Acute toxic effects of polyethylene microplastic on adult zebrafish

Highlights

• Different sizes of polyethylene (PE) pellets were exposed to adult zebrafish.

• Behavioral changes were noticed in fish exposed to PE.

• Level of cyp1a in the intestine (medium concentration) showed up-regulation.

• Level of vtg1 in the liver (medium and high concentration) also showed up-regulation.

Abstract

To identify the physical effects, behavioral changes, and gene expression profiles of the phase 1 detoxification-related gene (cyp 1a) and oogenesis-related gene (vtg 1) induced by microplastics, high-density polyethylene microplastics of various sizes were used because of their dominance in coastal areas and effluent samples in Hong Kong. Adult zebrafish were used as the model organism to identify the upper and lower boundaries of microplastics ingestion and were exposed to individual polyethylene microplastics in five size ranges (10–22 μm, 45–53 μm, 90–106 μm, 212–250 μm, and 500–600 μm) at a concentration of 2 mg/L for 96 h. To study behavioral changes and targeted gene expression profiles via real-time PCR (qPCR), a mixture of microplastics in three size ranges at effluent-related (11 particles/L), moderate (110 particles/L), and high concentrations (1,100 particles/L) were applied for 96 h. The zebrafish behavior was recorded by a video camera and by two observers (interrater reliability, >85%). The results implied that the upper and lower size boundaries for microplastic ingestion were 558.4 ± 26.2 μm (yellow) and 19.7 ± 3.1 μm (red), respectively. In addition, 61 ± 10% of fish in medium concentration treatments and 61 ± 10% of fish in high concentration treatments were found with the microplastic ingestion and remaining in their intestine. In addition, 28 ± 10% of fish in high concentration treatments were found with microplastic retaining in their gills (No. of fishes = 18 in each treatment). The presence of microplastics, which occupied 89 ± 6% of intestine area, reduced the voids inside the intestine for feed. The expression of cyp1a in the intestine (medium concentration) and vtg1 in the liver (medium and high concentration) showed significant up-regulation, and abnormal behavior (i.e., seizures and tail bent downward) was observed (medium and high concentration). In summary, the effects on the aryl hydrocarbon receptor (AHR) pathway, disruption of the oogenesis process, and neurotoxicity could be caused by acute exposure of adult zebrafish to microplastics.

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.