Microplastic ingestion and feeding ecology in three intertidal mollusk species from Lima, Peru

Introduction In 2017, annual global production of plastic reached close to 350 million tons (PlasticsEurope 2018)1, demonstrating its importance within our lifestyle. Plastics are synthetic organic polymers derived from petroleum that are versatile, lightweight, strong and durable, thus making ideally suited for a variety of applications and highly resistant to degradation (Derraik 2002, Rios et al. 2007, Andrady 2011). Plastics are a global issue and is perceived as one of the most severe forms of pollution in shorelines, oceans and freshwater bodies (Li et al. 2016a).


Introduction
In 2017, annual global production of plastic reached close to 350 million tons (PlasticsEurope 2018) 1 , demonstrating its importance within our lifestyle. Plastics are synthetic organic polymers derived from petroleum that are versatile, lightweight, strong and durable, thus making ideally suited for a variety of applications and highly resistant to degradation (Derraik 2002, Rios et al. 2007, Andrady 2011. Plastics are a global issue and is perceived as one of the most severe forms of pollution in shorelines, oceans and freshwater bodies (Li et al. 2016a).
Whilst there is not a scientific standard, microplastics have been attributed with different size-ranges (Cole et al. 2011). The present study refers to microplastics as anthropogenic particles ranging below 5 mm in diameter (Barnes et al. 2009). These are subdivided in two categories: primary microplastics, which are plastics that are manufactured to be of microscopic size (Cole et al. 2011) such as plastic scrubbers in skin cleaners as reported by Lei et al. (2017) and secondary microplastics, as consequence of the breakdown of larger plastic debris by degradation (Cole et al. 2011). The distribution of plastic debris in the ocean is irregular due to local wind and current conditions; it is apparent that microplastics have become both wide-spread and ubiquitous (Barnes et al. 2009, Cole et al. 2011). Due to their size, microplastics are prone to be ingested by marine biota; in addition to potential adverse effects from ingestion, toxic responses could also result from inherent contaminants leaching from the microplastics and extraneous hydrophobic pollutants adhered to the microplastics (Cole et al. 2011). Moreover, it is suggested that microplastics can be transferred within different food webs (De-la-Torre 2020), raising concerns regarding microplastic bioaccumulation and biomagnification in marine biota (Barboza et al. 2018).
Filter feeders are between the most vulnerable species to microplastic pollution in the marine environment. Bivalves are widely used as biomonitors in marine ecosystems due to their global distribution, accessibility and high tolerance to salinity (Li et al. 2016b). More specifically, mussels have been previously proposed by Li et al. (2019) as a global bioindicator of microplastic pollution. On the other hand, Polyplacophora and Gastropoda species are poorly studied. Rock grazers and detritivore species, like most marine chitons and snails, are also exposed to microplastics from the marine environment, therefore compromising their survival. Little is known about the relationship between microplastic ingestion and feeding ecology of coastal marine mollusks.
There is still scarce information assessing microplastic pollution in the marine environments of Peru. Thus, the objectives of the present study were (1) to report the incidence and characteristics of microplastic pollution in three mollusks from the intertidal rocky zone from Lima, Peru; and (2) investigate the relationship between microplastic content and feeding behavior in three different mollusk species.
were wiped clean. Glass and metal materials were used and plastic materials were avoided completely. Sampled organisms were stored in glass containers. For every batch of organisms treated, a blank control (distilled water) and a 10% KOH blank were prepared, vacuum filtrated and analyzed under the microscope.
Results were expressed in particles ind. -1 and particles g -1 (wet weight) ± standard error of the mean (SEM). Kolmogorov-Smirnov and Shapiro-Wilk tests invalidated the normal distribution of the data, thus non-parametric tests were used. Kruskal-Wallis test followed by Dunn's multiple comparisons test were conducted to compare microplastic abundance between mollusk species. Significance level was set to 0.05 for all the analyses. Statistical analysis was performed and graphs were created using GraphPad Prism (version 7.0 for Windows).

Results and discussion
All three mollusk species were contaminated with microplastics. Distilled water and 10% KOH blanks had a mean microplastic concentration of 0.50 ± 0.22 particles blank -1 , similar to Li et al. (2015). All particles in the blanks were identified as microfibers.

Materials and methods
Specimens of three mollusk species were collected from the intertidal rocky zone in Los Yuyos (12°09'11.7"S; 77°01'31.5"W) and Las Sombrillas (12°09'25.4"S; 77°01'35.1"W) beaches of Lima, Peru, both highly polluted with marine litter (De-la-Torre & Laura 2019) and microplastics (De-la-Torre et al. 2020). Importantly, these sites are considered unhealthy by local authorities due to anthropogenic pollution. Two sampling points were considered in the intertidal rocky shore of these locations. Specimens of filter feeder Semimytilus algosus (n= 45), and grazers Chiton granosus (n= 15) and Tegula atra (n= 15) were collected in three sampling campaigns throughout February 2019. Collected specimens were placed in glass containers and stored at -20 °C until further laboratory analysis.
The length and wet weight of each mollusk were recorded (Table 1). The soft tissues were extracted by dissecting the mollusks using a scalpel. For S. algosus three specimens were pooled. Then, the soft organic material was digested using Protocol 1b as described by Dehaut et al. (2016) with minor changes. In brief, the soft tissues were submerged in 15 ml of 10% (w/v) potassium hydroxide (KOH) in Pyrex screw cab test tubes and incubated over night at 60 °C. Digestion was followed by vacuum filtration of the supernatant solution through 20 µm pore glass fiber filter paper (Whatman PLC 122 United Kingdom). Filter papers were placed in closed glass petri dishes until further analysis.
Optical identification of microplastics was performed using an optical microscope (Krüss MBL2000) under 10-40 × magnification. To avoid false positives and negatives, microplastics were identified according to their physical characteristics, structure, color, morphology, and such (Desforges et al. 2014). Glass fibers were identified and discarded according to its description by Davidson & Dudas (2016). Microplastic abundance, type (fibers, beads, fragments and films) and color were recorded. All confirmed microplastics were photographed.
Following Hernandez-Milian et al. (2019), the term microplastic was used for anthropogenic particles smaller than 5 mm. The aim of this study was to investigate the relationship between anthropogenic particles content and feeding behavior in three mollusk types, therefore polymer identification by infrared spectrometry analysis was not necessary (Lusher et al. 2014, Hernandez-Milian et al. 2019. To reduce external contamination, the protocol described by Dioses-Salinas et al. (2020) was followed. In brief, cotton lab coats and latex gloves were worn at all times. All the equipment was rinsed with distilled water and all surfaces Table 1. Shell length and soft tissue wet weight of three rocky intertidal mollusks from Lima, Peru / Longitud de la concha y peso húmedo de los tejidos blandos de tres moluscos del intermareal rocoso en Lima, Perú Spheres and films were not observed in C. granosus and T. atra. Regarding microplastic color, red microplastics were dominant in C. granosus (46.22%) and T. atra (50%), while blue microplastics were dominant in S. algosus (35.23%) (Fig. 1b).
As wastewater treatment plants (WWTPs) play an important role in releasing microplastic to the marine environment (Sun et al. 2019), the proximity to the discharge point of La Chira, the largest WWTP in Lima, and active fishing activity may be the cause of abundant fiber particles, as fibers shed from laundering clothes (Browne et al. 2011) and degrade from fishing nets. Li et al. (2015) reported black, red and blue colors as the most frequent in bivalves, similar to Ding et al. (2018), indicating black, blue and green as the most popular. Indeed, our results have a similar approach to the color proportion reported in literature. Table 2. Abundance percentage of microplastic types in three species of intertidal mollusks. Fibers derive from textile shedding, fragments form from the breakdown of larger solid plastics, spheres are manufactured micro-sized and are found in cosmetics, and films derive from packaging or plastic bags / Porcentaje de abundancia de cada tipo de microplásticos en tres especies de moluscos intermareales. Las fibras se desprenden de los textiles, los fragmentos de la ruptura de plásticos sólidos más grandes, las esferas son fabricadas de tamaño microscópico y se encuentran en cosméticos y los films derivan de plástico de embalaje o bolsas In both sampling sites, T. atra specimens were found gliding on the surfaces of rocks and big boulders, while C. granosus inhabiting rock crevices. In several occasions both species were located close to each other or resting on the same boulder. On the contrary, S. algosus beds were separated by a few meters, colonizing isolated boulders partially buried in the sediment. C. granosus carry out foraging excursions of about 30-40 cm twice a day and has a tendency to come back into the rock crevices (Aguilera & Navarrete 2007), thus indicating a direct interaction with T. atra individuals. In situ observations determined that the foraging excursions by the sampled C. granosus specimens were carried out across T. atra gliding trace. Previous research (Gutow et al. 2019) revealed that gastropod pedal mucus retains suspended microplastics and foraging on the contaminated mucus promotes microplastic ingestion by marine grazers. Consequently, Polyplacophora and Gastropoda species from the intertidal rocky shore are exposed to microplastics adhered to pedal mucus. However, S. algosus are not subject to species interactions that may promote microplastic exposure. Once ingested, microplastics in marine mollusks are expected to scale along the food chain. De-la-Torre et al. (2019) suggested that high microplastic abundance in the gastrointestinal tracts of carnivorous fish were due to ingestion of contaminated mollusks.
It has been observed in other marine gastropod species (Littorina littorea) with similar feeding activity to T. atra that ingested microplastics through contaminated seaweed were mostly released through the feces and do not bioaccumulate rapidly (Gutow et al. 2015). This suggests T. atra may self-depurate from ingested microplastics and provoke a higher microplastic exposure to C. granosus when foraging, thus explaining why results indicated C. granosus as the most contaminated mollusk (Fig. 1a) and the microplastic type (Table 2) and color proportion similarities (Fig. 1b).
In the present study, the first evidence of microplastic ingestion by mollusks from Peru was presented. Microplastic concentrations in three mollusk species and physical characteristics of ingested microplastics were determined. The relationship between feeding ecology and microplastic exposure in three mollusk species was investigated and discussed. Further research must focus on determining the toxicity, chronical effects and physical impacts of microplastics in mollusks.