Co-exposure to nTiO2 impairs arsenic metabolism and affects antioxidant capacity in the marine shrimp Litopenaeus vannamei.

Aquatic animals are vulnerable to arsenic (As) toxicity. However, rarely does a contaminant occur alone in the aquatic environment. For this reason, this study was conducted to evaluate whether titanium dioxide nanoparticles (nTiO2) can interfere with the effects induced by As in Litopenaeus vannamei. Arsenic accumulation and metabolic capacity; expression and enzymatic activity of GSTΩ (glutathione-S-transferase omega isoform); antioxidant responses such as GSH, GR, and GST (reduced glutathione levels, glutathione reductase, and glutathione-S-transferase activity, respectively); and lipid peroxidation in the gills and hepatopancreas of shrimp were evaluated. The results are summarized as follows: (1) higher accumulation of As occurred in both tissues after exposure to As alone; (2) co-exposure to nTiO2 affected the capacity to metabolize As; (3) GSTΩ gene expression was not modified, but its activity was decreased by co-exposure to both contaminants; (4) As alone increased the GSH levels in the hepatopancreas, and co-exposure to nTiO2 reduced these levels in both tissues; (5) a decrease in the GST activity in the gills occurred with all treatments; (6) in the gills, GR activity was increased by As, and nTiO2 reversed this increase, whereas in the hepatopancreas co-exposure inhibited enzyme activity; (7) only in the hepatopancreas lipid damage was observed when animals were exposed to As or nTiO2 but not in co-exposure. The results showed that the As induces toxic effects in both tissues of shrimp and that co-exposure to nTiO2 can potentiate these effects and decrease the capacity to metabolize As, favoring the accumulation of more toxic compounds.

Circular Estimate Method (CEM) - a Simple Method to Estimate Caenorhabditis elegans Culture Densities in Liquid Medium.

BACKGROUND: Nematodes are used in many different fields of science, including environmental and biomedical research. Counting and/or estimating nematode numbers is required during research. Although being one of the most common procedures, this apparently simple task is a time-consuming process, prone to errors and concerns regarding procedure, reliability, and accuracy. When an estimate is necessary, there is a traditional manual counting procedure that in this study it will be called as "drop method" (DM). This popular method that extrapolates an animal count from a small drop of fluid shows a high coefficient of variation. To solve this problem, the present study used the free-living nematode Caenorhabditis elegans to develop a new estimation procedure that was based on a relationship between area and volume of a larger sample. RESULTS: The new method showed a low coefficient of variation and a close relationship between estimated and real counts of the total number of nematodes in large C. elegans suspensions. Reactive oxygen concentration was measured as an example of method application and to allow comparison between methods. CONCLUSION: The proposed method is accurate, facile and reproducible, requiring simple, inexpensive materials that make it an excellent alternative to the DM manual counting procedure. Although the DM is faster, its estimates are not as accurate or as precise as those of the new proposed method.

MitoTarget Modeling Using ANN-Classification Models Based on Fractal SEM Nano-Descriptors: Carbon Nanotubes as Mitochondrial F0F1-ATPase Inhibitors.

Recently, it has been suggested that the mitochondrial oligomycin A-sensitive F0-ATPase subunit is an uncoupling channel linked to apoptotic cell death, and as such, the toxicological inhibition of mitochondrial F0-ATP hydrolase can be an interesting mitotoxicity-based therapy under pathological conditions. In addition, carbon nanotubes (CNTs) have been shown to offer higher selectivity like mitotoxic-targeting nanoparticles. In this work, linear and nonlinear classification algorithms on structure-toxicity relationships with artificial neural network (ANN) models were set up using the fractal dimensions calculated from CNTs as a source of supramolecular chemical information. The potential ability of CNT-family members to induce mitochondrial toxicity-based inhibition of the mitochondrial H+-F0F1-ATPase from in vitro assays was predicted. The attained experimental data suggest that CNTs have a strong ability to inhibit the F0-ATPase active-binding site following the order oxidized-CNT (CNT-COOH > CNT-OH) > pristine-CNT and mimicking the oligomycin A mitotoxicity behavior. Meanwhile, the performance of the ANN models was found to be improved by including different nonlinear combinations of the calculated fractal scanning electron microscopy (SEM) nanodescriptors, leading to models with excellent internal accuracy and predictivity on external data to classify correctly CNT-mitotoxic and nonmitotoxic with specificity (Sp > 98.9%) and sensitivity (Sn > 99.0%) from ANN models compared with linear approaches (LNN) with Sp ≈ Sn > 95.5%. Finally, the present study can contribute toward the rational design of carbon nanomaterials and opens new opportunities toward mitochondrial nanotoxicology-based in silico models.

Fullerene (C60) particle size implications in neurotoxicity following infusion into the hippocampi of Wistar rats.

The buckminsterfullerene (C60) is considered as a relevant candidate for drug and gene delivery to the brain, once it has the ability to cross the blood-brain barrier. However, the biological implications of this nanomaterial are not fully understood, and its safety for intracerebral delivery is still debatable. In this study, we investigated if C60 particle size could alter its biological effects. For this, two aqueous C60 suspensions were used with maximum particle size up to 200nm and 450nm. The suspensions were injected in the hippocampus, the main brain structure involved in memory processing and spatial localization. In order to assess spatial learning, male Wistar rats were tested in Morris water maze, and the hippocampal BDNF protein levels and gene expression were analyzed. Animals treated with C60 up to 450nm demonstrated impaired spatial memory with a significant decrease in BDNF protein levels and gene expression. However, an enhanced antioxidant capacity was observed in both C60 treatments. A decrease in reactive oxygen species levels was observed in the treatments with suspensions containing particles measuring with up to 450nm. Thiobarbituric acid reactive substances, glutamate cysteine ligase, and glutathione levels showed no alterations among the different treatments. In conclusion, different particle sizes of the same nanomaterial can lead to different behavioral outcomes and biochemical parameters in brain tissue.

The role of lipoic acid in the protection against of metallic pollutant effects in the shrimp Litopenaeus vannamei (Crustacea, Decapoda).

The effects of cadmium (Cd) and arsenic (As), dosed alone or in combination have been poorly investigated in crustaceans. Besides, it is not known if dietary supplementation of exogenous antioxidants, like lipoic acid (LA), might prevent or even reverse toxic effects of Cd and As. The objective of the present study was to evaluate the role of lipoic acid in modulating biochemical responses after Cd and As exposures in Litopenaeus vannamei. Muscle from shrimp exposed to Cd alone or Cd+As showed a decrease in glutathione (GSH) levels, while the pre-treatment with LA reversed this situation. In this tissue, the pre-treatment with LA also induced an increase in glutathione-S-transferase (GST) activity in all groups. In hepatopancreas it was observed a marked accumulation of Cd and As, a decrease in the reactive oxygen species (ROS) concentration in response to Cd exposure alone (-LA); concomitant in the same group it was observed an increment of metallothionein-like content. As exposure induced an increase in GSH levels but LA reversed this increase. Also, LA showed to increase the GST activity in all groups treated. Besides, in this organ LA showed to augment total antioxidant competence. Obtained results indicate that LA can be used as a chemo-protectant against oxidative insults in shrimp.

Biochemical and physiological effects of multigenerational exposure to spheric polystyrene microplastics in Caenorhabditis elegans.

Although studies have already shown the effects of exposure to microplastics (MP) in different species, the effects over generations in these individuals remain poorly understood. Therefore, the present study aimed to evaluate the effect of polystyrene MP (spherical, 1 µm) on the responses of the free-living nematode Caenorhabditis elegans in a multigenerational approach over five subsequent generations. MP concentrations of both 5 and 50 µg/L induced a detoxification response, increasing glutathione S-transferase (GST) activity and inducing the generation of reactive oxygen species (ROS) and lipid peroxidation (TBARS). MP also demonstrated the ability to accumulate in the animal's body during the 96 h of each generational exposure, and possibly, this constant interaction was the main reason for the decreased response in physiological parameters as in the exploratory behavior (body bending) of nematodes, and in the reproduction, being this last parameter most negatively affected during the five exposed generations, with a reduction of almost 50% in the last generation. These results emphasize the importance of multigenerational approaches, highlighting their advantage in the assessment of environmental contaminants.

Polystyrene microplastic alters the redox state and arsenic metabolization in the freshwater bivalve Limnoperna fortunei.

Most organisms possess the capacity to metabolize arsenic (As) accumulating compounds to less toxic forms, thus minimizing the adverse effect induced by this metalloid. However, other contaminants may to interfere with As metabolism, contributing to the accumulation of more toxic compounds. Microplastics (MPs) are omnipresent in aquatic environment and may induce toxicological effects (alone or in combination with other contaminants) on living organisms. Therefore, the objective of the present study was to evaluate the effect of the exposure of the freshwater clam Limnoperna fortunei to a combination of MP (4 and 40 µg/L of polystyrene microbeads, 1.05 µm) and As (50 µg/L) for 48 h, evaluating the accumulation and metabolization of As and oxidative stress parameters, such as catalase (CAT), glutathione-S-transferase activities, total antioxidant competence, reduced glutathione (GSH), and lipid damage in the gills and digestive glands. Results revealed that low MP concentration disrupts the redox state of the digestive gland by a decrease in the antioxidant activity (CAT and total antioxidant capacity). GSH levels in the gills of animals exposed to MP (4 µg/L) alone and the combination of MP + As increased, concomitant with an increase in the percentage of toxic compounds, indicating the effect of MP on As metabolism. Although, few studies evaluated the effect of coexposure to MP + As by considering metabolization of metalloid in freshwater bivalve, our results revealed that exposure to MP reduced the metabolization capacity of As, favoring the accumulation of more toxic compounds besides the MP alone, which showed a pro-oxidant effect in L. fortunei.

Multigenerational effects of co-exposure to dimethylarsinic acid and polystyrene microplastics on the nematode Caenorhabditis elegans.

In the current study, we assessed the impact of DMA (dimethylarsinic acid) and MPs (microplastics) interactions in C. elegans over the course of five generations. We found that the redox state of the organisms changed over generations as a result of exposure to both pollutants. From the third generation onward, exposure to MPs reduced GST activity, indicating reduced detoxifying abilities of these organisms. Additionally, dimethylarsinic exposure decreased the growth of organisms in the second, fourth, and fifth generations. In comparison to isolated pollutants, the cumulative effects of co-exposure to DMA and MPs seem to have been more harmful to the organisms, as demonstrated by correlation analysis. These findings demonstrate that DMA, despite being considered less hazardous than its inorganic equivalents, can still have toxic effects on species at low concentrations and the presence of MPs, can worsen these effects.

Oxidative effects of consuming microplastics in different tissues of white shrimp Litopenaeus vannamei.

In this study, we evaluated the effect of microplastic (MP, polystyrene, 1.1 µm) exposure through diet at two different levels (40 and 400 µg MP/kg of ration) in the shrimp Litopenaeus vannamei for seven days. After the exposure period, oxidative stress parameters, histological alterations, and MP accumulation in different shrimp tissues (gut, gills, hepatopancreas, and muscle) were also evaluated. The results showed that MP was detected in the gills, muscles, and hepatopancreas. In addition, in the gut, gills, and hepatopancreas, disruption in redox cells was observed. Also, lipid and DNA damage was evident in the hepatopancreas. Histopathological analysis revealed edema in the intestine, hepatopancreas, and in the muscle. Granuloma formation with infiltrated hemocytes occurred in the intestine and hepatopancreas. These results show that MP exposure can affect the health and welfare of L. vannamei and may also affect the final consumers once MP is accumulated.

Arsenite and arsenate toxicity in the earthworm Eisenia andrei (Bouché 1972) in natural soil and tropical artificial soil.

Inorganic forms of As (arsenite - As(III) and arsenate - As(V)) are prevalent in soil and recognized for their high toxicity. Once in the soil, these forms of As can compromise key organisms for ecological processes, such as earthworms. The aim of the study was to evaluate the toxicity of arsenite and arsenate in the Californian earthworm Eisenia andrei exposed in natural soil and tropical artificial soil (TAS). Adverse effects were evaluated using avoidance test, acute toxicity test, and a sublethal concentration test to assess biochemical parameters. LC50 values for arsenite were 21.27 mg/kg in natural soil and 19.0 mg/kg in TAS and for arsenate were 76.18 mg/kg in natural soil and above 120 mg/kg in TAS. In the avoidance test, this behavior was shown to be significantly higher in the natural soil and for earthworms exposed to arsenite, while total antioxidant capacity, glutathione levels, lipid damage, and DNA damage were significantly higher in animals exposed to arsenite, but without differences in relation to the two types of soil tested. Animals exposed to As(V) showed increased activity of enzymes related to glutathione metabolism. The results obtained in the present study show the impact of As exposure on the health of the Californian earthworm E. andrei, especially in the form of arsenite, and alert the public authorities that legal limits should, whenever possible, consider the soil properties and also the different chemical species of the contaminants.

Fullerene and omega-3 and omega-6 fatty acids on fish brain antioxidant status.

The carbon nanomaterial fullerene (C(60)) can act as anti or pro-oxidant. The aim of this study was to evaluate, in cell suspensions of carp brains (Cyprinus carpio, Cyprinidae), the effect of C(60) after a pre-treatment with polyunsaturated fatty acid (PUFAs) such as omega-3 (docosahexaenoic acid, DHA) and omega-6 (linoleic acid, LA). Assays consisted of a pre-treatment with PUFA (48 h) and then exposure to C(60) (2 h). Cell viability and total anti-oxidant capacity did not differ (p > 0.05). A reduction (p < 0.05) was observed in reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) concentration in fish brain cells pre-exposed with PUFA groups and then exposed or not with C(60). An antioxidant effect of C(60) was evident since in control group (cells not pre-exposed to PUFA), a significant (p < 0.05) reduction of intracellular ROS concentration was observed, although this reduction was not enough to reduce the TBARS levels. Cysteine levels presented a reduction (p < 0.05) in all groups exposed to C(60). For glutathione (GSH), an increase (p < 0.05) was registered in cells exposed to C(60) without PUFAs pre-treatment and in the C(60) group pre-treated with DHA. Overall C(60) appears to play an antioxidant role that is modulated by PUFA, taking into account its effects on intracellular ROS concentration and MDA levels. Results also suggest that C(60) influences GSH synthesis, as showed for the augmented levels of this antioxidant and also for the lowering of the intracellular cysteine concentration.

Comparison of the toxic effects of organic and inorganic arsenic in Caenorhabditis elegans using a multigenerational approach.

Although arsenic (As) is a persistent contaminant in the environment, few studies have assessed its effects over generations, as it requires an animal model with a short lifespan and rapid development, such as the nematode Caenorhabditis elegans. Furthermore, few studies have evaluated the effects of As metabolites such as dimethylarsinic acid (DMAV), and several authors have considered DMA as a moderately toxic intermediate of As, although recent studies have shown that this chemical form can be more toxic than inorganic arsenic (iAs) even at low concentrations. In the present study, we compared the toxic effects of arsenate (AsV) and DMAV in C. elegans over 5 subsequent generations. We evaluated biochemical parameters such as reactive oxygen species (ROS) concentration, the activity of antioxidant defense system (ADS) enzymes such as catalase (CAT) and glutathione-S-transferase (GST), and nonenzymatic components of ADS such as reduced glutathione (GSH) and protein-sulfhydryl groups (P-SH). Exposure to 50 µg L-1 of AsV led to an increase in ROS generation and GSH levels together with a decrease in GST activity, while exposure to DMAV led to an increase in ROS levels, with an increase in lipid peroxidation, CAT activity, and a decrease in GSH levels. In addition, both treatments reduced animal growth from the third generation onward and caused disturbances in their reproduction throughout all 5 generations. This study shows that the accumulated effects of DMA need to be considered; it highlights the importance of this type of multigenerational approach for evaluating the effects of organic contaminants considered low or nontoxic.

Arsenic toxicity in mammals and aquatic animals: a comparative biochemical approach.

Arsenic (As) is a widespread pollutant in the world and its toxicity is related to its chemical form, with inorganic forms being considered more toxic than the organic form, and huge differences in effects and processes of metabolism. This paper reviews the potential biochemical mechanisms of uptake of arsenic by aquaporins, capacity for metabolism and cellular efflux of As. It is known that As can affect signaling pathways since it can activate proteins such as ERK2, p38 and JNK, as shown in mammals. A comparison between phosphorylation sites of these proteins is presented in order to determine whether the same effect triggered by As in mammals might be observed in aquatic animals. The toxicity resulting from As exposure is considered to be linked to an imbalance between pro-oxidant and antioxidant homeostasis that results in oxidative stress. So, present review analyzes examples of oxidative stress generation by arsenic. Biotransformation of As is a process where firstly the arsenate is converted into arsenite and then transformed into mono-, di-, and trimethylated products. In the methylation process, the role of the omega isoform of glutathione-S-transferase (GST) is discussed. In addition, a phylogenetic tree was constructed for aquaporin proteins of different species, including aquatic animals, taking into account their importance in trivalent arsenic uptake.

Oxidative stress in Ruditapes philippinarum after exposure to different graphene oxide concentrations in the presence and absence of sediment.

The use of carbon nanomaterials (CNMs) is growing in different technological fields, raising concern on their potential impacts on the environment. Given its diverse nanothenological applications, graphene oxide (GO) stands out among the most widely used CNMs. Its hydrophilic capacity enables it to remain stable in suspension in water allowing that GO can be accessible for accumulation by aquatic organisms through ingestion, filtration and superficial dermal contact when present in aquatic ecosystems. Considering that the effects induced to aquatic organisms may depend on environment characteristics, such as temperature, salinity, water pH as well as the presence/absence of sediment, the present study aimed to investigate the influence of sediment on the impacts caused by GO exposure. For this, oxidative stress parameters were measured in the clam Ruditapes philippinarum, exposed to different GO concentrations (0.01, 0.1 and 1 mg/L), in the presence and absence of sediment, for a 28-days experimental period. The results here presented showed that regardless the presence or absence of sediment, most of the biochemical parameters considered were altered when clams were exposed to the highest concentration. The present findings further revealed that in the presence of sediment, clams mostly invested in non-enzymatic defenses (such as reduced glutathione, GSH), while animals exposed to GO in the absence of sediment favored their enzymatic antioxidant defense capacity (catalase, CAT and superoxide dismutase, SOD). This study highlights the relevance of environmental variations as key factors influencing organisms' responses to pollutants.

Impact of different crystalline forms of nTiO2 on metabolism and arsenic toxicity in Limnoperna fortunei.

Arsenic (As) is a ubiquitous contaminant in the environment and it is known to induce oxidative stress in aquatic organisms. In an attempt to remove As from water, some studies have suggested the titanium dioxide nanomaterial (nTiO2) as a promising alternative. However, it has been observed that nTiO2 can induce toxicity alone or in combination with metals, and this toxicity is dependent on its crystalline form of nanomaterial (mainly rutile as nTiO2R and anatase as nTiO2A, respectively). Considering that both (nTiO2 and As) can occur together, the objective of this study was to evaluate if co-exposure to rutile and anatase may influence accumulation, metabolisation, and toxicity of arsenite (As+3) in the golden mussel Limnoperna fortunei after 48 h of co-exposure to nTiO2 (1 mg/L) and As (50 µg/L). Accumulation and chemical speciation of As in organisms were determined. Also, biochemical analyses, such as the activity of the enzymes glutathione S-transferase omega (GSTΩ), catalase (CAT) and glutathione S-transferase (GST), as well as lipid peroxidation (LPO) were investigated. Results showed that co-exposure to nTiO2A + As changed accumulation pattern of metalloid in gills and digestive gland. Both crystalline forms of nTiO2 affected the metabolisation capacity favoring the accumulation of more toxic As compounds and nTiO2A alone or in combination with As showed induce oxidative stress in gills of L. fortunei. In this way, it has a high potential risk of the co-exposure of these contaminants to aquatic organisms, and it also needs to consider the nanomaterial (nTiO2) properties and their application in the environmental remediation, carefully and judiciously.

Graphene oxide and GST-omega enzyme: An interaction that affects arsenic metabolism in the shrimp Litopenaeus vannamei.

Arsenic (As) is one of the most widespread contaminants; it is found in almost every environment. Its toxic effects on living organisms have been studied for decades, but the interaction of this metalloid with other contaminants is still relatively unknown, mainly whether this interaction occurs with emerging contaminants such as nanomaterials. To examine this relationship, the marine shrimp Litopenaeus vannamei was exposed for 48 h to As, graphene oxide (GO; two different concentrations) or a combination of both, and gills, hepatopancreas and muscle tissues were sampled. Glutathione S-transferase (GST)-omega gene expression and activity were assessed. As accumulation and speciation (metabolisation capacity) were also examined. Finally, a molecular docking simulation was performed to verify the possible interaction between the nanomaterial and GST-omega. The main finding was that GO modulated the As toxic effect: it decreased GST-omega activity, a consequence related to altered As accumulation and metabolism. Besides, the molecular docking simulation confirmed the capacity of GO to interact with the enzyme structure, which also can be related to the decreased GST-omega activity and subsequently to the altered As accumulation and metabolisation pattern.

Antioxidant responses in different body regions of the polychaeta Laeonereis acuta (Nereididae) exposed to copper.

Antioxidant enzymes, total antioxidant capacity (TOSC) and concentration of reactive oxygen species (ROS) were measured in anterior (A), middle (M) and posterior (P) body regions of Laeonereis acuta after copper (Cu; 62.5 microg/l) exposure. A catalase (CAT) activity gradient observed in control group (lowest in A, highest in P) was not observed in Cu exposed group. Glutathione-S-transferase (GST) activity in A region of Cu group was higher than in A region of the control group. DNA damage (comet assay) was augmented in the A region of Cu group. Since copper accumulation was similar in the different body regions, sensitivity to copper in A regions seems to be related to lowest CAT activity. In sum, copper exposure lowered TOSC, a result that at least in part can be related to lowering of antioxidant enzymes like CAT. DNA damage was induced in the anterior region, where a lower CAT activity was observed.

Genotoxic effect of dimethylarsinic acid and the influence of co-exposure to titanium nanodioxide (nTiO2) in Laeonereis culveri (Annelida, Polychaeta).

Few data are available about the effect of dimethylated forms (DMA) on aquatic organisms. As rarely a contaminant occurs alone, studies evaluating the combined effect of different contaminants in aquatic organisms are needed. In fact, the presence of nanomaterials, such as titanium dioxide nanoparticles (nTiO2), in the aquatic environment is now a reality due to its intensive production and use. So, this study evaluated the toxicological effects of DMA in an acute exposure condition and considered the potential influence of nTiO2 on the effects induced by DMA in the polychaete, Laeonereis culveri. The animals were exposed over 48 h to DMA (50 and 500 µg/l) alone or in combination with nTiO2 (1 mg/l). Biochemical parameters such as concentration of reactive oxygen species (ROS), glutathione-S-transferase (GST) activity, levels of reduced glutathione levels (GSH) and macromolecular (lipid and DNA) damage were evaluated, as well the DNA repair system. In addition, the accumulation of total As and the chemical speciation of the metalloid in the organisms was determined. The results showed that: (1) only the group exposed to 500 µg of DMA/l accumulated As and when co-exposed to nTiO2, this accumulation was not observed. (2) The levels of ROS increased in the group exposed to 50 µg/l of DMA alone and the effect was reversed when this group was co-exposed to nTiO2 (3) None of the treatments showed altered GST activity or GSH levels. (4) All groups that received nTiO2 (alone or in combination with DMA) showed lipid peroxidation. (5) The exposure to DMA (both concentrations) alone or in combination with nTiO2 induced DNA damage in L. culveri. These results showed that DMA exhibits a genotoxic effect and that co-exposure to nTiO2 had an influence on its toxicity. So the occurrence of both contaminants simultaneously can represent a threat to aquatic biota.

Different crystalline forms of titanium dioxide nanomaterial (rutile and anatase) can influence the toxicity of copper in golden mussel Limnoperna fortunei?

Although some studies have showed the effects of different crystalline structures of nTiO2 (anatase and rutile) and their applicability in several fields, few studies has analyzed the effect of coexposure with other environmental contaminants such as copper. Thus, the objective of this study was to evaluate if the coexposure to nTiO2 (nominal concentration of 1 mg/L; anatase or rutile) can increase the incorporation and toxic effect induced by Cu (nominal concentration of 56 µg/L) in different tissues of Linmoperna fortunei after 120 h of exposure. Our results showed that the coexposure increased the accumulation of Cu in the gills and adductor muscle independently of the crystalline form and can positively or negatively modulate the antioxidant system, depending on the tissue analyzed. However, exposure only to rutile nTiO2 induced damage in the adductor muscle evidenced by the infiltration of hemocytes in this tissue. Additionally, histomorphometric changes based on fractal dimension analysis showed that coexposure to both forms of nTiO2 induced damage in the same tissue. These results suggest that both crystalline forms exhibited toxicity depending on the analyzed tissue and that coexposure of nTiO2 with Cu may be harmful in L. fortunei, indicating that increased attention to the use and release of nTiO2 in the environment is needed to avoid deleterious effects in aquatic biota.