Does temperature influence on biomarker responses to copper exposure? The invasive bivalve Limnoperna fortunei (Dunker 1857) as a model.

Biomarkers are useful tools for assessing the early warning effects of pollutants. However, their responses can be influenced by confounding factors. In this study, we investigated the influence of temperature on multiple biomarkers in the invasive freshwater bivalve Limnoperna fortunei exposed to copper (Cu). The mussels were exposed to low and high environmental Cu concentrations at two temperatures (15 °C and 25 °C). After 96 h, the oxidative stress, neurotoxicity, and metabolic parameters were assessed. Our results showed that temperature is a key factor influencing biomarker responses in mussels, with higher glutathione S-transferase activity and lower energy reserves at cold temperature. In addition, the effects of Cu were greater at the highest concentration at 15 °C (increased lipid peroxidation and cholinesterase activity). Overall, these findings suggest that cold stress increases the susceptibility of L. fortunei to metal effects and highlight the importance of including temperature in toxicity testing and biomonitoring. In addition, using the invasive bivalve L. fortunei as a model could prove valuable in its role as a sentinel species for other organisms.

Non-lethal biomarkers as promising tools for fish health assessment: In situ exposure to bifenthrin as a case study.

Non-lethal biomonitoring should provide an innovative approach to establish bioethical protocols for the management of both aquaculture and wild fisheries resources. We aimed to assess non-lethal biomarkers in Piaractus mesopotamicus caged in a rice field during a bifenthrin (BF) application. We analyzed parameters related to the immune system, energy metabolism and oxidative stress in fish skin mucus and blood plasma. Fish exposed to BF showed a significant increase in skin mucus glucose levels and the enzymatic activities of protease, alkaline phosphatase and superoxide dismutase. Regarding plasmatic parameters, BF increased the levels of glucose, total protein and albumin, but decreased triglycerides. In addition, increased activities of lysozyme and alkaline phosphatase were found in the blood plasma of exposed fish. Our results indicated an increased energy demand, altered immune function and a mild oxidative stress response in fish exposed in situ to BF. We have shown that skin mucus and blood plasma are very promising matrices for the development of non-lethal biomarkers to assess fish health in a stressed environment.

Metal-based nanomaterials in aquatic environments: What do we know so far about their ecotoxicity?

The wide range of applications of nanomaterials (NM) in different fields has led to both uncontrolled production and release into environmental compartments, such as aquatic systems, where final disposal occurs. Some efforts have been made to estimate their concentrations in environmental matrices; however, little is known about the actual effects of environmental NM concentrations on biota. The aims of the present review are to (i) expose the state of the art of the most applied NM and their actual concentrations regarding how much is being released to the aquatic environment and which are the predicted ones; (ii) analyze the current literature to elucidate if the aforementioned conditions were proven to cause deleterious effects on the associated organisms; and (iii) identify gaps in the knowledge regarding whether the actual NM concentrations are harmful to aquatic biota. These novel materials are expected to being released into the environment in the range of hundreds to thousands of tons per year, with Si- and Ti-based NM being the two most important. The estimated environmental NM concentrations are in the low range of ng to µg/L, except for Ti-based ones, which concentrations reach values on the order of mg/L. Empirical information regarding the ecotoxicity of environmental NM concentrations mainly focused on metal-based NM, however, it resulted poor and unbalanced in terms of materials and test species. Given its high predicted environmental concentration in comparison with the others, the ecotoxicity of Ti-based NM has been well assessed in algae and fish, while little is known regarding other NM types. While only a few marine species were addressed, the freshwater species Daphnia magna and Danio rerio accounted for the majority of studies on invertebrate and fish groups, respectively. Most of the reported responses are related to oxidative stress. Overall, we consider that invertebrate groups are the most vulnerable, with emphasis on microcrustaceans, as environmentally realistic metal-based NM concentration even caused mortality in some species. In the case of fish, we assumed that environmental concentrations of Ti-based NM represent a growing concern and threat; however, further studies should be carried out by employing other kinds of NM. Furthermore, more ecotoxicological information is needed in the case of carbon-based NM, as they are expected to considerably increase in terms of released amounts and applications in the near future.

Environmental factors modify silver nanoparticles ecotoxicity in Chydorus eurynotus (Cladocera).

Silver nanoparticles (AgNPs) are among the most produced nanomaterials in the world and are incorporated into several products due to their biocide and physicochemical properties. Since freshwater bodies are AgNPs main final sink, several consequences for biota are expected to occur. With the hypothesis that AgNPs can interact with environmental factors, we analyzed their ecotoxicity in combination with humic acids and algae. In addition to the specific AgNPs behavior in the media, we analyzed the mortality, growth, and phototactic behavior of Chydorus eurynotus (Cladocera) as response variables. While algae promoted Ag+ release, humic acids reduced it by adsorption, and their combination resulted in an intermediated Ag+ release. AgNPs affected C. eurynotus survival and growth, but algae and humic acids reduced AgNPs lethality, especially when combined. The humic acids mitigated AgNP effects in C. eurynotus growth, and both factors improved its phototactic behavior. It is essential to deepen the study of the isolated and combined influences of environmental factors on the ecotoxicity of nanoparticles to achieve accurate predictions under realistic exposure scenarios.

Nanosilica and copper ecotoxicity in Gambusia holbrooki fish.

When silica nanoparticles (SiNP) reach the water bodies interact with the already existing pollutants in the environments. This study aimed to evaluate the ecotoxicity of SiNP under the presence/absence of Cu in mosquitofish (Gambusia holbrooki). Fish were exposed to 0, 10 and 100 mg SiNP L-1, alone or mixed with Cu (0.25 mg L-1). After 96 h, the amount of colony forming units (CFU) of bacteria living on the skin mucus was analysed, and oxidative stress, tissue damage enzymes, and neurotoxicity were evaluated. We observed a reduction in CFU when Cu was present in the media. The liver was the target organ, evidencing a decrease in tissue damage enzymatic activities, activation of the antioxidant system in all treatments, and lipid oxidative damage when the SiNP and Cu were mixed. Overall, SiNP ecotoxicity was proved, which could also be enhanced by the presence of ubiquitous elements such as metals.

Nanosilica size-dependent toxicity in Ceriodaphnia reticulata (Cladocera).

Silica nanoparticles (SiNP) are the most produced nanomaterials due to their variety of applications. When released to environments, surface water bodies are their main final sink. SiNP toxicity is still inconclusive and may vary according to particle properties such as their size. We analyzed the size-related effects of SiNP (22 and 244 nm) on mortality, life history traits, and oxidative stress in the cladoceran Ceriodaphnia reticulata. The smaller SiNP (LC5072 h: 105.5 µg/ml) were more lethal than the larger ones (LC5072 h >500 µg/ml). The 22 nm-sized SiNP decreased the number of molts and neonates, increased superoxide dismutase and inhibited glutathione S-transferase activities, while larger SiNP did not exert substantial effects on the organisms at the tested concentrations. In conclusion, SiNP toxicity depended on their size, and this information should be considered for regulatory purposes and to the development of safe-by-design nanoproducts to ultimately guarantee the environment protection.

Nanotechnology-based pesticides: Environmental fate and ecotoxicity.

The imminent increase in global food demand inevitably leads to an increase in agricultural practices, with an emphasis on pesticide applications. Nanotechnology-based pesticides, or nanopesticides, have gained importance as they are more efficient and, in some cases, less toxic than their conventional counterparts. However, concerns about these novel products have arisen as evidence about their (eco)safety is controversial. This review aims to: (1) introduce the currently applied nanotechnology-based pesticides and their mechanisms of toxic action; (2) describe their fate when released into the environment, with an emphasis on aquatic environments; (3) summarize available research on ecotoxicological studies in freshwater non-target organisms through a bibliometric analysis; and (4) identify gaps in knowledge from an ecotoxicological perspective. Our results show that the environmental fate of nanopesticides is poorly studied and depends on both intrinsic and external factors. There is also a need for comparative research into their ecotoxicity between conventional pesticide formulations and their nano-based counterparts. Among the few available studies, most considered fish species as test organisms, compared to algae and invertebrates. Overall, these new materials generate toxic effects on non-target organisms and threaten the integrity of the environment. Therefore, deepening the understanding of their ecotoxicity is crucial.

Ecotoxicity of nanosilver on cladocerans and the role of algae provision.

Silver nanoparticles (AgNPs) are applied in diverse industries due to their biocide and physicochemical properties; therefore, they can be released into aquatic systems, interact with environmental factors, and ultimately exert adverse effects on the biota. We analyzed AgNPs effects on Ceriodaphnia reticulata (Cladocera) through mortality and life-history traits, considering the influence of food (Tetradesmus obliquus, Chlorophyceae) presence and concentration. C. reticulata was exposed to AgNPs in acute (absence and two algae concentrations plus five AgNPs treatments) and chronic assays (two algae concentrations plus three AgNPs treatments). AgNPs did not affect algae flocculation but increased Ag+ release, being these ions less toxic than AgNPs (as proved by the exposure to AgNO3). A reduction in AgNPs acute toxicity was observed when algae concentration increased. Acute AgNP exposure decreased C. reticulata body size and heart rate. The chronic AgNP exposure reduced C. reticulata molt number, growth, heart rate, and neonate size:number ratio, being these effects mitigated at the highest algae concentration. Increases in relative size and number of neonates were observed in AgNP treatments suggesting energy trade off. The increased Ag+ release with food presence suggests that the AgNP-algae interaction might be responsible of the decreased toxicity. Although algae reduced AgNP toxicity, they still exerted adverse effects on C. reticulata below predicted environmental concentrations. Since algae presence reduces AgNP effects but increases Ag+ release, studies should be continued to provide evidence on their toxicity to other organisms.

Avoidance of neonicotinoid-treated seeds and cotyledons by captive eared doves (Zenaida auriculata, Columbidae).

Farmland birds can be exposed to neonicotinoids through the ingestion of treated unburied seeds and cotyledons. The aim of this study was to evaluate the avoidance of sorghum with imidacloprid, clothianidin or thiamethoxam, soybean with imidacloprid, and soybean cotyledons with imidacloprid or thiamethoxam on eared doves (Zenaida auriculata). Doves were fed with test food (untreated and neonicotinoid-treated sorghum, soybean or soybean cotyledons) and maintenance food (seed mix) for 3-5 days to study the repellency (primary repellency and conditioned aversion) and anorexia caused by neonicotinoid-treated food, followed by a 7-day period on maintenance food to study the persistence of the anorexic effect after neonicotinoid exposure. Immediately afterward, the same doves were exposed to treated test food during a second period of 3-5 days to study the potential reinforcement of food avoidance. Finally, doves were fed with untreated test food to test the capacity of the pesticide to induce conditioned food aversion against untreated food in subsequent encounters. Intoxication signs and differences of body weight were determined. With sorghum, the three neonicotinoids produced a decrease in the consumption of treated seeds by >97% compared to control birds. However, this was not enough to prevent the death of 3/8 and 1/8 of the doves exposed to imidacloprid and clothianidin, respectively. Anorexia was clearly observed with neonicotinoid-treated sorghum. The birds did not avoid the untreated sorghum after exposure to the treated sorghum, indicating that avoidance is not generalized to the type of food without an associated sensory cue. The results obtained with soybean seeds and cotyledons were less conclusive because captive doves hardly consumed these foods, even without neonicotinoid treatment. The avoidance of sorghum seeds treated with neonicotinoids was insufficient to prevent poisoning and death of eared doves.

Building the Bridge From Aquatic Nanotoxicology to Safety by Design Silver Nanoparticles.

Nanotechnologies have rapidly grown, and they are considered the new industrial revolution. However, the augmented production and wide applications of engineered nanomaterials (ENMs) and nanoparticles (NPs) inevitably lead to environmental exposure with consequences on human and environmental health. Engineered nanomaterial and nanoparticle (ENM/P) effects on humans and the environment are complex and largely depend on the interplay between their peculiar properties such as size, shape, coating, surface charge, and degree of agglomeration or aggregation and those of the receiving media/body. These rebounds on ENM/P safety and newly developed concepts such as the safety by design are gaining importance in the field of sustainable nanotechnologies. This article aims to review the critical characteristics of the ENM/Ps that need to be addressed in the safe by design process to develop ENM/Ps with the ablility to reduce/minimize any potential toxicological risks for living beings associated with their exposure. Specifically, we focused on silver nanoparticles (AgNPs) due to an increasing number of nanoproducts containing AgNPs, as well as an increasing knowledge about these nanomaterials (NMs) and their effects. We review the ecotoxicological effects documented on freshwater and marine species that demonstrate the importance of the relationship between the ENM/P design and their biological outcomes in terms of environmental safety.

Metallic, metal oxide, and metalloid nanoparticles toxic effects on freshwater microcrustaceans: An update and basis for the use of new test species.

In this article, we performed a literature review on the metallic, metal oxide, and metalloid nanoparticles (NP) effects on freshwater microcrustaceans, specifically focusing on (i) the main factors influencing the NP toxicity and (ii) their main ecotoxicological effects. Also, given that most studies are currently developed on the standard test species Daphnia magna Straus, we analyzed (iii) the potential differences in the biological responses between D. magna and other freshwater microcrustacean, and (iv) the ecological implications of considering only D. magna as surrogate of other microcrustaceans. We found that NP effects on microcrustaceans depended on their intrinsic properties as well as the exposure conditions. Among the general responses to different NP, we identified body burial, feeding inhibition, biochemical effects, metabolic changes, and reproductive and behavioral alterations. The differences in the biological responses between D. magna and other freshwater microcrustacean rely on the morphology (size and shape), ecological traits (feeding mechanisms, life cycles), and intrinsic sensitivities. Thus, we strongly recommend the use of microcrustaceans species with different morphological, physiological, and ecological characteristics in future ecotoxicity tests with NP to provide relevant information with regulation purposes regarding the discharge of NP into aquatic environments. PRACTITIONER POINTS: Nanoparticles effects depend on intrinsic and external factors. Nanoparticles affect the morphology, physiology, and behavior. Effects on Daphnia differ from other microcrustaceans. The use of more diverse test species is suggested.

Ecotoxicity of silica nanoparticles in aquatic organisms: An updated review.

This review aims to (i) provide a current overview of the main characteristics of SiNP (physical and chemical properties, applications, and emissions), (ii) evaluate the scientific production up to date concerning SiNP, with focus on their toxic effects, through a bibliometric analysis, (iii) describe the main toxic mechanisms of SiNP, (iv) assess the current knowledge about ecotoxicity of SiNP on aquatic organisms (marine and freshwater), and (v) identify the main gaps in the knowledge of SiNP toxicity from an environmentally point of view. The scientific production of SiNP concerning their chemical and physical characteristics has increased exponentially. However, little information is available regarding their ecotoxicity. Particle functionalization is a key factor that reduces SiNP toxicity. Most of the studies employed standard species as test organisms, being the local/native ones poorly represented. Further studies employing long-term exposures and environmentally relevant concentrations are needed to deepen the knowledge about this emergent pollutant.

Biomarkers of exposure and effect in the armoured catfish Hoplosternum littorale during a rice production cycle.

Fish cultivation in rice fields is a valuable resource in some rural areas of the world. Fish is a source of protein and an additional source of income for local farmers. However, the use of pesticides may impact fish and consumer health. The aim of this study was to evaluate exposure and effect biomarkers in native fish inhabiting a rice field during a production cycle. Samples of fish, water and sediment from a rice field in Santa Fe, Argentina were collected during a cultivation season (at the beginning: November 2017, in the middle: December 2017 and at the end: February 2018). At each sampling period, fish biomarkers of effect (biometric indices, hematological parameters, energy reserves, oxidative stress and neurotoxicity) were assessed together with pesticide screening in water, sediment, and fish samples. Only herbicides were present in water and sediment samples in agreement with land treatment before rice sowing stage, where only herbicides were applied. In general, the greatest water concentrations of bentazone, glyphosate and aminomethylphosphonic acid (AMPA), and the lowest sediment glyphosate and AMPA levels were observed at the beginning of the farming cycle. Fish bioaccumulated AMPA residues at all sampling periods and showed biological responses to cope with a stressful environment. Alterations in hematological parameters, mobilization of energetic reserves and activation of the antioxidant system were detected. However, no oxidative damage nor neurotoxic effects were present along the production cycle. Under a real exposure scenario, the present work demonstrates that biological changes are induced in fish to cope with stressors present in a rice field. Fish-rice coculture is an efficient and ecologically sustainable approach to increase food supplies, and a better understanding of the effect of this particular environment on fish would allow a greater and safer development of this promising productive activity in South American rice producing countries.

Nanosilver and Silver Nitrate Toxicity in Ex Vivo-Exposed Gills of Fish and Mitigation by Humic Acids.

Silver nanoparticles (AgNP) are unique because of their biocide properties. Once released to environment, AgNP interact with the natural organic matter which impact on their fate, dispersion, and ultimate toxicity. We carried out an ex vivo exposure of gill of Corydoras paleatus fish to 100 µg L-1 of AgNP or AgNO3, alone and in combination with 10 mg L-1 of humic acids (HA), with the aim to evaluate the potential mitigation of HA on AgNP toxic effects. We analyzed Ag accumulation and oxidative stress biomarkers. The results showed high bioaccumulation after the AgNO3+HA exposure. An inhibition of glutathione-S-transferase enzymatic activity and depletion of reduced glutathione levels were registered after the AgNO3 exposure, and increased lipid peroxidation levels in the case of AgNP one. Oxidative responses were mitigated when the HA were present in the media. Overall, the knowledge about the fate of this emergent pollutant was deepened through this study.

Low temperature stress in a cultured fish (Piaractus mesopotamicus) fed with Pyropia columbina red seaweed-supplemented diet.

This study aimed to analyze the cold stress effects (in terms of hematology, energy reserves, and oxidative stress) in Piaractus mesopotamicus (pacú) and their mitigation by a Pyropia columbina red seaweed-supplemented diet. For this purpose, juvenile fish were fed with a control (CD) or a red seaweed-supplemented diet (RD) for 60 days, and then, the animals were exposed to a low temperature (14 °C) and a control temperature (24 °C) for 24 h. The cold shock generated an increase of hemoglobin levels in fish fed with both diets. In CD-fed fish, plasmatic triglycerides, cholesterol, and hepatic glycogen decreased after the thermal shock; meanwhile, the animals fed with RD showed decreased hepatic proteins, but increased cholesterol and hepatic glycogen. Regarding oxidative stress, antioxidant enzymes augmented their activity in the liver, intestine, and gills; meanwhile, lipid oxidative damage was observed in the liver and intestine of fish exposed to 14 °C and fed with both diets. Pacú was sensitive to cold shock, but no mitigation effects were observed in fish fed with the supplemented diet. Further research should target higher concentrations of P. columbina in supplemented diets to take advantage of this valuable resource.

Mitigation of silver nanoparticle toxicity by humic acids in gills of Piaractus mesopotamicus fish.

Silver nanoparticles (AgNPs) are one of the most produced nanoproducts due to their unique biocide properties. The natural organic matter has an important impact on nanoparticle's dispersion as it may alter their fate and transport, as well as their bioavailability and toxicity. Therefore, this study aimed to evaluate the mitigatory effect of humic acids (HAs) on AgNP toxicity. For this purpose, we carried out an ex vivo exposure of gill of Piaractus mesopotamicus fish to 100 µg L-1 of AgNPs or AgNO3, alone and in combination with 10 mg L-1 of HAs. In parallel, a complete AgNP characterization in the media, including the presence of HAs, was provided, and the Ag+ release was measured. We analyzed Ag bioaccumulation, antioxidant enzymes activities, lipid peroxidation, antioxidant capacity against peroxyl radicals, and reduced glutathione levels in fish tissue. Our results indicated the Ag+ release from AgNPs decreased 28% when the HAs were present in the media. The Ag accumulation in gill tissue exposed to AgNPs alone was higher than the AgNO3 exposure, and sixfold higher than the treatment with the HA addition. Moreover, after both Ag forms, the catalase enzyme augmented its activity. However, those responses were mitigated when the HAs were present in the media. Then, our results suggested the mitigation by HAs under the exposure to both Ag forms, providing valuable information about the fate and behavior of this emergent pollutant.

Effects of cold stress on juvenile Piaractus mesopotamicus and the mitigation by ß-carotene.

This study investigated the effects of cold stress on morphometrical and hematological biomarkers, energy metabolism, and oxidative stress in different tissues of P. mesopotamicus, and the protective role of ß-carotene. Fish were fed with a control diet (CD) and the same diet supplemented with 105 mg/kg ß-carotene (BD) for 60 days. After the feeding trial, fish fed CD or BD diets were exposed to control (24 °C) and low temperature (14 °C) for 24 h. Fish (CD and BD) exposed to thermal stress showed lower hepatosomatic index. The hemoglobin increased only in CD-fed fish exposed to 14 °C. Increased glycemia, plasmatic protein depletion, and decreased hepatic glycogen were observed in fish fed the CD, while only the lipid levels in liver were augmented in BD-fed fish exposed at 14 °C. Regarding the oxidative stress, increased antioxidant enzymes activity and lipid peroxidation were observed in CD-fed fish exposed to cold. The two-way ANOVA showed an interaction between dietary treatment and temperature for glucose and oxidative stress biomarkers, with the highest values recorded in 14 °C-exposed fish fed with the CD. Our study demonstrated that cold stress had the greatest impact on fish oxidative status, and ß-carotene reduces harmful effects induced by cold in P. mesopotamicus.

Nanoparticles Toxicity in Fish Models.

The increasing production and use of nanoparticles (NP) have raised concerns regarding the potential toxicity to human and environmental health. In this review, we address the up to date information on nanotoxicity using fish as models. Firstly, we carried out a systematic literature search (articles published up to February 2019 in the Scopus database) in order to quantitatively assess the scientific research on nanoparticles, nanotoxicity and fish. Next, we carried out a narrative synthesis on the main factors and mechanisms involved in NP toxicity in fish. According to the bibliometric analysis, there is a low contribution of scientific research on nanotoxicity compared with the general nanoparticles scientific production. The literature search also showed that silver and titanium NP are the most studied nanomaterials and Danio rerio is the fish species most used. In comparison with freshwater fish, the effects of nanomaterials on marine fish have been little studied. After a non-systematic literature analysis, we identified several factors involved in nanotoxicity, as well as the effects and main toxicity mechanisms of NP on fish. Finally, we highlighted the knowledge gaps and the need for future research.

Exposure to a nanosilver-enabled consumer product results in similar accumulation and toxicity of silver nanoparticles in the marine mussel Mytilus galloprovincialis.

The incorporation of silver nanoparticles (AgNPs) in commercial products is increasing rapidly. The consequent release of AgNPs into domestic and industrial wastewater raises environmental concerns due to their anti-microbial properties and toxicity to non-target aquatic organisms. The aim of the present study was to investigate the effects of nanArgen™ (Nanotek S.A.), a AgNP-enabled consumer product, in the marine bivalve Mytilus galloprovincialis. Two environmentally relevant concentrations of nanArgen™ (1 and 10 µg/L) were tested in vivo for 96 h, and Ag was quantified in mussel soft tissue and natural seawater (NSW). nanArgen™ suspensions were characterized via TEM, SEM, EDS, DLS, and UV-vis optical analysis. Several molecular and biochemical responses were investigated in exposed mussels: lysosomal membrane stability by Neutral Red Retention Time (NRRT) assay; micronucleus (MN) frequency in hemocytes; metallothionein (MT) protein content and gene expression (mt10 and mt20); catalase (CAT) and glutathione-S-transferase (GST) activities; malondialdehyde (MDA) accumulation in digestive glands; and efflux activity of ATP-binding cassette transport proteins (ABC) in gill biopsies. SEM, TEM and DLS analyses confirmed the presence of well-defined AgNPs in nanArgen™ which were roughly spherical with an average particle size of approx. 30 ± 10 nm. DLS analysis revealed the formation of AgNP aggregates in nanArgen™ suspension in NSW (Z-average of 547.80 ± 90.23 nm; PDI of 0.044). A significant concentration-dependent accumulation of Ag was found in mussels' whole soft tissue in agreement with a concentration-dependent decrease in NRRT and an increase of MN frequency in hemocytes and GST activities in digestive glands. A significant increase in MDA levels and MT via both molecular and biochemical tests, were also observed but only at the highest nanArgen™ concentration (10 µg/L). No changes were observed in CAT activities. ABC efflux activities in gill biopsies showed a significant decrease (p < 0.05) only at the lowest concentration (1 µg/L). On such basis, nanArgen™ is shown to be able to induce toxicity and Ag accumulation in marine mussels similarly to AgNPs and in short-term exposure conditions at environmentally relevant concentrations. AgNP-enabled products, instead of pristine AgNPs, should be the focus of future ecotoxicity studies in order to address any risks associated to their widespread use, disposal and uncontrolled release into the aquatic environment for non target species.