Dissolved Organic Matter and Lignin Modulate Aquatic Toxicity and Oxidative Stress Response Activated by Layered Double Hydroxides Nanomaterials.

Advanced nanomaterials can be released into the environment and can coexist with natural organic matter (NOM). However, evidence on the impacts of NOM on the environmental behavior and toxicity of advanced nanomaterials is still scarce. Here, we investigated the behavior and toxic effects of two layered double hydroxides (LDHs) nanomaterials with different metallic constituents (Mg-Al-LDH and Zn-Al-LDH) at relatively low exposure concentrations on a freshwater green alga (Chlorella pyrenoidosa) in the absence and presence of two types of NOM, namely dissolved organic matter (DOM) and dealkaline lignin (DL). The DOM or DL interaction with the LDHs at different mixture levels was shown to be an antagonistic effect on the growth inhibition toxicity to C. pyrenoidosa mainly. The estimation of the index of Integrated Biological Responses version 2 indicated that the joint interaction of the LDHs with DOM or DL occurred in the following order of frequency synergism > antagonism > additivity. Furthermore, the physicochemical characteristics of LDHs were crucial for illuminating the mechanism by which the DOM or DL modified the LDH-induced oxidative stress response. These findings highlighted the important role of NOM in the behavior and effect of LDHs as a representative of a new class of multifunctional nanomaterials in the freshwater environment.

The Joint Toxicity of Organic Three-dimensional Layered Double Hydroxide and Methyl Orange to Green Algae Chlorella Vulgaris.

Organic modified layered double hydroxides (O-LDHs), known as attractive adsorbents for organic pollutants, may pose severe toxicity to the aquatic organisms during their large-scale application. However, little information is available on the toxicity of O-LDHs and the joint toxicity with the coexisted organic pollutants. Herein, we employed organic three-dimensional layered double hydroxide (O3D-LDH) and methyl orange (MO) as representative to investigate the toxicity mechanisms of single substance and its binary mixture on green algae Chlorella vulgaris. Results showed that O3D-LDH and MO presented concentration-dependent toxicity, the binary mixture showed additive effect after exposed to low O3D-LDH concentration (≤ 50 mg/L), but antagonism was observed for the other. It revealed that the agglomeration of O3D-LDH and microalgae influenced chlorophyll content, eventually inhibiting the growth of algae. Overall, this toxicity investigation was critical for understanding the environmental risk of organic LDHs to provide theorical guidance for their practical application in the water purification.

Safety Assessment of Inorganic Hydroxides as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of inorganic hydroxides, which function in cosmetics primarily as pH adjusters. Representatives from the cosmetic industry have indicated these ingredients are used in depilating and hair waving/straightening formulations to raise pH values. The Panel considered relevant data related to these ingredients. The Panel concluded that these inorganic hydroxides are safe in hair straighteners and depilatories under conditions of recommended use; users should minimize skin contact. These ingredients are safe for all other present practices of use and concentration described in this safety assessment when formulated to be nonirritating.

Toxicity of Zn-Fe Layered Double Hydroxide to Different Organisms in the Aquatic Environment.

The application of layered double hydroxide (LDH) nanomaterials as catalysts has attracted great interest due to their unique structural features. It also triggered the need to study their fate and behavior in the aquatic environment. In the present study, Zn-Fe nanolayered double hydroxides (Zn-Fe LDHs) were synthesized using a co-precipitation method and characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and nitrogen adsorption-desorption analyses. The toxicity of the home-made Zn-Fe LDHs catalyst was examined by employing a variety of aquatic organisms from different trophic levels, namely the marine photobacterium Vibrio fischeri, the freshwater microalga Pseudokirchneriella subcapitata, the freshwater crustacean Daphnia magna, and the duckweed Spirodela polyrhiza. From the experimental results, it was evident that the acute toxicity of the catalyst depended on the exposure time and type of selected test organism. Zn-Fe LDHs toxicity was also affected by its physical state in suspension, chemical composition, as well as interaction with the bioassay test medium.

Developmental toxicity induced by Cu(OH)2 nanopesticide in zebrafish embryos.

The current study evaluates the adverse effects of Cu(OH)2 nanopesticide (CNPE) on the early life stages of zebrafish (Danio rerio). The developmental toxicity was determined using different parameters such as mortality (including LC50 ), hatching, heart rates, malformations, and alteration of the gene expressions. Zebrafish embryos (4 hpf-hours postfertilization) were exposed to 1.0, 2.0, 4.0, 8.0, and 16.0 mg/l CNPE doses until 96 hpf. The 96 hours LC50 was recorded at 6.258 mg/l. Seventy-two hpf total malformation index values for 2.0, 4.0, and 8.0 mg/l CNPE doses were 4.3, 7.2 and 7.9, respectively. 1.0 mg/l CNPE is not toxic for the zebrafish embryos/larvae. 2.0 to 8.0 CNPE doses caused some abnormalities in embryos/larvae morphology, including lack of body parts, tail deformities, chorda deformity, bubbled head, scoliosis, lordosis, weak or non-pigmentation, decreased heart rate and larva length. 16.0 mg/l CNPE caused mortality in 72 hpf. The expression levels of seven immune system-related genes (il-1ß, il-8, cebp, tlr4, hsp70, NF-kB, and mtf-1) were examined. The transcription level of il-1ß, il-8, tlr4, hsp70, and NF-kB genes significantly increased in the CNPE exposure groups. While the expression of the mtf-1 gene considerably decreased, the cebp gene expression level did not change in the 4.0 and 8.0 mg/l CNPE doses. In conclusion, CNPE could induce developmental toxicity with malformations in embryos/larvae and alter the gene expression.

Cytotoxicity of NiO and Ni(OH)2 Nanoparticles Is Mediated by Oxidative Stress-Induced Cell Death and Suppression of Cell Proliferation.

The use of nanomaterial-based products continues to grow with advancing technology. Understanding the potential toxicity of nanoparticles (NPs) is important to ensure that products containing them do not impose harmful effects to human or environmental health. In this study, we evaluated the comparative cytotoxicity between nickel oxide (NiO) and nickel hydroxide (Ni(OH)2) in human bronchoalveolar carcinoma (A549) and human hepatocellular carcinoma (HepG2) cell lines. Cellular viability studies revealed cell line-specific cytotoxicity in which nickel NPs were toxic to A549 cells but relatively nontoxic to HepG2 cells. Time-, concentration-, and particle-specific cytotoxicity was observed in A549 cells. NP-induced oxidative stress triggered dissipation of mitochondrial membrane potential and induction of caspase-3 enzyme activity. The subsequent apoptotic events led to reduction in cell number. In addition to cell death, suppression of cell proliferation played an essential role in regulating cell number. Collectively, the observed cell viability is a function of cell death and suppression of proliferation. Physical and chemical properties of NPs such as total surface area and metal dissolution are in agreement with the observed differential cytotoxicity. Understanding the properties of NPs is essential in informing the design of safer materials.

Layered Double Hydroxide Nanoparticles for Efficient Gene Delivery for Cancer Treatment.

The use of cationic polymer based gene delivery vectors has several limitations such as low transfection efficiency, high toxicity, and inactivation by serum. The present work provides an inorganic based nanocarrier for efficient gene delivery and a method for preparing the same through a facile coprecipitation technique. The vehicle showed high loading capacity of DNA and can release the loaded DNA in a controlled pH-responsive manner. The developed gene delivery vehicle offers remarkable protection against DNase I and also provides protection against thermal damage. This vehicle also demonstrated efficient cellular uptake performance. Transfection and expression of plasmid gene encoding GFP proteins is achieved successfully by this LDH based vehicle. More interestingly, the developed Li-Al LDH efficiently induces GFP-p53 mediated apoptosis in HeLa cells exclusively sparing the normal tissue cells like NIH-3T3. The study demonstrates the potential of the developed inorganic based nanocarrier as a promising nonviral gene vector for tumor treatment.

Interactions between Biological Cells and Layered Double Hydroxides: Towards Functional Materials.

This review highlights the current research on the interactions between biological cells and Layered Double Hydroxides (LDH). The as-prepared biohybrid materials appear extremely attractive in diverse fields of application relating to health care, environment and energy production. We describe how thanks to the main features of biological cells and LDH layers, various strategies of assemblies can be carried out for constructing smart biofunctional materials. The interactions between the two components are described with a peculiar attention to the adsorption, biocompatibilization, LDH layer internalization, antifouling and antimicrobial properties. The most significant achievements including authors' results, involving biological cells and LDH assemblies in waste water treatment, bioremediation and bioenergy generation are specifically addressed.

Engineered nanoparticles interact with nutrients to intensify eutrophication in a wetland ecosystem experiment.

Despite the rapid rise in diversity and quantities of engineered nanomaterials produced, the impacts of these emerging contaminants on the structure and function of ecosystems have received little attention from ecologists. Moreover, little is known about how manufactured nanomaterials may interact with nutrient pollution in altering ecosystem productivity, despite the recognition that eutrophication is the primary water quality issue in freshwater ecosystems worldwide. In this study, we asked two main questions: (1) To what extent do manufactured nanoparticles affect the biomass and productivity of primary producers in wetland ecosystems? (2) How are these impacts mediated by nutrient pollution? To address these questions, we examined the impacts of a citrate-coated gold nanoparticle (AuNPs) and of a commercial pesticide containing Cu(OH)2 nanoparticles (CuNPs) on aquatic primary producers under both ambient and enriched nutrient conditions. Wetland mesocosms were exposed repeatedly with low concentrations of nanoparticles and nutrients over the course of a 9-month experiment in an effort to replicate realistic field exposure scenarios. In the absence of nutrient enrichment, there were no persistent effects of AuNPs or CuNPs on primary producers or ecosystem productivity. However, when combined with nutrient enrichment, both NPs intensified eutrophication. When either of these NPs were added in combination with nutrients, algal blooms persisted for >50 d longer than in the nutrient-only treatment. In the AuNP treatment, this shift from clear waters to turbid waters led to large declines in both macrophyte growth and rates of ecosystem gross primary productivity (average reduction of 52% ± 6% and 92% ± 5%, respectively) during the summer. Our results suggest that nutrient status greatly influences the ecosystem-scale impact of two emerging contaminants and that synthetic chemicals may be playing an under-appreciated role in the global trends of increasing eutrophication. We provide evidence here that chronic exposure to Au and Cu(OH)2 nanoparticles at low concentrations can intensify eutrophication of wetlands and promote the occurrence of algal blooms.

Diquat associated with copper sources for algae control: Efficacy and ecotoxicology.

The aims of this research were to evaluate the efficacy of copper oxychloride (CuCl2.3Cu(OH)2), copper hydroxide (Cu(OH)2) and diquat (1.1'-ethylene-2.2'-bipyridyldiylium dibromide), isolated and in association with 0.1% of both copper sources, in the control of the unicellular algae Ankistrodesmus gracilis and the filamentous algae Pithophora kewesis, and to determine the acute toxicity of the tested chemicals in Hyphressobrycon eques, Pomacea canaliculata, Lemna minor and Azolla caroliniana. The efficacy was estimated by the methods of chlorophyll a and pheophytin a readings, changed into growth inhibition percentage. Both algae were exposed to the following concentrations: 0.2; 0.4; 0.8; 1.2 mg L(-1) of diquat and its association with the copper sources; and 0.1; 0.3; 0.5; 0.7; 1.0 and 1.5 mg L(-1) in the isolated applications of copper hydroxide and copper oxychloride. An untreated control was kept. The acute toxicity was estimatedby 50% lethal concentration (LC50). The copper sources were effective for A. gracilis control, at rates as high as 0.1 mg L(-1) (>95% efficacy). Isolated diquat and its association with copper hydroxide were both effective at rates as high as 0.4 mg L(-1), with 95 and 88% control efficacy, respectively. The copper oxychloride was effective at 0.2 mg L(-1), with 93% efficacy. None of the tested chemicals and associations was effective on P. kewesis control. The most sensitive non target organism to the tested chemicals was L. minor; the less sensitive was H. eques.

Toxicity of copper hydroxide, dithianon, fluazinam, tebuconazole and pyraclostrobin to Didymella applanata isolates from Serbia.

A study of the in vitro sensitivity of 10 isolates of Didymella applanata to copper hydroxide, dithianon, fluazinam, tebuconazole and pyraclostrobin, was conducted. The isolates were derived from diseased raspberry canes sampled during 2013 at five localities in western part of Serbia, known as the main raspberry growing region of the country. Prior to sensitivity testing experimental conditions for radial growth assay were optimized. The results showed that the temperature of 22 °C, oatmeal agar medium and 12/12 hrs light/ darkness light regimen provided the best conditions for sensitivity tests. Most of D. applanata isolates were sensitive to the tested fungicides. The narrowest range of EC50 values was recorded for tebuconazole (1.42-2.66 mg L(-1)). The widest range of EC50 values was obtained for pyraclostrobin, ranging from 0.17 mg L(-1) to 55.33 mg L(-1). The EC50 values for the studied isolates were 39.48-51.19 mg L(-1) for copper hydroxide, 12.12-18.73 mg L(-1) for dithianon and 5.72-42.56 mg L(-1) for fluazinam. According to resistance factor values, all D. applanata isolates were sensitive to copper hydroxide, dithianon and tebuconazole. Among tested isolates, six were highly resistant to pyraclostrobin (RFs in the range of 207.1-325.5) and two moderately resistant to fluazinam (RFs were 3 and 7.4), respectively.

Development of drug delivery systems based on layered hydroxides for nanomedicine.

Layered hydroxides (LHs) have recently fascinated researchers due to their wide application in various fields. These inorganic nanoparticles, with excellent features as nanocarriers in drug delivery systems, have the potential to play an important role in healthcare. Owing to their outstanding ion-exchange capacity, many organic pharmaceutical drugs have been intercalated into the interlayer galleries of LHs and, consequently, novel nanodrugs or smart drugs may revolutionize in the treatment of diseases. Layered hydroxides, as green nanoreservoirs with sustained drug release and cell targeting properties hold great promise of improving health and prolonging life.

Toxicity of ZnFe-SO4 layered double hydroxide in Tetradesmus obliquus and evaluation of some physiological responses of the microalgae for stress management.

Layered double hydroxides (LDHs), regarding their physical and structural properties, have different and wide applications industry and their increasing use may raise ecological and human health concerns. However, the potential toxicity mechanisms of LDHs in different organisms are still unclear. In the present work, after synthesizing of ZnFe-SO4 LDH and studying of its characterization by XRD, FT-IR, SEM, EDX-mapping, TEM and Raman, its toxicity in Tetradesmus obliquus was evaluated. According to experimental results, the growth of the algae and content of photosynthetic pigments were significantly decreased after treatment with 100 mg/L of ZnFe-SO4 LDH. The high dose exposure to the LDH also inhibited the activity of SOD and POD enzymes, possibly due to the LDH- catalyzed reactive oxygen species production. In addition, lipid peroxidation and the content of phenolic compounds, as no-enzymatic antioxidants were increased by enhancement of the LDH concentration. The rise of phenol, flavonoids and MDA contents could be regarded as some manifestations and responses to the toxic effects of the contaminant in the algae cells. The results provided a better understanding of the undesirable effects and toxicity of LDHs in aquatic organisms.

Ageing influences the toxicity of two innovative nanofertilizers to the soil invertebrates Enchytraeus crypticus and Folsomia candida.

The increasing global food demand is threatening the sustainability of agrifood production systems. The intensification of agricultural practices, with inadequate use of pesticides and fertilizers, poses major challenges to the good functioning of agroecosystems and drastically degrades the soil quality. Nanotechnology is expected to optimize the current farming practices and mitigate some associated impacts. Layered double hydroxides (LDHs) are a class of nanomaterials with high potential for use in agricultural productions, mostly due to their sustained release of nutrients. Considering its novelty and lack of studies on the terrestrial ecosystem, it is essential to assess potential long-term harmful consequences to non-target organisms. Our study aimed to evaluate the effect of Zn-Al-NO3 LDH and Mg-Al-NO3 LDH ageing on the survival and reproduction of two soil invertebrate species Enchytraeus crypticus and Folsomia candida. We postulated that the toxicity of nanomaterials to soil invertebrates would change with time, such that the ageing of soil amendments would mediate their impacts on both species. Our results showed that the toxicity of LDHs was species-dependent, with Zn-Al-NO3 LDH being more toxic to E. crypticus, while Mg-Al-NO3 LDH affected more F. candida, especially in the last ageing period, where reproduction was the most sensitive biological parameter. The toxicity of both nanomaterials increased with ageing time, as shown by the decrease of the EC50 values over time. The influence of LDH dissolution and availability of Zn and Mg in the soil pore water was the main factor related to the toxicity, although we cannot rule out the influence of other structural constituents of LDHs (e.g., nitrates and aluminium). This study supports the importance of incorporating ageing in the ecotoxicity testing of nanomaterials, considering their slow release, as effects on soil organisms can change and lead to more severe impacts on the ecosystem functioning.

Surface coated Eu(OH)3 nanorods: a facile synthesis, characterization, MR relaxivities and in vitro cytotoxicity.

The water-soluble and biocompatible D-glucuronic acid coated Eu(OH)3 nanorods (average thickness x average length = 9.0 x 118.3 nm) have been prepared in one-pot synthesis. The D-glucuronic acid coated Eu(OH)3 nanorods showed a strong fluorescence at approximately 600 nm with a narrow emission band width. A cytotoxicity test by using DU145 cells showed that D-glucuronic acid coated Eu(OH)3 nanorods are not toxic up to 100 microM, making them a promising candidate for biomedical applications such as fluorescent imaging. The minimum Eu concentration needed for a conventional confocal imaging was estimated to be approximately 0.1 mM. Therefore, D-glucuronic acid coated Eu(OH)3 nanorods can be applied to fluorescent imaging. However, a very tiny magnetization of approximately 1.2 emu/g at room temperature and at an applied field of 5 tesla was observed. As a result, very small r1 and r2 water proton relaxivities were estimated, implying that surface coated Eu(OH)3 nanorods are not sufficient for MRI contrast agents.

In-vitro cyto-toxicity, geno-toxicity, and bio-imaging evaluation of one-pot synthesized luminescent functionalized mesoporous SiO2@Eu(OH)3 core-shell microspheres.

Luminescent functionalized mesoporous SiO2@Eu(OH)3 core-shell microspheres (LFMCSMs) were prepared by coating of europium hydroxide (Eu(OH)3) shell on mesoporous silica (SiO2) nanospheres via a facile one-pot process at low temperature. The FETEM images revealed that a well-defined luminescent europium hydroxide shell was successfully grafted on the surface of mesoporous silica nanospheres. These experimental results showed that the LFMCSM has a typical diameter of ca. 392 nm consisting of the silica core with about 230 nm in diameter and europium hydroxide shell with an average thickness of about 162 nm. LFMCSMs exhibited strong red emission peak upon irradiation with ultraviolet light, which originated from the electric-dipole transition (5)D0 → (7)F2 (614 nm) of Eu(3+) ion. The biocompatibility of the synthesized LFMCSMs was evaluated in vitro by assessing their cytotoxic and genotoxic effect on human hepatoblastoma (HepG2) cells using MTT, TUNEL, fluorescent staining, DNA ladder and Gene expression assays respectively. FROM THE CLINICAL EDITOR: This paper describes the development of a one-pot synthesis of luminescent mesoporous SiO2@Eu(OH)3 core-shell microspheres and evaluates their favorable in vitro cyto-toxicity and geno-toxicity, and their applications in bio-imaging of these particles that emit bright red signal under UV exposure.

Study of environmental risks incurred by leakage of lithium cells to the food chain in a freshwater ecosystem.

Water flea (Daphnia magna) and fish (Carassius auratus) at trophic level were used for comprehensive evaluation of environmental risks incurred by manufactured nanomaterial (nNi(OH)2) as leaked from lithium cells to the food chain in freshwater ecosystem. The 48, 72 and 96 h acute toxicities of water suspensions of nNi(OH)2 to the flea and the fish were tested, using the immobilization and the mortality as toxicological endpoints. The results showed that the water flea was more highly sensitive to nNi(OH)2 than the fish. Then, the fish were exposed to 1.0 mg/L nNi(OH)2 for 6, 12, 24, 36, 48, 60, 72 and 96 h, and the relationship between the concentrations in the water and the fish were described by a bioconcentration factor (BCF). After calculation, lgBCF is 1.61. Reactive oxygen species (ROS) generation was studied after fish were exposed to 1.0 mg/L water suspensions of nNi(OH)2 for 24 h. As proved by electron paramagnetic resonance, nNi(OH)2 may induce the generation of hydroxyl radical in the fish, and nNi(OH)2 as concentrated in the fish may incur redox reaction and produce redox metabolic intermediates. As one of the important toxic mechanisms of nNi(OH)2 to the fish, the oxidative stress mechanism requires further study.

Harmonizing nanomaterial exposure methodologies in ecotoxicology: the effects of two innovative nanoclays in the freshwater microalgae Raphidocelis subcapitata.

Layered double hydroxides (LDHs) are innovative nanomaterials (NMs) with a typical nanoclay structure (height <40 nm) consisting of layers of metallic cations and hydroxides stabilized by anions and water molecules. Upon specific triggers, anions can exchange by others in the surrounding environment. Due to this stimuli-responsive behavior, LDHs are used as carriers of active ingredients in the industrial or pharmaceutical sectors. Available technical guidelines to evaluate the ecotoxicity of conventional substances do not account for the specificities of NMs, leading to inaccuracies and uncertainty. The present study aimed to assess two different exposure methodologies (serial dilutions of the stock dispersion vs. direct addition of NM powder to each concentration) on the ecotoxicological profile of different powder grain sizes of Zn-Al LDH-NO3 and Cu-Al LDH-NO3 (bulk, <25, 25-63, 63-125, 125-250, and >250 µm) in the growth of the freshwater microalgae Raphidocelis subcapitata. Results revealed that the serial dilutions methodology was preferable for Zn-Al LDH-NO3, whereas for Cu-Al LDH-NO3 both methodologies were suitable. Thus, the serial dilutions methodology was selected to assess the ecotoxicity of different grain sizes for both LDHs. All Zn-Al LDH-NO3 grain sizes yielded similar toxicity, while Cu-Al LDH-NO3 powders with smaller grain sizes caused a higher effect on microalgae growth; thus, grain size separation might be advantageous for future applications of Cu-Al LDH-NO3s. Considering the differences between exposure methodologies for the Zn-Al LDH-NO3, further research involving other NMs and species must be carried out to achieve harmonization and validation for inter-laboratory comparison.

Factors affecting the toxicity and oxidative stress of layered double hydroxide-based nanomaterials in freshwater algae.

Layered double hydroxide (LDH) nanomaterials are utilized extensively in numerous fields because of their distinctive structural properties. It is critical to understand the environmental behavior and toxicological effects of LDHs to address potential concerns caused by their release into the environment. In this work, the toxicological effects of two typical LDHs (Mg-Al-LDH and Zn-Al-LDH) on freshwater green algae (Scenedesmus obliquus) and the main affecting factors were examined. The Zn-Al-LDH exhibited a stronger growth inhibition toxicity than the Mg-Al-LDH in terms of median effect concentration. This toxicity difference was connected to the stability of particle dispersion in water and the metallic composition of LDHs. The contribution of the dissolved metal ions to the overall toxicity of the LDHs was lower than that of their particulate forms. Moreover, the joint toxic action of different dissolved metal ions in each LDH belonged to additive effects. The Mg-Al-LDH induced a stronger oxidative stress effect in algal cells than the Zn-Al-LDH, and mitochondrion was the main site of LDH-induced production of reactive oxygen species. Scanning electron microscope observation indicated that both LDHs caused severe damage to the algal cell surface. At environmentally relevant concentrations, the LDHs exhibited joint toxic actions with two co-occurring contaminants (oxytetracycline and nano-titanium dioxide) on S. obliquus in an additive manner mainly. These findings emphasize the impacts of the intrinsic nature of LDHs, the aqueous stability of LDHs, and other environmental contaminants on their ecotoxicological effects.

Tolerance and bioaccumulation of copper by the entomopathogen Beauveria bassiana (Bals.-Criv.) Vuill. exposed to various copper-based fungicides.

This work evaluates for the first time the relationships between copper-tolerance, -solubilization and -bioaccumulation in the entomopathogen Beauveria bassiana exposed to Bordeaux mixture, copper oxychloride or copper hydroxide. Bordeaux mixture was highly detrimental to fungus, by inhibiting the growth totally at the recommended dose (RD) and 2×RD. Copper hydroxide and copper oxychloride were found to be less toxic, reducing fungus growth, sporulation and conidial germination in an average of 29  %, 30 % and 58 %, respectively. These two copper forms were the easiest to solubilize, to precipitate and the most accumulated by B. bassiana, suggesting the involvement of all these processes on fungus copper-tolerance.