Susana I. L. Gomes

Mechanisms of response to silver nanoparticles on Enchytraeus albidus (Oligochaeta): survival, reproduction and gene expression profile.

Silver has antimicrobial properties and silver nanoparticles (Ag-NPs) have been some of the most widely used NPs. Information regarding their effects is still insufficient, in particular for soil dwelling organisms. The standard soil Oligochaete Enchytraeus albidus was used to study the effects of Ag in soils, using differential gene expression (microarray) and population (survival, reproduction) response to Ag-NPs (PVP coated) and AgNO₃. Results showed higher toxicity of AgNO₃ (EC₅₀<50 mg/kg) compared to toxicity of Ag-NPs (EC₅₀=225 mg/kg). Based on the biological and material identity, the difference in toxicity between Ag-NPs and AgNO₃ could possibly be explained by a release of Ag(+) ions from the particles or by a slower uptake of Ag-NPs. The indications were that the responses to Ag-NPs reflect an effect of Ag ions and Ag-NPs given the extent of similar/dissimilar genes activated. The particles characterization supports this deduction as there were limited free ions measured in soil extracts, maybe related to little oxidation and/or complexation in the soil matrix. The possibility that gene differences were due to different levels of biological impact (i.e. physiological responses) should not be excluded. Testing of Ag-NPs seem to require longer exposure period to be comparable in terms of effect/risk assessment with other chemicals.

Effect of Cu-nanoparticles versus one Cu-salt: Analysis of stress biomarkers response in Enchytraeus albidus (Oligochaeta)

Abstract In the present study, the main goal was to compare the effects of ionic copper versus copper nanoparticles in Enchytraeus albidus assessing the effect at the biomarker level, testing different concentrations and exposure times. Measured parameters were lipid peroxidation (LPO), total, reduced and oxidized glutathione content (TG, GSH and GSSG), the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione S-transferases (GSTs) and cholinesterases (ChEs). Results showed that both salt- and nano-copper caused oxidative stress and damage to E. albidus, as confirmed by LPO levels, and effects could be discriminated between the copper forms. Nevertheless and despite the visible discrimination between nano and the salt form (time and exposure dependent), there was no single or a set of biomarkers that provided the best discrimination.

Effect of Cu-nanoparticles versus Cu-salt in Enchytraeus albidus (Oligochaeta): differential gene expression through microarray analysis.

Despite increased utilization of copper (Cu) nanoparticles, their behaviour and effect in the environment is largely unknown. Enchytraeids are extensively used in studies of soil ecotoxicology. Ecotoxicogenomic tools have shown to be valuable in nanotoxicity interpretation. A cDNA microarray for Enchytraeus albidus has recently been developed, which was used in this study. We compared the gene expression profiles of E. albidus when exposed to Cu-salt (CuCl(2)) and Cu-nanoparticles (Cu-NP) spiked soil. Exposure time was 48 h with a concentration range of 400 to 1000 mg Cu/kg. There were more down-regulated than up-regulated genes. The number of differently expressed genes (DEG) decreased with increasing concentration for CuCl(2) exposure, whereas for Cu-NP, the number did not change. The number of common DEG decreased with increasing concentration. Differences were mainly related to transcripts involved in energy metabolism (e.g. monosaccharide transporting ATPase, NADH dehydrogenase subunit 1, cytochrome c). Overall, our results indicated that Cu-salt and Cu-NP exposure induced different gene responses. Indirect estimates of Cu-NP related ion-release indicated little or no free Cu(2+) activity in soil solutions. Hence, it was concluded that the Cu-NP effects were probably caused by the nanoparticles themselves and not by released ions.

Effects of silver nanoparticles to soil invertebrates: Oxidative stress biomarkers in Eisenia fetida

Silver nanoparticles (Ag-NPs) are among the most produced NPs worldwide having several applications in consumer products. Ag-NPs are known to cause oxidative stress in several organisms and cell lines, however comparatively less information is available regarding their effects on soil living invertebrates. The purpose of this study was to investigate if Ag-NPs cause oxidative stress on soil invertebrates. The model soil species Eisenia fetida was used. Our results showed that total glutathione (TG) is the first mechanism triggered by Ag-NPs, followed by glutathione peroxidase (GPx) and glutathione reductase (GR), however oxidative damage was observed for higher doses and exposure time (increased lipid peroxidation, LPO). AgNO3 exposure caused impairment in GPx and glutathione-S-transferase (GST), probably as result of the higher bioavailability of Ag in the salt-form. The current results indicate that effects are partly caused by Ag ions released from Ag-NPs, but specific particle effects cannot be excluded.

Cellular Energy Allocation to Assess the Impact of Nanomaterials on Soil Invertebrates (Enchytraeids): The Effect of Cu and Ag

The effects of several copper (Cu) and silver (Ag) nanomaterials were assessed using the cellular energy allocation (CEA), a methodology used to evaluate the energetic status and which relates with organisms’ overall condition and response to toxic stress. Enchytraeus crypticus (Oligochatea), was exposed to the reproduction effect concentrations EC20/50 of several Cu and Ag materials (CuNO3, Cu-Field, Cu-Nwires and Cu-NPs; AgNO3, Ag NM300K, Ag-NPs Non-coated and Ag-NPs PVP-coated) for 7 days (0-3-7d). The parameters measured were the total energy reserves available (protein, carbohydrate and lipid budgets) and the energy consumption (Ec) integrated to obtain the CEA. Results showed that these parameters allowed a clear discrimination between Cu and Ag, but less clearly within each of the various materials. For Cu there was an increase in Ec and protein budget, while for Ag a decrease was observed. The results corroborate known mechanisms, e.g., with Cu causing an increase in metabolic rate whereas Ag induces mitochondrial damage. The various Cu forms seem to activate different mechanisms with size and shape (e.g., Cu-NPs versus Cu-Nwires), causing clearly different effects. For Ag, results are in line with a slower oxidation rate of Ag-NMs in comparison with Ag-salt and hence delayed effects.

Cu-nanoparticles ecotoxicity--explored and explained?

The nano-form of copper (Cu-NPs) is already extensively used. In this paper the toxic effect of Cu in the worm Enchytraeus crypticus (Oligochaeta: Enchytraeidae) was assessed following exposure to (1) Cu-salt: freshly spiked soil with copper-nitrate, (2) Cu-NPs: freshly spiked soil with Cu nanoparticles (80nm), and (3) Cu-field: historically Cu contaminated soil (80years ago). Our main aims were to compare the three different exposure regimes and respective toxicity, and to determine how the oxidation state of the Cu and dissolution state of the particles differed. Characterization of in situ-exposure included identification of oxidation states with synchrotron generated X-ray absorption near-edge spectroscopy (XANES) analysis, activity of free Cu(2+) in soil-solution (Ion Selective Electrode), and the relative distribution of the labile Cu-fractions (Sequential Extraction). Freshly spiked Cu-salt was the most toxic for reproductive output of the worms, followed by Cu-NPs and then Cu-field. XANES indicated only one oxidation state (II) in Cu-salt and Cu-field soil, whereas in Cu-NPs soil it was present in all oxidation states (0, I and II). The partial oxidation of the Cu-NPs (in soil) was evident and with limited dissolution.

Non-avoidance behaviour in enchytraeids to boric acid is related to the GABAergic mechanism

Soil invertebrates, e.g. enchytraeids, are known to be able to avoid unfavourable conditions, which gives them an important ecological advantage. These organisms possess chemoreceptors that can detect stressors, which in turn activate responses such as avoidance behaviour. We studied the avoidance behaviour in response to boric acid (BA) using enchytraeids. Results showed not only no avoidance, but that increasing concentrations seemed to have an “attraction” effect. To study the underlying mechanism, a selection of genes targeting for neurotransmission pathways (acetylcholinesterase (AChE) and gamma-aminobutyric acid receptor (GABAr)) were quantified via quantitative real-time polymerase chain reaction (qPCR). Evidences were that BA is neurotoxic via the GABAergic system mechanism where it acts as a GABA-associated protein receptor (GABAAR) antagonist possibly causing anaesthetic effects. This is the first time that (non)avoidance behaviour in invertebrates was studied in relation with the GABAergic system. We strongly recommend the combination of such gene and/or functional assay studies with the avoidance behaviour test as it can bring many advantages and important interpretation lines for ecotoxicity with minor effort.

Shorter lifetime of a soil invertebrate species when exposed to copper oxide nanoparticles in a full lifespan exposure test

Toxicity tests that last the all life duration of the organisms are not common, instead, long-term tests usually include one reproductive cycle. In the present study we optimized and propose a lifespan (all life) term test using Enchytraeus crypticus (Oligochaeta). The effect of copper oxide nanoparticles (CuO-NPs) was assessed in this lifespan test and compared to copper salt (CuCl2), using the same effect concentrations on reproduction (EC50). Monitored endpoints included survival and reproduction over-time (202 days). Results from survival showed that CuO-NPs caused shorter life of the adults compared to CuCl2 (control LT50: 218 days > CuCl2 LT50: 175 days > CuO-NPs LT50: 145 days). The effect was even more amplified in terms of reproduction (control ET50: 158 days > CuCl2 ET50: 138 days > CuO-NPs ET50: 92 days). Results suggest that CuO-NPs may cause a higher Cu effect via a trojan horse mechanism. The use of lifespan tests brings a novel concept in soil ecotoxicity, the longevity. This is a particularly important aspect when the subject is nanomaterials toxicity, where longer term exposure time is expected to reveal unpredicted effects via the current short/long-term tests. The present study confirms this higher effect for CuO-NPs.

Comparative Responses of Helianthus annuus Plants and Calli Exposed to NaCI: II. Selection of Stable Salt tolerant Calli Cell Lines and Evaluation of Osmotic Adjustment and Morphogenic Capacity

Summary Salt tolerant cell lines of Helianthus annuus L. cv. SH222 were selected by sub-culturing cotyledon-derived calli on MSmod medium containing 0, 50, 100, 200 and 300 mmol/L NaCl. NaCl-adapted calli were compact and developed a dark green colour but sustained a regular growth on salt media. Calli adapted to 200 mmol/L (HA2) and to 300 mmol/L NaCI (HA3) had maximum growth rates at, respectively, 50 and 100 mmol/L NaCI; Na, Cl - and B contents increased in HA2 and HA3 calli , whereas K, Ca, NC 3 - , HPO 4 2- , Co, Fe and Mo levels decreased. There was an increase in glucose, fructose and sucrose levels in NaCl-adapted calli . Also, the contents of free amino acids increased significantly in adapted calli . This increase was mainly due to Pro followed by Ser and Gly. The presence of 50 mmol/L NaCl increased the rate of shoot formation, while higher concentrations inhibited it. Rooting was completely inhibited even at low concentrations of NaCI. Leaves from tolerant shoots (HA2-2 and HA2-5 lines) were transferred to MSmod medium to induce calli in the absence of NaCl. Calli from HA2-5 line sustained growth after transference to 200 mmol/L NaCl, indicating the persistence of salt tolerance at the mitotic level. It was possible to regenerate plants from HA2-5 shoots but plants were stunted and did not survive to acclimation.

High-throughput transcriptomics reveals uniquely affected pathways: AgNPs, PVP-coated AgNPs and Ag NM300K case studies

Understanding the mode of action of nanomaterials (NMs) aids in improving predictions and environmental risk assessment. In the present study, a high-throughput (HTP) microarray was used to study Enchytraeus crypticus gene expression. Four Ag materials (Ag NM300K, PVP-coated AgNPs, AgNPs, and AgNO3) were tested at reproduction effect concentrations, EC20 and EC50, to anchor gene expression responses to higher effect level. The results showed that while PVP-AgNPs and AgNPs had similar responses, Ag NM300K caused effects via a differentiated transcriptomic profile, with uniquely affected processes (e.g. transcytosis). For the AgNPs, the EC50 negatively affected apoptosis, which can lead to accumulation of abnormal cells and cause apical damage (reproduction). Mechanisms which are known to be related to Ag toxicity and which were observed here for the various Ag forms included apoptosis regulation, cell redox homeostasis, impairment of energy production and response to DNA damage. This HTP genomic tool enabled discrimination between Ag materials, which is not possible via standard tests (i.e. survival and reproduction endpoints). Moreover, gene expression analysis provided information regarding the mechanisms of toxicity of NMs and the pathways uniquely affected by NMs. An adverse outcome pathway (AOP) was drafted for the first time for Ag NMs; this AOP can and should be used as a basis for further research.