Humans produce hundreds of millions tons of plastics each year^[1,2]. A large portion of them is transported at sea via winds^[3], rivers^[4] and wastewaters for the smallest ones^[1] or is dumped or lost by commercial, fishing and recreational boats^[2,4], to the point that plastics dominate debris found at sea^[4]. The yearly increase in numbers of large plastics at sea contributes to the increase of microplastics amounts^[5]: nowadays, microplastics may represent the greatest part of marine plastics^[6].

     Microplastics (MPs) are small plastic particles^[3,5,6] that have received interest as environmental pollutants since around two decades^[3,5]. Nowadays, MPs are a major global marine pollution issue^[4,7]. They are everywhere, from shorelines^[1,3,5] and beaches worldwide^[4,8] to intertidal^[1] and deep sea sediments^[5,9], ocean gyres^[3,5,10,11] and even Arctic^[5] and Antarctic sea ice caps^[10] although we do not know to what extend these environments are contaminated^[1]. There are two types of MPs^{[3,5,6,10,12]}: primary MPs^[10,12] are pre-designed micrometer-size particles^[5,7] widely used in cosmetics^[3,6,13] especially in facial cleansers^[14], in medicine^[3] or as raw material and abrasives in industry^[13]. In contrast, secondary MPs come from the degradation of macroplastics in the wild^[3,12]. Due to a lack of consensus between scientists^[3,5] their size range is defined as extremely wide, from 1.6 μm^[3] up to <5mm in diameter^{[5,6,7,12,13,14,15,16]}. MPs come as beads, granules or microfibers^[3,5] that reduce in size overtime due to abrasion (for example wave-turbulence and sand^[5,13,14]) and UV photo-oxydation^{[3,5,10,13,14]}. Nylon^[3] lost at sea with fishing nets^[10], polyvinylchloride (PVC)^[3,12], polyester^[3,13] and high-density polyethylene^[13] produce heavy particles that sink to the Ocean bottom^[10]. MPs made of buoyant materials like polypropylene^[3,12,13], polystyrene and polyethylene^[3,14] usually become colonized by microorganisms and invertebrates overtime, which makes them denser than water^[3,10]; therefore, a great part of buoyant MPs eventually sinks and accumulates in sediments^[7,9,10] for hundreds of years^[4]. Thus, sediments contain a larger MP volume than sea surface^[5].

    Demersal fish^[17] and benthic invertebrates such as deposit-feeding polychaete worms, barnacles, amphipods^[18], crayfish^[19], bivalves^[13,20,21], crabs, bryozoans^[13], sea cucumbers^[12] and even coral polyps^[22] ingest MPs in the wild or in laboratory. Many species target specific particle sizes or shapes, so MP are likely to be ingested provided their size is similar to these species' preys^[12]. Also, various studies mention MP consumption by a wide range of planktonic animals and larvae in laboratory^[23,24,25] and by pelagic fish^[17]. Moreover, the transfer of MPs from preys to predators is possible^[20,26]. However, there is no information about non-trophic interactions between MPs and organisms.

    MPs can be harmful. Translocation, or the transfer of plastic particles from gut to other inner organs through the epithelium^[21], exists in mussels^[12,21], rodents and humans^[3]. MPs can last inside bodies for weeks^[21], and Setälä et al.^[26] suggest that delayed MP egestion could enhance possible chemical intoxications with pollutants adsorbed or included in them. Indeed, MPs can adsorb hydrophobic contaminants from water such as Persistent Organic Pollutants (POPs)^[27,28] and heavy metals^[29], concentrating them several times more than ambient waters^[28]. Also, MPs released these poisons into the polychaete worm Arenicola marina in laboratory once ingested^[15]. On the other hand, pollutants used as plastic additives and named "plasticizers"^[3,10,16,30] can also leach from MPs, causing potential harm to organisms if MPs are eaten^[3,28]. Besides, in theory, it is possible that granules and especially fibres^[12] stay blocked into digestive tracts of animals, impairing digestive functions^[7,12,22,26]. These are all consequences of MP ingestion, however, no information exists about effects of a prolonged external close contact with MPs.

    The extend and effects of MP interactions with benthic, sedentary or sessile organisms is not fully understood^[7,12]. Yet, the noxiousness of MPs may depend on species and their environment^[22]. Besides, macroplastics already in oceans will keep fragmenting into more and more MPs overtime^[5]. Therefore, in the context of climate change, chemical pollution and other various threats affecting marine wildlife, assessing the dangerousness of MPs for all marine organisms becomes vital^[4]. It is especially true for taxa with crucial ecological roles like polychaetes^[7,31].

    Polychaete worms (Annelida, Polychaeta) play a major role in many benthic communities as suspension- or deposit feeders, or as preys for various taxa^[7]. Cirratulids (Palpata, Canalipalpata, Terebellida, Cirratuliformia, Cirratulidae^[32]) are ubiquitous polychaetes that can be dominant in certain benthic communities^[32,33]. They can be millimeters to centimeters long^[32] and often live buried into soft sediments^[33]. When buried, their gills are often extended to the sediment-water interface^[32]. They feed on surface microdetritus^[32,33] by use of their proboscis or their palps^[32] (Figure 1). They play a crucial role in particle transfers from the upper part of sediments to lower layers by feeding on the water-bottom interface and defecating in their burrows, thus influencing the chemistry and ecology of sediments^[33]. Importantly, cirratulids and generally deposit-feeders are highly selective on particle size for feeding^{[12,31,32,33,34]}, however, they do not discriminate between natural and artificial particles^[33]. Thompson et al.^[18] proved thereby that A. marina eats MPs 20 μm to 2 mm in size. Moreover, Shull & Yasuda^[33] demonstrated Cirriformia grandis ingests glass beads 16 to 32 μm in diameter.

    Burrowing polychaetes, notably terebellids, build a mucus layer lining their entire burrow that could serve as a cement or as a support for symbiosis with bacterial communities ^[34]. This mucus is mainly composed of lipids, sugars and mucins (glycoproteins) with highly conserved chemical groups across animal phyla such as thiols^[34,35], so Cirriformia sp. probably produces a very similar mucus. It may help purifying organisms from environmental contaminants by binding proteins containing thiol groups and by being regularly shed^[35]. In fact, Mouneyrac et al.^[36] found that worms from heavy-metal contaminated areas were secreting significantly more mucus compared to those from pristine environments.

    Effects of MPs have been studied on errant polychaetes^{[7,12,15,18,37]} but never on cirratulids. Therefore, this study investigated the effects of MP size and external contact on Cirriformia sp., a cirratulid worm abundant in sediments of aquaria of the University of Queensland. I tested three bead sizes from the size range known to interact with Cirriformia species (10 and 20 μm)^[33] and other polychaetes (40 μm)^[18]. For ensuring sufficient skin contact with MPs, I set a much larger concentration than in other studies^[7,13,15,18] that may not exist in the wild. This study tested the effects of MPs on mucus production levels as an increase in mucus volumes could mean an intolerance to MPs, and induce a considerable loss of energy. Also, I arbitrarily used survival and worm motility, ability to dig, stay buried, feed and breath as indicators for energy levels and overall health state. 

   

    I identified the worms as Cirriformia sp. under microscope (Nikon Eclipse E200) and dissecting microscope (Olympus SZX9) with an interactive identification key^[38] and a book on Cirratulidae^[32]. I used the following physical characters for identifying the Cirriformia genus: absence of a tube, shape of the prostomium and the pygidium, head shape and mouth position, smooth epidermis, presence and distribution of branchiae, presence of longitudinal grooves on palps, absence of pygidial appendages, presence of aciculae^[38], body colour and the presence of curved spines on chaetigers^[32].

    I found no mention of asexual reproduction in the Cirriformia genus so I collected all worms in the same aquarium. All the worms were moving and apparently healthy when taken. I also collected sediment from this aquarium for use in the experiment. I did not put worms back into the aquarium after MP exposures to avoid MP contamination.

    a. Setup

    I allocated three wells per treatment (10, 20 or 40 μm microbeads mixed to sediments) and three wells as controls (no plastic in the sediment) in two Greener 6-well culture cells (Figure 2). I removed any unwanted worm from the sediment and I took out most of the water from it by gravity. I did not dry it because I wanted to keep its meiofauna alive for the worms to feed on. Then I added a homogenized ≈33% v/v bead-sediment mix (using polyethylene MICROBEADS Spheromers CA10, CA20 and CA40 beads, respectively) to the wells up to ≈50% of their height. I filled each well with seawater from the worms' aquarium, drop by drop for preventing bead resuspension and differential decantation of the mixes. I added one worm in each well. I covered them with a lid and I let them at room temperature for one week (oxygen can pass through the lids).

    b. Worm survival

    I described living worms as buried, breathing or feeding with palps outside sediments and moving when touched. In contrast, I considered dead the worms staying outside sediments, not feeding, not reacting to any contact and not trying to burrow, or those with decomposing bodies.

    c. Microscope calibration & measurement of mucus volumes

    I calibrated the micrometers of the microscopes at the objective x10 using the actual sizes of MP beads before estimating mucus volume around worms (in μm³) (correction of x1000 to obtain actual volumes). I estimated mucus volumes at the objective x10. I referred to the depth of field and to worms' diameters for having an idea about mucus thickness (expected to be a rough estimate). I measured both clean and bead-contaminated mucus volumes (for controls, clean and muddy mucus volumes). I did not take sand particles in account for volume estimations.

    d. Measurement of relative energy levels

    I set a relative energy level scale in which: 3 means the worm is buried and gills or palps are visible out of sand and it reacts when I remove it from the sediment; 2 means the worm is out of sand but neatly moving when touched; 1 means the worm is out of sand and barely moving when touched; 0 means the worm is dead.

    I corrected total mucus volumes with the calibration factor on Excel, and performed the statistical analyses on R 3.3.0. I verified the homogeneity of variances in data distributions using Bartlett's K-squared test and the gaussian distribution of residuals using Shapiro tests (α=0.05). A Kruskal-Wallis test investigated the effect of plastic contamination on relative energy levels per treatment (Shapiro: P<0.05). Also, I investigated the effect of MP size on mucus production with a One-way type I ANOVA (α=0.05) (Bartlett's test: P=0.56, Shapiro test: P=0.07). Variances among energy levels distributions being significantly different, I could not perform any ANCOVA on total mucus volumes vs. energy levels depending on treatment effect.

    The experiment consisted in studying effects of three different MP bead sizes (10, 20 and 40 μm in diameter) on mucus secretion by Cirriformia sp. In all the treatments, all the mucus produced by each worm was bound either to microplastics, mud and sand particles, or to mud and sand in controls, thus forming cloud-like build-ups. So even in controls there was no clean mucus around the worms. Therefore, I only investigated whether worms produced more mucus in environments contaminated by MP.

    Worms in the three contaminated environments did not produce significantly more mucus, either compared to each other or to uncontaminated controls, at 95% of confidence (ANOVA F_3,8=1.42, P=0.307) (Figure 3). Therefore, microplastic size does not explain variations in mucus production. Mean mucus volumes produced per treatment and their standard errors are presented in Figure 4.

    Each worm had at least one large sand particle embedded in its mucus, especially when coiled. However, branchiae, palps, heads and mouths were always clean and free of MPs. Besides, worms moved to sediment-free environments either remained in their mucus clouds, or shedded them very quickly and efficiently.

    The goal was to investigate effects of the three different MP sizes chosen (10, 20 and 40 μm in diameter) on Cirriformia sp. vitality. There was no significant difference in worms' relative energy levels between the three contaminated environments or compared to controls, at 95% of confidence (Kruskal-Wallis=1.267, P=0.74) (Figure 5). Besides, the weakest individual (energy level = 1) was found in a control, so the control had the lowest mean (Figures 5 and 6). 

    All the worms survived in all the treatments and in controls. So none of the MP sizes tested can kill Cirriformia sp. after one week. This result must be tested for longer exposure times by setting experiments at least three or four weeks long and by using much more replicates and worms per treatment to increase the statistical power (beyond the possibilities of the course).

    The results suggest that Cirriformia sp. individuals naturally produce considerable amounts of mucus (see Figure 4). Clean mucus was absent from controls, so the mucus is naturally very sticky. Besides, heads and gills were always clean, suggesting worms naturally bind every type of particle around them with no consequences on breathing or feeding. The fact that worms could actively shed their mucus strengthens this conclusion. Therefore, the results indicate that MP beads do not affect mucus production in Cirriformia sp. more than any other kind of natural particle.

    However, the fact that the mucus seems to attach plastic and natural sediments equally efficiently could be potentially harmful in the wild. Indeed, Lalonde et al. proved that this mucus binds heavy metal cations like cadmium ions very durably due to the easy ionization of its functional groups (e.g. thiols) into anionic compounds at seawater pH^[34]. MPs can adsorb and concentrate POPs^[27,28] and heavy metals, and may release plasticizers^[3,28]. Therefore, if MPs stick strongly to worm mucus, there is a possibility for heavy metals to concentrate in it and to accumulate in the environment near burrows when worms shed their mucus layers. Moreover, mucus lipids^[34] could bind hydrophobic contaminants, for example POPs, with similar consequences. If such a mechanism was confirmed in Cirriformia sp., it could facilitate MP-bound heavy metal and other pollutants burial into sediments with unknown effects on these worms, bacterial communities and meiofauna. From an other perspective, it would also decrease heavy metal concentrations in benthic waters. Also, in this experiment worms could attach much larger sand particles than the MP sizes used; so the mucus of Cirriformia sp. is likely to stick to much larger MPs potentially carrying considerable quantities of contaminants.

    Even at much higher MP concentrations than in previous studies (Thompson put A. marina in sediments contaminated at 1.5 g MPs/L^[18]), worms in average had a similar vitality level in contaminated and pristine environments; also, there was no significant energy level difference between worms treated with different MP sizes; besides, the weakest worm belonged to controls. Therefore, selected MP sizes have no externally visible acute effect on Cirriformia sp. These results are coherent with the null impact of MP size on survival.

    However, this experiment does not fully prove the innocuousness of MPs on Cirriformia sp., for four main reasons. First, because of time constraints I could only assess worm health based on specific, measurable indicators: survival, motility and behavioral traits. They do not reflect the impact of MPs on worms' body surface at a cellular or molecular level.

    Also, the microbeads I used were devoid of POPs or heavy metals, so the worms were not exposed to these pollutants. Besides, even if MPs used may have contained plasticizers, they may have lacked time or suitable conditions for releasing them. Therefore, at sea and at longer (or chronic) exposures, these MP sizes could still have a harmful effect on Cirriformia sp.

    Cirriformia sp. is microphagous^[32] and likely size-selective according to previous studies on the Cirriformia genus, on Cirratulidae and deposit-feeders^{[12,31,32,33]}. Moreover, the bead size range eaten by Cirriformia grandis^[33] partly falls into the size range set for the experiment on Cirriformia sp. Consequently, this species may ingest MPs of the size range set in this study. However, I could not test whether worms ate MPs during the experiment. MP ingestion could induce a wide range of health issues I had to overlook in my study on Cirriformia sp. Indeed, von Moos et al.^[13] deduced that MPs could be either physically damaging or intrinsically toxic, from inflammations provoked in mussel tissues by additive-free beads comprising the size range set in my experiment (0-80 μm in diameter) and of the same material as in my study (polyethylene). Besides, 130 μm MPs provoked severe energy and weight losses in A. marina likely because they required much greater efforts and longer time for being digested, and because worms were foraging during significantly less time^[7]. Finally, MPs could potentially clog worms' guts^[3]. Cirriformia sp. might be affected in all these ways. Moreover, MPs coated with hydrophobic POPs and heavy metals or treated with plasticizers could diffuse their pollutants in Cirriformia sp. via gut surfactants and proteins^[31], poisoning the worms as proven for A. marina^[15]. Besides, translocations could occur, further increasing pollutant transfers^[26]. Finally, ingestion of MPs by Cirriformia sp. could result in problems of deep-sediment plastic and pollutant contaminations because burrowed cirratulids defecate at depth^[31,33] and can be very abundant^[33]. This potential issue needs to be studied.

   Finally, I submitted Cirriformia sp. to only one plastic type (polyethylene) whereas a great variety of MPs are present at sea. Whether buoyant or not, all plastic types can eventually reach the benthos^[10]. Plastics may be toxic regardless of their size^[13], so effects of different plastic types on Cirriformia sp. should be investigated as well. Cirriformia sp. individuals are more or less translucent under light microscope; therefore in the future, MP uptake by Cirriformia sp. could be visualized by using fluorescent plastic beads^[23,26] coupled with CARS inverted light fluorescent microscopy^[23]. It would save time by avoiding dissections and prevent outside-in MP contaminations, inevitable with worms covered with such amounts of contaminated mucus.