Comparative effects of polyvinyl chloride microplastics on the brittle star Ophiactis virens and the amphibian Xenopus laevis.

In this study, we investigated the effects of PVC microplastics (PVC-MPs) using two different animal models: the brittle star Ophiactis virens, and the African clawed frog Xenopus laevis. This is the first study using an environmental relevant sample of PVC-MPs obtained through mechanical fragmentation of a common PVC plumbing pipe. Exposure experiments on brittle star were performed on the adult stage for a duration of 14 days, while those on African clawed frog were performed on the embryogenic developmental stage according to the standardized FETAX protocol (Frog Embryo Teratogenesis Assay-Xenopus). For both models, different endpoints were analysed: mortality, developmental parameters, behavioural assays and histological analyses on target organs by optical and electronic microscopy. Results showed that the concentration of 0.1 µg mL-1 PVC do not cause any adverse effects in both models (common NOEC concentration), while exposure to 1 µg mL-1 PVC adversely affected at least one species (common LOEC concentration). In particular arm regeneration efficiency was the most affected parameters in O. virens leading to a significantly lower differentiation pattern at 1 µg mL-1 PVC. On the contrary, in X. laevis larvae histopathological analyses and behavioural tests were the most susceptible endpoints, exhibiting several abnormal figures and different swimming speed at 10 µg mL-1 PVC. Histopathological analyses revealed a higher abundance of degenerating cells, pyknotic nuclei and cellular debris in the gut of exposed larvae in respect to control. The comparative analyses performed in this work allowed to characterize the specificity of action of the PVC-MPs on the two species, underlining the importance of exploring a large spectrum of endpoints to offer adequate protection in the emerging fields of microplastic research.

Indigo-dyed cellulose fibers and synthetic polymers in surface-feeding seabird chick regurgitates from the Gulf of Alaska.

We provide evidence of anthropogenic materials ingestion in seabirds from a remote oceanic area, using regurgitates obtained from black-legged kittiwake (Rissa tridactyla) chicks from Middleton Island (Gulf of Alaska, USA). By means of GPS tracking of breeding adults, we identified foraging grounds where anthropogenic materials were most likely ingested. They were mainly located within the continental shelf of the Gulf of Alaska and near the Alaskan coastline. Anthropogenic cellulose fibers showed a high prevalence (85 % occurrence), whereas synthetic polymers (in the micro- and mesoplastics dimensional range) were less frequent (20 %). Most fibers (60 %) were blue and we confirmed the presence of indigo-dyed cellulosic fibers, characteristic of denim fabrics. In terms of mass, contamination levels were 0.077 µg g-1 wet weight and 0.009 µg g-1 wet weight for anthropogenic microfibers and synthetic polymers, respectively. These results represent the only recent report of contamination by anthropogenic fibers in seabirds from the Gulf of Alaska.

Anti-predator behavioral responses of Italian agile frog tadpoles (Rana latastei) exposed to microplastics.

Microplastics (MPs) are nowadays abundant, persistent, and ubiquitous in the environment, representing a new threat for terrestrial, marine, and freshwater ecosystems. Although anuran populations and species are globally declining, the effect of MP exposure on this taxon has been poorly investigated. With the aim of assessing the effects of microplastic exposure on the defensive responses of Italian agile frog (Rana latastei) tadpoles, we exposed them to three different concentrations (1, 7, and 50 mg L-1) of a mixture of plastic polymers (HPDE, PVC, PS, and PES) for 2 weeks. Then, we measured the total distance covered by individual tadpoles before and after exposure to tadpole-fed dragonfly larvae (Aeshna cyanea) cues. As expected, predation risk sharply lowered the total distance travelled by tadpoles; however, MP concentration did not affect their defensive performances. We also collected data on tadpole development, activity, and mortality. In contrast with previous experiments, neither tadpole growth nor mortality varied with MP concentration. Our results indicate that the intensity of MP effects on growth and development may depend on tadpole size, with large tadpoles being less susceptible to the negative effects of MP exposure.

Microplastic contamination in terrestrial ecosystems: A study using barn owl (Tyto alba) pellets.

Microplastics (MPs) are recognised as an emerging environmental problem that needs to be carefully monitored. So far, MPs have been widely recorded in marine and freshwater ecosystems. Still, few studies have focused on MP occurrence in terrestrial ecosystems, although soils are suspected to be one of the main MP reservoirs. To test a non-invasive method for assessing MP contamination in terrestrial ecosystems, we analysed the pellets of a top terrestrial predator, the barn owl (Tyto alba). Sixty pellets were collected from three agricultural areas (20 pellets each) and analysed to assess both barn owl diet and MP content. Thirty-four MPs were confirmed by micro-Fourier Transform Infrared Spectroscopy (µ-FTIR) analysis in 33% of the pellets (min-max 1-5 MPs per pellet). Most of the detected items were microfibres (88.2%). Polyethylene terephthalate, polyacrylonitrile and polyamide were the most abundant polymers. One of the three sites was significantly less contaminated. In the two sites with the highest MP occurrences, barn owl diet was characterised by predation on synanthropic rodents, particularly brown rats (Rattus norvegicus), which may indicate habitat degradation and increased exposure to MPs. Analyses also suggest that Savi's pine vole (Microtus savii) is the prey least at risk of MP contamination, probably due to its strictly herbivorous diet. We argue that the analysis of barn owl pellets may represent a cost-effective method for monitoring MP contamination in terrestrial ecosystems.

Detection and formation mechanisms of secondary nanoplastic released from drinking water bottles.

Since nanoplastics are currently considered potentially hazardous to the environment and human health, reliability of studies on nanoplastic exposure becomes crucial. However, analytical challenges limit our understanding of their formation and detection, thus hampering their biological interactions assessment. Here we provide a combined approach to quantitatively and qualitatively detect the release of nanoplastics in water matrix and, in particular, to measure direct exposure of consumers by simulated use of drinking water plastic bottles. We measured that the polyethylene sealing of the bottles released particles with a size distribution ranging from few hundreds nanometers up to about one micron and estimated a mass release in the order of few tenths of nanograms per opening/closing cycle. We observe that mechanical stress alters the physical-chemical characteristics of the generated secondary nanoplastics and degrades the material properties compared to the original bulk source, thus complicating their spectroscopic chemical identification. Our findings demonstrate that understanding material degradation processes is therefore crucial for identifying and quantifying nanoplastics in real samples. Moreover, methods allowing quantitative studies on the release of nanoplastic as a source of exposure are considered essential for proper assessment of their potential health hazards and to promote improvements in consumer products plastic packaging design.

Differential effects of microplastic exposure on anuran tadpoles: A still underrated threat to amphibian conservation?

Microplastics (MPs) have been reported to threaten a wide variety of terrestrial, marine, and freshwater organisms. However, knowledge about the effects of MPs on anuran amphibians, one of the most threatened taxa worldwide, is still limited. To assess the effects of MPs on the growth and survival of the Italian agile frog (Rana latastei) and green toad (Bufotes balearicus), we exposed tadpoles to three different concentrations (1, 7, and 50 mg L-1) of an environmental relevant mixture of microplastics (HPDE, PVC, PS and PES), recording data on their activity level, weight and mortality rates. While the effects of MPs on green toad tadpoles were negligible, Italian agile frog tadpoles were severely affected both in terms of growth and activity level, with high mortality rates even at the lowest MP density (1 mg L-1). Our results suggest that MP contamination of freshwater habitats may contribute to the ongoing decline of anuran amphibians.

Human airway organoids and microplastic fibers: A new exposure model for emerging contaminants.

Three-dimensional (3D) structured organoids are the most advanced in vitro models for studying human health effects, but their application to evaluate the biological effects associated with microplastic exposure was neglected until now. Fibers from synthetic clothes and fabrics are a major source of airborne microplastics, and their release from dryer machines is poorly understood. We quantified and characterized the microplastic fibers (MPFs) released in the exhaust filter of a household dryer and tested their effects on airway organoids (1, 10, and 50 µg mL-1) by optical microscopy, scanning electron microscopy (SEM), confocal microscopy and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). While the presence of MPFs did not inhibit organoid growth, we observed a significant reduction of SCGB1A1 gene expression related to club cell functionality and a polarized cell growth along the fibers. The MPFs did not cause relevant inflammation or oxidative stress but were coated with a cellular layer, resulting in the inclusion of fibers in the organoid. This effect could have long-term implications regarding lung epithelial cells undergoing repair. This exposure study using human airway organoids proved suitability of the model for studying the effects of airborne microplastic contamination on humans and could form the basis for further research regarding the toxicological assessment of emerging contaminants such as micro- or nanoplastics.

Following the fate of microplastic in four abiotic and biotic matrices along the Ticino River (North Italy).

Microplastics (MPs) are emerging contaminants in freshwater systems that have already attracted much scientific interest, but little attention has been paid to a multi-matrix analysis of MP occurrences along the length of a river. The present research provides the first record of MP contamination of four abiotic and biotic matrices from a river ecosystem simultaneously analysed. MPs were isolated and identified by micro-Fourier Transform Infrared (µ-FTIR) spectroscopy from samples collected along the Ticino River in North Italy during spring 2019. Abiotic samples were surface water (n = 18, 33 MPs m-3) and sediment (n = 18, 11 MPs kg-1), while biological samples consisted of stomach and gut content of fish (n = 18, wels catfish, Silurus glanis, 0.032 MPs g-1) and macroinvertebrates (n = 90, caddisfly larvae, Hydropsychidae, 0.03 MPs mg-1). MPs were found in biota from all stations; 44% of fish and 61% of macroinvertebrate samples contained MPs. The calculated unit-consistent concentration ratios indicate that both S. glanis and Hydropsychidae larvae had a consistent higher amount of MPs than their respective medium (sediment and water), strongly suggesting an efficient uptake pathway into organisms. MP levels in surface water, sediment, fish and macroinvertebrates were not correlated and did not increase with the river's length. From our mass balance calculations, the Ticino River transports a consistent amount of MP (yearly load of 3.40 × 1011 ± 1.1 × 1011 MPs) to the Po River. This MP load was almost half than an estimated MP load from wastewater treatment plants (WWTPs). On that basis and supported by the finding that MP concentration in sediment was mostly opposed to that in surface water but was on average 750-fold higher compared to the water matrix, we surmise that the complex hydrological network of the Ticino River retains a consistent amount of MPs which might build up over time.

Combined Effects of Pesticides and Electromagnetic-Fields on Honeybees: Multi-Stress Exposure.

Honeybee and general pollinator decline is extensively reported in many countries, adding new concern to the general biodiversity loss. Many studies were addressed to assess the causes of pollinator decline, concluding that in most cases multi-stress effects were the most probable ones. In this research, the combined effects of two possible stress sources for bees, pesticides and electromagnetic fields (multi-stress conditions), were analyzed in the field. Three experimental sites were chosen: a control one far from direct anthropogenic stress sources, a pesticide-stress site and multi-stress one, adding to the same exposure to pesticides the presence of an electromagnetic field, coming from a high-voltage electric line. Experimental apiaries were monitored weekly for one year (from April 2017 to April 2018) by means of colony survival, queen activity, storage and brood amount, parasites and pathogens, and several biomarkers in young workers and pupae. Both exposure and effect biomarkers were analysed: among the first, acetylcholinesterase (AChE), catalase (CAT), glutathione S-transferase (GST) and alkaline phosphatase (ALP) and Reactive Oxygen Species (ROS); and among the last, DNA fragmentation (DNAFRAGM) and lipid peroxidation (LPO). Results showed that bee health conditions were the worst in the multi-stress site with only one colony alive out of the four ones present at the beginning. In this site, a complex picture of adverse effects was observed, such as disease appearance (American foulbrood), higher mortality in the underbaskets (common to pesticide-stress site), behavioral alterations (queen changes, excess of honey storage) and biochemical anomalies (higher ALP activity at the end of the season). The overall results clearly indicate that the multi-stress conditions were able to induce biochemical, physiological and behavioral alterations which severely threatened bee colony survival.

Occurrence of microplastics in pellets from the common kingfisher (Alcedo atthis) along the Ticino River, North Italy.

Previous research has reported avian plastic ingestion in marine bird species. Yet, while research attention on plastic pollution is shifting from marine to freshwater ecosystems, very few information on plastic ingestion is available for freshwater birds. Here, we examined the presence of microplastic in regurgitated pellets of the common kingfisher (Alcedo atthis) collected along the Ticino River (North Italy). In total, 133 kingfisher's pellets were examined between March and October 2019 from 54 transects along the river. Plastic elements were detected and identified by visual inspection followed by µ-FTIR and SEM-EDS. Overall, we found 12 (micro)plastics from at least three different polymers in 7.5% of the pellets. This study provides the first report of plastic uptake of this bird species. It highlights the importance of spectroscopic techniques in plastic monitoring studies in order to avoid misidentification of items found. Documenting the presence of plastic ingestion by top carnivores such as fish-eating birds is necessary to understand the pervasiveness and impact of (micro)plastic pollution in food webs of freshwater ecosystems.

Does mechanical stress cause microplastic release from plastic water bottles?

Plastic particle ingestion has become of concern as a possible threat to human health. Previous works have already explored the presence of microplastic (MP) in bottled drinking water as a source of MP intake. Here, we consider the release of MP particles from single-use PET mineral water bottles upon exposure to mechanical stress utilizing SEM plus EDS, which allows the implementation of morphological and elemental analysis of the plastic material surface and quantification of particle concentrations in sample water. The aim of this study was to better evaluate the sources of MP intake from plastic bottles, especially considering the effect of daily use on these bottles such as the abrasion of the plastic material. For that, we analysed MP release of PET bottlenecks and HDPE caps on their surfaces after a series of bottle openings/closings (1 x, 10 x, 100 x). Furthermore, we investigated, if the inner surface of the PET bottles released MPs, counted particle increase of the water and identified MPs in the PET bottled water after exposing the bottles to mechanical stress (squeezing treatment; none, 1 min, 10 min). The results showed a considerable increase of MP particle occurrence on the surface of PET and HDPE material (bottlenecks and caps) after opening and closing the bottles. After 100 times the effect was impressive, especially on caps. Moreover, great differences exist in cap abrasion between brands which uncovers a discrepancy in plastic behavior of brands. Interestingly, particle concentrations in the bottled mineral water did not significantly increase after exposure to mechanical stress (squeezing treatment). The morphological analysis of the inner wall surface of the bottles supported this observation, as no stress cracks could be detected after the treatment, implying that the bottles itself are not a consistent source of MP particles after this extent of mechanical stress. However, chances of MP ingestion by humans increase with frequent use of the same single-use plastic bottle, though only from the bottleneck-cap system.

Predation on Amphibians May Enhance Eurasian Otter Recovery in Southern Italy.

Mediterranean freshwaters undergo extreme seasonal variation in water flow, which, exacerbated by water withdrawal for agriculture or hydroelectric purposes, may affect fish communities and thus prey availability for semi-aquatic predators, such as Eurasian otter Lutra lutra. To investigate the role played by food availability on the ongoing recovery of an otter population at the southernmost limit of its Italian range, we assessed otter diet by the analysis of 357 spraints collected from 2014 to 2017 on eight rivers, and both fish and amphibian availability by, respectively, electrofishing and visual encounter surveys. Fish and amphibians formed the bulk of otter diet, the latter resource contributing as much as fish to otter diet in spring. Use by otters of both fish and amphibians depended only fish availability, suggesting that amphibians constituted an alternative resource to be exploited in conditions of fish shortage. Accordingly, electrofishing showed that fish biomass may barely be sufficient to sustain the current otter population. ATR-FT-IR spectroscopy allowed to point out for the first time the occurrence of amphibian eggs in otter spraints, although the co-occurrence of anuran bones did not allow to discriminate between direct and passive predation. Overall results indicate that the expansion or even survival of this small otter population may depend on the effective management of water resources and reinforcement of fish assemblages.

Polystyrene microplastics did not affect body growth and swimming activity in Xenopus laevis tadpoles.

A growing number of studies have highlighted the contamination and the effects towards organisms of diverse microplastics (µPs) in the marine environment. Surprisingly, although the main sources of µPs for marine environments are inland surface waters, the information on the occurrence and the effects of µPs in freshwater ecosystems is still scant. Thus, the aim of the present work is to investigate the ingestion and possible adverse effects due to the exposure to polystyrene µPs (PSµPs; Ø = 3 µm) on tadpoles of the Amphibian Xenopus laevis. Larvae at the developmental stage 36, prior to mouth opening, were exposed under semi-static conditions to 0.125, 1.25, and 12.5 µg mL-1 of PSµPs, and allowed to develop until stage 46. At the end of the exposure, the digestive tract and the gills from exposed and control tadpoles were microscopically examined, as well as changes in body growth and swimming activity. PSµPs were observed in tadpoles' digestive tract, but not in the gills, from each tested concentration. However, neither body growth nor swimming activity were affected by PSµPs exposure. Our results demonstrated that PSµPs can be ingested by tadpoles, but they did not alter X. laevis development and swimming behavior at least during early-life stages, also at high, unrealistic concentrations.

Comparative toxicity of three differently shaped carbon nanomaterials on Daphnia magna: does a shape effect exist?

The acute toxicity of three differently shaped carbon nanomaterials (CNMs) was studied on Daphnia magna, comparing the induced effects and looking for the toxic mechanisms. We used carbon nano-powder (CNP), with almost spherical primary particle morphology, multi-walled carbon nanotubes (CNTs), tubes of multi-graphitic sheets, and cubic-shaped carbon nanoparticles (CNCs), for which no ecotoxicological data are available so far. Daphnids were exposed to six suspensions (1, 2, 5, 10, 20 and 50 mg L-1) of each CNM, and then microscopically analyzed. Ultrastructural analyses evidenced cellular uptake of nanoparticle in CNP and CNT exposed groups, but not in samples exposed to CNCs. Despite this difference, very similar effects were observed in tissues exposed to the three used CNMs: empty spaces between cells, cell detachment from the basal lamina, many lamellar bodies and autophagy vacuoles. These pathological figures were qualitatively similar among the three groups, but they differed in frequency and severity. CNCs caused the most severe effects, such as partial or complete dissolution of the brush border and thinning of the digestive epithelium. Being the cubic shape not allowed to be internalized into cells, but more effective than others in determining physical damages, we can conclude that shape is an important factor for driving nanoparticle uptake by cells and for determining the acute toxicological endpoints. Shape also plays a key role in determining the kind and the severity of pathologies, which are linked to the physical interactions of CNMs with the exposed tissues.

Benzoylecgonine exposure induced oxidative stress and altered swimming behavior and reproduction in Daphnia magna.

Several monitoring studies have shown that benzoylecgonine (BE) is the main illicit drug residue commonly measured in the aquatic system worldwide. Few studies have investigated the potential toxicity of this molecule towards invertebrate and vertebrate aquatic non-target organisms focusing on effects at low levels of the biological organization, but no one has assessed the consequences at higher ones. Thus, the present study was aimed at investigating the toxicity of a 48-h exposure to two concentrations of BE, similar to those found in aquatic ecosystems (0.5 µg/L and 1.0 µg/L), on the cladoceran Daphnia magna at different levels of the ecological hierarchy. We relied on a multi-level approach focusing on the effects at biochemical/biomolecular (biomarkers), individual (swimming activity) and population (reproduction) levels. We measured the amount of reactive oxygen species and of the activity of antioxidant (SOD, CAT, and GPx) and detoxifying (GST) enzymes to assess if BE exposure can alter the oxidative status of D. magna specimens, while the lipid peroxidation (TBARS) was measured as a marker of oxidative damage. Moreover, we also measured the acetylcholinesterase (AChE) activity because it is strictly related to behavioral changes in aquatic organisms. Changes in swimming behavior were investigated by a video tracking analysis, while the consequences on reproduction were assessed by a chronic toxicity test. Our results showed that BE concentrations similar to those found in aquatic ecosystems induced oxidative stress and inhibited AChE activity, affecting swimming behavior and the reproduction of Daphnia magna individuals.

Chronic toxicity effects of ZnSO4 and ZnO nanoparticles in Daphnia magna.

The chronic toxicity of ZnSO4 and ZnO nanoparticles has been studied in Daphnia magna also considering the life cycle parameters beyond the standard 21-day exposure time. Specimens have been individually followed until the natural end of their life, and some of them sampled for microscopic analyses at 48h, 9 and 21 days. Despite the low level of exposure (0.3mg Zn/L), ultrastructural analyses of the midgut epithelial cells revealed efficient internalization of nanoparticles between 48h and 9d, and translocation to other tissues as well. At 21d, the most affected fields have been recorded for both compounds; in particular samples exposed to ZnO nanoparticles showed swelling of mitochondria, while those exposed to ZnSO4 had a great number of autophagy vacuoles. The life cycle parameters resulted altered as well, with a significant inhibition of reproduction in both groups, when compared to controls. After the 21-day exposure, some interesting results were obtained: animals, previously exposed to nanoZnO at low concentrations, showed a complete recovery of the full reproduction potential, while those previously exposed to ZnSO4 presented a dose-dependent and compound-specific reduction in lifespan. Based on the results from the present research and the effects of the same chemicals at higher doses, it can be concluded that the soluble form plays a key role in ZnO nanoparticle cytotoxicity, and that the nanoparticulate form is able to locally increase the amount of Zn inside the cell, even within the ovary. It's worth noting that ZnO nanoparticles have been internalized despite the very low concentration used: this raises concern about the possible environmental implications which may derive from their use, and which in turn must be carefully considered.

Role of soluble zinc in ZnO nanoparticle cytotoxicity in Daphnia magna: A morphological approach.

The role of soluble zinc has been determined in Daphnia magna by a morphological approach, integrating a previous paper in which the ultrastructural damages to gut epithelial cells have been studied after ZnO nanoparticles exposure. In the present paper, the toxicity and morphological effects of soluble zinc from ZnSO4 have been determined in a 48-h acute exposure test. Daphnids have been exposed to six nominal zinc concentrations (0.075, 0.15, 0.3, 0.6, 1.2, and 2.4mg Zn/L) and then fixed for microscopic analyses. Data from the acute toxicity tests gave an EC50 value of 0.99mg/L and showed that no immobilization appeared up to 0.3mg Zn/L. Ultrastructural analyses of samples from the two highest concentrations showed large vacuolar structures, swelling of mitochondria, multilamellar bodies, and a great number of autophagy vacuoles. These findings have been compared to those from our previous study, and similarities and/or differences discussed. Based on the overall results it can be concluded that dissolved zinc ions played a key role in ZnO nanoparticle toxicity and that the morphological approach is an extremely useful tool for comparing toxicological effects as well. A possible common toxic mechanism of soluble zinc and ZnO nanoparticles was also proposed.

Polychlorinated biphenyls (PCBs) in air and soil from a high-altitude pasture in the Italian Alps: evidence of CB-209 contamination.

This study analyses the seasonal trend of polychlorinated biphenyls (PCB) concentrations in air and soil from a high-altitude mountain pasture in the Italian Alps. PCB concentrations in soil were generally comparable to background levels and were lower than those previously measured in the same area. Only CB-209 unexpectedly showed high concentrations with respect to the other congeners. GC-MS-MS identification was very clear, rising a new problem of increasing PCB contamination concerning only CB-209, which is not present in commercial mixtures used in the past in Italy and Europe. Considering all of the congeners, seasonal PCB trends were observed both in air and in soil that were related to the temperature and precipitation measured specifically in the study area. Highly significant relationships were found between the temperature-normalised concentrations in soil and the precipitation amounts. A north/south enrichment factor was present only in soil with rapid early summer re-volatilisation kinetics from soil to air and autumn re-deposition events from air to soil. Fugacity ratio calculations confirmed these trends. Surface soils respond rapidly to meteorological variables, while subsurface soils respond much more slowly. Seasonal trends were different for the northern and southern sides of the mountain. A detailed picture of the interactions among temperature, precipitation, mountain aspects and soil features was obtained.

Predicting PCB concentrations in cow milk: validation of a fugacity model in high-mountain pasture conditions.

A fugacity model reported in the literature was applied to a high-altitude pasture in the Italian Alps. The model takes into account three compartments (digestive tract, blood and fat tissues) in unsteady-state conditions using food as the contamination source. Disregarding biotransformation inside cow tissues, the predicted concentrations of 14 polychlorinated biphenyls (PCBs) in milk were in good agreement with the observed data, especially for congeners known for their resistance to biotransformation (e.g., CB-138 and 153). In contrast, the predicted concentrations were clearly overestimated for congeners with high biotransformation susceptibilities. Therefore data measured in milk and faeces were used to calculate the first-order-biotransformation rate constants in dairy cows. The PCB absorption efficiency observed for pasture conditions was lower than that observed in the cowshed. The final version of the model included biotransformation and observed PCB absorption and was able to predict PCB concentrations in cow milk with mean differences between the predicted and measured data below ± 20% for most congeners.

Toxic effects and ultrastructural damages to Daphnia magna of two differently sized ZnO nanoparticles: does size matter?

The toxic effects of two differently sized ZnO nanopowders have been studied in Daphnia magna using advanced microscopy techniques. Five nanoZnO suspensions (0.1, 0.33, 1, 3.3 and 10 mg/L) were tested. The results of the 48-h acute toxicity tests performed with ZnO < 100 nm (bZnO) and ZnO < 50 nm (sZnO) showed slight effects, with EC50 values of 3.1 and 1.9 mg/L for bZnO and sZnO, respectively. Specimens exposed to 1 and 3.3 mg/L have been microscopically analysed and nanoparticles (NPs) from both concentrations have been found into midgut cells: i) in the microvilli; ii) in endocytic vesicles near the upper cell surface; iii) in some endosomes, as well as in mitochondria, in multivesicular and multilamellar bodies; iv) into the enterocytes' nuclei; v) free in the cytoplasm; vi) in the paracellular space between adjacent cells; vii) into the folded basal plasma membrane, and viii) in the gut muscolaris, suggesting that not only both nanoZnOs are able to interact with the plasmatic membrane of D. magna enterocytes, but also that they are capable to cross epithelial barriers. The ultrastructural changes increased with increasing concentrations and the worst morphological fields came from samples exposed to 3.3 mg/L of both nanoZnOs. Morphological effects were qualitatively similar between the two nanomaterials, but they appear to be much more frequent for sZnO NPs. Data from ICP-OES analyses demonstrated that the maximum Zn(++) concentration in our tested suspensions was 0.137 mg/L, which is well below the reported NOEC for the soluble Zinc. The corresponding Zn-salt exposures (0.1 mg/L Zn(++)) gave 0% of immobilized daphnids for both NPs suggesting that in our test medium nanoZnO toxicity is not driven by their solubilized ions. The large presence of NPs inside midgut cells after only 48-h exposure to nanoZnOs and their effects on the intestinal cells highlighted the toxic potential of these nanomaterials, also suggesting that studies on chronic effects are needed.