Exposure of marine mussels Mytilus spp. to polystyrene microplastics: Toxicity and influence on fluoranthene bioaccumulation☆
Graphical abstract
Introduction
Pollution of the oceans by microplastics, defined as plastic particles of size below <5 mm (NOAA, 2008), originate from the accidental release of primary manufactured plastic particles of micrometric size used in many industrial and household activities (blasting, exfoliates, toothpastes, synthetic clothing), as well as from the fragmentation of larger plastics in the environment (Andrady, 2011). Quantitative studies on micro-debris in open oceans and in intertidal zones in the vicinity of industrial cities have confirmed the ubiquitous nature of microplastics (Eriksen et al., 2014). According to these authors, microplastics represent more than 92% of the total plastic debris (>0.33 mm) floating at sea, estimated at 5.25 trillion particles in worldwide marine environments. Ingestion of microplastic by marine organisms leading to substantial impacts on major physiological functions such as respiration, nutrition, reproduction, growth and survival has been shown in marine vertebrates and invertebrates (for review see Wright et al., 2013). In addition to physical injuries, the ability of microplastics to efficiently adsorb persistent organic pollutants (POP) has led to an increasing concern related to a potential role of microplastics as vector of POP into marine organisms (Cole et al., 2011, Ivar do Sul and Costa, 2014, Koelmans et al., 2014). Desorption of POP from microplastics was demonstrated to be enhanced under in vitro simulated digestive conditions (Bakir et al., 2014). In vivo experiments conducted on fish (Oliveira et al., 2013, Rochman et al., 2013), mussels (Avio et al., 2015) and lugworms (Besseling et al., 2013) revealed the transfer of chemicals after ingestion of contaminated microplastics, as well as combined effects of both contaminants on neurotransmission, energy production and oxidative metabolism. However, recent studies questioned the importance of such transfer in natural conditions given (i) the baseline contamination levels of seawater and marine organisms and (ii) the low proportion of microplastics in comparison with other suspended particles (organic matter, plankton, detritus, etc.) capable of transferring pollutants probably more efficiently due to their higher abundance in marine ecosystems (Herzke et al., 2016, Koelmans et al., 2016). Therefore, laboratory studies aiming to understand the relative sorption of POP to microplastics in comparison to other occurring media in marine ecosystems are needed to clarify their respective role as vector of organic pollutant for marine organisms.
The present study aims to investigate experimentally (i) the affinity of fluoranthene (FLU) for polystyrene microparticles (micro-PS) in comparison to phytoplankton by assessing its partition among seawater, micro-PS, and marine algae Chaetoceros muelleri used as a food source for mussels; (ii) whether the presence of loaded micro-PS alongside with contaminated algae and seawater may affect FLU bioaccumulation and depuration in marine mussels Mytilus spp., a common biological model in ecotoxicological studies (Kim et al., 2008); and (iii) the effects of micro-PS exposure alone or in combination with FLU on various physiological parameters at tissue, cellular and molecular levels to provide a comprehensive assessment of pollutant-related effects (Lyons et al., 2010). Fluoranthene was selected as (i) it is a model PAH belonging to the list of priority substances in water policy of the European Commission (Directive 2008/105/EC) and (ii) it constitutes one of the most abundant PAH found in the aquatic environment and in molluscs (Baumard et al., 1998, Bouzas et al., 2011). It is noteworthy that in most of the cited studies, as well as in our work, animals were acclimatized and then reared in “clean water” (seawater filtered on active carbon filters in our case) and exposures were performed in clean and controlled laboratory conditions. This is far from what may happen in natural environments where a wide range of confounding factors is likely to occur (and influence for instance the interaction between fluoranthene and polystyrene microplastics). However, due to the high complexity characterizing natural environments, controlled laboratory experiments remain necessary as a step by step approach for understanding processes, to assess the weight of each factor (in this case microplastics, food and fluoranthene) and sort out complexity of environmental pollution.
Section snippets
Mussel collection and acclimatization
Mussels (58.6 ± 9.6 mm, mean ± SD) were collected at the Pointe d’Armorique in the Bay of Brest (48°19’20.29″N, Brittany, France), a site known to exhibit low PAH concentrations (Lacroix et al., 2015). The sampling site is located within a zone of overlap between Mytilus edulis and Mytilus galloprovincialis (Bierne et al., 2003), the mussel population is thus considered as a “species complex” (Lacroix et al., 2014a), and is referred to as Mytilus spp. Mussels were acclimatized in a flow-through
Total RNA extraction and cDNA synthesis
An aliquot of 50 mg of grounded tissue was homogenized in 0.5 ml of Tri Reagent (Ambion) using a Precellys®24 grinder coupled to a Cryolys® cooling system (Bertin technologies) for total RNA extraction. An aliquot of 40 μg RNA was then treated with the RTS DNase™ Kit (1U/3 μg total RNA, Mo Bio). RNA purity and concentration were measured using a Nanodrop spectrophotometer (Thermo Scientific) and RNA integrity was assessed using RNA nanochips and Agilent RNA 6000 nanoreagents (Agilent
Statistical analyses
All quantitative variables were analyzed using a two-way ANOVA in order to determine possible interactive effects between the two independent variables called factors (microplastics and fluoranthene) on each parameter that constitutes the dependent variable (Sokal and Rohlf, 1981). Normality was assumed and homogeneity of variance was verified with Cochran’s test (data were log10 transformed when homogeneity of variance was not achieved). Percentages of phagocytic and of dead hemocytes were
Fluoranthene partition in algal cultures
No difference was observed between 45 min and 5 h in the quantity of FLU measured in each fraction (F2, Fd and Fp) suggesting that sorption equilibrium occurred. In the FLU condition (i.e. no micro-PS added in the algal culture), fluoranthene was mainly associated with algae (89%) in comparison with the fraction of FLU dissolved in water (11%) (Table 2). Algae exhibited a Log KpA of 4.84. In micro-PS + FLU condition, the fraction of FLU dissolved in water was similar (12%) but the fraction of
Micro-PS exhibited high sorption capacity for fluoranthene
The present study evidenced that micro-PS exhibited higher sorption capacity for fluoranthene than marine algae C. muelleri as indicated by the partition coefficient log Kp values, and this confirmed a strong affinity of fluoranthene for polystyrene, especially when considering the relative mass proportion of algae and micro-PS fed to the mussels (289:1) in the context of our study. Polyethylene (PE) and polyvinylchloride (PVC) also demonstrated high sorption capacity, as expressed with Log Kp
Conclusion
Despite a high sorption of fluoranthene on micro-PS, this did not enhance fluoranthene bioaccumulation in the specific conditions of this experiment; i.e. when mussels were also exposed to fluoranthene via water and micro-algae. Micro-PS concentration used in this study (0.032 mg L−1) was lower than those used in most studies on marine invertebrates (range 0.8–2500 mg L−1) (Avio et al., 2015, Besseling et al., 2013, Wegner et al., 2012, Von Moos et al., 2012) and was in the range of the highest
Acknowledgements
This study was partly funded by the MICRO EU Interreg-funded project (MICRO 09-002-BE). The authors are grateful to M. Van der Meulen, L. Devriese, D. Vethaak, T. Maes, and D. Mazurais for their valuable support and helpful discussions and also the anonymous reviewers of the paper who greatly helped improving the scientific quality of the manuscript. We also thank Pr R. Whittington for his helpful revision of the English and his comments on the manuscript.
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