Elsevier

Environmental Pollution

Volume 235, April 2018, Pages 918-930
Environmental Pollution

Microplastic accumulation patterns and transfer of benzo[a]pyrene to adult zebrafish (Danio rerio) gills and zebrafish embryos

https://doi.org/10.1016/j.envpol.2018.01.028Get rights and content

Highlights

  • Microplastics did not adhere strongly or accumulate at high amounts in adult zebrafish gills.

  • Particles only superficially adhered to the mucus layer on the filaments.

  • The gill EROD assay showed a clear trend to CYP 1A induction via exposure to ben-zo[a]pyrene-coated microplastics.

  • Microplastics and associated benzo[a]pyrene did not induce morphological effects in the fish embryo toxicity test (FET).

  • Fluorescence tracking revealed the transfer of benzo[a]pyrene from microplastics to the fish embryo.

Abstract

Since only a few studies have investigated effects of microplastics (MPs) by routes other than ingestion, this study was designed to analyze the accumulation patterns and transfer of toxic substances associated with microplastic exposure by simple attachment to (1) adult zebrafish (Danio rerio) gills and (2) zebrafish embryos. Two sizes of fluorescently labelled polymers (1–5 and 10–20 μm) loaded with the model polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) were used to analyze fate, accumulation and transfer of microplastic-associated persistent organic pollutants (POPs) on gills and embryos.

Results indicate that microplastics did not permanently accumulate at high amounts in adult zebrafish gills after 6 nor 24 h of incubation: Most particles only superficially adhered to the mucus layer on the filaments, which is constantly being excreted. In contrast, the smaller and heavier MPs (1–5 μm) accumulated in high numbers on the surface of zebrafish egg chorions. In both exposure scenarios, transfer of BaP could be visualized with fluorescence microscopy: A prominent BaP signal was visible both in gill filaments and arches after 6 and 24 h incubation and in zebrafish embryos after exposure to BaP-spiked microplastics. Furthermore, the gill EROD (Ethoxyresorufin-O-deethylase) assay showed a clear trend to CYP 1A (Cytochrom P450 1 A) induction via exposure to BaP-spiked microplastics. However, BaP from spiked microplastics did not reach sufficiently high concentrations to be able to induce morphological effects in the fish embryo toxicity test (FET). In contrast, control exposure to waterborne BaP did induce effects in the FET.

As a conclusion, microplastics can also transfer POPs not only via ingestion, but also by simple attachment to epithelia or via the water column. However, further studies are needed to clarify if these interactions are of environmental concern relative to waterborne exposure to toxic substances.

Introduction

Microplastics, plastic particles smaller than 5 mm (Andrady, 2011, Barnes et al., 2009, Cole et al., 2011) or 1 mm (Galloway and Lewis, 2016) − depending on definition − represent an increasingly widely discussed issue with respect to environmental pollution (Galloway and Lewis, 2016, Barboza and Gimenez, 2015, Thompson, 2015, Thompson et al., 2004, Eerkes-Medrano et al., 2015, do Sul and Costa, 2014). Recently, along with plastic debris, microplastics were even generally referred to as a ‘planetary boundary threat’ (Galloway and Lewis, 2016). Deriving from larger plastic items or produced as such small particles (Cole et al., 2011), microplastics are thought to have a variety of impacts on the environment (Thompson, 2015, Eerkes-Medrano et al., 2015). Numerous studies have shown interactions between these newly introduced particles and aquatic organisms (Wright et al., 2013). Detritus feeders, filter feeders as well as predators and even corals were shown to ingest and be effected by different types and sizes of microplastics, depending on natural feeding habits (Browne et al., 2008, Browne et al., 2013, Carlos de Sa et al., 2015, Cole et al., 2013, Hall et al., 2015, Lusher et al., 2013, Rochman et al., 2013, Setala et al., 2014, Sussarellu et al., 2016). Thus, there is no doubt that microplastics are being ingested by a wide variety of aquatic organisms.

Furthermore, since environmental microplastics found worldwide on beaches have been shown to carry considerable amounts of different highly toxic substances (Frias et al., 2010, Fries et al., 2013, Rios et al., 2007), it is of special importance to know if such substances might be transferred to organisms in interaction with microplastics. Numerous recent studies have made attempts to evaluate if these substances desorb from microplastics following ingestion and if this might pose an additional threat to organisms in aquatic environments. In addition to mathematically-based hypotheses, various experimental approaches have been used, from chemical analyses of whole tissues to more sensitive assays such as enzymatic tests, histological analyses and fluorescence tracking (Rochman et al., 2013, Batel et al., 2016, Chua et al., 2014, Gouin et al., 2011, Koelmans et al., 2016).

As a matter of fact, in all of these studies the major focus was set on oral ingestion of microplastics, and only a few studies included other pathways for microplastic exposure. Watts et al. (2014) reported that, upon exposure, clean microspheres were not only found in the intestinal tract, but also in the gills of the shore crab (Carcinus maenas) until 21 days post exposure (Watts et al., 2014). Lu et al. (2016) exposed zebrafish (Danio rerio) to very high amounts of nano- (70 nm) and micro- (5 and 20 μm) polystyrene particles and found 5 μm particles in gills, gut and liver after 7 days of exposure, while 20 μm particles were found in gills and gut only (Lu et al., 2016). Furthermore, among others, they found that microplastics induced an inflammatory response, lipid accumulation in the liver and modified metabolic profiles. Nobre et al. (2015) analyzed the leaching of pollutants from plastic pellets to embryos of the sea urchin (Lytechinus variegatus) via the water column and found that especially plastic additives from virgin pellets caused anomalous embryonic development (Nobre et al., 2015). Plastic pellets from beach samples did not induce any toxic effects. In most cases, however, in contrast to intestinal uptake of microplastics, alternative pathways of microplastic exposure and subsequent effects on aquatic organisms have been neglected so far.

Therefore, the present study was designed to focus on the accumulation, behavior and effects of microplastics on epithelia or outer surfaces of organisms in general. Due to their great surface, fish gills carry a high potential for microplastic accumulation and attachment in all aquatic organisms. Due to the constant filtration of water, high microplastic concentrations might also pose an additional threat to the gills of aquatic organisms, e.g. if microplastics accumulate and transfer harmful substances to the gills. On the other hand, fish eggs with their lipophilic chorion might also represent a potential surface for increased deposition and accumulation of microplastics. Therefore, both adult zebrafish gills and zebrafish eggs with fish embryos were studied as alternative pathways for the potential transfer of POPs via microplastics.

Benzo[a]pyrene was used as a model polynuclear aromatic hydrocarbon (PAH), since it has been shown (1) to induce EROD activity in zebrafish gills (Jönsson et al., 2009), (2) to cause morphological effects in the fish embryo toxicity test (FET) (Huang et al., 2014, Weigt et al., 2011) and, given its autofluorescence properties, (3) to be traceable by means of epifluorescence microscopy (Batel et al., 2016).

Section snippets

Material

Adult zebrafish aged 24 months were obtained from the breeding and maintenance facilities of the Aquatic Ecology and Toxicology Group at the Center for Organismal Studies Heidelberg (licensed by regional animal welfare authorities under 35–9185.64/BH Braunbeck). Temperature was maintained at 26.0 ± 1.0 °C, and fish were kept under a constant artificial dark/light regimen of 8/16hrs. Constant filtration in combination with permanent flow-through conditions (two-fold water exchange per day)

Accumulation of microplastics on adult zebrafish gills

Fish were exposed to microplastic particles of 1–5 μm or 10–20 μm in diameter with an approximate number of 5 × 106 (1–5 μm) and 1.2 × 106 (10–20 μm) particles/L for 6 and 24 h. In freshly dissected gills (Fig. 1 A, B), particles were detected between gill filaments at 6 and 24 h at similar concentrations. In order to analyze the exact pattern of MP accumulation in gills and surrounding tissue, whole heads were fixed and analyzed histologically. Most sections showed only few particles,

Discussion

This study was designed to analyze the potential effects of microplastics and associated POPs by attachment to outer epithelia: in this case on adult zebrafish gills and zebrafish embryos. Microplastic particles can adsorb to outer epithelia of aquatic organisms and transfer significant amounts of associated POPs via external attachment. POPs associated with microplastics may then desorb into the water column when the milieu is changed due to e.g. mucus secretion by aquatic organisms. This

Conclusions

Results in both adult zebrafish gills and zebrafish embryos indicate that POPs adsorbed to microplastic particles might not only be transferred via microplastic ingestion, but also via simple microplastic attachment and re-solution of POPs into the water column. Although present at lower numbers than expected, microplastic particles are constantly filtered through zebrafish gills and can also accumulate at considerable numbers on the outer surface of zebrafish eggs. Upon exposure to BaP-coated

Acknowledgements

The first author is grateful for a scholarship by the German Academic Scholarship Foundation (Studienstiftung des Deutschen Volkes) and funding by the German Federal Ministry for Science and Research (BMBF) under contract no. 03F0735A within the Joint Programming Initiative Healthy and Productive Seas and Oceans (JPI Oceans) project EPHEMARE (“Ecotoxicological effects of microplastics in marine ecosystems”). Special thanks are due to the Aquatic Ecology and Toxicology group of the University of

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