0.01.
previous studies of aquatic organisms. Quinn et al. (2008) demonstrated that exposure to 50 mg L–1 CBMZ for 96 h significantly reduces feeding activity in Hydra attenuata, and that exposure to 10 mg L–1 ibuprofen for 96 h significantly reduces the time for prey ingestion. However, our results are in contrast to those of Richards et al. (2004), who reported that food intake increases in Wistar rats exposed to 2.5 mg kg–1 DCF for 10 d, resulting from effects on stress and pro-inflammatory activation. The difference in observed effects of DCF on feeding behavior of medaka and Wistar rats may be due to species or dose differences. Nevertheless, decreased feeding by fish in natural environments, due to long-term exposure to DCF and/or CBMZ at subchronic levels, may have far-reaching effects on growth, reproduction, and population success. Effects of exposure to PPCPs on swimming speed and feeding behavior may be interrelated, at least in some cases (Schmidt et al., 2004). The decreased feeding rate of medaka exposed to CBMZ could certainly be explained by the simultaneous reduction in swimming speed. De Lange et al. (2006) similarly speculated that reduced locomotion of Gammarus pulex exposed to CBMZ or ibuprofen may interfere with feeding behavior. In contrast to
CBMZ, exposure to DCF or TCS did not have consistent effects on SS and TE in medaka: DCF decreased feeding behavior without significantly affecting swimming speed; TCS decreased swimming speed, but did not affect feeding behavior. These results suggest that TCS, DCF and CBMZ may affect different behaviors through different neuron-dependent factors, such as appetite. As for swimming speed, differential effects of TCS, DCF and CBMZ on feeding behavior suggest different mechanisms of action. For example, inhibition of feeding behavior by CBMZ and DCF may be related to serotonin (5-HT). Serotonin functions as a neurotransmitter and regulates a wide range of behaviors, including feeding activity (Barton et al., 2002; Fent et al., 2006). Dailey et al. (1998) suggested that toxic effects of CBMZ on feeding behavior of Sprague–Dawley rats may be related to the inhibition activity of serotonin. DCF (10–300 lM) inhibited serotonin-induced reproducible levels in pig ureter (Mastrangelo et al., 2000). The relationships between DCF and CBMZ toxicity, serotonin, and feeding behavior in aquatic organisms are worthy of future research. The PPCP concentrations used in the present study were 10% of the 96 h LC50 values calculated for medaka in our previous study
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(Nassef et al., 2009). Our present results clearly showed that these lower concentrations of TCS, DCF and CBMZ have significant effects on swimming and feeding behaviors of O. latipes, suggesting that behavior is a more sensitive indicator of toxicity than mortality. Previous research demonstrated that a variety of contaminants disrupt normal fish behavior after exposures much less severe than those causing significant mortality (Hernando et al., 2006). Thus, behavioral indicators are useful for assessing sublethal impacts of pollutants. Furthermore, the present study demonstrated that objective characterization of behavioral data using computational methods can provide real-time, on-line tools for monitoring TCS, DCF or CBMZ toxicity. The chronic exposure of aquatic organisms to a mixture of PPCPs in nature may result in greater ecological risk than exposure to individual chemicals at comparable concentrations (Schwaiger et al., 2004). Thus, mixtures of PPCP residues may cause adverse effects on aquatic ecosystems, if WWTP effluents are discharged without dilution or without the use of appropriate removal technologies (Hernando et al., 2006). In summary, exposure of medaka to CBMZ, DCF, or TCS had deleterious effects on feeding behavior and swimming speed. In addition, behavioral changes may provide a sensitive indicator for assessing the toxicity of TCS, DCF and CBMZ to aquatic organisms. Additional research on mixtures of TCS, DCF and CBMZ at low concentrations is needed to further our understanding of how these chemicals affect aquatic species in contaminated environments. References Andrew, S.K., James, D.S., Geoffrey, T.G., Timothy, C.A.M., Colin, H., 2004. A videobased movement analysis system to quantify behavioral stress responses of fish. Water Res. 38, 3993–4001. Barton, B.A., Morgan, J.D., Vijayan, M.M., 2002. Physiological and condition-related indicators of environmental stress in fish. In: Adams, S.M. (Ed.), Biological Indicators of Aquatic Ecosystem Stress. American Fisheries Society, Bethesda, MD, pp. 111–148. Bendz, D., Paxéus, N.A., Ginn, T.R., Loge, F.J., 2005. Occurrence and fate of pharmaceutically active compounds in the environment, a case study: Höje River in Sweden. J. Hazard. Mater. 122 (3), 1995–2204. Cabrera, C., Ortega, E., Lorenzo, M.L., López, M.D.C., 1998. Cadmium contamination of vegetable crops, farmlands, and irrigation waters. Rev. Environ. Contam. Toxicol. 154, 55–81. Dailey, J.W., Reith, M.E., Steidley, K.R., Milbrandt, J.C., Jobe, P.C., 1998. Carbamazepine-induced release of serotonin from rat hippocampus in vitro. Epilepsia 39 (10), 1054–1063. De Lange, H.J., Noordoven, W., Murkc, A.J., Lürling, M., Peeters, E.T.H.M., 2006. Behavioural responses of Gammarus pulex (Crustacea, Amphipoda) to low concentrations of pharmaceuticals. Aquat. Toxicol. 78, 209–216. Dove, A., 2006. Drugs down the drain. Nat. Med. 12, 376–377. Dussault, E.B., Balakrishnan, V.K., Sverko, E., Solomon, K.R., Sibley, P.K., 2008. Toxicity of human pharmaceuticals and personal care products to benthic invertebrates. Environ. Toxicol. Chem. 27, 425–432. Fent, K., Weston, A.A., Caminada, D., 2006. Ecotoxicology of human pharmaceuticals. Aquat. Toxicol. 76, 122–159. Ferrari, B., Paxéus, N., Giudice, R.L., Pollio, A., Garrica, J., 2003. Ecotoxicological impact of pharmaceuticals found in treated wastewaters: study of carbamazepine, clofibric acid, and diclofenac. Ecotoxicol. Environ. Saf. 55, 359–370. Gao, X.M., Chuang, D.M., 1992. Carbamazepine-induced neurotoxicity and its prevention by NMDA in cultured cerebellar granule cells. Neurosci. Lett. 135, 159–162. Gaworecki, K.M., Klaine, S.J., 2008. Behavioral and biochemical responses of hybrid striped bass during and after fluoxetine exposure. Aquat. Toxicol. 88, 207–213. Gokcimen, A., Rag˘betli, M.Ç., Basß, O., Tunc, A.T., Aslan, H., Yazici, A.C., Kaplan, S., 2007. Effect of prenatal exposure to an anti-inflammatory drug on neuron number in cornu ammonis and dentate gyrus of the rat hippocampus: a stereological study. Brain Res. 1127, 185–192. Hernando, M.D., Mezcua, M., Fernandez-Alba, A.R., Barcelo, D., 2006. Environmental risk assessment of pharmaceutical residues in wastewater effluents, surface waters and sediments. Talanta 69, 334–342. Jensen, A., Bro-Rasmussen, F., 1992. Environmental cadmium in Europe. Rev. Environ. Contam. Toxicol. 125, 101–181. Jones, K.H., Hill, S.A., 1974. The toxicology absorption and pharmacokinetics of amoxicillin. Adv. Clin. Pharmacol. 7, 20–27. Kang, I.J., Moroishi, J., Nakamura, A., Nagafuchi, K., Kim, S.G., Oshima, Y., 2009. Biological monitoring for detection of toxic chemicals in water by the
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