Anita Jemec Kokalj

Plastic bag and facial cleanser derived microplastic do not affect feeding behaviour and energy reserves of terrestrial isopods

Current data regarding the effects of microplastic (MP) on terrestrial organisms are very scarce. Isopods play an important role in plant litter decomposition processes and are commonly used test species in terrestrial ecotoxicity studies. Their altered feeding behaviour and energy reserves are established biomarkers of adverse effects upon stressor exposure. For this study we assessed the effects of MP derived from plastic bag film (mean size 183±93μm) and particles from a facial cleanser (mean size 137±51μm) on the terrestrial isopod, Porcellio scaber. Isopods were exposed to MP via feeding on food pellets (4mgg-1 dry weight; 0.4% w w-1) for 14days under laboratory conditions. A control group was exposed to food pellets with no MP added. In line with previously suggested modes of MP action on animal ingestion, we assessed the food ingestion rate, defecation rate, food assimilation rate and efficiency, body mass change, mortality and energy reserves (proteins, carbohydrates, and triglycerides) in the digestive glands (hepatopancreas) of individual isopods. Contrary to our expectations, no effects on either end-point were observed under the given exposure conditions. Further work should be carried out to investigate the potential longer-term effects of such exposure. We conclude that 14days exposure to plastic bag and facial cleanser MP is not severely hazardous to isopods.

Cerium(IV) oxide nanoparticles induce sublethal changes in honeybees after chronic exposure

The high annual production and use of cerium(IV) oxide nanoparticles (nCeO2s) may lead to their atmospheric release and substantial deposition on plants. This poses a potential threat to pollinators. We investigated the effects of nCeO2-spiked food (2–500 mg L−1) on summer and winter honeybees (Apis mellifera carnica) after chronic 9 days oral exposure. Acetylcholinesterase (AChE) and glutathione S-transferase (GST) activities were measured in different body compartments (heads, thoraces, and haemolymph). The activity of AChE was assessed in salt-soluble (SS) (containing soluble and membrane AChE) and detergent-soluble (DS) (predominantly membrane-bound AChE) fractions. Exposure of honeybees to nCeO2-spiked food had no significant effects on survival up to 500 mg L−1 (<10%), while significant biochemical alterations were evidenced already at 2 mg L−1. In summer honeybees, a significant increase in the activities of AChE in the SS fraction and GST was found, while AChE activity in DS fractions was decreased at nearly all exposure concentrations. An exception was the 250 mg L−1 exposure, where AChE activity in DS fractions was increased. The alteration of AChE in the DS fraction could be symptomatic for the affected neuronal system, while alterations of GST activity indicate detoxification processes. An apparent difference in response to nCeO2 was evidenced between the summer and winter honeybees, which is in line with their different physiology. We ascribe most of the observed effects to particulate nCeO2 because a negligible presence of Ce ion species was found in their food. We conclude that nCeO2 release into the environment, especially atmospherically deposited material, is a potential risk to honeybees.

The use of multiwell culture plates in the duckweed toxicity test—A case study on Zn nanoparticles

Extensive production of nanomaterials of various properties needs to be coupled with rapid toxicity testing in order to provide information about their potential risks to the environment and human health. Miniaturization of toxicity tests may accelerate economical testing of nanomaterials, but is not a common practice. We describe a case study to miniaturize a commonly used toxicity test with plant duckweed Lemna minor. 6-well, 12-well and 24-well culture plates were used to assess their potential use for the duckweed toxicity test with potassium chloride as reference material. The results were compared to the standard test design using 100u202fmL glass beakers. The comparison showed that the best agreement was with the 6-well vessels. This set-up was further used for toxicity testing of zinc oxide nanoparticles (ZnO NP) and zinc chloride. Zinc was not adsorbed onto either glass or plastic walls of the miniaturized system. We assume that in both vessels a fast agglomeration and settling of ZnO NP took place. Linear regression and statistical testing indicated a good correlation between the toxicity results obtained in the standard test and miniaturized 6-well vessels. The miniaturization of the test system for assessing the biological effect of nanomaterials on Lemna minor could become an appropriate alternative to the traditionally used high volume vessels.

The Biological Fate of Silver Nanoparticles from a Methodological Perspective

We analyzed the performance and throughput of currently available analytical techniques for quantifying body burden and cell internalization/distribution of silver nanoparticles (Ag NPs). Our review of Ag NP biological fate data shows that most of the evidence gathered for Ag NPs body burden actually points to total Ag and not only Ag NPs. On the other hand, Ag NPs were found inside the cells and tissues of some organisms, but comprehensive explanation of the mechanism(s) of NP entry and/or in situ formation is usually lacking. In many cases, the methods used to detect NPs inside the cells could not discriminate between ions and particles. There is currently no single technique that would discriminate between the metals species, and at the same time enable localization and quantification of NPs down to the cellular level. This paper serves as an orientation towards selection of the appropriate method for studying the fate of Ag NPs in line with their properties and the specific question to be addressed in the study. Guidance is given for method selection for quantification of NP uptake, biodistribution, precise tissue and cell localization, bioaccumulation, food chain transfer and modeling studies regarding the optimum combination of methods and key factors to consider.

Environmental Impacts of Engineered Nanomaterials—Imbalances in the Safety Assessment of Selected Nanomaterials

For this Editorial, we, the guest editors, performed a brief review with the aim of setting the framework for the Special issue on the Environmental Impacts of Nanomaterials [...].

Integration of behavioral tests and biochemical biomarkers of terrestrial isopod Porcellio scaber (Isopoda, Crustacea) is a promising methodology for testing environmental safety of chars

We investigated how different carbonized materials, or chars (olive mill pomace biochar (BC) and urban greens pruning residues hydrochar (HC)) affect an important member of soil fauna, the terrestrial isopod Porcellio scaber. The selection behavior of isopods towards chars after a 48xa0h exposure was studied in (i) soil-char amended tests with single and multiple choices, and (ii) tests with chars offered as pure material. Finally, we exposed the isopods to char-amended soils for a period of 14 days to follow the effect on food consumption, body mass and activities of enzymes that are commonly altered upon stressor exposure (acetylcholinesterase, AChE, and glutathione S-transferase, GST). We showed that isopods are able to select between char amended and un-amended soil and different forms of char amendments: a clear preference for BC, and avoidance of HC were evidenced. The preferences remained the same when the chars were sterilized leading to the conclusion that initial microorganism composition was not the reason for selection, but selection was governed by other chars physico-chemical properties. It remains to be elucidated which of these properties were the dominant reason for the selection. We also showed that isopods intentionally use BC as food at a similar rate to alder leaves. Medium-term exposure to HC resulted in adverse effects on isopods because it led to reduced feeding and growth, in addition to increasing GST activity, although no alterations in AChE activity were found. We suggest that behavioral tests with P.xa0scaber could be used as a fast, reliable and economically feasible screening method for determining the safety of chars for the soil environment. Results represent significant contribution in the field of char toxicity testing, highlighting the importance of tests with isopods as important members of soil meso fauna, with the aim of influencing environmental policies and quality standards.

The significance of nanomaterial post-exposure responses in Daphnia magna standard acute immobilisation assay: Example with testing TiO 2 nanoparticles

One of the most widely used aquatic standarized tests for the toxicity screening of chemicals is the acute toxicity test with the freshwater crustacean Daphnia magna, which has also been applied in the toxicity screening of manufactured nanoparticles (NPs). However, in the case of non-soluble NPs most of the results of this test have showed no effect. The aim of the work presented here was to modify the standardized test by the least possible extent to make it more sensitive for non-soluble particles. The standard acute immobilisation assay with daphnids was modified by prolonging the exposure period and by measuring additional endpoints. Daphnids were exposed to TiO2 NPs in a standard acute test (48h of exposure), a standard acute test (48h of exposure) followed by 24h recovery period in clean medium or a prolonged exposure in the NPs solutions totaling 72h. Together with immobility, the adsorption of NPs to body surfaces was also observed as an alternative measure of the NPs effects. Our results showed almost no effect of TiO2 NPs on D. magna after the 48h standard acute test, while immobility was increased when the exposure period to TiO2 NPs was prolonged from 48h to 72h. Even when daphnids were transferred to clean medium for additional 24h after 48h of exposure to TiO2 NPs the immobility increased. We conclude that by transferring the daphnids to clean medium at the end of the 48h exposure to TiO2 NPs, the delayed effects of the tested material can be seen. This methodological step could improve the sensitivity of D. magna test as a model in nanomaterial environmental risk assessment.

Different response of acetylcholinesterases in salt- and detergent-soluble fractions of honeybee haemolymph, head and thorax after exposure to diazinon

Organophosphate pesticide diazinon is a specific inhibitor of acetylcholinesterase (AChE), which is a common neurotoxicity biomarker in environmental studies. In honeybees, AChE exists in two forms having different physiological roles, one existing as a soluble form and the other as membrane-bound. In most studies AChE activity has been analysed without paying considerable attention to different forms of AChE. In this study, we exposed honeybees Apis mellifera carnica for 10days to diazinon via oral exposure and analysed the total AChE activities in salt soluble (SS) and detergent soluble (DS) fractions. We assumed that SS fraction would preferentially contain the soluble AChE, but the DS fraction would contain only membrane AChE. On the contrary, our results showed that SS and DS fractions both contain soluble and membrane AChE and the latter has considerably higher activity. Despite this we obtained a differential response of AChE activity in SS and DS fractions when exposed to diazinon. The head/thorax AChE activity in DS fraction decreased, while the head/thorax AChE activity in SS fraction increased at sublethal concentrations. The AChE activity in honeybee hemolymph shown here for the first time is a salt soluble enzyme. Its activity remained unaltered after diazinon treatment. In conclusion, we provide evidence that varying results regarding AChE activity alterations upon stressor exposure are obtained when extracted through different procedures. In further environmental studies with honeybees this differential response of AChE activity should be given considerable attention because this affects the outcome of ecotoxicity study.

Screening study of four environmentally relevant microplastic pollutants: Uptake and effects on Daphnia magna and Artemia franciscana

This study investigated four different environmentally relevant microplastic (MP) pollutants which were derived from two facial cleansers, a plastic bag and polyethylene textile fleece. The mean size range of the particles (according to number distribution) was 20-250u202fμm when measured as a powder and 0.02-200u202fμm in suspension. In all MP exposures, plastic particles were found inside the guts of D.xa0magna and A.xa0franciscana, but only in the case of daphnids a clear exponential correlation between MP uptake in the gut and the size of the MP was identified. Exposure tests in which the majority of the MP particles were below 100u202fμm in size also had higher numbers of daphnids displaying evidence of MP ingestion. As the average MP particle size increased, the percentage of daphnids which had MP in their gut decreased. Using a number distribution value to measure particle size when in a suspension is more experimentally relevant as it provides a more realistic particle size than when samples are measured as a powder. Generally, artemias had fewer MP particles in the gut, than the daphnids, which could be explained by their different food size preferences. No acute effects on D.xa0magna were found, but the growth of A.xa0franciscana was affected. We conclude that zooplankton crustacean can ingest various MPs but none of the exposures tested were highly acutely hazardous to the test species. In addition, no delayed lethal effects in a 24u202fh post-exposure period were found.

The in vivo effects of silver nanoparticles on terrestrial isopods, Porcellio scaber, depend on a dynamic interplay between shape, size and nanoparticle dissolution properties

The present work aims to study the effects that acute exposure to low concentrations of silver nanoparticles (AgNPs) cause in digestive glands of terrestrial isopods (Porcellio scaber). The experiments were designed to integrate different analytical techniques, such as transmission electron microscopy, atomic absorption spectroscopy, proton induced X-ray emission, and Fourier transform IR imaging (FTIRI), in order to gain a comprehensive insight into the process from the AgNPs synthesis to their interaction with biological tissues in vivo. To this aim, terrestrial isopods were fed with AgNPs having different shapes, sizes, and concentrations. For all the tested conditions, no toxicity at the whole organism level was observed after 14 days of exposure. However, FTIRI showed that AgNPs caused detectable local changes in proteins, lipids, nucleic acids and carbohydrates at the tissue level, to an extent dependent on the interplay of the AgNPs properties: shape, size, concentration and dissolution of ions from them.