Effects of ibuprofen, diclofenac and paracetamol on hatch and motor behavior in developing zebrafish (Danio rerio)
Graphical abstract
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used pain relief medicines, among which ibuprofen, diclofenac and paracetamol are commonly prescribed on a daily basis. They are frequently prescribed by doctors (Paul and Chauhan, 2005), and can also be sold as nonprescription drugs. In addition, such compounds are difficult to be removed in sewage treatment plants (Nakada et al., 2006, Zhou et al., 2009, Lin et al., 2009, Maskaoui and Zhou, 2010), with as low as 0% removal for diclofenac indicating their persistent behavior. As a result, residues of NSAIDs pollution have been widely detected in natural water around the world. Weigel et al. (2002) detected concentrations of 0.6 and 6.2 ng/L in German coastal water. Maskaoui and Zhou (2010) reported wide occurrence of diclofenac in river water and groundwater, with concentrations up to 93 ng/L in river water and 4 ng/L in groundwater. Yan et al. (2015) also reported up to 70 ng/L of paracetamol and 10 ng/L of ibuprofen and diclofenac in the Yangtze River Estuary, China. Multiple studies have shown their toxicity on aquatic life (Brun et al., 2006, Khetan and Collins, 2007, Gamarra et al., 2015), thus there is a growing concern about their persistence and potential adverse effects on aquatic organisms.
Acute and chronic exposure experiments using NSAIDs have been conducted on Zebrafish, a well-established toxicological animal model (Chou et al., 2010, Chen et al., 2017). Peng et al. (2010) reported high dose of paracetamol caused kidney damage in Zebrafish embryo. Ji et al. (2013) found that low dose of ibuprofen significantly decreased the number of eggs produced by adult zebrafish, and contributed to estrogenic effects. Gröner et al. (2015) also observed that diclofenac at environmentally relevant concentrations induced the expression of estrogenic biomarker vitellogenin (VTG), and enzymes related to biotransformation phases I (cytochrome P4501A), phases II (glutathione S-transferase) and phases III (multidrug resistance protein). Most of these studies also revealed a significant delay in hatching, but the cause was not fully explained. Thus it is necessary to investigate the potential effects of NSAIDs on embryo early motion.
Behavioral endpoint analysis is critical in ecotoxicology study. Behavior anomalies such as decrease in motion are direct reflections of low level endogenous changes (i.e. hormone disorder) caused by stress e.g. pollution, and can be linked to high level changes such as reproductive failure (Kane et al., 2004). Behavior anomalies can also happen without any obvious morphological deformations or survival rate decrease, thus quantifiable behavior changes have proved to be sensitive supplement information for traditional toxicology study (Little and Finger, 1990). The developmental motor behavior of zebrafish embryo has already been well characterized. Transient spontaneous movement, twitching response to touch, and the ability to swim were determined as three major endpoints for quantitative behavior study (Saint-Amant and Drapeau, 1998). Previous studies have used them to investigate the developmental neurotoxicity of pollutants such as polybrominated diphenyl ethers (Chen et al., 2012a, Chen et al., 2012b) and bisphenol A (Wang et al., 2013). In this study, zebrafish embryos were used as a model to assess the potential behavior changes caused by commonly used NSAIDs. Three widely used NSAIDs, paracetamol, ibuprofen and diclofenac were chosen as representatives. The hatch rate, spontaneous movement, swimming ability, as well as morphological index and several neuron related genes were tested. The objective of this study was therefore to quantify the extent to which these common NSAIDs could cause embryo behavior defects, elaborate underlying mechanisms, and further our understanding about the ecological risk of NSAIDs in the aquatic environment.
Section snippets
Zebrafish maintenance and embryo collection
Wild-type AB strain zebrafish were used in this experiment. Parent fish were raised and kept in an automatic circulation system, using water which was dechlorinated and carbon filtered. The water properties were monitored and maintained at a temperature of 28 ± 1 °C and dissolved oxygen >7 mg/L. Adult fish were fed twice a day with dry flakes and fresh brine shrimp (Artemia nauplii). For embryo collection, 14 h/10 h (light/dark) cycle was used to meet the spawning condition. One day before
Actual exposure concentrations
Of all the exposure experiments conducted in this study, the exposure media were changed twice a day (every 12 h). In order to identify any possible concentration changes, the actual concentrations of the exposure media were tested at around 60 hpf to 72 hpf. For ibuprofen with 5 μg/L, 50 μg/L and 500 μg/L nominal concentrations, the detected concentrations were 5.70 μg/L, 58.38 μg/L and 521.94 μg/L, respectively. For diclofenac, its nominal concentrations of 5 μg/L, 50 μg/L and 500 μg/L
Discussion
In this study, zebrafish embryo was used as a model animal to investigate the effect of three commonly used NSAIDs (ibuprofen, diclofenac and paracetamol) on the early behavior of teleost fish. No significant increase in embryo death rate or morphology defects was found by ibuprofen, diclofenac and paracetamol exposure. Previous studies on diclofenac and paracetamol showed similar results (Peng et al., 2010, Hallare et al., 2004, Lee et al., 2011). However, a study by David and Pancharatna
Conclusions
In summary, this study investigated the potential effects of three common NSAIDs, ibuprofen, diclofenac and paracetamol on zebrafish embryos. Both ibuprofen and diclofenac caused significant hatch delay due to the suppression of overall embryo motion. Paracetamol showed no observable effect, possibly due to its different mode of action. Additionally, the locomotion tests showed that ibuprofen affected larval motion mainly under dark condition, while diclofenac caused similar effect in the
Acknowledgements
This research was supported by the National Key R&D Programme of China (2016YFC1402402), and the State Key Laboratory of Estuarine and Coastal Research (2016RCDW02).
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