Using single-species and algal communities to determine long-term adverse effects of silver nanoparticles on freshwater phytoplankton.

The physical and chemical properties of silver nanoparticles (AgNPs) have led to their increasing use in various fields such as medicine, food, and industry. Evidence has proven that AgNPs cause adverse effects in aquatic ecosystems, especially when the release of Ag is prolonged in time. Several studies have shown short-term adverse effects of AgNPs on freshwater phytoplankton, but few studies have analysed the impact of long-term exposures on these populations. Our studies were carried out to assess the effects of AgNPs on growth rate, photosynthesis activity, and reactive oxygen species (ROS) generation on the freshwater green algae Scenedesmus armatus and the cyanobacteria Microcystis aeruginosa, and additionally on microcystin (MC-LR) generation from these cyanobacteria. The tests were conducted both in single-species cultures and in phytoplanktonic communities exposed to 1 ngL-1 AgNPs for 28 days. The results showed that cell growth rate of both single-species cultures decreased significantly at the beginning and progressively reached control-like values at 28 days post-exposure. This effect was similar for the community-cultured cyanobacteria, but not for the green algae, which maintained a sustained decrease in growth rate. While gross photosynthesis (Pg) increased in both strains exposed in single cultures, dark respiration (R) and net photosynthesis (Pn) decreased in S. armatus and M. aeruginosa, respectively. These effects were mitigated when both strains were exposed under community culture conditions. Similarly, the ROS generation shown by both strains exposed in single-species cultures was mitigated when exposure occurred in community cultures. MC-LR production and release were significantly decreased in both single-species and community exposures. These results can supply helpful information to further investigate the potential risks of AgNPs and ultimately help policymakers make better-informed decisions about their utilization for environmental restoration.

Adverse effects of iron-based nanoparticles on freshwater phytoplankton Scenedesmus armatus and Microcystis aeruginosa strains.

Zero-valent nano-iron particles (nZVI) are increasingly present in freshwater aquatic environments due to their numerous applications in environmental remediation. However, despite the broad benefits associated with the use and development of nZVI nanoparticles, the potential risks of introducing them into the aquatic environment need to be considered. Special attention should be focused on primary producer organisms, the basal trophic level, whose impact affects the rest of the food web. Although there are numerous acute studies on the acute effects of these nanoparticles on photosynthetic primary producers, few studies focus on long-term exposures. The present study aimed at assessing the effects of nZVI on growth rate, photosynthesis activity, and reactive oxygen activity (ROS) on the freshwater green algae Scenedesmus armatus and the cyanobacteria Microcystis aeruginosa. Moreover, microcystin production was also evaluated. These parameters were assessed on both organisms singly exposed to 72 h-effective nZVI concentration for 10% maximal response for 28 days. The results showed that the cell growth rate of S. armatus was initially significantly altered and progressively reached control-like values at 28 days post-exposure, while M. aeruginosa did not show any significant difference concerning control values at any time. In both strains dark respiration (R) increased, unlike net photosynthesis (Pn), while gross photosynthesis (Pg) only slightly increased at 7 days of exposure and then became equal to control values at 28 days of exposure. The nZVI nanoparticles generated ROS progressively during the 28 days of exposure in both strains, although their formation was significantly higher on green algae than on cyanobacteria. These data can provide additional information to further investigate the potential risks of nZVI and ultimately help decision-makers make better informed decisions regarding the use of nZVI for environmental remediation.

The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and members of these diverse forms persist within infected cell reservoirs, even during antiretroviral therapy (ART). Characterizing the diverse viral sequences that persist during ART is critical to HIV cure efforts, but our knowledge of on-ART proviral evolutionary dynamics remains incomplete, as does our understanding of the differences between the overall pool of persisting proviral DNA (which is largely genetically defective) and the subset of intact HIV sequences capable of reactivating. Here, we reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study (WIHS) who experienced HIV seroconversion. We measured diversity, lineage origins and ages of proviral sequences (env-gp120) sampled up to four times, up to 12 years on ART. We used the same techniques to study HIV sequences emerging from the reservoir in two participants. Proviral clonality generally increased over time on ART, with clones frequently persisting across multiple time points. The integration dates of proviruses persisting on ART generally spanned the duration of untreated infection (though were often skewed towards years immediately pre-ART), while in contrast, reservoir-origin viremia emerging in plasma was exclusively "younger" (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained highly stable during ART in all but one participant in whom, after 12 years, there was evidence that "younger" proviruses had been preferentially eliminated. Analysis of within-host recombinant proviral sequences also suggested that HIV reservoirs can be superinfected with virus reactivated from an older era, yielding infectious viral progeny with mosaic genomes of sequences with different ages. Overall, results underscore the remarkable genetic stability of distinct proviral sequences that persist on ART, yet suggest that replication-competent HIV reservoir represents a genetically-restricted and overall "younger" subset of the overall persisting proviral pool in blood.

Regulation of growth-related genes by nutrition in paralarvae of the common octopus (Octopus vulgaris).

The common octopus (Octopus vulgaris) is a species of great interest to the aquaculture industry. However, the high mortalities registered during different phases of the octopus lifecycle, particularly the paralarvae stage, present a challenge for commercial aquaculture. Improvement of diet formulation is seen as one way to reduce mortality and improve growth. Molecular growth-markers could help to improve rearing protocols and increase survival and growth performance; therefore, over a hundred orthologous genes related to protein balance and muscle growth in vertebrates were identified for the common octopus and their suitability as molecular markers for growth in octopus paralarvae explored. We successfully amplified 14 of those genes and studied their transcription in paralarvae either fed with artemia, artemia + zoea diets or submitted to a short fasting-refeeding procedure. Paralarvae fed with artemia + zoea had higher growth rates compared to those fed only with artemia, as well as a significant increase in octopus mtor (mtor-L) and hsp90 (hsp90-L) transcription, with both genes also up-regulated during refeeding. Our results suggest that at least mtor-L and hsp90-L are likely linked to somatic growth in octopus paralarvae. Conversely, ckip1-L, crk-L, src-L and srf-L had expression patterns that did not match to periods of growth as would be expected based on similar studies in vertebrates, indicating that further research is needed to understand their function during growth and in a muscle specific context.

Global impact of diet and temperature over aquaculture of Octopus vulgaris paralarvae from a transcriptomic approach.

Common octopus, Octopus vulgaris, is an economically important cephalopod species. However, its rearing under captivity is currently challenged by massive mortalities previous to their juvenile stage due to nutritional and environmental factors. Dissecting the genetic basis and regulatory mechanism behind this mortality requires genomic background knowledge. A transcriptomic sequencing of 10 dph octopus paralarvae from different experimental conditions was constructed via RNA-seq. A total of 613,767,530 raw reads were filtered and de novo assembled into 363,527 contigs of which 82,513 were annotated in UniProt carrying also their GO and KEGG information. Differential gene expression analysis was carried out on paralarvae reared under different diet regimes and temperatures, also including wild paralarvae. Genes related to lipid metabolism exhibited higher transcriptional levels in individuals whose diet includes crustacean zoeas, which had an impact over their development and immune response capability. High temperature induces acclimation processes at the time that increase metabolic demands and oxidative stress. Wild individuals show an expression profile unexpectedly similar to Artemia fed individuals. Proteomic results support the hypothesis revealed by transcriptional analysis. The comparative study of the O. vulgaris transcriptomic profiles allowed the identification of genes that deserve to be further studied as candidates for biomarkers of development and health. The results obtained here on the transcriptional variations of genes caused by diet and temperature will provide new perspectives in understanding the molecular mechanisms behind nutritional and temperature requirements of common octopus that will open new opportunities to deepen in paralarvae rearing requirements.

Heavy metals immobilization capability of two iron-based nanoparticles (nZVI and Fe3O4): Soil and freshwater bioassays to assess ecotoxicological impact.

The contamination by heavy metals constitutes an environmental problem of great importance in the last decades, and demands of society for clean environments are increasingly evident. To achieve this goal, several strategies have appeared for the in situ remediation of soil contamination caused by heavy metals. This study evaluated two types of iron-based nanoparticles, zero-valent iron nanoparticles (nZVI) and Fe3O4 nanoparticles, for the effective immobilization of Furthermore, we conducted a set of ecotoxicological bioassays: Microtox® Test, Caenorhabditis elegans Test, and Phytoplankton Toxicity Tests, on selected soil and aquatic test organisms to both, i) evaluate the potential ecotoxicological risks associated with nanoparticles treatment, and ii) to define sensitive organisms to be used as suitable bioindicators of heavy metals pollution. The application of 5% nZVI significantly reduced the amount of bioavailable heavy metals, which was effective from an ecotoxicity point of view as a reduction of the toxicity of was observed. Among the bioassays used, C. elegans seems the most effective reference organism in detecting changes in the toxicity of and therefore, C. elegans was found to be a sensitive heavy metals pollution bioindicator. When the Combination index (CI) was obtained to determine combined heavy metals interactions, the results indicated that toxicity would be higher than that expected for Pb, Cd and Zn individually considered, due to the proved antagonistic interactions of those toxicants. The obtained results suggested that nZVI nanoparticles are susceptible to be used as a soil remediation strategy for heavy metal pollution, although a short reactive lifespan must be considered, and therefore its effectiveness in long periods remains to be elucidated.

Potential risk of acute toxicity induced by AgI cloud seeding on soil and freshwater biota.

Silver iodide is one of the most common nucleating materials used in cloud seeding. Previous cloud seeding studies have concluded that AgI is not practically bioavailable in the environment but instead remains in soils and sediments such that the free Ag amounts are likely too low to induce a toxicological effect. However, none of these studies has considered the continued use of this practice on the same geographical areas and thus the potential cumulative effect of environmental AgI. The aim of this study is to assess the risk of acute toxicity caused by AgI exposure under laboratory conditions at the concentration expected in the environment after repeated treatments on selected soil and aquatic biota. To achieve the aims, the viability of soil bacteria Bacillus cereus and Pseudomonas stutzeri and the survival of the nematode Caenorhabditis elegans exposed to different silver iodide concentrations have been evaluated. Freshwater green algae Dictyosphaerium chlorelloides and cyanobacteria Microcystis aeruginosa were exposed to silver iodide in culture medium, and their cell viability and photosynthetic activity were evaluated. Additionally, BOD5 exertion and the Microtox® toxicity test were included in the battery of toxicological assays. Both tests exhibited a moderate AgI adverse effect at the highest concentration (12.5µM) tested. However, AgI concentrations below 2.5µM increased BOD5. Although no impact on the growth and survival endpoints in the soil worm C. elegans was recorded after AgI exposures, a moderate decrease in cell viability was found for both of the assessed soil bacterial strains at the studied concentrations. Comparison between the studied species showed that the cyanobacteria were more sensitive than green algae. Exposure to AgI at 0.43µM, the reference value used in monitoring environmental impact, induced a significant decrease in photosynthetic activity that is primarily associated with the respiration (80% inhibition) and, to a lesser extent, the net photosynthesis (40% inhibition) in both strains of phytoplankton and a moderate decrease in soil bacteria viability. These results suggest that AgI from cloud seeding may moderately affect biota living in both terrestrial and aquatic ecosystems if cloud seeding is repeatedly applied in a specific area and large amounts of seeding materials accumulate in the environment.

Residual impact of aged nZVI on heavy metal-polluted soils.

In the present study, the residual toxicity and impact of aged nZVI after a leaching experiment on heavy metal (Pb, Zn) polluted soils was evaluated. No negative effects on physico-chemical soil properties were observed after aged nZVI exposure. The application of nZVI to soil produced a significant increase in Fe availability. The impact on soil biodiversity was assessed by fluorescence in situ hybridization (FISH). A significant effect of nZVI application on microbial structure has been recorded in the Pb-polluted soil nZVI-treated. Soil bacteria molecular response, evaluated by RT-qPCR using exposure biomarkers (pykA, katB) showed a decrease in the cellular activity (pykA) due to enhanced intracellular oxidative stress (katB). Moreover, ecotoxicological standardised test on Caenorhabditis elegans (C. elegans) showed a decrease in the growth endpoint in the Pb-polluted soil, and particularly in the nZVI-treated. A different pattern has been observed in Zn-polluted soils: no changes in soil biodiversity, an increase in biological activity and a significant decrease of Zn toxicity on C. elegans growth were observed after aged nZVI exposure. The results reported indicated that the pollutant and its nZVI interaction should be considered to design soil nanoremediation strategies to immobilise heavy metals.

Impact of Ag and Al2O3 nanoparticles on soil organisms: in vitro and soil experiments.

In vitro analyses were conducted to assess the impact of Al2O3 and Ag nanoparticles on two common soil bacteria, Bacillus cereus and Pseudomonas stutzeri. Al2O3 nanoparticles did not show significant toxicity at any dose or time assayed, whereas exposure to 5 mg L(-1) Ag nanoparticles for 48 h caused bactericidal effects. Moreover, alterations at the morphological level were observed by transmission electron microscopy (TEM); Ag but not Al2O3 nanoparticles evoked the entrance of B. cereus cells in an early sporulation stage and both nanoparticles penetrated P. stutzeri cells. At the molecular level, a dramatic increase (8.2-fold) in katB gene expression was found in P. stutzeri following Al2O3 nanoparticles exposure, indicative of an oxidative stress-defence system enhancement in this bacterium. In the microcosm experiment, using two different natural soils, Al2O3 or Ag nanoparticles did not affect the Caenorhabditis elegans toxicity endpoints growth, survival, or reproduction. However, differences in microbial phylogenetic compositions were detected by fluorescence in situ hybridization (FISH). The use of katB- and pykA-based sequences showed that the microbial transcriptional response to nanoparticle exposure decreased, suggesting a decrease in cellular activity. These changes were attributable to both the nanoparticles treatment and soil characteristics, highlighting the importance of considering the soil matrix on a case by case basis.

Transcriptional and proteomic stress responses of a soil bacterium Bacillus cereus to nanosized zero-valent iron (nZVI) particles.

Nanosized zero valent iron (nZVI) is emerging as an option for treating contaminated soil and groundwater even though the potentially toxic impact exerted by nZVI on soil microorganisms remains uncertain. In this work, we focus on nanotoxicological studies performed in vitro using commercial nZVI and one common soil bacterium (Bacillus cereus). Results showed a negative impact of nZVI on B. cereus growth capability, consistent with the entrance of cells in an early sporulation stage, observed by TEM. Despite no changes at the transcriptional level are detected in genes of particular relevance in cellular activity (narG, nirS, pykA, gyrA and katB), the proteomic approach used highlights differentially expressed proteins in B. cereus under nZVI exposure. We demonstrate that proteins involved in oxidative stress-response and tricarboxilic acid cycle (TCA) modulation are overexpressed; moreover proteins involved in motility and wall biosynthesis are repressed. Our results enable to detect a molecular-level response as early warning signal, providing new insight into first line defense response of a soil bacterium after nZVI exposure.

Assessing the impact of zero-valent iron (ZVI) nanotechnology on soil microbial structure and functionality: a molecular approach.

In this work, nanoscale zero-valent iron (NZVI) particles have been used as an immobilisation strategy to reduce Pb and Zn availability and mobility in polluted soils. The application of NZVI to two soil microcosms (MPb and MZn) at a dose of 34 mg g(-1) soil efficiently immobilised Pb (25%) and zinc (20%). Exposure to NZVI had little impact on the microbial cellular viability and biological activity in the soils. Three bacterial genes (narG, nirS and gyrA) were used as treatment-related biomarkers. These biomarkers ruled out a broad bactericidal effect on the bulk soil microbial community. A transcriptome analysis of the genes did not reveal any changes in their expression ratios after the NZVI treatment: 1.6 (narG), 0.8 (nirS) and 0.7 (gyrA) in the MPb microcosm and 0.6 (narG), 1.2 (nirS) and 0.5 (gyrA) in the MZn microcosm. However, significant changes in the structure and composition of the soil bacteria population were detected by fluorescence in situ hybridisation. Thus, our results showed that NZVI toxicity could be highly dose and species dependent, and the effective applicability of the proposed molecular approach in assessing the impact of this immobilisation strategy on soil microbial population.

A new mathematical model to evaluate simazine removal in three different immobilized-biomass reactors.

A new mathematical model based on the cinetical Langmuir equation is developed to interpret and predict the effectiveness of simazine (SZ) removal in immobilized-biomass reactor (IBR), to consider herbicide-support affinity (Cx), and herbicide-cell affinity (Cy). Three solid supports: sepiolite monolith, granular sepiolite, and alginate were used in pilot-scale reactors that were inoculated with Klebsiella planticola DSZ. The abiotic process was analysed by measuring the SZ sorption capacity of the reactor supports. Sepiolite monolith showed the maximum value for herbicide-support affinity (28.02+/-0.9%). The effectiveness of the biotic process was estimated considering the formation of biomass and SZ biodegradation. Granular sepiolite showed either higher affinity with SZ and viability rate (0.90) throughout the process, and SZ removal rate was 3.39+/-0.06 mg/h. The mathematical model presented in this paper provides useful insights into the interpretation of experimental data as well as prediction for the implementation of biological reactors.