Conversely, while nCuO did not cause changes in mitochondrial membrane potential , CuSO4 significantly decreased MMP even at 1 mg/L. Interestingly, nCuO and CuSO4 acted as a chemosensitizers and increased the developmental toxicity of vinblastine, a cell division inhibitor . In an evaluation of 24 different metal oxides, nCuO was found to rank among the top most toxic within this group of NPs . Human bronchial epithelial and murine myeloid cell lines were used to assess oxidative stress and acute pulmonary inflammation. Exposure to nCuO resulted in a marked decline in cell viability even at 0.4 μg/L and a dose-dependent decline in the cellular energy levels . Additionally, nCuO also affected ROS production, intracellular calcium flux, mitochondrial membrane potential, and surface membrane permeability in BEAS-2B and RAW 264.7 cells . To corroborate the in vitro assays, in vivo toxicity assays of acute pulmonary inflammation were conducted with C57 BL/6 mice. nCuO induced statistically significant increases in neutrophil cell counts, monocyte chemo attractant protein-1 , and interleukin-6 levels, compared to less toxic NPs, indicating a strong immune system defense response.Pseudofeces can be ingested by other organisms in the water column or sediments, potentially resulting in Cu bio-availability and bio magnification. Given the likelihood that a fraction of Cu NPs will pass through WWTPs , the microbial community in a decentralized wastewater treatment system was subjected to 3 individual experiments lasting 3 weeks to a low uniform dose of different Cu particles: nCu, nCu2-b, and μCu . The three particles disrupted septic tank function,hydroponic grow system which was unable to meet treatment objectives in terms of 5- day biochemical oxygen demand and pH . There were also noticeable fluctuations in microbial community structure and phenotypes. However, 3 weeks after the end of exposure, the treatment system returned to baseline conditions .
This indicates that while a regular low dose of Cu from the NPs can result in sporadic system disruption, overall the treatment system maintains proper function. The Cu NPs were transformed to copper phosphate and sulfurized compounds, reducing the toxicity of the effluent by 95% or more . To more broadly assess the nanotoxicity of Cu NPs, dozens of studies with different organisms, including many in the USEPA ECOTOX database, were evaluated . Only acute toxicity in freshwater was considered, given the paucity in marine in general and freshwater chronic toxicity data.This information was used to construct species sensitivity distributions for freshwater organisms . The toxicity threshold was much higher for nCuO relative to nCu or Cu+ 2 ions . The SSDs for nCu and Cu+ 2 almost overlapped, indicating similar toxicity thresholds . Of the species used to develop the nCuO and nCu SSDs, the LC50 values tend to be lowest for various daphnia species , indicating high sensitivity, while crustaceans and fish tolerated higher concentrations of nCuO and nCu . The lower toxicity of nCuO in freshwater may reflect its slower dissolution at low ionic strengths and Cu+ 2 complexation in the presence of organic matter . Other studies indicate that the toxic effect observed from nCuO strongly correlates with the fraction of NP dissolved in aquatic media . In freshwater and marine systems, nCu and nCuO were found to cause some toxicity across a range of toxicological endpoints at concentration < 1 mg/L , while others only found toxicity at greater exposure concentrations .While lettuces did not exhibit any reduction in catalase activity, indicating no increased formation of ROS, alfalfa tissues exhibited a considerable change at 10 mg/L nCuO, and in alfalfa roots at 5 mg/L nCu2-a and μCuO . Surprisingly, ascorbate peroxidase activity in lettuce roots increased in all treatments except μCuO, and it also increased in alfalfa roots in all treatments except the two nCu2 nanopesticides. These early growth stage results indicate some noticeable effects from all Cu NPs, although no clear pattern was exhibited with regards to toxicity ranking based on physiological or biochemical indicators. Lettuce grown from seed to maturity in soil, and exposed through foliar spray to nCu2-b during the last 4 weeks before harvesting at 1050 mg/L to 2100 mg/L, exhibited no visible damage . In fact, leaf biomass, which is the harvestable product, increased significantly . However, metabolomics analysis revealed that nCu2-b altered metabolite levels in the lettuce leaves .
The tricarboxylic cycle and several amino acid-related biological pathways were disturbed. Some antioxidant levels were significantly decreased compared to the control, indicating that oxidative stress and a defense response occurred. Nicotianamine, a chelator, increased by 12 to 27 times compared to the control, which may represent a detoxification mechanism. The up-regulation of polyamines and increased potassium may mitigate oxidative stress and enhance tolerance . These changes in antioxidants within the leaves may alter their nutritional value.There were no visible signs of toxicity in the leaves. In contrast, for nCu applied to C. sativus, significant translocation of Cu was observed. When plants were exposed to 10 mg/L to 20 mg/L nCu in hydroponic media, Cu uptake in the roots was greater than in stems and leaves , but in all cases there was significant increase relative to unexposed controls . When plants were exposed to 200–800 mg nCu/kg in soil, Cu was also highest in the roots, but there was significant translocation to stems, leaves and even cucumber fruits . However, the translocation factor was lower in plants exposed to nCu and those only exposed to bio-available Cu in the soil , suggesting that not all the Cu taken up in the roots was available for translocation when it was supplied as nCu . Although the plants showed no visible damage, exposure to nCu reduced photosynthetic rate , and increased transpiration rate . The concentrations of Na, P, S, K, Mo, Fe and Zn decreased in all cucumber tissues in exposed plants . Formation of Cu-phosphate complexes at the root surface decreased P uptake. In addition, meta bolomics demonstrated that C. sativa engages an active defense mechanism in the root zone against nCu stress: up-regulation of amino acids to sequester/exclude Cu/nCu; down-regulation of citric acid to reduce the mobilization of Cu ions; ascorbic acid up-regulation to combat reactive oxygen species; and upregulation of phenolic compounds to increase antioxidant activity . nCu spiked in soil also up- or down-regulated 15 metabolic pathways in cucumber fruits: carbohydrate metabolism , amino acid synthesis and metabolism , and pathways related to lipid metabolism, biosynthesis of other secondary metabolites, and energy metabolism . Despite these metabolic changes, root, stem, leaf and fruit bio-masses were not impacted by exposure to nCu, so the effect may be only on fruit quality and needs further exploration . While crop plants are the intended recipients of nanopesticides and are more likely to be exposed to Cu NPs in biosolids, impacts to surrounding wild plants were also considered.
A study compared the effects of nCu2-b on a wild herbaceous annual plant , radish , and wheat under the same conditions. The uptake and toxicity of nCu2-b grown in potting, grassland, or agricultural soils were found to be dependent on plant species, soil type, soil nutrient levels, and illumination intensity . Exposure to soil nCu2-b concentrations as low as 10 mg/L caused stunted growth in C. unguiculata and resulted in a complete cessation of photosynthesis during the period of peak growth in individuals under high stress growth conditions . These plants also had elevated fractions of oxidized photosystem II reaction centers, which is consistent with known mechanisms of photosynthetic disruption caused by ionic Cu . Uptake and translocation patterns also suggest that nCu2-b underwent partial or total dissolution in the potting soil used , and were being taken up as ionic Cu rather than as particulate Cu2 . However, C. unguiculata, radish, and wheat grown in grassland or agricultural soils had leaf Cu concentrations 1 to 2 orders of magnitude lower than plants grown in potting soil, with the highest Cu concentrations in roots. Few physiological impacts due to nCu2- b exposure were seen in these plants, possibly as a result of decreased translocation to leaves. One exception was seen in wheat grown in grassland soil under low illumination levels during the 8th week of exposure to 25 mg nCu2-b/L·week, which had decreased PsiI quantum yield efficiencies, photochemical quenching, electron transport rates,hydroponic growing systems and ratios of oxidized PsiI reaction centers, compared to control plants. Additionally, radish plants grown under high light conditions and exposed to nCu2-b had significantly larger hypocotyls than control plants, but there was significantly lower grain yield in wheat plants grown under the same conditions. In summary, although effects were observed in seedlings and young plants when exposed to the various Cu particles and dissolved Cu2+, mature plants generally exhibited fewer apparent symptoms, and in several cases produced more biomass, particularly in tissues with commercial value . The terrestrial plants tolerated the exposure to Cu NPs better than aquatic organisms, with higher levels needed to produce measurable effects. This may be due to lower dissolution of Cu in terrestrial environments due to interactions with soil and natural organic matter , and the need for translocation through the root systems. In general, exposure to Cu compounds resulted in alteration of metabolite profiles, inducing anti-oxidant response, potential defense mechanisms , reduced photosynthetic rates and increased transpiration rates in some species. There was no clear trend in toxicity ranking, in some cases nCu being the most toxic, but in many others nCuO was as toxic or more. Only a few studies have been conducted that directly test the toxicity of terrestrial organisms to Cu NPs in soil media. Generally, growth inhibition does not occur until relatively high exposure concentrations, indicating that other toxic endpoints would not occur at likely environmental concentrations for either nCu or nCuO. Rousk et al. found an acute toxic response to soil bacteria from nCuO that did not occur upon exposure to bulk CuO. The growth inhibition EC50 was found to be around 2254 mg/kg for soil bacteria .
Dimkpa et al. investigated the effects of nCuO on sand grown wheat and beans and found both reduced root and shoot growth at 500 mg/kg, with again a greater effect seen from the nano scale rather than the bulk particles . These effects were not observed until relatively high exposure concentrations were reached . Unrine et al. found no adverse effect to E. fedita to nCu at exposure concentrations as high as 65 mg/kg across a range of ecologically relevant endpoints . A number studies have been conducted on the sensitivity and toxicity of microbes and plants to nCuO, however the majority of these were tested in growth media and not in soil. For example, Baek and An found the 24-hr microbial growth inhibition EC50 to fall between 28.6 and 65.9 mg/L . Future studies should consider the effects of Cu NP speciation and transformation, once they are released from the product into different environmental matrices, on toxicity. For example, the toxicity of most Cu NPs was decreased by > 95% after passing through the WWTP, and the speciation was very different from the input NPs. Studies are needed that go beyond the original, pristine NPs. Further work is needed to understand Cu NP toxicity in soils and in particular microbial communities.Aquaporins , also known as MIPs , are integral membrane proteins that increase the permeability of membranes to water, as well as small uncharged molecules. Of all kingdoms, the plant kingdom contains the largest known AQP family consisting over 30 members. There are AQPs in Arabidopsis , in maize and in tomato , tonoplast intrinsic proteins , NOD26-like intrinsic proteins , small basic intrinsic proteins and X intrinsic proteins. Plant PIPs can be divided into two major groups, PIP1 and PIP2, on the basis of their sequences and water-channel activity. PIP2 proteins exhibit high levels of water-channel activity in Xenopus oocytes and yeast vesicles; whereas PIP1 proteins often have relatively low permeability to water. Evidence for the role of PIP1 aquaporin in planta has come from mutant analyses and the manipulation of PIP1 expression in plants. Analysis of Arabidopsis mutants has shown that AtPIP1,2 can account for a significant portion of aquaporin-mediated leaf water transport. The antisense expression of AtPIP1,2 in Arabidopsis has been associated with reductions in the membrane hydraulic conductivity of isolated protoplasts and decreased total root hydraulic conductivity.