Both Zn biofortification and nutrient distribution pattern in cherry tomato plants are influenced by the application of ZnO nanofertilizer.

A pot experiment was conducted to determine the influence of commercial nanoparticles (ZnO-NPs) at different doses for use as nanofertilizer on nutrient uptake and its distribution in cherry tomato (Solanum lycopersicum L var. cerasiforme) plants in an acidic (soil pH 5.5) and calcareous soil (soil pH 8.5) from the Mediterranean area. We determined crop yield; macro- (N, P, K, Mg, S and Ca) and micro-nutrient (B, Cu, Fe, Mn, Na and Zn) concentrations in the different parts of the crop (root, stem, leaves and tomato fruits) and the extent of nutrient translocation to the aerial part of the plant. The concentrations of macronutrients N, P, K and Mg in tomato fruits grown in both soils can be considered adequate in terms of nutritional requirements. However, the Ca concentration in tomato fruits grown in the calcareous soil did not reach the required concentration to be considered sufficient. This effect was related to the characteristics of this calcareous soil. Although different concentrations of ZnO-NPs did not affect Fe and Na concentrations in tomato fruit, B concentration in tomato fruits increased with the application of ZnO-NPs. In addition, Cu concentration decreased with the application of ZnO-NPs compared to treatments without any Zn application (Nil-ZnO NP) in the calcareous soil. Manganese concentrations decreased with ZnO-NPs application in both soils. The effect of the application of ZnO-NPs depends on soil characteristics. Zinc applied as a nanofertilizer in the form of ZnO-NPs can be used to increase the crop yield and to obtain an adequate Zn biofortification in cherry tomato crop. The Zn concentrations in tomato fruits reached ranges of 4.5-4.8 mg Zn kg-1 in the acidic soil and 2.5-3,5 mg Zn kg-1 in the calcareous soil. Nutrient concentrations in these fruits following biofortification are adequate for human consumption.

Zn concentration decline and apical endpoints recovery of earthworms (E. andrei) after removal from an acidic soil spiked with coated ZnO nanoparticles.

ZnO nanoparticles (ZnO-NPs) can reach soil in both deliberate and non-deliberate ways, which leads to contamination. Notwithstanding knowledge about ZnO-NPs impacts on earthworms inhabiting these soils is limited and gaps appear in the recovery of damaged functions after their migration to unpolluted environments. To estimate these impacts, earthworms (Eisenia andrei) were exposed to different concentrations of coated ZnO-NPs (20, 250, 500, 1000 mgZnkg-1) in an acidic agricultural soil (pH 5.4) for 28 days. Subsequently, earthworms were placed in the same unpolluted soil to study the depletion of Zn accumulated and the recovery potential of the affected functions for another 28-day period.In the exposure phase, ecotoxicological responses were dose-dependent. Mortality and growth were affected at 500 and 1000 mg kg- 1, and the reproduction was impaired from 250 mgZnkg- 1 compared to control (54% fecundity and 80% fertility reduction). Zn uptake increased with coated ZnO-NPs in soil but it did not exceed 163 mgZnkg- 1 earthworm. During the recovery period, the Zn in earthworms were similar to the control regardless of the initially Zn accumulated. Reproduction parameters returned to the control values in the animals pre-exposed to 250 mgZnkg- 1 as coated ZnO-NP. In the earthworms preexposed to the two highest doses, growth and fertility were stimulated compared to the control when placed in clean soil, but not fecundity. However, the total hatchlings number did not reach the control figures after 28 days, but probably would for in longer times, which would be key for maintaining earthworm populations.

Study of Zn availability, uptake, and effects on earthworms of zinc oxide nanoparticle versus bulk applied to two agricultural soils: Acidic and calcareous.

The increasing use of zinc oxide nanoparticles (ZnO NPs) in agriculture renders it necessary to evaluate their impact on soil non-target organisms. This work studies Zn availability to earthworms from the ZnO (NP and bulk) applied to two agricultural soils with a different pH at 20, 225, 500, and 1000 mg Zn kg-1. Zn uptakes and the effects on Eisenia andrei, grown under controlled conditions, were determined. Effects were assessed at three levels: organisms, mortality, growth and reproduction; biochemical, catalase and glutathione S-transferase activities, malondialdehyde (MDA), and protein content; cellular in coelomocytes, reactive oxygen species (ROS) generation, lysosomal membrane alterations (RN) and mitochondrial dysfunction (MTT). Available Zn was 100-fold higher in acidic than in calcareous soil and did not differ among ZnO (NP or bulk). Zn in worms was auto-regulated regardless of the soil Zn concentration, pH and ZnO size. Effects on mortality and weight were observed only in the acidic soil at the highest concentration, ZnO NPs reduced survival and body weight, while ZnO bulk reduced body weight. Reproduction parameters in acidic soil were: EC50 (fecundity) 277 and 256 mg Zn kg-1 and EC50 (fertility) 177 and 179 mg Zn kg-1 for ZnO NPs and bulk, respectively, with no found NP-specific effects. No responses of enzymatic activities, MDA and MTT were detected. ROS and RN were altered in the coelomocyte cells of earthworms in the two soils, but effects depended on ZnO size suggesting nanospecific effects. Soil pH governs toxicity more than ZnO size regardless of body Zn concentration.

Effect of ageing of bare and coated nanoparticles of zinc oxide applied to soil on the Zn behaviour and toxicity to fish cells due to transfer from soil to water bodies.

This study evaluated the influence of ageing of ZnO nanoparticles (NPs) applied to soil on the potential availability and chemical speciation of Zn, and also of their toxicity to aquatic organisms due to transfer of contaminants from soil to water. To this end, soil samples were spiked with two types of bare nanoparticles: b1ZnO NPs (rod- and elongated-shaped) and b2ZnO NPs (near-spherical shaped) and ZnO NPs coated with (3-aminopropyl)triethoxysilane (cZnO NPs) within the 0-800 mg Zn kg-1 soil dose range, and were left to age for 0, 30, 60 and 90 days. The available concentration and speciation of Zn in soil were determined by the DGT (diffusive gradients in thin films) technique and sequential extraction procedures, respectively. The toxicity of the aqueous extracts from the ZnO NP-treated soils was assessed in vitro in established fish cell lines (RTG-2). The highest distribution percentages of the applied Zn occurred in the organically complexed (OC), followed by the exchangeable (EXC) fraction, for all NP types, applied doses and incubation times. The toxicity of NPs depended on their intrinsic properties: b1ZnO NPs affected the membrane function, reductase enzyme activity and, to a lesser extent, reactive oxygen species (ROS) levels of fish cells, whereas b2ZnO NPs and cZnO NPs affected mainly ROS generation. Ageing increased Zn soil availability, but toxicity to fish cells showed no trend over time. The particle dissolution of ZnO NPs did not explain the observed toxicity, hence a nanoparticles-specific effect should be assumed. The findings of this study seem to indicate that the transfer of ZnO NP from contaminated soils to aquatic ecosystems should be addressed.

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.

Influence of Zn-contaminated soils in the antioxidative defence system of wheat (Triticum aestivum) and maize (Zea mays) at different exposure times: potential use as biomarkers.

In this study, we evaluated the antioxidant responses of wheat and maize growing in Zn-treated soils (200, 450 and 900 mg kg(-1)) at different exposure times (7, 14, 21 and 35 days). The Zn concentration in the plants increased with an increase in the Zn concentration in the soil, thereby causing an increase in the accumulation of Mg and Mn. The emergence of wheat and the growth of maize were inhibited by Zn. The chlorophyll levels increased in wheat, whereas the opposite effect was observed in maize. Regarding enzymatic activities, Zn only provoked pronounced increases in the ascorbate peroxidase activity in maize at the early exposure times and occasionally in the superoxide dismutase (14 days) and catalase (7 and 35 days) activities in wheat. The most notable effect of the exposure of plants to Zn was an inhibition of antioxidative activities after 35 days in both plant species. The reduced glutathione levels increased in wheat and maize after 35 days and the protein levels in wheat after 7 and 35 days. The only significant alteration of lipid peroxidation was a decrease in the malondialdehyde level in wheat after 35 days. Results of this work suggest that Zn may generate oxidative stress by interfering with the plant antioxidant defence system (peroxidases, catalases and superoxide dismutase) responsible for free radical detoxification. The enzymatic activities, particularly ascorbate peroxidase, and the content of reduced glutathione could be considered good biomarkers of serious stress by Zn in soils.

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.

Assessing the ecotoxicological effects of long-term contaminated mine soils on plants and earthworms: relevance of soil (total and available) and body concentrations.

The interactions and relevance of the soil (total and available) concentrations, accumulation, and acute toxicity of several essential and non-essential trace elements were investigated to determine their importance in environmental soil assessment. Three plant species (T. aestivum, R. sativum, and V. sativa) and E. fetida were simultaneously exposed for 21 days to long-term contaminated soils collected from the surroundings of an abandoned pyrite mine. The soils presented different levels of As and metals, mainly Zn and Cu, and were tested at different soil concentrations [12.5, 25, 50, and 100% of contaminated soil/soil (w/w)] to increase the range of total and available soil concentrations necessary for the study. The total concentrations in the soils (of both As and metals) were better predictors of earthworm uptake than were the available concentrations. In plants, the accumulation of metals was related to the available concentrations of Zn and Cu, which could indicate that plants and earthworms accumulate elements from different pools of soil contaminants. Moreover, Zn and Cu, which are essential elements, showed controlled uptake at low concentrations. The external metal concentrations predicted earthworm mortality, whereas in plants, the effects on growth were correlated to the As and metal contents in the plants. In general, the bioaccumulation factors were lower at higher exposure levels, which implies the existence of auto-regulation in the uptake of both essential and non-essential elements by plants and earthworms.

Risk assessment of an abandoned pyrite mine in Spain based on direct toxicity assays.

This research reports the risk assessment of an abandoned pyrite mine using direct toxicity assays of soil and groundwater samples taken at the site. The toxicity of As and heavy metals from mining soils to soil and aquatic organisms was studied using the Multispecies Soil System (MS-3) in soil columns. Ecotoxicological assessment was performed with soil samples diluted with a control soil at concentrations of 12.5, 25, 50 and 100% test soil/soil (w/w). In this way, changes in the mobility and bioavailability of soil contaminants due to changes in geochemical soil properties via soil dilution were studied. The toxicity of water samples was tested on algae and Daphnia magna. The assessment of the mining area indicated that the current presence of As and heavy metals at the site may cause injuries to soil and aquatic organisms in the entire research area. Moreover, this investigation demonstrated that changes in geochemical conditions can increase the availability of arsenic and, consequently, the environmental risk of these soils. A good correlation was not found between toxicity parameters and the concentrations of soil contaminants based on total and extracted element concentrations. This finding reinforces the usefulness of direct toxicity assays for evaluating environmental risk.

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.

Screening risk assessment tools for assessing the environmental impact in an abandoned pyritic mine in Spain.

This paper describes a new methodology for assessing site-specific environmental impact of contaminants. The proposed method integrates traditional risk assessment approaches with real and variable environmental characteristics at a local scale. Environmental impact on selected receptors was classified for each environmental compartment into 5 categories derived from the whole (chronic and acute) risk assessment using 8 risk levels. Risk levels were established according to three hazard quotients (HQs) which represented the ratio of exposure to acute and chronic toxicity values. This tool allowed integrating in only one impact category all the elements involved in the standard risk assessment. The methodology was applied to an abandoned metal mine in Spain, where high levels of As, Cd, Zn and Cu were detected. Risk affecting potential receptors such as aquatic and soil organisms and terrestrial vertebrates were assessed. Whole results showed that impact to the ecosystem is likely high and further investigation or remedial actions are necessary. Some proposals to refine the risk assessment for a more realistic diagnostic are included.