Science of the Total Environment 842 (2022) 156923 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Antagonistic in vivo interaction of polystyrene nanoplastics and silver compounds. A study using Drosophila Mohamed Alaraby a,b, Doaa Abass a,b, Aliro Villacorta a,c, Alba Hernández a, Ricard Marcos a, ⁎ a Group of Mutagenesis, Department of Genetics and Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Campus of Bellaterra, 08193 Cerdanyola del Vallès, Barcelona, Spain b Zoology Department, Faculty of Sciences, Sohag University, 82524 Sohag, Egypt c Facultad de Recursos Naturales Renovables, Universidad Arturo Prat, Iquique, Chile H I G H L I G H T S G R A P H I C A L A B S T R A C T • The potential interaction between nanopolystyrene and silver was evaluated in Drosophila. • The coexposure increased the silver inter- nalization regarding silver exposure alone. • TEM+EDX analysis showed the absorption of silver compounds by nanopolystyrene. • Coexposure reduces the levels of oxidative stress and genotoxicity, regarding silver effects. • The antagonistic interaction could be due to the reduced bioavailability of silver once absorbed by polystyrene. A R T I C L E I N F O A B S T R A C T Editor: Damià Barceló Since heavy metals and micro−/nanoplastics (MNPLs) can share common environmental niches, their potential inter- actions could modulate their hazard impacts. The current study was planned to evaluate the potential interactions be- Keywords: tween silver compounds (silver nanoparticles or silver nitrate) and two different sizes of polystyrene nanoplastics Nanoplastics (PSNPLs) (PS-50 and PS-500 nm), administered via ingestion to Drosophila larvae. While egg-to-adult survival was Polystyrene not affected by the exposure to silver compounds, PSNPLs, or their coexposures, the combined treatments succeeded Silver nanoparticles Carriers to restore the delay of fly emergence induced by silver compounds. Transmission electron microscopy (TEM) and Size inductively coupled plasma mass spectrometry (ICP-MS) showed the ability of PSNPLs to transport silver compounds Uptake (regardless of their form) across the intestinal barrier, delivering them into the hemolymph of Drosophila larvae in a Oxidative stress concentration exceeding that mediated by the exposure to silver compounds alone. The molecular response (gene ex- Genotoxicity pression) of Drosophila larvae greatly fluctuated, accordingly if exposures were administered alone or in combination. Antagonistic interaction Although PSNPLs produced some oxidative stress in the hemocytes of Drosophila, especially at the highest dose Drosophila melanogaster (1 mM), higher levels were observed after silver exposure, regardless of its form. Interestingly, the oxidative stress of silver, especially that produced by nano‑silver, drastically decreased when coexposed with PSNPLs. Similar effects were observed regarding the DNA damage induced in Drosophila hemocytes, where cotreatment decreased the geno- toxicity induced by silver compounds. This antagonistic interaction could be attributed to the ability of tiny plastic specks to confine silver, avoiding its bioavailability, and diminishing their potential impacts. ⁎ Corresponding author at: Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus de Bellaterra, 08193 Cerdanyola del Vallès, Barcelona, Spain. E-mail addresses: mohamed.alaraby@science.sohag.edu.eg (M. Alaraby), ricard.marcos@uab.es (R. Marcos). http://dx.doi.org/10.1016/j.scitotenv.2022.156923 Received 8 April 2022; Received in revised form 27 May 2022; Accepted 20 June 2022 Available online 23 June 2022 0048-9697/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). M. Alaraby et al. Science of the Total Environment 842 (2022) 156923 1. Introduction Since silver materials and MNPLs can share a common environment, some types of interactions can take place (Bhagat et al., 2021). Therefore, The increasing levels of micro/nanoplastics (MNPLS) in our environ- our aim is to investigate the potential interactions between two different ment arise many questions about their potential hazards. Its consideration sizes of polystyrene nanoplastics (50 and 500 nm) and two silver com- as contaminants of emergent concern requires generating scientifically pounds as silver nanoparticles (AgNPs, about 10 nm) and silver nitrate sound data to understand the potential health risks associated with their ex- (as a source of silver ions). To detect such interactions, we have used posure (Mitrano et al., 2021). Although microplastics were environmen- Drosophila melanogaster to evaluate a wide range of effects including uptake tally detected early in 1990 (Ryan and Moloney, 1990), their extensive and toxicity, as well as changes in the expression of genes involved in differ- research skyrocketed recently (Bhagat et al., 2021) and, among the differ- ent functional pathways, oxidative stress, and genotoxicity. It is important ent questions raised by the environmental presence of MNPLs, their possi- to indicate that we have previously described the physical interaction ble interactions with other environmental pollutants stand out. between AgNPs and PSNPLs, as well as their biological effects using It has been reported that MNPLs can act as sponge adsorbing pathogens human intestinal Caco-2 cells (Domenech et al., 2021). In addition, we (Viršek et al., 2017), pharmaceuticals (Razanajatovo et al., 2018), pesti- have successfully used the Drosophila larvae as a useful in vivo model to de- cides (Fajardo et al., 2022), and heavy metals (Zhou et al., 2020; Khalid termine the effect of the ingested nanomaterials and their effects on the et al., 2021), among others compounds. This would be a consequence of intestinal components (Alaraby et al., 2018). their large surface area and hydrophobicity (Bhagat et al., 2021). Regarding heavy metals, they are major contaminants of soil and water (Zhou et al., 2020) and, consequently they share environmental niches with environ- 2. Materials and methods mental MNPLs. Nevertheless, limited knowledge is available on their inter- actions, as well as on the biological impacts resulting from their combined 2.1. Silver and polystyrene nanoparticles characterization exposures (Cao et al., 2021). The interactions between metals-plastics can induce substantial changes to the physicochemical properties of plastics, al- AgNPs were purchased from nanoComposix (San Diego, CA, USA), with tering their internalization and accumulation in the exposed organisms standard properties according to the manufacturer (diameter: 6.6 ± (Hansjosten et al., 2022). Moreover, the mechanisms controlling the ad- 1.8 nm; surface area: 76.1 m2/g; solvent: USP purified water). Two different sorption mechanisms are not completely specified (Liu et al., 2022) sizes of polystyrene nanoparticles (PSNPLs) were obtained from Spherotech whether they are synergistic (Qiao et al., 2019; Lee et al., 2019), additive (Lake Forest, IL, USA), PS-50 (80 nm; reference SPH-PP-008-10), and PS- (Ma et al., 2016), or antagonistic (Oliveira et al., 2018). 500 (430 nm, reference SPH-PP-05-10). According to the manufacturer The interaction between MNPLs and environmental pollutants (includ- the PSNPLs were prepared through polymerization of styrene monomer ing heavy metals) can be modulated by different physicochemical parame- using potassium persulfate as a polymerization initiator, resuspended in de- ters, and also by the MNPLs size. Differences in size between MPLs and ionized water, and sodium azide (0.02 %) was added as a bacteriostatic. NPLs can also modulate their interaction with environmental pollutants The other chemicals, including AgNO3 (ref. 209,139), were from Sigma due to differences in surface area, bioavailability, and adsorption efficiency Chemical Co. (St. Louis, MO, USA). To further characterize AgNPs and (Gigault et al., 2021). Due to the technical difficulties of detecting/quanti- PSNPLs, a Malvern Zetasizer Nano-ZS zen3600 instrument (Malvern, UK) fying NPLs in environmental matrices, most environmental studies have was used to determine the hydrodynamic size and the zeta potential; trans- used MPLs as a model of MNPLs. Nevertheless, the small size of NPLs mission electron microscopy (TEM; Jeol 1400; Jeol Ltd., Tokyo, Japan) was makes easier their uptake by the organisms and, consequently, they could used to show size, morphology, and degree of aggregation; energy- become more hazardous. For this reason, studies using NPLs are usually car- dispersive X-ray spectroscopy (EDX) spectrum was used to evaluate the ried out in laboratory conditions and using both in vitro and in vivo sorption of silver particles upon polystyrene using a TEM (200 kV) Jeol approaches. Studies using whole organisms can produce data more useful 2011 device (Jeol Ltd., Tokyo, Japan). The functional groups of polystyrene to be extrapolated for risk assessment approaches. were determined by Fourier-transform infrared spectroscopy (FTIR) using a Nevertheless, most of the in vivo studies aiming to detect potential inter- Hyperion 2000 micro-spectrometer (Bruker, Billerica MA, USA). This anal- actions MNPLs/ environmental pollutants focus on aquatic animals, mainly ysis was carried out at the Molecular Spectroscopy and Optical Microscopy because marine MNPLs are considered to act as very important vectors of facility at the Institut Català de Nanociència i Nanotecnologia (ICN2). To as- many marine pollutants (Amelia et al., 2021). Nevertheless, MNPLs are sess the composition, interferograms were analyzed and contrasted with widely spread in all environmental niches including also continental wa- previous reports. To achieve the desired concentrations, PSNPLs were ters, air, and soils. This would mean that other in vivo models, out of the dispersed in Mili-Q water. aquatic organisms, should also be used/explored. In that context, Drosophila stands out as a powerful in vivo model to determine the potential hazards induced by environmental nanoparticles, including nanoplastics (Alaraby 2.2. Drosophila melanogaster exposure et al., 2016a, 2021). Using Drosophila as an in vivo experimental model our aim is to evaluate The wild-type Canton-S strain was used for all experiments. The ex- potential interactions between polystyrene nanoplastics (PSNPLs), used as perimental design includes treatments of Drosophila with two different a model of nanoplastic, and silver as a model of metal pollutant. Although concentrations of silver compounds (AgNPs and AgNO 3) and two of when thinking about MNPLs pollutants we imagine those resulting from PSNPLs (PS-50 and PS-500) alone, or with the different combinations the degradation of plastic goods (secondary MNNPLs), MNPLs are also cre- (i) AgNPs+PS-50, (ii) AgNPs+PS-500, (iii) AgNO 3 + PS-50, (iv) ated at these sizes for specific purposes (primary MNPLs). Thus, for exam- AgNO3 + PS-500. Two exposure scenarios were established, (i) a low ple, polystyrene micro and nanobeads are created/used for different exposure where 0.2 mM of all the components were used, and (ii) a purposes like for the exfoliating agents present in personal care products high exposure where 1 mM was used. The equivalent concentrations such as creams, and toothpaste, among other uses (Silva et al., 2018). Nev- of 0.2 and 1 mM for nanopolystyrene correspond to 52 and 260 μg/g ertheless, independently of their origin, as primary or secondary MNPLs, all food; for silver NPs they correspond to 54 and 270 μg/g food; and for sil- finally are present in the different environmental compartments as ver nitrate they correspond to 85 and 425 μg/g food. Stock solutions environmental pollutants. The antibacterial activity and the high electrical were freshly prepared before the experiments and diluted to prepare and thermal conductivity of silver compounds have extended their use in all proposed doses. Particle suspensions were vortexed before treat- many fields including catalysts, cosmetics, electronics, and textiles. Conse- ments to obtain an optimum distribution of nanoparticles. The chosen quently, they are polluting many environments, including aquatic ones, and concentrations were based on the reported by us in previous studies are present in the respective sediments (Zhao et al., 2021). (Alaraby et al., 2019, 2022). 2 M. Alaraby et al. Science of the Total Environment 842 (2022) 156923 2.3. Toxicity mass spectrometry (ICP-MS) in an Agilent, Model 7500ce device was used to measure silver content in each of the tested samples. Toxicity was evaluated according to the ability to move from egg to adult stages. To proceed, five replicates of groups of 50 eggs of Canton-S 2.5. Alteration of gene expression levels flies were seeded in food vials containing 4 g of instant medium (Carolina Biological Supply, Burlington, NC, USA), previously wetted with 10 mL of Exposure to xenobiotic agents could mediate changes at the molecu- various concentrations (0.0, 0.2, and 1 mM) of silver (AgNPs/AgNO3), or lar level, affecting the expression levels of genes involved in different PSNPLs (PS-50/PS-500) whether alone or in combination. Vials were left pathways. Such effects correspond with their potential mode of action. at 25 °C, 60 % humidity, and (12 h/12 h light/dark) conditions. MiliQ To this end, a battery of gene markers representative of different water was used as a control solution. The vials were checked daily till pathways is included. As representative of general stress markers, the adult emergence to check for any differences in the development of treated Heat-shock-protein-70 (Hsp70, NM_169441.2) and the Heat-shock-pro- flies. The emerged adults were collected, investigated, and counted to es- tein-83 (Hsp83, NM_001274433.1) were included; as representative of tablish comparisons with the untreated adults. Moreover, adults were in- oxidative stress markers, the Catalase (Cat, NM_080483.3) and the Cu, vestigated to record any morphological abnormalities. According to the Zn Superoxide dismutase 2 (Sod2, NM_057577.3) were selected; as repre- toxicity information, the concentrations to be used in the other experiments sentative of intestinal genes stimulated by physical stress, the Dual were decided. oxidase (Duox, NM_001273039.1) and the Prophenoloxidase 2 (PPO2, NM_136599.4) were chosen; finally, the 8-oxoG glycosylase1 (Ogg1, 2.4. AgNPs and PSNPLs internalization NM_132271.5) was selected as a DNA repair gene. Gene expression changes were determined as previously published (Alaraby et al., This section is proposed to answer several questions regarding the abil- 2018). Briefly, 30 third-instar larvae (120 h-old) were homogenized in ity of PSNPLs to penetrate the intestinal barrier, as well as their ability to ice-cold TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) and manipu- transfer heavy metals into the organism. Thus, the whole intestinal journey lated according to the manufacturer protocol to obtain pure mRNA, of PSNPLs alone, or mixed with silver compounds, was highlighted using which was asserted using RNase-free DNase I (DNA-freeTM kit; Ambion, TEM; while inductively coupled plasma mass spectrometry (ICP-MS) was Paisley, UK). The quantity of the obtained mRNA was determined using used to determine the larval body internalization. Furthermore, changes a Nanodrop device and converted into cDNA using the Transcriptor First in the expression levels of genes related to “physical stress”, as indicative Strand cDNA Synthesis Kit (Roche) protocol and stored at −20 °C. cDNA of “intestinal damage” were also determined. samples were amplified by real-time RT-PCR using a Light Cycler 480 (Roche, Basel, Switzerland). To prepare RT-PCR samples for each gene, 10 μL of reaction volume was used containing 4 μL of cDNA 2.4.1. TEM investigation (10 ng/μL) mixed with 5 μL of SYBER Green mix, and 1 μL of 10 μM Drosophila larvae at the fifth day of exposure were investigated to gene-specific primers (forward and reverse); the housekeeping β-actin highlight the ability of MNPLs internalization, and the transferring of was used as control. RT-PCR reaction conditions: pre-incubation for silver materials. The exposed larvae were carefully collected, cleaned, 5 min at 95 °C, 1 cycle, and the amplification was repeated 45 times and dissected in phosphate buffer solution (PB; 0.1 M, pH 7.4). Drops (10 s at 95 °C, 15 s at 61 °C, 72 °C for 25 s). of hemolymph samples were poured upon TEM grids and left for further investigation, while the extracted intestines were manipulated follow- 2.6. Detection of oxidative stress induction ing our previous protocol (Alaraby et al., 2015). The intestine samples were directly immersed in a fixation solution for 2 h (1 % glutaralde- Drosophila larvae’ hemocytes have been the cell model used to detect hyde and 4 % paraformaldehyde, prepared in 0.15 M PB at pH 7.4), the potential oxidative stress induction with different exposure conditions. post-fixed in osmium tetroxide 1 % (w/v) containing 0.8 % (w/v) potas- On the fifth day of exposure, larvae were collected, washed, and dissected. sium hexacyanoferrate, and washed four times with deionized water The hemolymph of untreated larvae was divided into two groups; the first and sequential dehydration in acetone at 4 °C. Eponate 12™ resin (Ted group was used as negative control and the second group was treated Pella Inc., Redding, CA, USA) was used to embed the samples and poly- with 0.5 mM H2O2 and used as the positive control. Hemolymph samples merize them at 60 °C for 48 h. Next, semi-thin sections (1 μm thick) were were treated with 5 μM 6-carboxy-2,7′-dichlorodihydro-fluorescein cut with a Leica ultracut UCT microtome (Leica Microsystems GmbH, diacetate (DCFH-DA) and incubated for 30 min at 24 °C. The fluorescent he- Wetzlar, Germany) and stained with 1 % (w/v) toluidine blue to select mocytes were detected by a fluorescent microscope with an excitation of the proper areas of the intestine. Ultimately, ultrathin sections 485 nm and an emission of 530 nm (green filter). The effects were quanti- (100 nm in thickness) were obtained via cutting the resin blocks with fied with the ImageJ program. a diamond knife (45o, Diatome, Biel, Switzerland) and placed on non- coated 200 mesh copper grids and contrasted with uranyl acetate for 2.7. DNA damage induction 30 min, and Reynolds lead citrate for 5 min. TEM (Jeol 1400,100 kV) equipped with a CCD Gatan ES1000w Erlangshen Camera (Gatan Hemocytes of larvae exposed to different conditions were used as cell Inc., Pleasanton, CA, USA) was used to rummage sections for Silver models to detect DNA damage via the comet assay, according to our previ- and polystyrene. ously developed method (Alaraby et al., 2021). The 72 h-old larvae reared upon normal medium were collected, cleaned, and transferred into new 2.4.2. Internalization by ICPMS vials containing instant medium, previously mixed with 10 mL of the differ- This technique was proposed to evaluate if MNPLs act as heavy metal ent doses of the designed treatments. Nine exposure conditions were evalu- carriers, modulating their internalization, and also as an indirect ated: AgNPs, AgNO3, PS-50, PS-500, AgNPs+PS-50, AgNPs+PS-500, approach to detect MNPLs' internalization via absorbing metals such AgNO3 + PS-50, AgNO3 + PS-500, and 4 mM EMS as positive control, in as silver. Fifth-days treated larvae, in addition to untreated ones, were addition to the negative control. Exposures last for 24 h and three replicates collected, carefully washed, and transferred into a new untreated in- were carried out. Next, pooled larvae (50 larvae) of each treated group, stant food medium for 6 h, to clean their gastrointestinal tract’ lumen were picked up and dissected in 100 μL of filtered and cold autoclaved from silver. Next, larvae were collected again and stored at −80 °C for phosphate buffer solution. To proceed, a sample of 10 μL of hemolymph further analysis. Three replicates of larval samples (50–100 mg) of containing about 10,000 cells was carefully mixed with 90 μL of 0.75 % each dose were digested in a microwave oven in an acidic solution of low-melting agarose at 37 °C. Seven μL of hemocytes/agarose mixture (6 HNO 3/H2 O 2 (1:1) in a Teflon container. Inductively coupled plasma drops) were dropped on the hydrophilic surface of an ice-cold Gelbond® 3 M. Alaraby et al. Science of the Total Environment 842 (2022) 156923 film (GBF) (Life Sciences, Lithuania) in triplicate. To avoid artificial 2.8. Statistical analysis DNA damage, all next procedures were performed under dim light. The GBFs were submerged in freshly cooled lysis solution (2.5 M NaCl, To address the normality and the homogeneity of the data the 0.1 M Na2EDTA, 0.2 M NaOH, 0.01 M Tris, 1 % Triton X-100, and 1 % N- Kolmogorov-Smirnov & Shapiro-Wilk test and Levene's test were applied. lauryl sarcosinate, pH 10) for 1 h at 4 °C for cells lysis. The GBFs The data showing normal distribution and equal variance were analyzed were washed for 5 min with cold electrophoresis buffer (0.001 M EDTA, with the Student t-test and the one-way ANOVA, while those with skewed 0.3 M NaOH, pH 13.2) before incubation for 25 min in a horizontal gel- distribution and unequal variance were analyzed with nonparametric ap- electrophoresis tank, filled with cold electrophoresis buffer to permit proaches (Mann-Whitney U test). Significant differences were considered unwinding DNA. Electrophoresis was directly performed for 20 min at at P ≤ 0.05. Data were calculated as mean ± standard error (SE). conditions of 20 V and 300 mA at 4 °C. The GBFs were neutralized with two washes of PBS for 5 min each, followed by 1 min in distilled 3. Results water, then fixed in absolute ethanol for at least 2 h, fixed for 5 min in 70 % and 100 % ethanol, respectively, then they air-dried for 2 h. The 3.1. Characterization of AgNPs and PSNPLs GBFs were stained with 25 mL of TE-buffer (10 mM Tris; 1 mM EDTA, pH 7.5) containing 1000× diluted SYBERGold fluorochrome for 20 min, The size-frequency distribution of AgNPs and PSNPLs was determined with brief shaking, then washed with water to remove stain excess, via representative TEM images, as shown in supplementary data (Fig. S1). and left to dry overnight. DNA damage of hemocytes was measured using The average diameter of AgNPs is <10 nm (7.2 ± 2.4), while plastic parti- the Komet 5.5 Image-Analysis System (Kinetic Imaging Ltd., Liverpool, cles whether PS-50 or PS-500 have wider diameters (43.6 ± 12.2 and UK) fitted with an Olympus BX50 fluorescence microscope equipped 415.0 ± 12.9, respectively). Further approaches were conducted to de- with a 480–550-nm wide-band excitation filter, and a 590-nm barrier filter. scribe their morphology, degree of aggregation, and charge Fig. 1 (A-H). The percentage of DNA in the tail of the nucleoids was measured in three TEM images clearly showed differences in the morphology of PSNPLs replicates of 100 randomly selected cells per dose, and compared with alone (A and D) with those combined with AgNPs (B and E). AgNPs are ob- that of the negative control. served stacked upon the outer border of the plastic nanospheres of PS-50 Fig. 1. TEM image of PS-50 (A), AgNPs adsorbed upon PS-50 edge (B), Energy dispersive X-ray spectroscopy (EDX) of AgNPs+PS-50 (C). TEM image of PS-500 (D), AgNPs adsorbed upon PS-500 (E), Energy dispersive X-ray spectroscopy (EDX) of AgNPs+PS-500 (F). Hydrodynamic size and Zeta potential values (G). The FTIR spectra for PS-50 and PS-500 (H). 4 M. Alaraby et al. Science of the Total Environment 842 (2022) 156923 regularly like ring lobes (B), as well as with large size plastic particles in PS- negative control. It should be remembered that in Section 2.2 we have indi- 500, but with low regular distribution (E). The silver nature was cated the relationship between the concentrations expressed in mM, and confirmed by EDX (C and F). The evaluated hydrodynamic size of AgNPs the amount of μg of the different compounds per g of food medium. The is very small (12.84 ± 7.33) in comparison with that of plastic particles adults were collected and counted to calculate the percentage of viability (73.74 ± 1.88 and 488.91 ± 8.62) and agrees with their TEM sizes. Inter- regarding the negative control. The emerged adults were investigated for estingly, the hydrodynamic size of plastic and silver combination any morphological changes. It is noticeable that the PSNPLs/silver (AgNPs+PS-50 and AgNPs+PS-500) is lower than PSNPLs alone (58.4 ± coexposure reduced the faint pigmentation caused by silver exposure, 1.29 and 434.4 ± 10.02), respectively (G). Both AgNPs and PSNPLs especially at the higher tested scenario (1 mM). Generally, the treated/ dispersions show high values of zeta potential indicating they are well untreated adults emerged through two days, the eleventh and twelfth dispersed. The negative charge of PSNPLs can be attributed to sulfate from seeding eggs. Interestingly, PSNPLs exposure, when coadministered groups of potassium persulfate used in their synthesis. The FTIR spectra with the silver compounds, improves adult emergence by reducing the of both PS-50 and PS-500 agree with the expected for polystyrene (H). delay induced by silver exposure Fig. 2 (C and D). They show peaks around 3000 cm−1 indicative of CH2 and a mono- For the AgNPs (0.2 mM) experiment, cotreatments with PS-50 and PS- substituted benzene ring, while saturated C\\H of methylene stretches 500 significantly reduce delay in adult emergence moving from 61 % of are below 3000 cm−1 and the unsaturated C\\H from the benzene ring emerged adults on day 11th to 97 % and 94 %, respectively. Similarly, stretches fall above 3000 cm−1. The other strong peaks are apparent AgNPs (1 mM) induced a similar trend but with less effect moving from around 1500 cm−1 confirming the benzene ring that typically falls from 65 % to 82 % and 76 %, respectively. Regarding AgNO3 exposures, these 1620 to 1400 cm−1. Additionally, benzene fingers as a series of weak effects are clearly observed at the concentration of 1 mM where the bands which fall from 1650 to 2000 cm−1 are related to the mono- emergence of 7 % of adults at the eleventh is significantly enhanced to substituted benzene ring of polystyrene. 46 % and 77 % after coexposure with PS-50 and PS-500, respectively. 3.2. Drosophila viability after exposures 3.3. Internalization of silver compounds and PSNPLs Silver compounds, regardless of their form, and PSNPLs regardless of 3.3.1. Silver content detection in larval body by using ICP-MS their size, whether administrated alone or in combination failed to induce One open question is if PSNPLs can act as carriers of environmental pol- significant decreases in Drosophila viability Fig. 2 (A and B). The experiment lutants, including metals, modulating their uptake. To such end, we have was carried out using five replicates of 250 eggs for each concentration sce- determined if silver concentrations inside Drosophila larvae differ when nario (0.2 mM and 1 mM) as well as for untreated medium, used as the larvae are exposed to only AgNPs or AgNO3, regarding their coexposures A B AgNO₃ PS-50 AgNPs PS-50 PS-500 PS-50+AgNO₃ PS-500 PS-50+AgNPs PS-500+AgNO₃ PS-500+AgNPs 200 200 150 % of survival 150 % of survival 100 100 50 50 0 0 0.2 1.0 0.2 1.0 Concentration (mM) Concentration (mM) C D 11th 12th 11th 12th a a a b b 100 100 80 80 Emergence % Emergence % 60 60 40 40 20 20 0 0 0.2 mM 1 mM 0.2 mM 1 mM Fig. 2. Egg-to adult survival rate of Drosophila flies treated with AgNPs/AgNO3, PSNPLs (PS50/PS500) and their combinations (A and B). Percentages of emergence (emergence at 11th day and 12th day) of Drosophila flies treated with AgNPs+PSNPLs (C) or AgNO3 + PSNPLs (D), regarding those treated with AgNPs or AgNO3, respectively. Statistically significant (P ≤ 0.05), (a) vs (0.2 AgNPs/AgNO3) and (b) vs (1 AgNPs/AgNO3). 5 M. Alaraby et al. Science of the Total Environment 842 (2022) 156923 A B AgNPs AgNO₃ AgNPs+PS-50 AgNO₃+PS-50 AgNPs+PS-500 AgNO₃+PS-500 50 ac 50 Ag conc.(µg/mg) a Ag conc.(µg/mg) 40 ac 40 a 30 ac 30 20 a 20 a a ab ab 10 a a 10 0 0 0.2 1.0 0.2 1.0 Concentration (mM) Concentration (mM) Fig. 3. Silver concentration inside Drosophila larvae tissues after exposure to AgNPs and AgNPs+ PSNPLs (A) or to AgNO3 and AgNO3 + PSNPLs (B). Statistically significant (P ≤ 0.05), (a) vs negative control (0.04 μg/mg), (b) vs (0.2 mM AgNPs/AgNO3) and (c) vs (1 mM AgNPs/AgNO3). with PSNPLs. As indicated in Fig. 3 (A and B, respectively) the silver content ingestion route were described by using TEM images. The different evalu- of cotreated larvae is significantly higher than in the silver (AgNPs or ated stages comprise their interaction with midgut lumen components, AgNO3) treatments alone. The effect is dose-dependent, reaching very sig- their interaction with the enterocytes constituting the intestinal barrier, nificant effects at the higher dose (1 mM) of both silver compounds. At and the interaction with hemocoel cavity components (including hemo- that dose, PSNPLs (PS-50 and PS-500) clearly magnified the levels of inter- cytes) once PSNPLs cross the intestinal barriers (Figs. 4, 5, and 6, respec- nalized silver when coexposed. Thus, the internal levels of silver after tively). AgNPs (10.70 μg/mg) and AgNO3 (24 μg/mg) exposures are increased in their coexposures with PS-50 (22.67 and 32.33 μg/mg) and PS-500 (38 and 44 μg/mg), respectively. It is interesting to point out that these effects 3.3.2.1. PSNPLs (PS-50 and PS-500) internalization. Fig. 4 describes the in- are more marked when the coexposure involves the large size of PSNPLs ternalization of PS-50 (A, B, and C) and PS-500 (D, E, and F) inside the (PS-500). Drosophila larvae body. Both plastic particles PS-50 and PS-500 were ob- served distributed inside the midgut lumen (A and D), before penetrating the peritrophic membrane to reach intestinal enterocyte cells (B and 3.3.2. PSNPLs/silver internalization detection by using TEM E) and, finally, reached the hemocoel compartment once translocated TEM was used to monitor the internalization of PSNPLs (PS-50 and PS- through the intestinal barrier (C and F). It is interesting to note that PS-50 500) alone or in combination with AgNPs. To get a better description of the are distributed in Drosophila larval midgut lumen in batches showing aggre- uptake process, the different stages of the journey of PSNPLs through the gation (A) while PS-500 are found to be individually distributed (D). The Fig. 4. TEM images of PS-50 (A, B, and C) and PS-500 (D, E, and F) internalization in Drosophila larvae. In the midgut lumen (A and D), inside the cytoplasm of midgut enterocytes (B and E) and in the hemolymph (C and F). 6 M. Alaraby et al. Science of the Total Environment 842 (2022) 156923 Fig. 5. TEM images of AgNPs+PS-50 internalization through Drosophila’ larvae. Distributed inside midgut lumen (A), inside or surrounding microbiota of midgut lumen (B and C) or inside cytoplasm of midgut enterocytes (D). AgNPs whether located individually (E) or attached to PS-50 (F) in hemolymph of treated larvae. success of PSNPLs to be internalized inside the larval tissue could facilitate indicated in Fig. 5. Complexes are observed inside the midgut lumen (A), their mission of acting as a potential carrier facilitating silver particles to where AgNPs were clearly observed at the outer surface of PS-50 particles internalize. that are distributed inside the midgut lumen. AgNPs+PS-50 also appear inside (or surrounding) the microbiota of the midgut lumen (B and C) or 3.3.2.2. PS-50/AgNPs internalization. TEM is also a useful tool to follow up distributed in the cytoplasm of midgut enterocytes (D). The investigation on the internalization of AgNPs+PS-50 at different biological stages as of hemolymph samples of treated larvae with AgNPs/PS-50 showed the Fig. 6. TEM images of AgNPs+PS-500 internalization through Drosophila’ larvae. The complex is distributed inside the midgut lumen near to peritrophic membrane (A), surrounded completely by intestinal vacuole (B) moving among enterocytes' microvilli (C), inside cytoplasm of midgut enterocytes, and penetrating cells borders (D), damaging cellular mitochondria (E) and, finally, reaching the hemolymph (F). 7 M. Alaraby et al. Science of the Total Environment 842 (2022) 156923 presence of Ag nanoparticles whether located individually (E) or attached deregulation after the exposure to AgNPs/AgNO3 or PSNPLs alone or ad- to PS-50 (F). ministered as a combination. It must be indicated that the highest expres- sion level was associated with the exposure to the highest AgNO3 dose 3.3.2.3. PS-500/AgNPs internalization. The large size of PS-500 facilitates (1 mM), which is reflected in the high expression levels observed in its their detection through the ingestion process. Thus, TEM images of coexposure with PSNPLs. Similarly, the Hsp83 gene also showed deregula- AgNPs+PS-500 through their journey until their internalization in the lar- tion but milder, and this expression was not affected by the applied dose of vae body are presented in Fig. 6. Thus, PS-500 as large spherical particles AgNPs/AgNO3. To address the antioxidant response at the molecular level, are observed inside the midgut lumen near to peritrophic membrane of the expression of Sod2 and Cat, as reliable genes, was examined. Although the larvae intestine (A). In this case, AgNPs are not clearly observed due there is an opposite effect between AgNPs/AgNO3 and PSNPLs regarding to the high contrast of plastic particles with their black color. Inside the the expression of Sod2, their coexposure returned its expression to the lumen, PS-500/AgNPs complexes were surrounded completely by intesti- basal level. The expression levels of Cat were low in AgNPs, PSNPLs, or in nal vacuoles (B) before penetrating the peritrophic membrane to move the coexposure experiments. It is important to point out that the coexposure among enterocytes' microvilli (C). The existence of these large plastic parti- reduced the Cat expression levels produced by the exposure to the highest cles in the cytoplasm of the intestinal cells, and due to their kinetics energy, dose of AgNPs (1 mM). Regarding the effects of the Cat expression levels mediate harmful impacts appearing as holes in the cell borders (D) and in after the AgNO3 exposures, they were significantly increased at the highest the mitochondria (E). Finally, the PS-500 journey overcomes the intestinal dose, in contrast with the low levels observed with the PSNPLs exposure. barrier, translocating and reaching the hemolymph (F). Interestingly, the coexposure (PSNPLs+AgNO3) mediated a significant up-regulation of the Cat expression levels, independently of the PSNPLs 3.4. Gene expression changes size. It is notorious the induced de-regulation of the intestinal physical stress genes (Duox and PPO2) after exposure to PSNPLs, mainly at the One of the major aims of this study is to unveil the in vivo molecular higher size (PS-500) for PPO2. This downregulation was partially recovered responses to the coexposure of plastic particles with heavy metals. Accord- in the combination with silver compounds mainly for Duox. All this would ingly, we have evaluated changes in the expression levels of genes involved point out that the physical stress induced in the intestinal barrier by PSNPLs in different pathways/mechanisms, and the obtained results are presented is partially recovered when combined with silver compounds. Genotoxicity, in Fig. 7. As observed, the heat-shock protein gene (Hsp70) showed and especially oxidative DNA damage, is an important target to evaluate AgNPs PS-50 AgNO₃ PS-50 AgNPs PS-50 Hsp70 Hsp70 PS-500 Hsp83 AgNO₃ PS-50 PS-500 PS-50+AgNPs PS-50+AgNO₃ PS-500 PS-50+AgNPs Hsp83 PS-500 PS-50+AgNO₃ PS-500+AgNPs PS-500+AgNO₃ PS-500+AgNPs PS-500+AgNO₃ 100 800 a a 100 250 a 600 ab 80 a 80 200 % of mRNA a a a 400 a a b 60 a 100 200 60 150 a a a 80 ac 40 40 100 60 a ac a ac a a ab 40 a 20 ab ac ab 20 ab 50 ac 20 ac ac ab ab 0 0 0 0 0.2 1.0 0.2 1.0 0.2 1.0 0.2 1.0 Sod2 Sod2 Cat Cat 250 100 400 ac 100 a ab a a 200 80 a 80 300 % of mRNA b 150 ab 60 60 a a 200 c a a 100 ac 40 a 40 a ab ac ac 100 ac ac ac ac ab ab 50 ab ac 20 20 ab ac ab ab 0 0 0 0 0.2 1.0 0.2 1.0 0.2 1.0 0.2 1.0 Duox Duox PPO2 PPO2 250 ab 300 100 100 a a a 200 80 80 a a % of mRNA a a a a ac 200 a a 150 60 60 ac a c a a 100 40 40 a 100 ac ac a ab ab ac ab 50 ab ab 20 ab 20 ab 0 0 0 0 0.2 1.0 0.2 1.0 0.2 1.0 0.2 1.0 Ogg1 Ogg1 250 150 c a 200 100 % of mRNA 150 a a a a 100 a ac 50 a ac ab ac 50 ac ac a ac ab ab 0 0 0.2 1.0 0.2 1.0 Concentration (mM) Concentration (mM) Fig. 7. Gene expression levels in Drosophila’ larvae after exposure to AgNPs, PSNPLs and AgNPs+PSNPLs or to AgNO3, PSNPLs and AgNO3 + PSNPLs. General stress genes (Hsp70 and Hsp83), antioxidant genes (Sod2 and Cat), intestinal physical stress genes (Duox and PPO2), and DNA repair gene (Ogg1). Statistically significant (P ≤ 0.05), (a) vs negative control, (b) vs (0.2 mM AgNPs/AgNO3) and (c) vs (1 mM AgNPs/AgNO3). The lack of a letter in a column indicates no statistical differences. 8 M. Alaraby et al. Science of the Total Environment 842 (2022) 156923 A B AgNPs PS-50 AgNO₃ PS-50 PS-500 PS-50+AgNPs PS-500 PS-50+AgNO₃ PS-500+AgNPs PS-500+AgNO₃ a 900 a 600 a a % of ROS 400 600 % of ROS a a ac a a ab ac b ac a ac 200 b 300 b c a c b 0 0 0.2 1 0.5 mM H₂O₂ 0.2 1 0.5 mM H₂O₂ Concentration (mM) Concentration (mM) Fig. 8. Oxidative stress induced in hemocytes of Drosophila larvae exposed to AgNPs, PSNPLs and AgNPs+PSNPLs (A) or AgNO3, PSNPLs and AgNO3 + PSNPLs (B). Statistically significant (P ≤ 0.05), (a) vs negative control, (b) vs (0.2 mM AgNPs/AgNO3) and (c) vs (1 mM AgNPs/AgNO3). The lack of a letter in a column indicates no statistical differences. the harmful effects of exposures. Herein the expression of Ogg1 was investi- 3.6. Genotoxic impact evaluated in the comet assay gated after silver and plastic particles exposure. It is noticeable that the up- regulation of the Ogg1 gene after the exposure to 1 mM of AgNPs was The genotoxicity of silver (AgNPs/AgNO3), PSNPLs (PS-50 and PS- reduced in the coexposure with PSNPLs at the same dose. Similarly, the 500), and their coexposures were determined using the comet assay PSNPLs coexposure with AgNO3 at the highest dose (1 mM) reduced the as a tool to detect DNA strand-breaks induction. The obtained results effects induced by AgNO3 alone. are indicated in Fig. 9, and DNA damage induction was determined as the percentage of DNA present in the tail section of the comet images. 3.5. Oxidative stress induction From these results, several points need to be highlighted. The first point is that both silver compounds induced significant increases in Oxidative stress induction has been extensively proposed as the main the levels of DNA breaks, AgNO 3 inducing more damage mainly at factor standing behind the detrimental impact of nanomaterials. Herein, the highest concentration. The second point is that both PSNPL sizes silver compounds exposures produced a significant elevation of the oxida- (PS-50 and PS500) also induced significant increases in the levels tive stress levels in the hemocytes of the exposed larvae, in comparison of DNA damage, PS-50 being more genotoxic than PS-500. Third, with untreated ones and in all studied exposure scenarios (Fig. 8). Although and more relevant, the combination of both PSNPLs with the two PSNPLs were able to induce some oxidative stress at the high dose (1 mM), selected silver compounds significantly reduced its potential DNA this effect could not be detected at the low concentration scenario damaging effects. These results are very representative of the antagonis- (0.2 mM). Importantly, when the oxidative stress levels induced by the tic relationship established when both sets of compounds are evaluated. combinations AgNPs/AgNO3 + PSNPLs are compared with those induced by AgNPs/AgNO3 alone a significant reduction is observed confirming 4. Discussion their antagonistic effects. These decreases are observed in all the cases with the exception of the AgNO3 exposure in the low exposure The behavior of MNPLs in the different environmental compartments scenario. Interestingly, both PS-50 and PS-500 show similar behavior in is considered an emerging topic, mainly due to their ability to absorb the coexposure scenario. certain hazardous pollutants (including heavy metals) sharing the same A B AgNPs PS-50 PS-500 PS-50+AgNPs AgNO₃ PS-50 PS-500+AgNPs PS-500 PS-50+AgNO₃ 300 PS-500+AgNO₃ a 300 a % of DNA in tail % of DNA in taill 200 a a a a ab 200 a a a ab ab ac ab ac acac ab ab ac 100 c c 100 0 0 0.2 1.0 4 mM H₂O₂ 0.2 1.0 4 mM H₂O₂ Concentration (mM) Concentration (mM) Fig. 9. DNA damage levels in Drosophila larvae hemocytes after exposure to AgNPs, PSNPLs, and AgNPs+PSNPLs (A) or AgNO3, PSNPLs, and AgNO3 + PSNPLs (B). Statistically significant (P ≤ 0.05), (a) vs negative control, (b) vs (0.2 mM AgNPs/AgNO3) and (c) vs (1 mM AgNPs/AgNO3). 9 M. Alaraby et al. Science of the Total Environment 842 (2022) 156923 environments (Zhou et al., 2022). Such absorption capacities can signifi- Despite the importance of tracking the internalization of nanoplastics cantly modulate the potential impacts of such pollutants, by changing materials and their sorbent pollutants, the available data at present remain their physicochemical properties (Bhagat et al., 2021). Nonetheless, there insufficient, especially at the in vivo level (Paul et al., 2020). Accordingly, is limited information regarding the hazardous scenario resulting from we wish to emphasize the interest of our work using TEM methodology the MNPLs/pollutants combinations, and the potential mechanisms as an essential tool to follow up the complete journey of the complexes' involved in such types of interactions (Cao et al., 2021). In our case, the PSNPLs/Ag-compounds from the larvae mouth, to their interactions physicochemical properties of PSNPLs indicate that both plastic sizes (PS- with gut components, their interaction with the intestinal barrier, their 50 and PS-500) are well dispersed and with a negative charge similar to translocation through the barrier to reach hemolymph (as homologous of that of AgNPs. The interaction of PSNPLs and AgNPs was confirmed mor- blood), and interacting with hemocytes (as homologous of lymphocytes). phologically by TEM images, and chemically through TEM-EDX analysis. Interestingly, nanoplastics followed the same journey pattern as observed Fortunately, the smaller size of silver nanoparticles (7 nm) revealed how for different nanomaterials (Alaraby et al., 2018, 2021). The use of hemo- they can be sorted regularly stack upon the outer border of the spherical cytes as a target is relevant, not only because of the functionality of such particles of PSNPLs, especially for the smaller ones (PS-50). In addition, cells but because indicating that the complexes are able to cross the intesti- the TEM images picked up by the high TEM resolution showed the ability nal barrier and spread by organs and tissues. Thus, MNPLs associated with of silver particles to infiltrate inside PSNPLs, especially for the larger ones benzo(a)pyrene were observed to affect mussel hemocytes indicating their (PS-500), presumable by forming sandwiched π-stacking structures with ability to cross the intestinal barrier (Katsumiti et al., 2021). Although the side benzene rings of polystyrene (Feng et al., 2022). PSNPLs, regardless of their size, are able to internalize into the intestinal, At present, there are many questions regarding the potentially harmful as also demonstrated in zebrafish (Sendra et al., 2021), the internalization impacts of nanoplastics exposure. It is necessary to know if such effects are pathways would depend on their size. Thus, while small particles PS-50 related to its polymer structure (physical stress) or owing to the adsorption uptake mainly via clathrin- and caveolae-mediated pathways, PS-500 of various pollutants (chemical stress). In addition, we need to know if the would uptake via macropinocytosis (Liu et al., 2021a). In our case, PS- behavior of the combination MNPLs-pollutants is synergistic or antagonis- 500 were observed surrounded completely by vacuoles, pointing out a tic and if such interactions are size- or dose-dependent. All of these potential role of midgut vacuoles in the PSNPLs uptake/translocation in questions drive our work and are discussed in the current study. Firstly, it Drosophila larvae (Alaraby et al., 2021). The internalization of PS-500 is interesting to point out that the survival rates were not affected whether inside the enterocytes of Drosophila larvae was associated with damaged Drosophila larvae were exposed to silver compounds (AgNPs/AgNO3) or cell borders, as well as damaged mitochondria, which was also observed PSNPLs (PS-50/PS-500) administrated each alone or together, at doses up in human intestinal Caco-2 cells (Xu et al., 2021). All of this would be to 1 mM. A recent in vitro study using human intestinal Caco-2 cells, showed indicative of the physical stress induced by MNPLs exposure. that exposures to AgNPs/AgNO3 alone or in combination with PSNPLs To address the physical stress induced by MNPLs as well as the chemical reduced cell viability, although PSNPLs alone failed to induce any toxic stress attributed to the attached silver compounds, changes in the impacts at all studied doses (Domenech et al., 2021). Contrarily, herein expression levels of a wide battery of genes were investigated. This we have observed that coexposures were able to restore the reduced approach is useful for the detection of mild effects. Thus, the expression pigmentation of emerged Drosophila adults induced by silver, especially levels of the highly conserved stress genes Hsp70 and Hsp83 were altered after AgNO3 exposures. Moreover, PSNPLs when coadministered with after silver exposures in comparison with PSNPLs exposure. Nevertheless, silver decrease the emergence delay resulting from exposure to silver no relevant changes were observed in the coexposures, regarding those alone regardless of its form. These effects are the first indication of an induced by silver compounds alone. The negative impact of silver (regard- antagonistic interaction between silver and MNPLs. This behavior has less of its form) on the expression of the Hsp genes in Drosophila has already also been recently reported by Zhang et al. who found that PSMPLs been reported, in particular for Hsp70 (Ahamed et al., 2010; Alaraby et al., exposure reduces the toxic effects of silver in Escherichia coli, Selenastrum 2019). The expression of the antioxidant genes Sod2 and Cat were greatly capricornutum, Daphnia magna, and zebrafish. The authors attributed this down-regulated with exposure to PSNPLs alone, which could be attributed behavior to the attachment of MPLs capacity, which could modify silver to the oxidative stress produced by PSNPLs exposure (Liu et al., 2020; chemical speciation (Zhang et al., 2021). The reduced toxicity of the Li et al., 2020a). Regarding the effects of coexposures, is relevant to point complexes' PSNPLs/micropollutants might be attributed to the reduced out the increased expression levels observed for Cat after the high exposure bioavailability of the adsorbed micropollutants (Verdú et al., 2022). This scenario with AgNO3. In such conditions, the effects induced by the sorption ability does not reduce the ability of silver to be incorporated cotreatment were significantly higher than those induced by the silver into the organisms as observed using ICP-MS, where PSNPLs significantly compound. These results would point to an interaction effect of the increased the presence of silver in Drosophila larvae tissues, especially coexposure. The expression levels of the genes Duox and PPO2, as under the high dose (1 mM) scenario. This would support the proposal responding to the induction of intestinal physical stress, have shown to that nanoplastics can act as heavy metal vectors (Maity et al., 2021). be sensitive to any stress introduced by nano-scaled materials upon the Interestingly, this increase in the internalization inside the body tissue is intestinal barrier (Alaraby et al., 2016b, 2020). In our study, they were mainly observed after the cotreatment with PS-500. This means that PS- de-regulated with exposures to silver or PSNPLs, indicating their 500 can transfer more silver (regardless of its form) into Drosophila tissues interaction with the intestinal barrier as confirmed by the TEM and ICP- rather than PS-50. This disagrees with the proposal that heavy metals' MS results. Regarding the effects of coexposures, interactions were adsorption is increased as the surface of plastic particles increases observed for Duox under coexposures with AgNPs at the low exposure (Guo et al., 2020). At this point, it must be pointed out that while PS-50 scenario, with higher expression levels than those observed for AgNPs are observed in aggregated batches PS-500 are distributed individually, alone. Regarding the PPO2 gene, the interactions were observed under showing some roughness when present inside the midgut lumen of the high exposure scenario but, in this case, the coexposures induced Drosophila larvae. Thus, our findings would agree with the proposal that expression levels than the AgNPs alone. Altogether, these data confirm there are positive correlations between roughness and heavy metal adsorp- the differential effects induced by the coexposures. Finally, changes in the tion (Qi et al., 2021). It is important to indicate that the pH changes expression levels of the Ogg1 gene were determined, as a potential response occurring inside the Drosophila midgut (Overend et al., 2016) might affect to oxidative stress damaging DNA. From the set of obtained data, we large plastic particles more than smaller ones. The pH conditions can can point out those observed after AgNPs exposure under the highest control the adsorption capacity of plastic particles to heavy metals by concentration scenario. In such conditions, the coexposures reduced deprotonating functional groups on the nanoplastics surface, increasing significantly the expression levels induced by AgNPs alone. Interestingly, the electronegativity and adsorption sites that facilitate the adsorption of this observed effect match well with the data obtained in the detection heavy metals (Lin et al., 2021). of the intracellular levels of ROS. 10 M. Alaraby et al. Science of the Total Environment 842 (2022) 156923 The induction of oxidative stress is considered one of the mechanisms CRediT authorship contribution statement involved in the hazardous effects of nanomaterials (Mendoza and Brown, 2019). In our study, the exposure to both silver and PSNPLs mediated RM and AH planned the experiments. MA, DA and AV carried out the oxidative stress induction, but to different degrees. It has been reported experimental part. MA and DA analyzed the data, carried out the statistical that silver compounds, independently of their form, induced oxidative analysis, and prepared tables/figures. MA, DA, RM, and AH wrote the final stress in different organisms including Drosophila (Alaraby et al., 2019; manuscript. Singh et al., 2021), while for MNPLs these effects have been recently re- viewed (Kumar et al., 2022). It is important to note that cotreatments succeeded to diminish the oxidative stress induced by silver, especially by Declaration of competing interest AgNPs, independently of the exposure scenario. This also occurs after AgNO3 exposure, but only under the high exposure scenario. All this The authors declare they have no actual or potential competing finan- confirms the antagonistic effects of coexposures. Although Bhagat et al. cial interests. (2021) in their review found more than ten studies pointing out the antagonistic interaction of MNPLs with other pollutants, our study is the Acknowledgments first one showing the effects on oxidative stress levels. There is a strong link between oxidative stress and genotoxicity; thus, This project has received funding from the European Union's Horizon any disturbance in the redox balance can mediate a genotoxic impact 2020 research and innovation program under grant agreement No (AshaRani et al., 2009; Dutta et al., 2018). In our study, silver compounds 965196 (PlasticHeal). This work was partially supported by the Spanish (regardless of their form) caused pronounced DNA damage levels in the Ministry of Science and Innovation [PID2020-116789, RB-C43]. hemocytes of Drosophila larvae. This is something well-known in many M. Alaraby held a postdoctoral fellowship from the Cultural Affairs organisms, including Drosophila (Alaraby et al., 2019). More interesting Sector and Missions (Ministry of Higher Education), Egypt. are the results showing that PSNPLs (regardless of their form) also act A. Villacorta was supported by a PhD fellowship from the National as genotoxic agents. Only a very recent paper reported the genotoxic Agency for Research and Development (ANID), CONICYT PFCHA/ effects of PSMPLs in Drosophila but it used the wing-spot assay, which mea- DOCTORADO BECAS CHILE /2020-72210237. sures the induction of somatic mutation or somatic recombination, and microplastics measuring between 4 and 20 μm (Demir, 2021). Neverthe- less, MNPLs induced genotoxic damage in different human hematopoietic References cells (Rubio et al., 2020), as well as in mussel hemocytes (Gonçalves Ahamed, M., Posgai, R., Gorey, T.J., Nielsen, M., Hussain, S.M., Rowe, J.J., 2010. Silver nano- et al., 2022). But no studies on the cotreatment effects have been addressed particles induced heat shock protein 70, oxidative stress and apoptosis in drosophila until now. Herein, coexposure of PSNPLs (bot sizes) with silver compounds melanogaster. Toxicol. Appl. Pharmacol. 242 (3), 263–269. https://doi.org/10.1016/j. 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