Full Length ArticlePolystyrene nanoplastics (20 nm) are able to bioaccumulate and cause oxidative DNA damages in the brain tissue of zebrafish embryo (Danio rerio)⋆
Graphical abstract
Introduction
Usage of synthetic polymers such as plastics has been increasing globally day by day, although its debris has resulted in serious health and environmental problems. Although the hazards caused by macro size plastics have been well studied, the presence of microplastics which smaller size formed of degradation of macroplastics where in the ocean, freshwater, coastal lanes, embouchure and marine sediments is an increasingly important concern. On the other hand, recent studies have shown that these degradation process of plastic particles does not remain at the micrometre level and continues to disintegrate at the nano level (Lambert and Wagner, 2016; Lehner et al., 2019). The size of formatted particles depends on the type and severity of external forces. It has also been suggested that plastic particles with lengths in two or three dimensions between 1 and 100 nm can be formed in the aquatic environment (Chen et al., 2017b; Klaine et al., 2008; Koelmans et al., 2015). Studies have shown that the main sources of nano-sized plastics are (i) degradation process of plastic materials (Lambert and Wagner, 2016; Mattsson et al., 2015b) which is the mass source, (ii) personal care and cosmetic products (Besseling et al., 2014) and (iii) producing process of engineered plastics (Lu et al., 2008). Eventually, it can be concluded that nano-sized particles from any size of the particle would spread to the transient environment. Unfortunately, today it is difficult to say anything about the continuously increasing amount of nano-sized plastic particles in the environment. The lack of adequate analytical methods makes it difficult to quantify the number of nanoplastics in nature despite this potential toxic effect (Koelmans, 2015). The worrying part is that ‘smaller particles are generally more toxic than the corresponding bulk material at the same mass concentration’ (Mattsson et al., 2017).
A limited number of studies performed on the toxic effects of different sized plastic particles which have become an important health and environmental threat have shown that nanoplastics may cause toxicity in living organisms. On a general review of the studies, it would be concluded that nanoplastic exposure has resulted in accumulation within the body (Mattsson et al., 2017; Pitt et al., 2018a, b; Veneman et al., 2017), compromising immune responses (Greven et al., 2016), induction liver lesions (Lu et al., 2016b), ultimately affecting behavior, physiology, and metabolism (Mattsson et al., 2015a). Furthermore, mechanistic parts of some studies have shown that nano-sized plastic particles can transport through the food web (Mattsson et al., 2017), translocate between organs in the body (Farrell and Nelson, 2013) and transfer from mothers to offspring (Pitt et al., 2018b).
As a matter of fact, while the biological effects of these nanoplastics, metal and carbon-based ones have been the subject of more research, little is known about nanoplastics such as polystyrene. Polystyrene, which is used to make foodservice and food packaging in addition to many other usages, is able to transport into food chains directly (Lu et al., 2016b). In this respect, polystyrene is one of the most widely used plastics among thermoplastic polymers and one of the primary components of plastic debris observed in the environment (Lu et al., 2016b). For instance, Šilc et al. (2018) reported that, of a total of 120 samples (water and sediment), 80.6 % contained plastic debris, and 38.7 % of the samples were composed of polystyrene plastics in the southern Adriatic Sea. In addition, Polystyrene was reported to be the second most common form (25 %) found in floating plastic debris in the Tamar Estuary in South England (Sadri and Thompson, 2014). Furthermore, many studies have been conducted to evaluate the toxic effects of both nano and micro size polystyrene on various animal models (Khan et al., 2015; Lee et al., 2019; Mattsson et al., 2017; Pitt et al., 2018a). In a zebrafish study, 7-day exposure to 70 nm Polystyrene was reported to cause inflammation and lipid accumulation (Lu et al., 2016a). In another study, Pitt et al. (2018a) reported that Polystyrene in zebrafish embryos able to pass into the yolk sac and migrate through the gastrointestinal tract to the gallbladder, liver, pancreas and heart. In the same study, they suggested that the embryos exposed to Polystyrene showed hypoactive locomotor activity at the end of 144 hpf (hours post fertilisation), thus affecting behaviour. Indeed, studies in different animal models (Mattsson et al., 2017; Wegner et al., 2012) suggest that polystyrene nanoplastics (PNP) can affect the neurological system and cause behavioral disorders, but neurotoxic effects are not examined in detail. Together, this kind of studies suggests that PNP can cause a toxic effect on living organisms. But, more detailed and molecular studies are needed for precautions that can be taken against PNP which is a global health threat today. In this context, it is important that the detection at the molecular level of destruction in the location where to reach of nano-sized particles, especially in the brain.
Although the potential toxicity of PNP was mainly focused on adult fish, a limited number of studies have attempted to determine toxicity in developmental stages. One of these studies was performed by van Pomeran and stated that PNPs could not cross the chorion layer of embryos which are still in development stage. For the elimination of this condition, zebrafish embryos were injected by van Pomeren et al. (2017) via microinjection into the yolk sac PNP (700 nm size). The same investigators found that the injected PNPs were spread by blood and deposited, particularly in the heart area.
Zebrafish have become a popular animal model also on neurological and brain investigation in addition to toxicity studies, due to the development of laser scanning microscopy, behavioural assays, and progress in genetic engineering (Matsui, 2017). Because zebrafish embryos are forced to live dependent on yolk up to 5–6 dpf (days post fertilization), they can be defined as “closed systems” in which food intake is not influenced by outside (Fraher et al., 2016). Therefore, in present study, we have chosen zebrafish animal model and microinjection method to modelling for PNP intake with food and for the fate of PNP transferred maternally. Here, we have aimed to detect whether can reach and bioaccumulate the microinjected PNP to the brain, in case they reach and bioaccumulate, whether PNP cause oxidative DNA damage in the location of bioaccumulation, and body malformations, apoptosis and oxidative stress in the whole body during the embryonic development stage of zebrafish.
Section snippets
Characterization of polystyrene nanoplastics (PNP)
Fluorescent polystyrene nanoplastics (PNP) were purchased from Thermo Fisher Scientific Co. (Cat. No: R25). The 1 % solid particles have been labelled red fluorescent dye (542/612 nm) with a density of 1.06 g/cm3. They are packaged in deionized water with trace amounts of surfactant and preservative to inhibit aggregation and promote stability. Previous studies have shown that trace amount of this kind of surfactants not toxic for zebrafish embryo (Pitt et al., 2018a). We have also injected to
Characterization of polystyrene nanoplastics (PNP)
The polystyrene nanoplastics have been screened and imaged by TEM. According to TEM images, the size of PNP has been confirmed as 20 ± 3.2 (SD) nm (Fig. 1A). Additionally, the mean size of PNP has been measured as 18.43 ± 0.28 (SD) nm from zetasizer analyses (Fig. 1B). It has been also confirmed the spectral properties of PNP with LSM (Fig. 1C).
Survival and hatching rates of embryo/larvae
To evaluate developmental toxicity effects of PNP on zebrafish, the survival and hatching rates have been determined. As shown in Fig. 2A, survival rate
Discussion
Although the dangers posed by macro and microplastics in organisms are relatively well known, nanoplastics are becoming more and more research as they are potentially more dangerous (Koelmans et al., 2015). This study contains strong evidence that PNPs injected to yolk sac are localized to specific tissues in zebrafish larvae and therefore cause toxic effects at the tissue level. The following sections discuss our results in detail and the potential impacts associated with exposure to PNPs, a
Conclusion
Here we have demonstrated that 20 nm diameter PNP which are intake with food or transferred maternally can reach the brain and bioaccumulate there, moreover can lead to oxidative DNA damage in the brain regions where it bioaccumulates. As a result of these, we have detected excessive ROS and apoptosis in especially the brain. But more studies need to clarify the transfer mechanism of PNP to the brain. This study once again showed the danger dimensions of plastic wastes and nano-sized particles
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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This paper includes PhD thesis of Teoman Özgür SÖKMEN