Uptake and effects of different concentrations of spherical polymer microparticles on Artemia franciscana

Ecotoxicology and Environmental Safety 176 (2019) 211–218

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Uptake and effects of different concentrations of spherical polymer microparticles on Artemia franciscana

T

Diogo Peixotoa,∗, João Amorima, Carlos Pinheiroa, Luís Oliva-Telesa,b, Inmaculada Varóc, Renato de Medeiros Rochad, Maria Natividade Vieiraa,b a

CIIMAR, Interdisciplinary Centre of Marine and Environmental Research - University of Porto, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal Department of Biology, Faculty of Sciences of University of Porto, Rua do Campo Alegre s/n, Edifício FC4 2.47, 4169-007, Porto, Portugal c Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Ribera de Cabanes, Castellón, 12595, Spain d Department of Geography, Federal University of Rio Grande do Norte - UFRN, Campus de Caicó, Rua Joaquim Gregório, s/n, Penedo, CEP 59300-000, Caicó, RN, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Microplastic Artemia franciscana Growth Mortality Reproductive success

Artemia cysts have a huge economic importance for the aquaculture sector due to the fact that they are used as live feed for larviculture. Microplastics (MPs) are common and emergent pollutants in the aquatic environments, with unknown and potential long-term effects on planktonic species such as Artemia spp. When used as live feed, Artemia could transfer contaminants to fish along the food chain, with possible adverse effects on human health through their consumption. This study aims to assess the uptake of different concentrations of spherical polymer microparticles (FRM) (1–5 μm diameter) and their associated chronic effects on feeding, growth, mortality, and reproductive success from juvenile to adult stage of brine shrimp Artemia franciscana. Individuals were exposed for 44 days to 0.4, 0.8 and 1.6 mg.L−1 of FRM. No significant detrimental effects on growth, ingestion and mortality rates of A. franciscana were observed in all tested conditions. However, reproductive success was strongly affected by the increase of MP concentrations. The results of the present study showed that A. franciscana juveniles and adults were able to survive different experimental MP concentrations, but their reproductive success and progeny were significantly impacted by exposure to FRM particles.

1. Introduction

seafood safety. Microplastics (MPs), a type of plastic particles with a linear dimension inferior to 5 mm, are undeniably polluting aquatic environments as principal marine debris, with concentrations comparable to those of plankton (Arthur et al., 2009; Cole et al., 2014; De Revisão et al., 2011; Koelmans et al., 2015). These synthetic microparticles occur in marine environments in several forms: primary MPs, which are specifically manufactured for their abrasive qualities (e.g., microbeads and industrial scrubbers); secondary MPs, which are originated from the breakdown or degradation of larger plastic pieces (e.g., sunlight and wave action), or from direct input (e.g., discarded plastic items, cosmetic products and synthetic textiles); and tertiary MPs, which include any preproduction pellets used to mould plastic goods (Carbery et al., 2018; Cole et al., 2014; De Revisão et al., 2011; Koelmans et al., 2015; Smith et al., 2018). Currently, MPs are responsible for the contamination of all food chains, from the smallest planktivorous organisms to the largest fish, reaching the highest trophic levels, including humans (Bessa et al., 2018; Cole et al., 2013; Pacheco et al., 2018; Peixoto et al., 2019).

Since their invention, plastics have empowered innovation by allowing the development of products and solutions that otherwise would not exist (PlasticsEurope, 2017). Nowadays, these synthetic polymers have become key raw-materials in strategic sectors, such as packaging, construction, transportation, and manufacturing, due to their low-price, lightweight and durability, among other properties (Peixoto et al., 2019; Thompson et al., 2004; Wang et al., 2016). Additionally, plastics have contributed positively for the European economy, giving direct employment to 1.5 million people, with a turnover of 350 billion euros, and a trade balance close to 15 billion euros (PlasticsEurope, 2017). The annual worldwide plastic production has been increasing since the 50s, from 1.5 million tonnes to approximately 280 million tonnes in 2016, with 10% being estimated to enter the oceans and seas (Lebreton et al., 2017; PlasticsEurope, 2017; Revel et al., 2018). Plastic contamination of aquatic environments continues to increase and there are significant knowledge gaps on their occurrence and possible effects on

∗

Corresponding author. Av. General Norton de Matos s/n, 4450-208, Matosinhos, Portugal. E-mail addresses: [email protected] (D. Peixoto), [email protected] (J. Amorim), [email protected] (C. Pinheiro), [email protected] (L. Oliva-Teles), [email protected] (I. Varó), [email protected] (R. de Medeiros Rocha), [email protected] (M.N. Vieira). https://doi.org/10.1016/j.ecoenv.2019.03.100 Received 19 December 2018; Received in revised form 18 March 2019; Accepted 25 March 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

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days. All rearing parameters were carefully chosen, based on results from Browne et al. (2008), Santos et al. (2018) and Varó et al. (2015).

Several studies have already reported that MPs can be ingested by commercially important marine organisms, such as fish, bivalves and crustaceans, as well as by several species of marine zooplankton (Barboza et al., 2018c, 2018d; Browne et al., 2008; Derraik, 2002; Eriksson and Burton, 2003; Foekema et al., 2013; Fossi et al., 2014, 2012; Li et al., 2018, 2015; Lima et al., 2015; Murphy et al., 2017; Murray and Cowie, 2011; Neves et al., 2015; Pellini et al., 2018; Pinheiro et al., 2017; Thompson et al., 2004; Ward and Shumway, 2004; Watts et al., 2014). The transfer of MPs along marine trophic webs can result in significant ecological consequences, especially due to their ability to adsorb persistent, bioaccumulative and toxic contaminants from the environment, such as hazardous chemicals (Bakir et al., 2016; Karami et al., 2017; Koelmans et al., 2016), microorganisms (Foulon et al., 2016; Karami et al., 2017), metals (Brennecke et al., 2016; Fonte et al., 2016; Karami et al., 2016; Khan et al., 2015; Luís et al., 2015; Ma et al., 2016; Oliveira et al., 2013), polycyclic aromatic hydrocarbons (PAHs) (Karami et al., 2016; Ma et al., 2016; Oliveira et al., 2013), toxins (Fonte et al., 2016), and pharmaceuticals (Fonte et al., 2016), which could ultimately pose a threat to the environment and the food safety of fisheries and aquaculture products. Nonetheless, aquaculture production continues to grow faster than any other animal food-producing sector, with Artemia spp. as the main live feed organism for rearing early stages of molluscs, crustaceans, and fish (Le et al., 2018; Ohs et al., 2009; Soltanian, 2007). Adding to this problematic, the extent and magnitude of potential long-term effects of MPs is limited and poorly understood in these organisms (Foley et al., 2018; Pacheco et al., 2018), as well as its possible effects in the aquaculture sector. These zooplanktonic organisms are used as model-organisms in several scientific fields of study (e.g., ecology, physiology, genetics, and ecotoxicology) due to their easy adaptability to diverse environments, resistance to manipulation, short life-cycle, wide geographic distribution, large offspring production and well-known biology (Manfra et al., 2015; Soltanian, 2007; Varó et al., 2015). In A. franciscana, micro- and nanoparticle ingestion has been mainly assessed by short-term assays in nauplii stages (Arulvasu et al., 2014; Ates et al., 2013a, 2013b; Batel et al., 2016; Bergami et al., 2016; Gambardella et al., 2014; Manfra et al., 2012; Rodd et al., 2014). Recently, Wang et al. (2019) studied the acute (24 h) and chronic (14 days) effects of microparticles (1–20 μm diameter) on A. parthenogenetica nauplii and juveniles, with no apparent impacts on the survival, growth and development. Nonetheless, and after histological examination, abnormal ultrastructure of intestinal epithelium cells were observed in organisms exposed to polystyrene microspheres. Indeed, better knowledge is needed to assess the potential adverse effects of MPs in different life stages of brine shrimp, as well as the reproductive success, in order to study and predict possible transfer of contaminants to food chains. To provide some insights into this topic, the aim of this study was to assess, for the first time, the uptake of different concentrations of spherical polymer microparticles (FRM) (1–5 μm diameter) and their associated long-term effects on feeding, growth, mortality, and reproductive success from juvenile to adult stage of brine shrimp A. franciscana.

2.2. Microplastic contamination and diet formulation FRM (Cospheric LLC®, USA; lot number: 4-1006-1053) were used as representative of MPs with 1–5 μm diameter. These red opaque spheres, with 1.3 g cm−3 density, can be detected by spectrofluorimetry (excitation and emission wavelength of 575 and 607 nm, respectively), allowing for easy quantification in test medium and detection inside of the individuals of A. franciscana. Moreover, FRM particles were also selected given their widespread use and application (e.g., biotechnology, medical and scientific research), being a suitable model of primary MPs, used in cosmetics and personal care products (Martins and Guilhermino, 2018). Four different experimental concentration of FRM (0, 0.4, 0.8 and 1.6 mg.L−1 FRM) were tested. FRM concentrations were selected based on ecologically relevant concentrations, and the results of preliminary tests. From the selected FRM concentrations, 0.4 mg.L−1 can be considered ecologically relevant in marine environments (Barboza et al., 2018d; Goldstein et al., 2012). Nevertheless, the remaining concentrations (0.8 and 1.6 mg.L−1 FRM) were selected considering a scenario where MP pollution steadily increases, and particles become more available to marine organisms. 2.3. Experimental design A total of 180 juvenile individuals (12 days after hatching; DAH) were randomly selected from the same culture, measured, and arbitrarily distributed into 12 conical flasks of 200 mL (15 individuals per conical flask). Experimental FRM concentrations tested were randomly assigned, in triplicates, and individuals were fed every two days. All test flasks were kept at the same conditions (25 °C, 14:10 h photoperiod) used in the rearing, and were covered to prevent water evaporation. To assess the reproductive success of the individuals (number of offspring), the trial duration was for 44 days (56 DAH). Reproductive and mortality rates were assessed every day. A. franciscana individuals were considered dead if they did not display any movement for 10 s of observation under a binocular microscope (Persoone and Wells, 1987). Alongside with the total medium renewal, ingestion rates were assessed every two days (Manfra et al., 2012; Savorelli et al., 2007). The MPs in suspension were quantified in a Neubauer improved cell counting chamber (hematocytometer), under a Leica DMLB (PL FLUOTAR 40×/ 0.70) fluorescence optical microscope, with the aim of knowing the real number of particles available to the brine shrimp. Additionally, individuals from each tested condition were photographed under an automated fluorescence microscope Olympus BX64, equipped with a UC90 Olympus camera. Light field images and dark light images were taken and overlaid in order to directly identify the presence, and consequently the uptake and distribution, of MPs in different body parts of A. franciscana. These images were edited using Cellsens Entry Imaging Software 1.11 (Olympus Corporation). Individual growth was assessed at 12, 19, 25, 31 and 38 DAH, after MP exposure, and was obtained by photographing each individual under a stereo microscope Zeins Stemi 2000-C, with a USB digital camera Leica EC3 through Leica Application Suite (LAS v4.12) that was subsequently analysed using ImageJ (version 1.50b). Individual total body length was measured from the cephalic region to the furca.

2. Materials and methods 2.1. Artemia franciscana rearing A. franciscana were hatched from commercial cysts (San Francisco Bay Brand, California, USA), following the general procedures described in Varó et al. (1998, 2015). Briefly, newly hatched nauplii (0 h old) were reared until the juvenile stage (12 days old), under constant conditions of temperature (25 ± 0.5 °C), salinity (artificial seawater – ASW, 35 g.L−1 TropicMarin® Sea Salt – Italy) and photoperiod (14:10 h light:dark), with CO2-enriched air bubbling. Culture were fed with the microalgae Phaeodactylum tricornutum at a final density of 100.000 cells.mL−1. Complete medium renewal was performed every 2

2.4. Statistical analysis The possible effects of both MP concentration and exposure time, on growth and MP ingestion by A. franciscana, were examined using a oneway analysis of variance (ANOVA) (dependent variables: growth and MPs ingestion; fixed factor: day = 38 DAH for growth, and day = 56 DAH for MP ingestion; covariate: MP concentration), followed by post 212

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Fig. 1. Different concentrations of 1–5 μm diameter microspheres (FRM) in A. franciscana individuals after 5 (A–D) and 24 h (E–H) exposure. Images A, B, C, E, F and G represent the digestive tract, and images D and H represent the head. A. franciscana juvenile exposed during 5 h to 0.4 mg L−1 FRM (A), 0.8 mg L−1 FRM (B) and 1.6 mg L−1 (C and D). A. franciscana adult exposed during 24 h to 0.4 mg L−1 FRM (E), 0.8 mg L−1 FRM (F) and 1.6 mg L−1 (G). A. franciscana adult exposed during 24 h to the control treatment (H).

and fluorescence microscopy, in the digestive tract (Fig. 1 A–G), head (Fig. 1 D and H) and in the faecal pellets (not shown) of the individuals exposed to FRM. Quantification of the MPs suspended in the water was performed by fluorescence microscopy, with MPs being found in all experimental groups, except in control (0 mg.L−1 FRM). As expected, every two days, significant differences in the number of ingested particles were observed between the FRM concentrations tested (Table 1). The number of ingested particles was calculated by the difference between the particles in suspension and the concentration previously added. Overall, the concentration of ingested MPs, independently of the concentration added, was superior to 80% for all treatments (Table 1).

hoc Tukey's multiple comparisons test, when statistically significant differences were found in ANOVA (Guilhermino et al., 2018). Values of accumulated mortality, at 44 days of exposure (56 DAH), were analysed by Pearson's chi-square test and by Cox regression model. The reproductive success (total offspring) was analysed using regression models (dependent variable: total offspring; fixed factor: day = 56 DAH). Statistical significance was accepted at p < 0.05 for all analyses. Statistical analyses were performed using the TIBCO® Statistica 12.

3. Results 3.1. MP ingestion by Artemia franciscana MP ingestion by A. franciscana individuals was confirmed by light 213

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Table 1 Concentration of ingested MPs (mg.L−1) by A. franciscana in each treatment (average of all replicates). Tested mediums were fully renewed every two days: 0 mg.L−1 FRM; 0.4 mg.L−1 FRM; 0.8 mg.L−1 FRM, 1.6 mg.L−1 FRM. DAH

0 mg.L−1 FRM

15 17 19 21 24 27 29 31 34 36 38 40 42 45 47 49 54 56

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.4 mg.L−1 FRM 0d 0d 0d 0d 0d 0c 0c 0b 0d 0c 0d 0d 0d 0c 0d 0d 0d 0d

0.37 0.35 0.37 0.37 0.37 0.28 0.29 0.27 0.25 0.33 0.28 0.30 0.28 0.27 0.33 0.33 0.32 0.31

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.8 mg.L−1 FRM 0.04c 0.06c 0.02c 0.01c 0.02c 0.10b, 0.04b, 0.06b 0.08c 0.05b, 0.05c 0.12c 0.02c 0.04b, 0.03c 0.08c 0.10c 0.10c

c c

c

c

0.73 0.59 0.66 0.71 0.75 0.50 0.47 0.44 0.71 0.74 0.69 0.68 0.66 0.63 0.74 0.57 0.68 0.66

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.6 mg.L−1 FRM 0.01b 0.14b 0.10b 0.10b 0.03b 0.19b 0.22b 0.29b 0.06b 0.03a, 0.08b 0.03b 0b 0.07b 0.03b 0.01b 0.14b 0.03b

b

1.44 1.53 1.48 1.45 1.37 1.05 1.22 1.27 1.13 1.10 1.23 1.48 1.29 1.15 1.11 1.32 1.28 1.22

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04a 0.01a 0.10a 0.22a 0.08a 0.06a 0.20a 0.20a 0.10a 0.37a 0.17a 0.05a 0.06a 0.22a 0.08a 0.02a 0a 0.06a

Values are presented as mean ± standard deviation (n = 3 for all the tested conditions). Values in same the same row without a common superscript letter differ significantly (ANOVA and Tukey's test, p < 0.05).

3.2. Microplastic effects on A. franciscana's growth and mortality The effects of different MP concentrations, on A. franciscana's growth (mean length, mm), are represented by the growth curves in Fig. 2. Growth was not significantly (p = 0.880) affected by any concentration of FRM tested (0, 0.4, 0.8, 1.6 mg.L-1) after 26 days of exposure (38 DAH) (Fig. 2). Growth was not assessed after 38 DAH, because individuals had already achieved their full development. Nonetheless, the highest growth value was obtained in the control group (0 mg.L−1 FRM) at 38 DAH (7.51 ± 0.43 mm). Survival of A. franciscana after long-term exposure to different MP concentrations is shown in Fig. 3, for the totality of the MP exposure assay (44 days; 56 DAH). Pearson's chi-square test showed that mortality during the assay was not significantly (p = 0.6038) affected by the tested MP concentrations. These results may suggest that mortality observed was not related to the MP effects.

Fig. 3. Survival of A. franciscana after long-term exposure (44 days; 56 DAH) to 0, 0.4, 0.8, 1.6 mg.L−1 FRM.

(r = 0.9997; p = 0.0227; r2 = 0.9995), suggesting that the total offspring of A. franciscana decreased with the increase of MP concentration. Indeed, the model showed that the total offspring was lower at 0.4 mg.L−1 FRM, follow by 0.8 and by 1.6 mg.L−1 FRM.

3.3. Reproductive success Data from A. franciscana's reproductive success is presented in Fig. 4. The influence of different FRM concentrations on the reproductive success (total offspring) of A. franciscana was analysed by regression models. The tested models were linear, cubic and quadratic. The regression model that better fitted within these variables was the cubic regression model, expressed by the following function: y = 391.6379-1.2374*x1-0.0001*x3 (Fig. 4). Herein, it was possible to observe that a significant relation existed between FRM concentrations and the total offspring in the three replicates of each condition

4. Discussion The effects of MPs in several invertebrate marine species are already reported in the literature, but few studies have investigated their effects on the most common species used as live feed (A. franciscana) for rearing early stages of molluscs, crustaceans, and fish at aquaculture industries. Recent studies have shown that A. franciscana nauplii can ingest and egest MP beads with 1–5 and 10–20 μm diameter (Batel et al., 2016), and ingest MP fibres with 10 × 40 μm in size (Cole et al., 2016). Another study reported that A. franciscana nauplii and juveniles had the ability to ingest 50 nm NH2 and 40 nm COOH coated polystyrene (PS) nanoparticles (NPs) (Bergami et al., 2017, 2016). However, to the best of our knowledge, the present study is the first to investigate the long-term effects caused by MPs on multi-effect criteria, such as feeding, mortality, growth, and reproductive success, from juvenile to adult stage, of A. franciscana. Our results indicate that the ingestion of MPs was superior to 80% of the concentration of FRM present in the medium during all exposure time. Plastic particles were found throughout the digestive tract of the organisms (Fig. 1), confirming A. franciscana's ability to uptake MPs ranging from 1 to 5 μm in size, with very few free particles present in the medium. Additionally, the ingested MP beads aggregated into the faecal pellets, sinking to the bottom of conical flasks in all MP

Fig. 2. Mean growth curves for A. franciscana after 26 day (38 DAH) of exposure to different concentrations of FRM (0, 0.4, 0.8, 1.6 mg.L−1 FRM). 214

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Fig. 4. Nonlinear regression fitted to the data of total offspring in the three replicates of the different FRM concentrations tested (0, 0.4, 0.8, 1.6 mg.L−1 FRM).

treatments 0.4, 0.8 and 1.6 mg.L−1 FRM. Batel et al. (2016) reported similar results, with extremely high MP ingestion by A. franciscana nauplii and a low number of MPs in the water column. Indeed, Reeve (1963a, b), reported that A. franciscana can ingest indigestible sand particles ten times faster than algae of the same size, forming faecal pellets of undigested food (Evjemo and Olsen, 1999; Reeve, 1963a, b) that sink very quickly to the bottom. Additionally, the uptake and egestion of non-plastic nanomaterials by different brine shrimp Artemia species has been recently described (Arulvasu et al., 2014; Ates et al., 2013b, 2013a; Bergami et al., 2017; Gambardella et al., 2014; Pretti et al., 2014; Rodd et al., 2014). In marine environments, zooplankton faecal pellets play an essential role in the transport of nutrients, carbon, and energy to benthonic systems (Cole et al., 2016; Gauld, 1957; Turner, 2002). Given this fact, sinking zooplankton faecal pellets might promote the transport of anthropogenic pollutants, including polycyclic aromatic hydrocarbons (PAHs) (Prahl and Carpenter, 1979), hydrocarbon petroleum residues (Sleeter and Butler, 1982), and floating MPs (Cole et al., 2016) to deeper waters, removing them from surface waters and feeding zones, avoiding their re-ingestion (Gauld, 1957; Marshall and Orr, 1955). The baseline for growth and survival of A. franciscana are well known in the literature (Evjemo and Olsen, 1999; Pinto et al., 2014, 2013; Reeve, 1963a, b; Santos et al., 2018; Varó et al., 2015). The observed growth values in the control group were higher than that obtained by Santos et al. (2018) (at 36 DAH. Bergami et al. (2017) reported that PS-NH2 NPs were able to disrupt the physiology and the energy flow in developing A. franciscana, over a long-term exposure (14 days). Nonetheless, and similarly to the results obtained by Bergami et al. (2017), growth was not affected by the presence of MPs at the concentrations tested, since no differences were found between exposed and control organisms (Fig. 2). For this reason, it can be reasonable to hypothesize that in the present study, the observed growth was directly related to the P. tricornutum concentrations added as food base (Reeve, 1963a, b), and that individual growth might not be the most sensitive endpoint to study MP contamination. The mortality results obtained followed the same trend, with this parameter not showing differences with the increase of MP concentration. Bergami et al. (2016) and Gambardella et al. (2014) found similar mortality values in their assays, after short-term exposure (48 h) to 5000, 10, 000, 25, 000, 50, 000, and

100, 000 mg.L−1 of NH2 and COOH coated PS NPs, and 10, 100, 1000 mg.L−1 of metal oxide NPs (tin(IV) oxide (stannic oxide (SnO2)), cerium(IV) oxide (CeO2) and iron(II, III) oxide (Fe3O4) NPs), respectively. In their works, Bergami et al. (2017), Besseling et al. (2014) and Cole et al. (2013), hypothesized that the accumulation of NPs in the digestive tract, as a result of prolonged exposure, might limit food intake and significantly affect growth and development of branchiopod species, such as D. magna and A. franciscana. However, in our work, long-term exposure to MPs with 1–5 μm in size, did not affect the growth and survival of A. franciscana. On the contrary, the obtained results suggest a clear trend in regards to MP contamination and reproductive success in A. franciscana. The total offspring (total nº of nauplii) were significantly hampered with increasing MP concentrations, from 0.4 to 1.6 mg.L−1. The differences observed in reproductive success among treatments suggested that the presence of FRM particles in the medium may negatively impact the reproductive effort of A. franciscana, and in a dose-dependent manner. Martins and Guilhermino (2018) reported similar results with D. magna, where FRM (1–5 μm) caused a fertility reduction at a concentration of 0.1 mg.L−1, leading to a lower total offspring than the values registered in the control group. Additionally, these authors also reported high mortality among the offspring. In our study, and given the observed dose-dependent response, the negative impacts at the ecologically relevant concentration (0.4 mg.L−1) were not so severe when directing comparing to the other two FRM concentrations tested (0.8 and 1.6 mg.L−1). However, the results from the lowest MP concentration were still significantly different from the control group. Nonetheless, we need to take into consideration that the majority of marine plastics are believed to originate from land-based sources, including surface waters (Lasee et al., 2017; Peixoto et al., 2019; Wagner et al., 2014). Freshwater systems, such as rivers, lakes, and wetlands are considered to be a significant transport pathway of MPs to the oceans (Lebreton et al., 2017; Pinheiro et al., 2017; Wagner et al., 2014). Lasee et al. (2017) reported that the average concentration of MPs collected in samples from urban lakes in Texas (USA), ranged from 0.31 mg.L−1 to 1.98 mg.L−1, while in wetlands this value ranged from 0.64 mg.L−1 to 1.79 mg.L−1. The highest MP concentrations found by Lasee et al. (2017) were more than double of the highest concentration used in our study (1.6 mg.L−1 FRM), suggesting that future works might need to 215

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estuarine ecosystems under global change scenarios” that is also funded by the Lisboa 2020 programme (LISBOA-01-0145-FEDER-016885). The study was also supported by the Strategic Funding UID/Multi/04423/ 2013 through national funds provided by FCT and ERDF in the framework of the programme Portugal 2020 to CIIMAR.

take into account the constant increase of MP concentrations found in both freshwater and marine environments. Moreover, and as evidenced by Martins and Guilhermino (2018), even low contamination may result in significant detrimental results, with D. magna continuously exposed to only 0.1 mg.L−1 of MPs being at a significant risk of extinction, after only two generations. Our results are in agreement with previous studies with the same type MPs (Martins and Guilhermino, 2018; Ogonowski et al., 2016; Pacheco et al., 2018), as well as with other types of nanoplastics (NPs-PS) (Besseling et al., 2014), showing that these can negatively affect the reproductive success and population fitness in zooplanktonic organisms (i.e. D. magna). In realistic scenarios, such as during long-term exposure in the natural environment, processes of trophic web transfer (biomagnification) and bioavailability cannot be excluded upon the evidence of MP ingestion by marine zooplankton, as already hypothesized by several authors (Andrady, 2017; Avio et al., 2017; Barboza et al., 2018a, 2018b; Batel et al., 2016; Bergami et al., 2017, 2016; Cole et al., 2011, 2016; Fossi et al., 2014, 2012; Galloway and Lewis, 2016; Karami et al., 2017; A. Lusher et al., 2017; A.L. Lusher et al., 2017; Peixoto et al., 2019; Watts et al., 2014; Wright et al., 2013), with the potential to reach high trophic levels, including humans. The transfer of MPs along marine trophic webs can promote significant ecological consequences, due to the ability of MPs to adsorb persistent, bioaccumulative and toxic contaminants from the environment (Martins and Guilhermino, 2018). MPs can harm marine organisms and humans through bioaccumulation and biomagnification phenomena (Sharma and Chatterjee, 2017; Wang et al., 2016). For this reason, it is important to further improve the knowledge base in this thematic, and continue to study the effects of MPs in natural environments, as well as ways to limit their input in both fresh- and saltwater ecosystems (including hypersaline water bodies). The results of the present study show that juvenile and adult individuals of A. franciscana are able to ingest and egest MPs when continuously exposed (long-term exposure) to ecologically relevant concentrations (0.4 mg.L−1), as well as to relatively high concentrations (0.8 and 1.6 mg.L−1). In addition, their ingestion (at these concentrations) did not appear to significantly affect the growth and survival of A. franciscana. Nonetheless, these concentrations caused a decrease in reproductive success (total offspring) of A. franciscana individuals, which may lead to a reduction in population's size. The use of long-term end-points seems to be a more suitable tool for determining the impact of MPs on brine shrimp Artemia, as a suitable model organism for studying the impact of MPs in aquatic ecosystem, especially in hypersaline ecosystem where they occur. Consequently, these results raise a great concern regarding the long-term exposure of animals and human populations to MPs. Indeed, these highlight the urgent need for more studies regarding the MP phenomenon and their negative effects on wild and aquaculture organisms, and their possible impacts on human health and welfare, through their consumption. MP contamination of aquatic environments continues to increase, despite the significant knowledge gaps in their occurrence, as well as their possible effects on seafood safety.

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Conflicts of interest Declarations of interest: none. Acknowledgements This study was funded by the “Fundação para a Ciência e a Tecnologia, I.P. (FCT), Portugal, with national funds (FCT/MCTES, “orçamento de Estado”, project reference PTDC/MAR-PRO/1851/ 2014), and the European Regional Development Fund through the bib_COMPETE_2020COMPETE 2020 programme (POCI-01-0145FEDER-016885) through the project “PLASTICGLOBAL – Assessment of plastic-mediated chemicals transfer in food webs of deep, coastal and 216

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