Histological, enzymatic and chemical analyses of the potential effects of differently sized microplastic particles upon long-term ingestion in zebrafish (Danio rerio)
Introduction
In microplastic (MP) research, special attention has been paid to MP ingestion by aquatic organisms. MP ingestion has been shown in several laboratory studies (Cole et al., 2013; Rochman et al., 2013; Setala et al., 2014) and field studies (Cole and Galloway, 2015; Desforges et al., 2015; Frias et al., 2014; Goldstein and Goodwin, 2013; Nadal et al., 2016; Rummel et al., 2016; Steer et al., 2017), although in wild fish this phenomenon has been claimed to only rarely occur (Hermsen et al., 2017). However, in the oceans, fish and other biota do interact with plastic debris: Carson (2013) analyzed environmental plastic debris and found bite marks characteristic of fish on 15.8% of the particles investigated, suggesting that fish in natural environments frequently accidentally take plastics as potential prey (Carson, 2013). Furthermore, (very) small MPs might accumulate along trophic food webs and reach higher trophic consumers at relative high amounts by prey ingestion (Setala et al., 2014; Desforges et al., 2015; Au et al., 2017; Farrell and Nelson, 2013). In laboratory studies, Cedervall et al. (2012) found that nano-sized polystyrene particles were transferred from algae via zooplankton to fish (Cedervall et al., 2012); effects were evident in cholesterol distribution between muscle and liver, and fish displayed behavioral changes while feeding on zooplankton.
Various studies showed that ingested MPs into the intestinal tract were even transferred to other organs and tissues: Browne et al. (2008) analyzed MP uptake in mussels and detected MPs in the hemolymph and the digestive gland (Browne et al., 2008; von Moos et al., 2012), Brennecke et al. (2015) found MP transfer to organs in crab (Brennecke et al., 2015) and Avio et al. (2015) even found a 400 μm MP particle in the liver of a wild fish (Avio et al., 2015), although such an observation appears anatomically and physiologically questionable. For the transfer a rigid plastic particle from the intestinal lumen to surrounding tissues and organs, cellular uptake by enterocytes has to occur in a first instance, followed by transfer to the blood circulation. However, transfer of a foreign particle to any tissue would likely cause an inflammatory response of the surrounding tissue, which has not been documented in any of these studies. Moreover, the mean size of blood vessels in fish in livers is approx.100 μm and usually does not exceed 250 μm (Hinton et al., 2017). As a consequence, cellular uptake and transfer of MPs across organs most likely only seems possible for very small or nano-sized plastic particles.
Another controversial issue is the potential of MPs to facilitate the transfer of persistent organic pollutants (POPs) to biota. Many laboratory studies provided evidence that certain amounts of POPs bound to MPs might be delivered to different organisms due to a change of lipophilicity of the milieu upon ingestion (Rochman et al., 2013; Bakir et al., 2014; Batel et al., 2016; Chua et al., 2014; Sleight et al., 2017; Teuten et al., 2007; Teuten et al., 2009; Wardrop et al., 2016). However, other scientists argue that – under environmental conditions − the relative importance of MPs to act as vectors for POPs is rather low, given the total number of MP particles compared to the overall number of natural particles (Guven et al., 2018; Beiras et al., 2019; Beiras and Tato, 2019). Likewise, given the chemical equilibrium levels in biota pre-exposed to POPs in natural environments, desorption of POPs from MPs is quite unlikely (Bakir et al., 2016; Gouin et al., 2011; Koelmans et al., 2016; Lohmann, 2017). Nevertheless, it is important to clarify and understand the processes underlying adsorption to MPs and desorption of POPs from MPs, especially when considering the continuous increase in the number and surface of particles with decreasing size (Eriksen et al., 2014; Ivleva et al., 2017). In fact, after the establishment of new extraction and identification techniques, Ivleva et al. (2017) found a rapid increase of very small MPs (<15 μm) and concluded that very small MPs and nanoplastics (<1 μm) might be of higher concern than larger MPs (Ivleva et al., 2017). Smaller MPs and nanoplastics are thus likely to accumulate in food webs at amounts higher than anticipated and might deliver higher amounts of POPs than previously assumed, especially because of higher surface-to-volume ratios.
An issue of major concern is the fact that part of the communications published in recent years applied questionable methods and exposure scenarios and may thus have produced false-positive effects. One example is Lu et al. (2016), who showed MP accumulation in fish gills, liver and intestines (Lu et al., 2016). In fact, especially histological analyses of this paper were criticized by the scientific community (Baumann et al., 2016). Still, this publication is continuously being cited and has become one of the most influential publications in the ECHA restriction proposal for intentionally added microplastics, released in January 2019 (see Annex XV restriction report, Proposal for Restriction on Intentionally added Microplastics, European Chemicals Agency). It is of utmost importance that questionable methods and insecure results in scientific publications are questioned and false outcomes are corrected with additional high-quality work.
Against this background, the present study was designed to analyze MP uptake and resulting effects in fish based on stringent methodology. Both the effects of size and feeding route of environmentally relevant MP concentrations were analyzed with focus on effects in chemical, enzymatic and histopathological analyses. In a long-term 21-day feeding study, 4–6 μm polyethylene (PE) particles were ingested by Artemia spec. nauplii, which were then fed to adult zebrafish (Danio rerio). In contrast to this trophic transfer scenario, a parallel experiment offered 125–500 μm PE particles directly by mixing with dry flake food. Both polyethylene particle types were labelled by the same supplier, suggesting the same composition. All particles were spiked with the same amount of benzo(a)pyrene (BaP) as a model lipophilic contaminant. Thus, size-dependent effects (very small MPs vs. larger MPs) and exposure routes (food chain vs. direct feeding) could be compared directly to test the hypothesis that smaller MPs accumulate at higher numbers in food webs and transfer more POPs to higher trophic organisms than larger MPs. Effects of the different MPs were evaluated via cytochrome P450 1A (CYP1A) induction (ethoxyresorufin-O-deethylase assay, EROD) and histopathological changes in fish liver, gonads and intestine, as well as chemical analysis of BaP in fish tissues. In order to identify potential MP transfer to fish tissues other than the intestinal tract, particular attention was given to good laboratory procedures to avoid cross contamination between organs and artefacts.
Section snippets
Chemicals
All chemicals used were purchased at the highest purity available from Sigma-Aldrich (Deisenhofen, Germany), unless stated otherwise.
Microplastics spiking
Two differently sized low-density polyethylene (LDPE) MPs (4–6 μm and 125–500 μm, Micropowders, Tarrytown, NY, US) were used.
Prior to use, the MP particles were analyzed for background contamination with benzo(a)pyrene (BaP): related analyses were conducted according to Larsson et al. (2013) (Larsson et al., 2013). Two replicates with 0.25 g of plastic were
Results
During the entire duration of the experiment, one fish in one of the replicate tanks for the MP control group of 4–6 μm died with no signs of severe disease; otherwise, no disease- or exposure-related effects were visible in fish. No change in fish behavior was observed.
Discussion
This study was designed to analyze effects of MP size and feeding route in long-term exposure scenarios with fish. In a three-week exposure of adult zebrafish (Danio rerio), MP concentrations of 1% (1 mg in 100 mg dry food) per day were fed via dry food (125–500 μm polyethylene particles) or via live Artemia nauplii (4–6 μm polyethylene particles). Both MP types were purchased from the same provider and spiked with identical amounts of benzo(a)pyrene (BaP; 16 μg/g). Thus, the two sizes and
CRediT authorship contribution statement
Annika Batel:Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft, Writing - review & editing.Lisa Baumann:Methodology, Investigation, Formal analysis, Writing - original draft.Camilla Catarci Carteny:Methodology, Investigation, Formal analysis, Writing - original draft.Bettie Cormier:Resources, Writing - original draft.Steffen H. Keiter:Resources, Writing - original draft.Thomas Braunbeck:Supervision, Writing - original draft, Funding acquisition.
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.
Acknowledgments
This study has been funded by the German Federal Ministry for Science and Research (BMBF) under contract no. 03F0735A, the Belgian BELSPO grant number BR/154/A1/EPHEMARE, the Swedish FORMAS grant number 2015-01865, and the French IdEx grant from University of Bordeaux, all within the “Joint Programming Initiative Healthy and Productive Seas and Oceans” (JPI Oceans) project EPHEMARE (“Ecotoxicological Effects of Microplastics in Marine Ecosystems”).
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