Ingested microscopic plastics translocate from the gut cavity of juveniles of the ascidian Ciona intestinalis

Abstract Small plastic particles, named microplastics, are abundant in the marine environment and can be ingested by marine organisms. Species with different feeding strategies can be differently affected by the presence of microplastics. Moreover, the impact of these particles can depend on their size. In this study, we analyzed the effects of 1 µm polystyrene particles on larval and juvenile development in the ascidian Ciona intestinalis. As previously reported for 10 µm beads, smaller particles caused a delay in the growth of juveniles, even if this delay was registered only at the highest concentration tested. Instead, larval development was not affected by the presence of microplastics. Histological analysis of juveniles revealed that 1 µm particles, after ingestion, can translocate from the gut to the hemocoelic cavity in just 8 days. As a defense mechanism, plastic spheres can also be phagocytized from specific circulating cells with phagocytic activity. Microplastics confirmed their potential as a threat to marine wildlife, interfering with food uptake and growth.


Introduction
Microscopic plastics, 1 µm-1 mm diameter (microplastics, MPs), are abundant and widespread marine pollutants of increasing environmental and economic concern. They derive from the fragmentation of larger plastic debris, such as plastic bottles and bags, and may also be directly produced by cosmetic industries (O'Brine & Thompson 2010;Browne et al. 2011;Davidson 2012;Napper et al. 2015).
Different species display disparate aptitudes in ingestion and retention of MPs, as demonstrated by the differences in stomach content reported in various invertebrates sampled in the same locality. For example, Ascidia spp. specimens retained a number of MPs five-fold higher than bivalve species (Crassostrea gigas; Mytilus galloprovincialis; Anomia ephippium) (Bonello et al. 2018).
In fact, the ability to select particles varies among species and depends on their buccal specialization and feeding mechanisms. No effect of MPs on survival and growth has been reported when crustaceans or fish, which have selective feeding strategies, have been analyzed (Fernández 1979;Paffenhofer & Vansant 1985). Similarly, polychaete worms are able to ingest and expel plastic microspheres without any apparent detrimental effects (Cole et al. 2011). On the contrary, filter-feeders are more sensitive to microplastics pollution as filter-feeding is a less selective strategy than predation (Gallo & Tosti 2015;Rummel et al. 2016).
The effects of MPs oral uptake vary among taxa ranging from damaging and blocking the feeding appendages and the digestive system (Laist 1997;Derraik 2002;Murray & Cowie 2011), to limiting the food intake and ultimately causing fertility reduction (Mato et al. 2001;Oehlmann et al. 2009;Talsness et al. 2009;Teuten et al. 2009). The size of microplastics plays an important role in determining their effects on marine organisms. The crustacean Mysis relicta showed different performances in ingesting MPs of different sizes, the smallest (0.75 µm) and largest (40-50 µm) being less favored than intermediate sizes (Bigelow & Lasenby 1991). Moreover, it has been demonstrated that large microplastics (>10 µm) accumulate in the digestive tracts and are eventually discarded with the feces (Cole et al. 2011), while only few studies have investigated whether small microplastics (<10 µm) can translocate from the gut cavity to the circulatory system and body tissues. Mussels (Mytilus edulis) exposed to 3 µm and 9.6 µm polystyrene beads for 3 days presented MPs particles of both sizes in their circulatory system, where they persisted for over 48 days (Browne et al. 2008). This long permanence in the animal body greatly enhanced the possibility of trophic transfer throughout the food web and hindered the efforts to elucidate MPs fate and impacts on the marine ecosystem.
Ascidians are filter-feeding organisms evolutionarily close to vertebrates (Delsuc et al. 2006), and they are an important component of benthic assemblages worldwide (Zega et al. 2009). Adults and juveniles of the ascidian Ciona intestinalis employ the pharyngeal basket to filter a huge amount of water per day, estimated around 46.4 ml/min for adults with a total dry weight of 0.84 g (Randløv & Riisgård 1979), and they appeared to be unable to discriminate between food and inorganic particles (Messinetti et al. 2018). In a previous paper, we tested the effects of 10 µm diameter polystyrene beads in C. robusta and demonstrated that exposure to these MPs was not detrimental during larval and juvenile development. In laboratory conditions, juveniles could efficiently ingest them even when they are present at low concentrations (0.125 µg/ml). At high concentrations (12.5 and 25 µg/ml), 10 µm MPs persisted in the digestive system and affected the growth-rate during metamorphosis, probably by decreasing the energy intake (Messinetti et al. 2018).
In this paper, we tested the effects of small polystyrene MPs (1 µm diameter) on larval and juvenile development of C. intestinalis in order to compare their effects with those obtained with larger MPs. Moreover, we tested whether small MPs can translocate in the circulating fluids.

Microplastics
Polystyrene spherical microparticles with a dark blue color and a diameter of 1 µm were used in the experiments; chemical and physical properties of the MPs were provided by the supplier (Sigma, Milano, Italy). The blue color allowed us to track the beads inside the transparent-tested animals. Polystyrene microbeads were preferred as they determine a negligible amount of styrene release when in aqueous suspension (Cohen et al. 2002). The commercial standard, an aqueous suspension of 50 mg particles/ml, was diluted 1:1000 in filtered seawater (FSW) to produce a stock suspension of 50 µg/ml of beads from which the final exposure suspensions were made. All the suspensions were freshly prepared each time and sonicated for 10 min before use to ensure a homogenous distribution of the beads in the medium. Based on previous work (Messinetti et al. 2018), four different MPs concentrations were tested: 0.125, 1.25, 12.5, and 25 µg/ml.

Ascidians
Adults of Ciona intestinalis were collected in the water near Roscoff (France) and maintained in aquaria at 18 ± 1°C. Constant light condition was preferred to promote gamete production and avoid spawning (Lambert & Brandt 1967). For each experiment, at least three adults were sacrificed. Eggs and sperms were obtained by dissection of gonoducts and crossfertilization was performed in vitro. Embryos were cultured at 18 ± 1°C in FSW until they reached the desired developmental stages (see below).

Development and survival rate
To test the effects of MPs presence on embryonic development, 30 embryos at two-cells stage (Hotta et al. 2007) were exposed to various bead concentrations in FSW. Samples were reared at 18 ± 1°C. Larval survival rate was evaluated when control embryos (CO), maintained in FSW, reached the hatching larva stage (,18 h post fertilization (hpf); Hotta et al. 2007). Each experimental group was carefully observed under a stereoscope, and the percentage of alive samples was calculated as: (number of alive sample/total exposed samples) x 100. Each experiment was performed in triplicate.

Metamorphosis
To determine the effects of small MPs on metamorphosis, 30 larvae for each treatment were transferred into 5.5 cm Petri dishes and allowed to attach to the substrate. After adhesion, FSW was replaced with the testing suspensions (0.125, 1.25, 12.5, and 25 µg/ml microbeads in FSW). Control animals (CO) were maintained in fresh FSW. One hundred microliters of a concentrated suspension of algae were added to each treatment group. The media were changed every day with freshly prepared ones. Animals were left to develop in the experimental conditions for four days. Then, each individual was observed under a stereoscope to estimate the proceeding of juvenile development. Metamorphosis in C. intestinalis proceeds following a series of events (Chiba et al. 2004), mainly consisting in tail reabsorption, organs rotation, and development of protostigmata or gill slits. By day 4, juveniles normally reach stage 4, characterized by completed organs rotation and the presence of two pairs of protostigmata. Metamorphosis is completed by day 14 (stage 8) when the two atrial siphons fuse together, and the organs and tissue are almost the same as those of the adult (for a comprehensive description of Ciona metamorphosis process see Chiba et al. 2004).
To evaluate MPs effects on metamorphosis, we assigned a developmental stage, roughly corresponding to those described in Chiba et al. (2004), to each individual, mainly evaluating organ rotation and the dimension of the axial complex (Messinetti et al. 2018). We also counted animals that adhered to the dish and died soon after. Each experiment was performed in triplicate.

Tracking the uptake of polystyrene microbeads
To determine whether polystyrene microspheres had accumulated in the gut, juveniles exposed for 8 days to 25 µg/ml microplastics were fixed in 4% paraformaldehyde in phosphate buffer and sectioned for detailed localization of microbeads. Briefly, animals were dehydrated in ethanol series, stained in alcoholic eosine and embedded in Technovit resins (Heraeus Kulzer, Werheim, Germany). Five micrometer sections were cut with a microtome, counterstained with ematossilin and mounted with entellan (Merck, Whitehouse Station, N.J.). Samples were observed under a light microscope and photographed using a Leica DFC-320-C camera.

Statistical analyses
To evaluate if the different MPs concentrations significantly affect animal survival and development, the analysis of variance (ANOVA), followed by HSD Tukey's post hoc test, was performed using R software (R-Core-Team 2018) and "agricolae" package (de Mendiburu 2015). A Cochran test was performed to test the homogeneity and normality of the variances and percentage data were transformed when they did not meet the assumptions of the analysis (normality and homoscedasticity).

Results
Exposure of ascidian embryos to different concentrations of 1 µm MPs from two cells to larval stage did not affect development and larval survival ( Figure 1A; ANOVA: F = 0.9721, p = 0.4644). Under laboratory conditions, 4 days after attachment, more than 90% of control juveniles reached stage 4 of development, characterized by completed organs rotation and the presence of two pairs of protostigmata. The percentage of juveniles that reached stage 4 after exposure to the MPs decreased with increasing bead concentrations and was significantly different between samples exposed to 25 µg/ml MPs and control juveniles ( Figure   The transparency of ascidian juveniles allowed to clearly observe the gut content in whole mount specimens (Figure 2A). Juveniles were able to efficiently ingest small microplastics, that appeared clearly visible in their gut cavity, and to egest them in the fecal pellets ( Figure 2B). When both MPs and algae were present in the medium, juveniles were not able to discriminate between them. Algae and MPs were both present in the gut cavity and in the fecal pellets of exposed individuals ( Figure 2C).
Histological analyses showed that ascidian juveniles exposed to microparticles had accumulated polystyrene microspheres in their gut cavity (Figure 3). The microparticles were easily recognizable for their shape, color, and dimensions. They were found in the pharynx, the esophagus, the stomach and the intestine ( Figure 3A-D). Some beads were present in the cytoplasm of fusiform cells localized in the wide hemocoelic cavity, characteristic of ascidian juveniles ( Figure  3C). These cells presented dark granules in their cytoplasm, of ,1 µm in diameter ( Figure 3C'', C''') and,  based on these characteristics, they were identified as granular amoebocytes with phagocytic activity (Rowley 1982). The microbeads were particularly abundant in the stomach where they appeared densely packed ( Figure 3D). Most interestingly, some microparticles were found outside the gut cavity, in the extracellular fluid, next to the stomach wall ( Figure 3D).

Discussion
The present work demonstrates that juveniles of Ciona intestinalis can efficiently ingest 1 µm microplastics which accumulate in stomach and intestine.
The presence of MPs in the gut delayed juvenile development probably due to lower food intake and insufficient energy supply. Control individuals, fed with algae could obtain all the energy necessary for the development, while juveniles that co-ingested MPs and algae obtained a lower amount of energy from their feeding activity, as their stomach was filled with MPs, and the amount of ingested algae was drastically reduced. Our data are consistent with those previously reported exposing ascidian juveniles to larger (10 µm) plastic particles (Messinetti et al. 2018). However, the effects on juvenile development were more severe when animals were fed with large MPs, probably because smaller ones can be more easily expelled. Altogether these results confirm that underfeeding may be a detrimental consequence of microplastics presence in the marine environment for filter feeder invertebrates.
Though MPs have been shown to accumulate in the gut cavity of several marine animals, particles translocation from the gut to the circulatory system is still poorly investigated in invertebrates. In our study, particles of polystyrene translocated from the gut cavity to the internal extracellular compartment in only 8 days. Particles translocation can occur through two alternative pathways: the paracellular pathway, implying the passage between cells, through intercellular junctions and spaces, and the transcellular pathway, involving the absorption of particles by the enterocytes, which release them through the basolateral membrane (for a review see Carr et al. 2012). In histological sections, we never observed MPs inside the enterocytes but always in strict contact with the gut basal membrane so that we cannot exclude either of the alternative mechanisms. Further works are required to elucidate the mechanism by which MPs cross the gut barrier and move to the internal compartment.
After translocation, phagocytosis may play an important role in recognizing MPs as nonself particles and in clearing them. In fact, we observed MPs inside the cytoplasm of fusiform circulating cells, probably phagocytes. In ascidians, several blood cell types were identified, including macrophage-like cells with large vacuoles and devoid of amoeboid activity and amoebocytes of the hyaline/microgranular type involved in phagocytosis (Ballarin et al. 1994). Granular amoebocytes are readily recognizable as they are the only amoebocyte type that contains large granules (0.5-1.5 µm in diameter; Rowley 1981). Since the MPs containing cells have also dark cytoplasmic granules, they could be reasonably identified as granular amoebocytes.
In the colonial ascidian Botryllus schlosseri, hyaline cells can ingest latex granules of 1 and 3 µm in diameter, confirming the capability of ascidian phagocytes to internalize particles of that size. Granulocytic hemocytes of Mytilus galloprovincialis are able to phagocytize polystyrene particles up to 800 nm in in vitro trials (Cajaraville & Pal 1995) suggesting that the surface properties of polystyrene beads can be recognized by invertebrate phagocytes.
Considering our results, we cannot exclude the possibility that ingestion and/or translocation of plastic into the animal body may induce toxicological effects. We treated the animals only for 8 days with one type of plastic particles; in the environment, animals are exposed to different kinds of particles characterized by different chemical properties. Plastics can potentially release various types of contaminants, especially the additives used in their production (Jang et al. 2016). Moreover, persistent organic pollutants may accumulate on plastic fragments and plastic pellets (Rios et al. 2007), possibly leading to further adverse effects after particles ingestion.
Therefore, future studies should be addressed to survey the toxicological effects induced by long-term exposure to various plastic particles usually found in marine habitats.

Funding
The ASSEMBLE PLUS Project funded by European Union's Horizon 2020 research and innovation programm under the grant agreement No 730984 Contract number SR010220187086.

Disclosure statement
No potential conflict of interest was reported by the authors.