Acute toxic effects of polyethylene microplastic on adult zebrafish
Graphical abstract
Introduction
The presence of microplastics has been well documented in the viscera, gills, and other tissues of aquatic organisms, including some commercially important bivalve mollusks, crustaceans, and fish (Cheung et al., 2018; Jovanović, 2017; Mak et al., 2019; Santillo et al., 2017). The potential effects of microplastics on the health of aquatic organisms have been characterized in a variety of species. These effects may be related to two pathways: i) physical effects from the stress of ingestion, including physical blockage (Wright et al., 2013), and a reduction of feeding and energy assimilation that result in reduced energy and fertility (Besseling et al., 2013; Duis and Coors, 2016); and ii) chemical effects from the leakage of additives from the plastic, such as plasticizers, or absorbed chemical contaminants, including polyaromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCB), and polybrominated diphenyl ethers (PBDEs) (Andrady, 2011; Cole et al., 2011; Ross and Morales-Caselles, 2015).
The physical attributes of microplastics, including size, color, and polymer type, are influential factors in their toxicity to aquatic organisms. Particles larger than 150 μm generally are not absorbed and produce only local inflammation (EFSA, 2016). However, smaller particles can induce systemic exposure and migrate to vital organs, such as in Mytilus edulis (Browne et al., 2008). In contrast, the variability in color preference of microplastics by aquatic organisms may be related to the likelihood of ingestion because of a resemblance to prey items. Red and green are equally preferred over yellow by zebrafish, while they have a strong aversion to blue (Avdesh et al., 2011). Similarly, juvenile common goby (Pomatoschistus microps) mistake white polyethylene (PE) particles for food more frequently than black or red particles (de Sa et al., 2015).
The chemical effects are the result of the release of plastic additives from various types of polymers, including PE with hindered amine light stabilizers, chromium (IV) oxide, oxide of Sulphur [aryl hydrocarbon receptor pathway (AHR) related] and antioxidant like 4-nonylphenol (endocrine disruptor); polyvinyl chloride (PVC), including phthalates such as Di(2-ethylhexyl) phthalate; di-isodecyl phthalate; and polycarbonate containing bisphenol A (an estrogenic compound) (Bakir et al., 2014a, b). These plastic additives have been linked to alterations in the oogenesis, possibly with the involvement of estrogen receptor pathway and detoxification processes in teleosts, possibly with the involvement of AHR pathway (Oehlmann et al., 2009; Lithner et al., 2011). However, the physical effects, behavioral changes, and alterations in biochemical pathways induced by microplastics remain poorly understood (Desforges et al., 2014).
The objectives of this study were to identify the physical effects, behavioral changes, and gene expression profiles of the phase 1 detoxification-related gene (cytochrome P450 1A, cyp 1a) and the oogenesis-related gene vitellogenin 1 (vtg 1) in zebrafish (Danio rerio) liver, intestine, and gills induced by various concentrations and colors of PE microplastics in various size ranges.
Our hypothesis was that the toxic effects in terms of blockage of the digestive system, the degree of severity and likelihood of occurrence of exhibited observed behavior, and the alteration in the targeted gene expression profiles (cyp 1A and vtg 1) would increase when the concentration of microplastics increased.
The adult zebrafish is used as a model organism for behavioral and gene expression studies because its genome is fully characterized and its physiology parallels that of humans (Chen et al., 2009; Peitsaro et al., 2007). In addition, zebrafish observed behavioral responses are characterized by lethargy, anxiety, reduced physiological function such as locomotor activity, food intake, reproductive performance, exploratory activity and social interaction. (Dantzer and Kelley, 2007; Grossberg et al., 2011; Haba et al., 2012; Hennesey et al., 2014; Kelley et al., 2003). These responses are sensitive and robust, appear to be evolutionarily conserved, and resemble those of mammalian species (Blaser et al., 2010; Champagne et al., 2010; Egan et al., 2009; Jesuthasan, 2012). Cyp 1a and vtg 1 were selected as the biomarkers; cyp1a is involved in the biotransformation of hazardous decomposition by-products, for example, AHR-compatible toxicants from microplastics (i.e., polyaromatic hydrocarbons) under the AHR pathway (Mazurais et al., 2015), and vtg 1 is involved in oogenesis in teleosts under hormonal control (Murata et al., 1997) and acts as an indicator of exposure to estrogenic or anti-estrogenic substances in aquatic environments, such as bisphenol A, S, and F (Arukwe and Goksoyr, 2003). This study was designed to test our hypothesis that acute exposure to microplastics such as PE can affect the aryl hydrocarbon receptor (AHR) pathway, disrupt oogenesis, and induce neurotoxic or behavioral changes in adult zebrafish.
Section snippets
Microplastics characterization before and after the exposure experiments
Spherical high-density PE microplastics of various colors and size ranges (of which 99.7% ± 0.6% of particles were 10–22 μm [red], 45–53 μm [blue], 90–106 μm [green], 212–250 μm [clear], or 500–600 μm [yellow], respectively) were supplied by Cospheric Ltd. The polymer type was verified by micro-Raman spectroscopy. After that, the morphology of the microplastics was characterized under a stereomicroscope (Euromex DZ stereo zoom 1:10 microscope) incorporating an 18-Mpix digital camera (Euromex
Chemical loads on used microplastics
The recovery rates of individual PAHs in spiked microplastics were 71 ± 12%. Concentrations of PAHs on MPs were below the detection limit (LOD) (Supplementary materials S5; S6).
Characterization of microplastics in exposure treatments
No significant morphological changes, including color, shape and size between ingested microplastics in both experiments and original microplastics were found, except 500–600 μm yellow microplastic in zebrafish intestine which showed slightly deformation (Supplementary materials S7; S8). The shape and size of ingested
Exposure under realistic conditions
The microplastic concentration used in the previous exposure experiments were far exceeds the levels which have been documented in the marine environment (Browne et al., 2008; Mazurais et al., 2015; Von et al., 2012). The documented toxicity effects, such as reduced feeding, survival, and fecundity, may not be suitable for extrapolation to wild organisms.
To simulate realistic conditions, we used PE because it is the dominant polymer type in effluent and surface water in Hong Kong (Mak et al.,
Conclusions
To conclude, the changes of expression levels in cyp 1a and vtg 1 provide cues in relation to the effects on the AHR pathway, disruption of the oogenesis process. In addition, sickness behaviors including seizures, indicating neurotoxicity, may be caused by acute exposure to microplastics. Further investigations, including behavioral changes end points (average velocity of zebrafish and total distance traveled), chronic microplastic exposure experiments, and expression profiles of several genes
Acknowledgments
We thank the School of Life Sciences, The Chinese University of Hong Kong (CUHK) for our research allowance and for SURP (Summer Undergraduate Research Program) support for Kristen Yeung, CUHK. We are grateful to Mr. Man Long Kwok (School of Life Sciences, CUHK) for technical assistance with real-time PCR. Mak CW is the recipient of a postgraduate studentship (2016-18) from CUHK.
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Current address: Chemistry Department & School of the Environment, University of Toronto, Ontario, Canada.