Responses of soil microbiome to copper-based materials (nano and bulk) for agricultural applications: An indoor-mesocosm experiment.

The foreseen increasing application of copper-based nanomaterials (Cu-NMs), replacing or complementing existing Cu-agrochemicals, may negatively impact the soil microbiome. Thus, we studied the effects on soil microbiome function and composition of nano copper oxide (nCuO) or copper hydroxide NMs in a commercial (Kocide®3000) or a lab-synthetized formulation (nCu(OH)2) or bulk copper hydroxide (Cu(OH)2-B), at the commonly recommended Cu dose of 50 mg(Cu)kg-1 soil. Microbial responses were studied over 28 days in a designed indoor mesocosm. On day-28, in comparison to non-treated soil (CT), all Cu-treatments led to a reduction in dehydrogenase (95% to 68%), arylsulfatase (41% to 27%), and urease (40% to 20%) activity. There was a 32% increase in the utilization of carbon substrates in the nCuO-treatment and an increased abundance of viable bacteria in the nCu(OH)2-treatment (75% of heterotrophic and 69% of P-solubilizing bacteria). The relative abundance of Acidobacteria [Kocide®3000, nCuO, and Cu(OH)2-B treatments] and Flavobacteriia [nCu(OH)2-treatment] was negatively affected by Cu exposure. The abundance of Cu-tolerant bacteria increased in soils treated with Kocide®3000 (Clostridia) and nCu(OH)2 (Gemmatimonadetes). All Cu-treated soils exhibited a reduced abundance of denitrification-related genes (0.05% of nosZ gene). The DTPA-extractable pool of ionic Cu(II) varied among treatments: Cu(OH)2-B > Kocide®3000 âˆ¼ nCuO>nCu(OH)2, which may explain changes on the soil microbiome composition, at the genera and OTU levels. Thus, our study revealed that Cu-materials (nano and bulk) influence the soil microbiome with implications on its ecological role. It highlights the importance of assessing the impact of Cu-materials under dynamic and complex exposure scenarios and emphasizes the need for specific regulatory frameworks for NMs.

Toxicity assessment of binary mixtures of BP3 with 4-MBC (UV-filters), and BP3 with DEET (insect repellent) using the aquatic midge Chironomus riparius.

Personal care products have various organic ultraviolet filters (UV filters) in their composition to increase protection against ultraviolet radiation. Some of these products also contain insect repellents in their formulations. Consequently, these compounds reach freshwater ecosystems, exposing aquatic organisms to a cocktail of anthropogenic contaminants. In this study, the joint effects of two most frequently detected UV filters (Benzophenone - 3 (BP3) and Enzacamene (4-MBC)) and joint effects of BP3 combined with an insect repellent (N, N diethyl-3-methylbenzamide - DEET) were evaluated using life-history traits of the aquatic midge Chironomus riparius such as emergence rate, time to emergence and imagoes body weight. The results showed synergistic effects between BP3 and 4-MBC for C. riparius emergence rate. Regarding the effects of BP3 and DEET mixture, our analysis suggests synergism in the case of males but antagonism in the case of females' time to emergence. Our results imply that the effects of UV filters present in sediments within chemical mixtures are complex and that the evaluation of effects using different life-history traits can yield different patterns of responses. This study demonstrates the importance of assessing the combined effects of pollutants used/found concomitantly in aquatic systems for a more accurate risk assessment, as individual chemical testing can underestimate the toxicity of organic UV filters.

Toxic effects of a mixture of pharmaceuticals in Mytilus galloprovincialis: The case of 17α-ethinylestradiol and salicylic acid.

The impact of pharmaceuticals on marine invertebrates has been a topic of rising concern, with an increasing number of studies regarding the impacts on bivalves. However, very few investigated the toxicity of mixtures of pharmaceuticals. This knowledge gap was investigated in the present study, where the toxicity of 17α-ethinylestradiol (EE2) and salicylic acid (SA) mixture was evaluated. To this end, Mytilus galloprovincialis mussels were chronically subjected to both pharmaceuticals, acting alone and in combination, and the effects at the cellular level were measured. The Independent Action (IA) model was performed aiming to compare obtained with predicted responses. The integrated biomarker response (IBR) index was used to assess the overall biochemical response given by mussels. The results obtained revealed that the most stressful condition was caused by the combined effect of EE2 and SA, with the highest metabolic capacity, antioxidant (catalase activity) and biotransformation (carboxylesterases activity) activation and cellular damage in organisms exposed to the mixture of both drugs in comparison to responses observed when each drug was acting alone. Predicted responses obtained from the IA model indicate that caution should be paid as frequent deviations to observed responses were found. This study highlights the need for future studies considering the mixture of pollutants, mimicking the actual environmental conditions.

The effects of different temperatures in mercury toxicity to the terrestrial isopod Porcellionides pruinosus.

Climate changes and metal contamination are pervasive stressors for soil ecosystems. Mercury (Hg), one of the most toxic metals, has been reported to interact with temperature. However, compared to aquatic biota, little is known about how temperature affects Hg toxicity and bioaccumulation to soil organisms. Here, toxicity and bioaccumulation experiments were replicated at 15 °C, 20 °C, and 25 °C to understand how sub-optimal temperatures affect the toxicokinetics and toxicodynamics of Hg via soil. Genotoxicity and energy reserves were also assessed to disclose potential trade-offs in life-history traits. Results underpin the complexity of temperature-Hg interactions. Survival was determined mainly by toxicokinetics, but toxicodynamics also played a significant role in defining survival probability during early stages. The processes determining survival probability were faster at 25 °C: General Unified Threshold of Survival (GUTS) model identified an earlier/steeper decline in survival, compared to 20 °C or 15 °C, but it also approached the threshold faster. Despite potentiation of Hg genotoxicity, temperature promoted faster detoxification, either increasing toxicokinetics rates or damage repair mechanisms. This metabolism-driven increase in detoxification led to higher depletion of energy reserves and likely triggered stress response pathways. This work emphasized the need for comprehensive experimental approaches that can integrate the multiple processes involved in temperature-metal interactions.

Terrestrial organisms react differently to nano and non-nano Cu(OH)2 forms.

The application of nanomaterials in agriculture is deemed as a promising strategy to increase the sector's sustainability. Nanopesticides are expected to improve solutions for pest/disease management, while reducing adverse effects to ecosystems, in accordance with the European Green Deal strategy. Hitherto, nanopesticide research has mostly focused on assessing effects to target species or crops, and less attention has been devoted to non-target soil species. In this study, we explored whether three copper hydroxide-based forms (nano and non-nano) show different toxicity and accumulation patterns in two terrestrial invertebrates, the isopod Porcellionides pruinosus and the mealworm larvae Tenebrio molitor. Toxicity and bioaccumulation experiments were performed and time-course toxicity and toxicokinetics analyzed and modelled. We found important differences in copper sensitivity and accumulation: T. molitor was more resistant and able to eliminate copper compared to P. pruinosus. The nanopesticide mostly elicited lower effects for both species. Slower toxicokinetics by the nanopesticide and lower bioaccumulative potential to P. pruinosus must be partially responsible for the differences in toxicity. Although no toxicokinetics differences were found in T. molitor between Cu forms, distinct negative effects on growth must reflect different energetic costs for copper regulation. Even though effects on toxicity and bioaccumulation are species-specific, copper hydroxide-based nanopesticides may cause less adverse effects to soil organisms than conventional products, being a good solution for reducing the environmental impact. CAPSULE: Copper hydroxide-based nanopesticide was less toxic and bioaccumulative to soil organisms than conventional copper hydroxide products, being a good solution for reducing the environmental impact.

Bioaccumulation but no biomagnification of silver sulfide nanoparticles in freshwater snails and planarians.

Bioaccumulation studies are critical in regulatory decision making on the potential environmental risks of engineered nanoparticles (NPs). The present study evaluated the toxicokinetics of silver, taken up from sulfide nanoparticles (Ag2S NPs; simulating an aged Ag NP form) and AgNO3 (ionic counterpart), in the pulmonate snail Physa acuta and the planarian Girardia tigrina. The snails were first exposed for 7 days to Ag-spiked water, along with the microalgae Raphidocelis subcapitata upon which they fed setting up a double route exposure, and subsequently provided as pre-exposed food to the planarians. Ag toxicokinetics and bioaccumulation were assessed in planarians and snails, and potential biomagnification from snail to planarian was evaluated. Gut depuration was also explored to understand whether it constitutes a factor likely to influence Ag toxicokinetics and internal concentrations in the test species. Both species revealed Ag uptake in Ag2S NP and AgNO3 treatments, with higher uptake from the latter. Uptake by the snails was probably via a combination of water exposure and ingested algae provided as food, but ingestion of algae possibly had higher relevance for Ag uptake from the Ag2S NPs compared to AgNO3. For planarians, diet probably was the most important exposure route since no Ag uptake was observed in previous waterborne exposures to Ag2S NPs. Kinetics and internal Ag concentrations did not significantly differ between depurated and non-depurated snails or planarians. The planarians fed on snails revealed no biomagnification. To the best of our knowledge this is the first study investigating the toxicokinetics and biomagnification of NPs in planarians, and with that providing important data on the kinetics and bioaccumulation of NPs in a relevant benthic species.

Mixture toxicity prediction of substances from different origin sources in Daphnia magna.

Due to several anthropogenic activities, water bodies have been heavily impacted by contaminants identified in aquatic ecosystems, including pharmaceuticals, personal care products, agricultural and industrial chemicals. Risk assessment based on chemical mixtures is still default in many monitoring studies, with decisions being based solely on a chemical-by-chemical basis. The present study aimed to improve risk assessment procedures in water bodies by focusing on mixtures of chemical substances of different origins. The goal was to analyze potential interactions occurring at different complexity levels (binary and quaternary mixtures) using standardised toxicity assays. Mixture toxicity effects were assessed using Daphnia magna as the model organism and the compounds sodium fluoride, boric acid, ammonium hydroxide and acetaminophen as general representatives of contaminants in the aquatic ecosystem. The results revealed interactions between the compounds, mainly showing antagonism but also dose level and dose ratio-dependent deviations. Overall antagonism was the dominant deviation pattern, particularly at low doses, though synergism was also detected at higher doses or specific ratios. Synergism at low doses was found for the binary mixture of ammonium hydroxide and acetaminophen, two common pollutants, which denotes an enhanced risk to aquatic ecosystems. Independent Action provided more accurate predictions for the quaternary mixture, whereas Concentration Addition overestimated the toxicity of the mixture. Regarding the environmental risk assessment of water bodies, the interaction between chemicals in a mixture should not be neglected. The complexity of the mixture interactions found in the present study highlights the importance of complementing chemical screenings of water bodies with mixture toxicity data, particularly when considering chemicals of multiple origins whose joint action remains unknown.

Gut and faecal bacterial community of the terrestrial isopod Porcellionides pruinosus: potential use for monitoring exposure scenarios.

This work aimed to characterize the gut and faeces bacterial communities (BC) of Porcellionides pruinosus using high-throughput sequencing. Isopods were collected from the field and kept in laboratory conditions similar to those normally applied in ecotoxicology tests. Faeces and purged guts of isopods (n = 3 × 30) were analysed by pyrosequencing the V3-V4 region of 16 S rRNA encoding gene. Results showed that gut and faecal BCs were dominated by Proteobacteria, particularly by an OTU (Operational Taxonomic Unit) affiliated to genus Coxiella. Diversity and richness values were statistically higher for faecal BC, mainly due to the occurrence of several low-abundance phylotypes. These results may reflect faecal carriage of bacterial groups that cannot settle in the gut. BCs of P. pruinosus comprised: (1) common members of the soil microbiota, (2) bacterial symbionts, (3) bacteria related to host metabolic/ecological features, and (4) bacterial etiological agents. Comparison of BC of this isopod species with the BC from other invertebrates revealed common bacterial groups across taxa. The baseline information provided by this work will assist the design and data interpretation of future ecotoxicological or biomonitoring assays where the analysis of P. pruinosus BC should be included as an additional indicator. CAPSULE: Terrestrial isopods bacterial communities might support ecotoxicological assays and biomonitoring processes as a valuable tool.

Site-specific hazard evaluation for improved groundwater risk assessment.

Groundwater represents one of the most important natural water resources worldwide. Contamination is a key driver in sustaining water quality to populations and the environment. Therefore, it is crucial to look at contamination and potential effects. Within the WaterJPI project "We-Need - WatEr NEEDs, Availability, Quality and Sustainability", the Cremona and the Bologna Aquifers (Italy) were chosen as case studies since both aquifers represent two major Italian water resources of the Emilia-Romagna region. Aiming for a site-specific groundwater hazard assessment, the groundwaters from the Cremona and Bologna aquifers were simulated by preparing synthetic waters, CSW and BSW, respectively. Boron (as boric acid), fluoride (as sodium fluoride), and ammonium (as ammonium hydroxide), detected in the aquifers, were used to assess potential negative impacts on groundwater systems using aquatic organisms. Acute toxicity tests with Daphnia magna and Fish Embryo Toxicity Tests (FET) with Danio rerio were performed in the CSW and BSW synthetic waters and respective culture media, and toxicity was assessed for each organism. Boron, fluoride and ammonium had no ecological hazard effects at the concentrations detected in the groundwaters. Besides, a crucial result from this study regards the use of different media when addressing toxicity assessment. In this case, it was observed that toxicity was media and organism dependent. Therefore, adapting testing protocols for higher relevance should be considered for site-specific hazard assessment.

Unravelling the molecular mechanisms of nickel in woodlice.

During the last few years, there has been an alarming increase in the amount of nickel (Ni) being released into the environment, primarily due to its use in the production of stainless steel but also from other sources such as batteries manufacturing and consequent disposal. The established biotic ligand models provide precise estimates for Ni bioavailability, in contrast, studies describing the mechanisms underpinning toxicological effect of Ni are scarce. This study exploits RNA-seq to determine the transcriptomic responses of isopods using Porcellionides pruinosus as an example of a terrestrial metal-resistant woodlouse. Furthermore, the recently proposed model for Ni adverse outcome pathways (Ni-AOP) presents an unprecedented opportunity to fit isopod responses to Ni toxicity and define Porcellionides pruinosus as a metalomic model. Prior to this study, P. pruinosus represented an important environmental sentinel, though lacking genetic/omic data. The reference transcriptome generated here thus represents a major advance and a novel resource. A detailed annotation of the transcripts obtained is presented together with the homology to genes/gene products from Metazoan and Arthropoda phylum, Gene Ontology (GO) classification, clusters of orthologous groups (COG) and assignment to KEGG metabolic pathways. The differential gene expression comparison was determined in response to nickel (Ni) exposure and used to derive the enriched pathways and processes. It revealed a significant impact on ion trafficking and storage, oxidative stress, neurotoxicity, reproduction impairment, genetics and epigenetics. Many of the processes observed support the current Ni-AOP although the data highlights that the current model can be improved by including epigenetic endpoints, which represents key chronic risks under a scenario of Ni toxicity.

Toxicokinetics of cadmium in Palaemon varians postlarvae under waterborne and/or dietary exposure.

The present study assessed cadmium uptake and depuration rates in the euryhaline estuarine shrimp Palaemon varians under different exposure routes. Postlarval shrimp were exposed for 4 d under different exposure routes: contaminated water, contaminated diet, and a 2-way exposure scenario where both contaminated water and diet were used. After exposure, postlarval shrimp were transferred to a clean medium and fed a noncontaminated diet for 96 h. Bioaccumulation via the different exposure routes was modeled with a standard first-order, one-compartment toxicokinetics model and one with an additional parameter reflecting an inert fraction or storage compartment. The simultaneous 2-way exposure (through water and diet) resulted in accumulation being almost twice as high as the sum of the individual exposure routes, thus indicating that accumulation from multiple routes may be more than additive. Cadmium uptake from water was faster than uptake from food maintained for 48 h at that same cadmium concentration. Shrimp were unable to eliminate cadmium from their body, showing no depuration during 96 h after exposure via different routes, thus suggesting that a longer depuration period is needed. Model comparisons did not provide a significantly better fit when the model included the presence of an inert fraction. The present study highlights the importance of assessing accumulation using multiple exposure routes compared with individual routes because the latter may underestimate bioaccumulation. Environ Toxicol Chem 2018;37:1614-1622. © 2018 SETAC.

Joint effects of chlorpyrifos and mancozeb on the terrestrial isopod Porcellionides pruinosus: A multiple biomarker approach.

The exposure to pesticides by nontarget soil biota has long been regarded as a serious downside of modern agricultural regimes and the subject of heated debate. Of utmost relevance is the exposure to pesticide mixtures because their effects have been shown to not necessarily reflect the individual toxicity of their components, and even the simple addition of effects may lead to consequences not clearly anticipated. In the present study, a multiple biomarker approach was employed to identify the mechanistic and time effects underlying several single and mixture treatments of chlorpyrifos (CPF) and mancozeb (MCZ) in juveniles and adults of the terrestrial isopod Porcellionides pruinosus. The effects of the individual pesticides and the mixtures at recommended doses were mostly transitory under these controlled conditions and one-pulse exposure. Whereas imbalances were identified on detoxification and oxidative stress-related enzymes, the isopods generally showed the ability to recover through the end of the experiment. However, juveniles displayed greater vulnerability than adults. Most of the differences between life stages occurred in energy-related parameters where distinct performances and stress-handling behaviors were observed, suggesting higher metabolic costs to juveniles. Our results stress that understanding the time dependence of the underlying mechanisms governing the joint effects of the pesticides can help in assessing and anticipating the effects of the pesticide mixtures. Moreover, we emphasize the importance of taking life stage-related differences into consideration when evaluating the environmental risks of pesticides and pesticide mixtures. Environ Toxicol Chem 2018;37:1446-1457. © 2018 SETAC.

Influence of environmental conditions on the toxicokinetics of cadmium in the marine copepod Acartia tonsa.

mMarine and estuarine ecosystems are highly productive areas that often act as a final sink for several pollutants, such as cadmium. Environmental conditions in these habitats can affect metal speciation, as well as its uptake and depuration by living organisms. The aim of this study was to assess cadmium uptake and depuration rates in the euryhaline calanoid copepod Acartia tonsa under different pH, salinity and temperature conditions. Cadmium speciation did not vary with changing pH or temperature, but varied with salinity. Free Cd2+ ion activity increased with decreasing salinities resulting in increased cadmium concentrations in A. tonsa. However, uptake rate, derived using free Cd2+ ion activity, showed no significant differences at different salinities indicating a simultaneous combined effect of Cd2+ speciation and metabolic rates for osmoregulation. Cadmium concentration in A. tonsa and uptake rate increased with increasing pH, showing a peak at the intermediate pH of 7.5, while depuration rate fluctuated, thus suggesting that both parameters are mediated by metabolic processes (to maintain homeostasis at pH levels lower than normal) and ion competition at membrane binding sites. Cadmium concentration in A. tonsa, uptake and depuration rates increased with increasing temperature, a trend that can be attributed to an increase in metabolic energy demand at higher temperatures. The present study shows that cadmium uptake and depuration rates in the marine copepod A. tonsa is mostly affected by biological processes, mainly driven by metabolic mechanisms, and to a lesser extent by metal speciation in the exposure medium.

Toxicity interaction between chlorpyrifos, mancozeb and soil moisture to the terrestrial isopod Porcellionides pruinosus.

A main source of uncertainty currently associated with environmental risk assessment of chemicals is the poor understanding of the influence of environmental factors on the toxicity of xenobiotics. Aiming to reduce this uncertainty, here we evaluate the joint-effects of two pesticides (chlorpyrifos and mancozeb) on the terrestrial isopod Porcellionides pruinosus under different soil moisture regimes. A full factorial design, including three treatments of each pesticide and an untreated control, were performed under different soil moisture regimes: 25%, 50%, and 75% WHC. Our results showed that soil moisture had no effects on isopods survival, at the levels assessed in this experiment, neither regarding single pesticides nor mixture treatments. Additivity was always the most parsimonious result when both pesticides were present. Oppositely, both feeding activity and biomass change showed a higher sensitivity to soil moisture, with isopods generally showing worse performance when exposed to pesticides and dry or moist conditions. Most of the significant differences between soil moisture regimes were found in single pesticide treatments, yet different responses to mixtures could still be distinguished depending on the soil moisture assessed. This study shows that while soil moisture has the potential to influence the effects of the pesticide mixture itself, such effects might become less important in a context of complex combinations of stressors, as the major contribution comes from its individual interaction with each pesticide. Finally, the implications of our results are discussed in light of the current state of environmental risk assessment procedures and some future perspectives are advanced.