Joint effects of zinc oxide nanoparticles and chlorpyrifos on the reproduction and cellular stress responses of the earthworm Eisenia andrei.

The co-exposure of soil organisms to ZnO nanoparticles (ZnO NPs) and pesticides is likely to take place in agricultural soils. However, the impacts of co-exposure on terrestrial ecosystems are virtually unknown. In this paper, Eisenia andrei was exposed for a 28-day period to serial concentrations of ZnO NPs and/or the organophosphate insecticide chlorpyrifos (CPF) in natural soil, and was evaluated for single and joint effects. Zn and CPF accumulation in earthworm tissue was also determined. In the single assay, ZnO NPs and CPF caused statistical significant effects on survival and growth, but mainly on reproduction. Significant reductions in fecundity and fertility were detected with EC50 values of 278 and 179 mg Zn/kg for ZnO NPs, and of 50.75 and 38.24 mg/kg for CPF, respectively. The most notable effect on biomarkers was the reduction in acetylcholinesterase (AChE) activity caused by CPF, which reflected the neurotoxicity of this compound. The results of the combined assay indicated that co-exposure to ZnO NPs and CPF increased adverse effects in E. andrei. According to the independent action model, the binary mixtures showed a synergism (a stronger effect than expected from single exposures) on earthworm reproduction, which became up to 84% higher than the theoretically predicted values. Zn, and especially CPF accumulation, were influenced by the co-exposure. These results underpin the need to consider the effects of mixtures of NPs and organic chemicals on soil to adequately make ecological risk assessments of NPs.

Soil pH effects on the toxicity of zinc oxide nanoparticles to soil microbial community.

We conducted an experiment with two agricultural soils with acidic and alkaline pH levels to assess the effects of zinc oxide nanoparticles (nZnO) on the bacterial community. The effect of the nZnO concentrations (0, 0.1, 1, 10, 100, 1000 mg Zn/kg soil) and contact time between nanoparticles and soil (180 days) was considered. We measured the microbial respiration rate, nitrogen transformation, enzymatic activities (dehydrogenase (DH), acidic phosphatase (ACP), and alkaline phosphatase (ALP)), and the community-level physiological profile (CLPP) soil parameters. Respiration potential and nitrogen transformation were negatively affected only at the highest nZnO concentration. The changes in enzymatic activities were very variable with time and between both soils. A stimulating effect of the nanoparticles on microbial activity was clearly shown at 30 days after the nZnO application in both soils, except for the 1000 mg/kg in calcareous soil, after which time in the latter, the functional richness of the bacterial community was reduced to virtually zero. However, values of the enzymatic activities demonstrated that there was self-adaptation of microbial communities over the study period (180 days). The nZnO 1000 mg/kg dose produced an increase in bacterial growth in the acidic soil, without apparent changes in their metabolic profiles over time. A good correlation was found between microbial respiration rates (calcareous and acidic soils) and microbial metabolic activity (acidic soil) based on extracted Zn concentrations. Our findings suggest the necessity of additional studies to examine the effects of nZnO in natural microorganism populations in soil with different pH levels for extended periods of time.

Comparative study of the phytotoxicity of ZnO nanoparticles and Zn accumulation in nine crops grown in a calcareous soil and an acidic soil.

The increasing use of zinc oxide nanoparticles (ZnO NPs) in agriculture and consumer products has created the need to evaluate their impact on crops. Nine crops were investigated: wheat, maize, radish, bean, lettuce, tomato, pea, cucumber, and beet. The toxic effects of ZnO NPs on seed germination, plant growth, and biochemical parameters, including photosynthetic pigments, protein and malondialdehyde (MDA) content, reactive oxygen species (ROS), enzymes of the antioxidant defence system, as well as the Zn translocation in the plants were investigated using pots containing non-contaminated or ZnO NP-contaminated soil at concentrations of 20, 225, 450, and 900 mg Zn kg-1. Two soils with different physicochemical properties, namely a calcareous soil and an acidic soil, were used. The total and available Zn in the soils were correlated with the Zn in the plants (roots and shoots) and the observed effects. In the calcareous soil, the available Zn was very low and the phytotoxicity was limited to a slight reduction in the biomass for wheat, cucumber, and beet at the highest concentration. Only beet showed an increase in photosynthetic pigments. The parameters related to oxidative stress were affected to different degrees depending on the crop, with the exceptions of maize, lettuce, pea, and beet. In the acidic soil, the available Zn was high, and the germination of bean, tomato, lettuce, and beet, and the growth of most of the crops were seriously affected. The calculated EC50 values (growth) in the acidic soil ranged from 110 to 520 mg Zn kg-1. The photosynthetic pigments and most of the markers of oxidative stress were negatively affected in maize, wheat, bean, and pea. However, these changes were not always associated with a decrease in plant weight. In summary, soil pH and plant species are key factors affecting the Zn availability and phytotoxicity of ZnO NPs.

Effects of aged ZnO NPs and soil type on Zn availability, accumulation and toxicity to pea and beet in a greenhouse experiment.

Most studies have assessed the toxicity of pristine NPs to plants without considering the likely changes that these NPs will undergo during their residence time in the soil. In this study, we assessed the effects of ZnO NPs (3, 20, and 225 mg Zn kg-1 soil) aged for a year in soil and after a previous crop on the Zn availability in soil, leaf accumulation and toxicity to green pea (Pisum sativum L.) and beet root (Beta vulgaris L). The effects were compared to bulk ZnO and ZnSO4 in two agricultural soils with different pH under greenhouse conditions. The Zn concentration in the plant leaf was 6-12-fold higher in acidic than in calcareous soil that could explain the different effects on plants caused by Zn applications depending on soil type. Thus, in acidic soil, ZnO NPs promoted ROS generation in both plant species with increases from 47% to 130%, increased the MDA content in pea up to 58 ±â€¯8% in plant exposed to ZnSO4 at 225 mg Zn kg-1 soil and altered the ratio of photosynthetic pigments in beet between 12% and 41%, suggesting distressed chloroplast constituents. In calcareous soil, the changes seemed to be related to the supply of Zn in Zn deficient soils, whose principal effect was the 20-65% decrease of ROS levels in treated plants. The available and leaf Zn concentrations did not differ among Zn sources. Likewise, ZnO NPs showed comparable toxic or stimulatory effects to ZnO bulk and Zn salt, with some exceptions where Zn ion showed the highest phytotoxicity and effectiveness as a micronutrient. According to our results, we cannot affirm that NPs pose a higher potential environmental risk than their bulk counterparts after one-year of residence time in soil.

Comparative effect of ZnO NPs, ZnO bulk and ZnSO4 in the antioxidant defences of two plant species growing in two agricultural soils under greenhouse conditions.

The present study has investigated the toxicity of ZnO NPs to bean (Phaseolus vulgaris) and tomato (Solanum lycopersicon) crops grown to maturity under greenhouse conditions using an acidic (soil pH5.4) and a calcareous soil (soil pH8.3). The potentially available Zn in the soils and the Zn accumulation in the leaves from NPs applied to the soil (3, 20 and 225mgZnkg-1) and changes in the chlorophylls, carotenoids and oxidative stress biomarkers were measured at 15, 30, 60 and 90days and compared with those caused by bulk ZnO and ZnSO4. The available Zn in the soil and the leaf Zn content did not differ among the Zn chemical species, except in the acidic soil at the highest concentration of Zn applied as Zn ions, where the highest values of the two variables were found. The ZnO NPs showed comparable Zn toxicity or biostimulation to their bulk counterparts and Zn salts, irrespective of certain significant differences suggesting a higher activity of the Zn ion. The treatments altered the photosynthetic pigment concentration and induced oxidative stress in plants. ROS formation was observed at Zn plant concentrations ranging from 590 to 760mgkg-1, but the effects on the rest of the parameters were highly dependent on the plant species, exposure time and especially soil type. In general, the effects were higher in the acidic soil than in the calcareous soil for the bean and the opposite for the tomato. The similar uptakes and toxicities of the different Zn forms suggest that the Zn ions derived from the ZnO NPs exerted a preferential toxicity in plants. However, several results obtained in soils treated with NPs at 3mgZnkg-1 soil indicated that may exist other underlying mechanisms related to the intrinsic nanoparticle properties, especially at low NP concentrations.

Ecotoxicological evaluation of sewage sludge contaminated with zinc oxide nanoparticles.

The objective of this work was to evaluate the ecotoxicological qualitative risk associated with the use of sewage sludge containing Zn oxide nanoparticles (ZnO-NPs) as soil amendment. A sludge-untreated soil and two sludge-treated soils were spiked with ZnO-NPs (0-1,000 mg/kg soil). Soil ecotoxicity was assessed with Eisenia fetida (acute and sublethal end points), and the unfilterable and filterable (0.02 µm) soil leachates were tested with a battery of biomarkers using Chlorella vulgaris, Daphnia magna, and the fish cell line RTG-2 (Oncorhynchus mykiss). The production of E. fetida cocoons in sludge-treated soils was lower than that in sludge-untreated soils. The highest effect in the algal growth inhibition test was detected in sludge-untreated soil, most likely caused by the loss of organic matter in these samples. The D. magna results were always negative. Toxic effects (lysosomal cell function and production of reactive oxygen species) in RTG-2 cells were only observed in sludge-treated soils. In general, the toxicity of ZnO-NPs in sludge-treated soils was similar to that of sludge-untreated soil, and the filterable leachate fraction [Zn salt (Zn(2+))] did not produce greater effects than the unfilterable fraction (ZnO-NPs). Thus, after the addition of ZnO-NP--enriched sewage sludge to agricultural soil, the risk of toxic effects for soil and aquatic organisms was shown to be low. These findings are important because repeated use of organic amendments such as sewage sludge may cause more and more increased concentrations of ZnO-NPs in soils over the long-term.

Evaluation of zinc oxide nanoparticle toxicity in sludge products applied to agricultural soil using multispecies soil systems.

To study the environmental impact of nanoparticles, the sludges of wastewater (WWTS) and water treatment (WTS) plants enriched with ZnO nanoparticles were added to agricultural soil, and the toxic effects of the nanoparticles were studied using a microcosm system based on the soil. The WWTS treated soils were characterised by statistically significant decreases (p<0.05) in Vicia sativa germination at the lowest (76.2%) and medium (95.2%) application rates, decreases in the fresh biomass for Triticum aestivum (19.5%), Raphanus sativus (64.1%), V. sativa (37.4%) and Eisenia fetida (33.6%) at the highest application rate and a dose-related significant increase (p<0.05) in earthworm mortality. In WTS amended soils, significant reductions (p<0.05) of the fresh biomass (17.2%) and the chlorophyll index (24.4%) for T. aestivum and the fresh biomass for R. sativus (31.4%) were only recorded at the highest application doses. In addition, the soil phosphatase enzymatic activity decreased significantly (p<0.05) in both WWTS (dose related) and WTS treatments. For water organisms, a slight inhibition of the growth of Chlorella vulgaris was observed (WWTS treated soils), along with statistically significant dose-related inhibition responses on total glutathione cell content, and statistically significant dose-related induction responses on the glutathione S-transferase enzyme activity and the reactive oxygen species generation on the RTG-2 fish cell line.

In vitro evaluation of cellular responses induced by ZnO nanoparticles, zinc ions and bulk ZnO in fish cells.

Zinc oxide nanoparticles (ZnO-NPs) are inevitably released into the environment and are potentially dangerous for aquatic life. However, the potential mechanisms of cytotoxicity of zinc nanoparticles remain unclear. Studying the toxicity of ZnO-NPs with In vitro systems will help to determine their interactions with cellular biomolecules. The aim of this study was to evaluate the cytotoxic potentials of ZnO-NPs in established fish cell lines (RTG-2, RTH-149 and RTL-W1) and compare them with those of bulk ZnO and Zn(2+) ions. Membrane function (CFDA-AM assay), mitochondrial function (MTT assay), cell growth (KBP assay), cellular stress (ß-galactosidase assay), reductase enzyme activity (AB assay), reactive oxygen species (ROS), total glutathione cellular content (tGSH assay) and glutathione S-transferase (GST) activities were assessed for all cell lines. ZnO-NPs cytotoxicity was greater than those of bulk ZnO and Zn(2+). ZnO-NPs induced oxidative stress is dependent on their dose. Low cost tests, such as CFDA-AM, ROS, GST activity and tGSH cell content test that use fish cell lines, may be used to detect oxidative stress and redox status changes. Particle dissolution of the ZnO-NPs did not appear to play an important role in the observed toxicity in this study.

Effects of individual and a mixture of pharmaceuticals and personal-care products on cytotoxicity, EROD activity and ROS production in a rainbow trout gonadal cell line (RTG-2).

The presence of pharmaceuticals and personal-care products (PPCPs) in aquatic environments is of concern. Although measured concentrations of individual substances are low, little consideration has been given to the likely chronic nature of the exposures or to the potential for mixture effects. The purpose of the present study was to use the RTG-2 rainbow trout cell line to analyse sub-lethal and cytotoxic effects of PPCPs present in a wastewater-treatment-plant (WWTP) effluents and their mixtures. Interactions with cytochrome P450 1A enzyme, oxidative stress, cellular senescence and cell viability were assessed using 7-ethoxyresorufin-o-deethylase (EROD), reactive oxygen species (ROS), ß-galactosidase (ß-gal) and neutral red (NR) uptake assays, respectively. Not all of the compounds that were tested exhibited significant effects. The lowest-observed-effect concentrations and half maximal effective concentrations (EC50 ) were within the range 0.15 to 784.47 µg l(-1) . Clear dose-response curves were found for cells exposed to different mixtures of PPCPs. The lowest-observed-effect concentrations and concentrations causing EC50 were within the range 0.05 to 54.61 µg l(-1) . Four out the seven tested mixtures induced EROD activity. ROS production was detected in two mixtures. The ß-gal inhibition response was observed in six out the seven tested mixtures and occurred at a higher concentration than was observed for EROD induction activity or ROS generation. The present study clearly shows that the stress response through which cells mount a homeostatic response to toxicants can be potentially used for an initial, rapid and cost-effective assessment of the complex mixtures of PPCP that present in WWTP effluents are difficult and expensive to analyse chemically.

Bioventing remediation and ecotoxicity evaluation of phenanthrene-contaminated soil.

The objectives of soil remediation processes are usually based on threshold levels of soil contaminants. However, during remediation processes, changes in bioavailability and metabolite production can occur, making it necessary to incorporate an ecotoxicity assessment to estimate the risk to ecological receptors. The evolution of contaminants and soil ecotoxicity of artificially phenanthrene-contaminated soil (1000 mg/kg soil) during soil treatment through bioventing was studied in this work. Bioventing was performed in glass columns containing 5.5 kg of phenanthrene-contaminated soil and uncontaminated natural soil over a period of 7 months. Optimum conditions of mineralisation (humidity=60% WHC; C/N/P=100:20:1) were determined in a previous work. The evolution of oxygen consumption, carbon dioxide production, phenanthrene concentration and soil toxicity were studied on sacrificed columns at periods of 0, 3 and 7 months. Toxicity to soil and aquatic organisms was determined using a multispecies system in the soil columns (MS-3). In the optimal bioventing treatability test, we obtained a reduction rate in phenanthrene concentration higher that 93% after 7 months of treatment. The residual toxicity obtained at the end of the treatment was not attributed to the low phenanthrene concentration, but to the ammonia used to restore the optimal C/N ratio.

Application of bioassays for the ecotoxicity assessment of contaminated soils.

The use of bioassays for soil characterization is receiving significant attention as a complementary tool to chemical analysis. Bioassays consist of direct toxicity assays of environmental samples that are transferred to the laboratory and analyzed for toxicity against selected organisms. Such soil samples contain the combination of the different pollutants present in situ and enable factors such as the bioavailability of contaminants or the interactions (synergic and antagonic) between them to be simultaneously studied.In this chapter, methods for soil toxicity assessment based in the guidelines developed by OECD for single substances are described. These methods have been adapted for their application to the assessment of complex matrices such as soils. The field sample can be tested undiluted and/or diluted with "uncontaminated" soil to create a pollution gradient. In the diluted samples, concentration/response relationships may be obtained. Toxicity assays to soil organisms include earthworms, plants, and microorganisms tests. In addition, toxicity assays with soil extracts are recommended. Assays of extracts with algae, daphnia, and fish (in vitro test using fish cell lines) are also described.

The Prestige oil spill: a laboratory study about the toxicity of the water-soluble fraction of the fuel oil.

The Prestige oil spill caused severe effects on the coastal fauna and flora due to direct contact of organisms with the fuel oil. However, the water soluble fraction (WSF) of the fuel oil can also provoke deleterious effects in the long term and even in regions not directly affected by the spill. Our objective was to determine the toxicity of the WSF using a battery of laboratory toxicity tests. To obtain a WSF in the laboratory, a sample of the spilled fuel was mixed with adequate medium, sonicated, agitated and filtered. No cytotoxic effects were detected in RTG-2 cells exposed to the WSF. In an algae growth inhibition test (OECD test guideline 201) the WSF did not affect the growth of Chlorella vulgaris. Furthermore, acute and reproductive toxicity tests (OECD test guideline 202) carried out using Daphnia magna did not indicate any deleterious effect of the WSF. In a bioassay designed in our laboratory, D. magna were fed with algae previously exposed to the fuel, but no toxic effects were detected. However, the WSF was able to induce a dose-dependent increase of ethoxyresorufin-O-deethylase activity in RTG-2 cells, indicating the presence of chemicals that could cause sub-lethal effects to organisms. After chemical analyses it was established that the final total quantity of polyaromatic hydrocarbons dissolved in medium was approximately 70 ng/ml. These low concentrations explain the observed lack of toxicity.

Ecological risk assessment of contaminated soils through direct toxicity assessment.

A microcosm (MS-3) with a multispecies soil system is introduced as an experimental tool for direct toxicity assessment of contaminated soils. The capacity of MS-3 to determine soil ecotoxicity potential was evaluated using samples from three sites contaminated with organic and/or inorganic compounds. Soils were toxic to soil-dwelling organisms (earthworm, plants, and microorganisms) and to aquatic organisms (algae and RTG-2 cell fish). As expected, responses varied substantially among different soils and organisms. The application of this evaluation system provided complementary information to the chemical characterization. For soils containing metals the toxic response was lower than predicted from total metal concentrations. For hydrocarbons, the toxicity response agreed with estimated values. The induction of EROD activity suggested the presence of dioxin-like compounds, which had not been addressed in the chemical characterization. The proposed multispecies system affords the measurement of 11 endpoints covering three soil and three aquatic taxonomic groups, reproduces soil conditions and gradients, and appears as an excellent complementary tool to chemical analysis for characterization of contaminated sites.

In vitro toxicity of antimicrobials on RTG-2 and RTL-W1 fish cell lines.

This study proposed a battery of endpoints based on in vitro fish cell lines. Two fish cell lines and four toxicity endpoints are considered. The tetracycline (TC), oxytetracycline (OTC), sulfachlorpyridazine (SCP), and septrin(®) (ST) were selected as model antimicrobials and chlorpyrifos as positive control. EROD was induced by septrin(®) (formulation containing sulfamethoxazole and trimethoprim) at concentrations higher than 20mg/L, but inhibited by tetracycline, oxytetracycline and chlorpyrifos. Dose dependent inhibition responses were observed for ß-galactosidase (ß-Gal) activity in cells exposed to septrin(®), tetracycline or oxytetracycline. The EROD EC50s ranged between 2.29×10(-2)mg/L (chlorpyrifos) and 167.63mg/L (tetracycline). The ß-Gal EC50s ranged between 22.1 (septrin(®)) and 84.59mg/L (tetracycline). Data suggest that in vitro testing using a battery of endpoints can be a cost-effective solution for screening the toxicity of antimicrobials on fish. The absence of in vitro effects at concentrations well above those expected/measured in the environment may replace the need for conducting acute lethality tests on fish.

Effect of the Host and Temperature on the Formation of Defective RNAs Associated with Broad bean mottle virus Infection.

ABSTRACT Previously, we demonstrated that Broad bean mottle virus (BBMV), a member of the genus Bromovirus, could accumulate RNA 2-derived defective interfering (DI) RNAs during infection. In this work, we study how host and environmental factors affect the accumulation of DI RNAs. Serial passages of BBMV through selected plant species reveal that, with low-multiplicity inocula, some systemic hosts (Vicia faba, Nicotiana clevelandii, and N. tabacum cv. Samsum) support DI RNA accumulation after the first passage cycle but other hosts (Phaseolus vulgaris, Pisum sativum, and Glycine max) do not. However, several passages with the high-multiplicity inocula can generate DI RNAs in pea plants. Local lesion hosts (Chenopodium quinoa, C. amaranticolor, and C. murale) remain free of the DI RNA components. The size of the de novo-formed DI RNAs depends on the host and on environmental conditions. For instance, broad bean plants cultivated in a greenhouse or in a growth chamber at 20 degrees C accumulated DI RNAs of 2.4 or 1.9 kb in size, respectively. A reverse trend was observed in pea plants. Lower temperatures greatly facilitated the formation of DI RNAs in broad bean and pea hosts after the first passage. The importance of these findings for the studies on DI RNAs are discussed.