Artisanal and small-scale gold mining and biodiversity: a global literature review.

Artisanal and small-scale gold mining (ASGM) is crucial to the livelihoods of close to 20 million people in over 80 countries, including 4-5 million women, mainly in rural areas with limited alternative economic prospects, particularly in developing countries. ASGM is largely informal, which can add to the challenge of addressing negative social and environmental effects including impacts on biodiversity. However, with proper guidance, ASGM can operate in a responsible manner, using cleaner production methods that minimize impacts on human health and the environment. This study presents and analyzes the interactions between ASGM and biodiversity based on new findings from 27 ASGM National Action Plans (NAPs) developed within the framework of Article 7 and Annex C of the Minamata Convention on Mercury, as well as a global literature review of more than 100 publications. In terms of key findings according to the literature reviewed, alongside other human occupation such as agriculture and industrial activities, ASGM also has an impact on the environment and biodiversity. The interrelationship between ASGM and biodiversity, including protected areas, is pervasive at every stage of ASGM operations, from extraction to mine closure, and generates significant impacts on the surrounding ecosystems. These impacts include, in descending order of most reported impacts: deforestation, soil degradation, chemical contamination of aquatic and terrestrial systems, and changes to the turbidity of watercourses. Tropical regions and key species such as amphibians and freshwater fish are among the most affected. Singly or combined, these environmental stressors lead to loss or deterioration of habitat and, by extension, indigenous biodiversity and ecosystem services. In addition, legal, institutional, and regulatory frameworks and related measures, inadequate or non-existent in some cases, may not necessarily support sustainable practices, often resulting in exploited sites abandoned without remediation, reclamation, rehabilitation, or restoration measures. To mitigate such impacts a key recommendation arising from the literature review is to strengthen the integration of the interrelationship between ASGM and biodiversity in the implementation of existing relevant national strategies, including those developed under the NAPs. The global literature review also highlights the importance of a multi-stakeholder, systemic approach combining the use of geospatial analysis, scientific and local knowledge, as well as the adaptation of the relevant frameworks, capacity building, and awareness raising. This approach can inform decision making with a view to developing sustainable initiatives that prevent and reduce the impacts of artisanal and small-scale gold mining on ecosystems, and that preserve biodiversity.

Asymmetrical Flow Field-Flow Fractionation Coupled to ICP-MS for Characterization of Trace Metal Species in the Environment from Macromolecular to Nano-Assemblage Forms: Current Challenges for Quantification.

Asymmetrical flow field-flow fractionation (AF4) is a powerful technique employed for the separation of macromolecules, nanoparticles, and their assemblages according to their hydrodynamic behavior. It is well known that at this size range, complex interactions can occur between components (e.g. surface adsorption, aggregation) controlling the fate of trace metals (TMs) bound to them. AF4 coupling to inductively coupled plasma mass spectrometry (ICP-MS) allows the quantification of metal-containing species at trace levels present in environmental and biological systems on a size-composition basis. The combination of AF4-ICP-MS with other online detectors provides additional information that allows the assessment of the origin of analytes present in mixtures and complex matrixes with minimal sample preparation, which is crucial for understanding the behavior of trace metal contaminants. Despite the increasing use of AF4-ICP-MS in environmental contexts, we acknowledge that the quantification of inorganic species using such combined techniques requires further development of standardized procedures and need certified reference materials. In this review, we also discuss critical endpoints within the ICP-MS instrument coupled to AF4 that need to be controlled before quantitative measurements can be validated. Then, we illustrate how the combination of different online detectors in addition to ICP-MS offers an integrated picture of natural components states, thus providing key information on the changes in behavior of trace metal species and metallic nanoparticles (MNPs) as observed in both environmental samples and biofluids.

Metabolomic Responses of Green Alga Chlamydomonas reinhardtii Exposed to Sublethal Concentrations of Inorganic and Methylmercury.

Metabolomics characterizes low-molecular-weight molecules involved in different biochemical reactions and provides an integrated assessment of the physiological state of an organism. By using liquid chromatography-mass spectrometry targeted metabolomics, we examined the response of green alga Chlamydomonas reinhardtii to sublethal concentrations of inorganic mercury (IHg) and monomethylmercury (MeHg). We quantified the changes in the levels of 93 metabolites preselected based on the disturbed metabolic pathways obtained in a previous transcriptomics study. Metabolites are downstream products of the gene transcription; hence, metabolite quantification provided information about the biochemical status of the algal cells exposed to Hg compounds. The results showed that the alga adjusts its metabolism during 2 h exposure to 5 × 10-9 and 5 × 10-8 mol L-1 IHg and MeHg by increasing the level of various metabolites involved in amino acid and nucleotide metabolism, photorespiration, and tricarboxylic acid (TCA) cycle, as well as the metabolism of fatty acids, carbohydrates, and antioxidants. Most of the metabolic perturbations in the alga were common for IHg and MeHg treatments. However, the exposure to IHg resulted in more pronounced perturbations in the fatty acid and TCA metabolism as compared with the exposure to MeHg. The observed metabolic perturbations were generally consistent with our previously published transcriptomics results for C. reinhardtii exposed to the comparable level of IHg and MeHg. The results highlight the potential of metabolomics for toxicity evaluation, especially to detect effects at an early stage of exposure prior to their physiological appearance.

Determination of the Intracellular Complexation of Inorganic and Methylmercury in Cyanobacterium Synechocystis sp. PCC 6803.

Understanding of mercury (Hg) complexation with low molecular weight (LMW) bioligands will help elucidate its speciation. In natural waters, the rate of this complexation is governed by physicochemical, geochemical, and biochemical parameters. However, the role of bioligands involved in Hg intracellular handling by aquatic microorganisms is not well documented. Here, we combine the use of isotopically labeled Hg species (inorganic and monomethylmercury, iHg and MeHg) with gas or liquid chromatography coupling to elemental and molecular mass spectrometry to explore the role of intracellular biogenic ligands involved in iHg and MeHg speciation in cyanobacterium Synechocystis sp. PCC 6803, a representative phytoplankton species. This approach allowed to track resulting metabolic and newly found intracellular Hg biocomplexes (e.g., organic thiols) in Synechocystis sp. PCC 6803 finding different intracellular Hg species binding affinities with both high and low molecular weight (HMW and LMW) bioligands in the exponential and stationary phase. Furthermore, the parallel detection with both elemental and molecular ionization sources allowed the sensitive detection and molecular identification of glutathione (GSH) as the main low molecular weight binding ligand to iHg ((GS)2-Hg) and MeHg (GS-MeHg) in the cytosolic fraction. Such a novel experimental approach expands our knowledge on the role of biogenic ligands involved in iHg and MeHg intracellular handling in cyanobacteria.

When Environmental Chemistry Meets Ecotoxicology: Bioavailability of Inorganic Nanoparticles to Phytoplankton.

The present review critically examines the state-of-the-art of the research concerning the likely environmental implications of engineered nanoparticles (ENPs) with specific emphasis on their interactions with phytoplankton in the aquatic environment. Phytoplankton plays a key role in the global carbon cycle and contributes to the half of the global primary production, thus representing some of the Earth ' s most critical organisms making the life on our planet possible. With examples from our own research and the literature, we illustrate what happens when aquatic organisms are unintentionally exposed to metal-containing ENPs, which are increasingly released into the environment from nano-enabled materials. We highlight the complexity of the ENPs behavior in the aquatic environment and focus on the three key steps of the bioavailability process: exposure availability, uptake availability and toxico-availability. The influence of the phytoplankton on the ENPs fate in the aquatic environment is discussed, too.

Insect Life Traits Are Key Factors in Mercury Accumulation and Transfer within the Terrestrial Food Web.

As plants and associated insects are at the bottom of some terrestrial food webs, they are the primary contributors to mercury (Hg) fluxes in ecosystems. In addition to the trophic position of these organisms, factors related to their life traits have been hypothesized to influence their exposure to Hg. This study investigates the transfer of Hg in a soil-nettle-insect system and the insect-related factors affecting their Hg concentrations in a revegetated chlor-alkali landfill. Twenty-three insect species were identified and classified according to their life traits, their relationship with nettle, and their morphological characteristics. We observed low total mercury (THg) concentrations in nettles, with only 1% methylmercury (MeHg) being detected, while concentrations ranged from 5 to 3700 µg/kg dry wt. in insects with a MeHg percentage of up to 75%. The nettle-related insects were primarily exposed to Hg through the food web with significant biomagnification, particularly at the level of secondary predators. Within the nettle-unrelated group, the insect habitat was the most explanatory factor, with the highest enrichment being for the insects that spent part of their cycle in direct contact with Hg sources. Therefore, these insects require special attention because they are an essential vector of Hg transfer for terrestrial top predators.

Dissolved Organic Matter and Associated Trace Metal Dynamics from River to Lake, Under Ice-Covered and Ice-Free Conditions.

The present study investigates the changes in dissolved organic matter (DOM) composition and its influences on trace metal dispersion from the Shuya River (SR) in the Petrozavodsk Bay of Lake Onega during ice-covered and ice-free periods. Humic substances (HS) found in the SR dominated the composition of DOM through the river-bay-lake continuum in both periods. When the bay was ice-covered, both the aromaticity and the size of HS varied in the water column according to a horizontal stratification and decreased in the bay, while under ice-free conditions, they decreased along the river-lake gradient, suggesting in both cases a decrease in the proportion of HS with high aromatic character. These findings were associated with an overall decrease in the proportion of HS components that have the highest molecular masses. The quantification of metal bound to HS revealed that these characteristics were associated with a decrease in the binding capacity of the HS for Fe and Al but not Cu while dispersing in the bay to the lake. Pb was found to bind on HS, but its behavior in the bay could not be related to the HS dispersion nor to the changes in HS properties.

Colloidal Size and Redox State of Uranium Species in the Porewater of a Pristine Mountain Wetland.

Uranium (U) speciation was investigated in anoxically preserved porewater samples of a natural mountain wetland in Gola di Lago, Ticino, Switzerland. U porewater concentrations ranged from less than 1 µg/L to tens of µg/L, challenging the available analytical approaches for U speciation in natural samples. Asymmetrical flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry allowed the characterization of colloid populations and the determination of the size distribution of U species in the porewater. Most of the U was associated with three fractions: <0.3 kDa, likely including dissolved U and very small U colloids; a 1-3 kDa fraction containing humic-like organic compounds, dispersed Fe, and, to a small extent, Fe nanoparticles; and a third fraction (5-50 nm), containing a higher amount of Fe and a lower amount of organic matter and U relative to the 1-3 kDa fraction. The proportion of U associated with the 1-3 kDa colloids varied spatially and seasonally. Using anion exchange resins, we also found that a significant proportion of U occurs in its reduced form, U(IV). Tetravalent U was interpreted as occurring within the colloidal pool of U. This study suggests that U(IV) can occur as small (1-3 kDa), organic-rich, and thus potentially mobile colloidal species in naturally reducing wetland environments.

Species-species interactions modulate copper toxicity under different visible light conditions.

Combination of biotic and abiotic factors influences the effects of naturally occurring or anthropogenic chemicals on photosynthetic microorganisms in the aquatic environment. Nonetheless, the combined effects of physical stressors and species-species interaction on chemicals' toxicity are still poorly understood. The present study examines the responses of the green alga Chlamydomonas reinhardtii and the cyanobacterium Synechocystis sp. alone and in mixtures to copper exposure under increasing visible light intensities. Cell growth, chlorophyll bleaching, oxidative stress and membrane permeability were determined by flow cytometry in both mono- and multi-species tests. The results revealed that species-species interactions influenced copper toxicity under different light regimes at 4 h and 48 h - exposure. For a given light condition, monocultures of Synechocystis sp. were more sensitive to copper than those of C. reinhardtii. In long-term incubation C. reinhardtii sensitivity to copper diminished in presence of Synechocystis sp. under low-intensity light, however it was enhanced under high-intensity light. The present results revealed the complex interplay between visible light intensity variations, species-species interaction and copper effects to phytoplankton in long- term exposure.

Molecular Effects, Speciation, and Competition of Inorganic and Methyl Mercury in the Aquatic Plant Elodea nuttallii.

Mercury (Hg) remains hazardous in aquatic environments because of its biomagnification in food webs. Nonetheless, Hg uptake and impact in primary producers is still poorly understood. Here, we compared the cellular toxicity of inorganic and methyl Hg (IHg and MeHg, respectively) in the aquatic plant Elodea nuttallii. IHg and MeHg regulated contigs involved in similar categories (e.g., energy metabolism, development, transport, secondary metabolism), but MeHg regulated more contigs, supporting a higher molecular impact than IHg. At the organism level, MeHg induced antioxidants, while IHg decreased chlorophyll content. The uptake of Hg and expression of a subset of contigs was subsequently studied in complex media. Measured uptake pointed to a contrasted impact of cell walls and copper (Cu) on IHg and MeHg. Using a speciation modeling, differences in uptake were attributed to the differences in affinities of IHg and MeHg to organic matter in relation to Cu speciation. We also identified a distinct gene expression signature for IHg, MeHg, and Cu, further supporting different molecular toxicity of these trace elements. Our data provided fundamental knowledge on IHg and MeHg uptake in a key aquatic primary producer and confirmed the potential of transcriptomics to assess Hg exposure in environmentally realistic systems.

Biological effects of four iron-containing nanoremediation materials on the green alga Chlamydomonas sp.

As nanoremediation strategies for in-situ groundwater treatment extend beyond nanoiron-based applications to adsorption and oxidation, ecotoxicological evaluations of newly developed materials are required. The biological effects of four new materials with different iron (Fe) speciations ([i] FerMEG12 - pristine flake-like milled Fe(0) nanoparticles (nZVI), [ii] Carbo-Iron® - Fe(0)-nanoclusters containing activated carbon (AC) composite, [iii] Trap-Ox® Fe-BEA35 (Fe-zeolite) - Fe-doped zeolite, and [iv] Nano-Goethite - 'pure' FeOOH) were studied using the unicellular green alga Chlamydomonas sp. as a model test system. Algal growth rate, chlorophyll fluorescence, efficiency of photosystem II, membrane integrity and reactive oxygen species (ROS) generation were assessed following exposure to 10, 50 and 500 mg L-1 of the particles for 2 h and 24 h. The particles had a concentration-, material- and time-dependent effect on Chlamydomonas sp., with increased algal growth rate after 24 h. Conversely, significant intracellular ROS levels were detected after 2 h, with much lower levels after 24 h. All Fe-nanomaterials displayed similar Z-average sizes and zeta-potentials at 2 h and 24 h. Effects on Chlamydomonas sp. decreased in the order FerMEG12 > Carbo-Iron® > Fe-zeolite > Nano-Goethite. Ecotoxicological studies were challenged due to some particle properties, i.e. dark colour, effect of constituents and a tendency to agglomerate, especially at high concentrations. All particles exhibited potential to induce significant toxicity at high concentrations (500 mg L-1), though such concentrations would rapidly decrease to mg or µg L-1 in aquatic environments, levels harmless to Chlamydomonas sp. The presented findings contribute to the practical usage of particle-based nanoremediation in environmental restoration.

Toward Quantitative Understanding of the Bioavailability of Dissolved Organic Matter in Freshwater Lake during Cyanobacteria Blooming.

Occurrence of cyanobacterial harmful algal blooms (CyanoHAB) can induce considerable patchiness in the concentration and bioavailability of dissolved organic matter (DOM), which could influence biogeochemical processes and fuel microbial metabolism. In the present study, a laboratory 4-stage plug-flow bioreactor was used to successfully separate the CyanoHAB-derived DOM isolated from the eutrophic Lake Taihu (China) into continuum classes of bioavailable compounds. A combination of new state-of-the-art tools borrowed from analytical chemistry and microbial ecology were used to characterize quantitatively the temporary evolution of DOM and to get deeper insights into its bioavailability. The results showed a total 79% dissolved organic carbon loss over time accompanied by depletion of protein-like fluorescent components, especially the relatively hydrophilic ones. However, hydrophilic humic-like fluorescent components exhibited bioresistant behavior. Consistently, ultrahigh resolution mass spectrometry (FTICR-MS) revealed that smaller, less aromatic, more oxygenated, and nitrogen-rich molecules were preferentially consumed by microorganisms with the production of lipid-like species, whereas recalcitrant molecules were primarily composed of carboxylic-rich alicyclic compounds. Moreover, the bioavailability of DOM was negatively correlated with microbial community diversity in the bioreactor. Results from this study provide deeper insights into the fate of DOM and relevant biogeochemical processes in eutrophic lakes.

Non-invasive continuous monitoring of pro-oxidant effects of engineered nanoparticles on aquatic microorganisms.

Engineered nanomaterials (ENMs) are key drivers for the development of highly sophisticated new technologies. As all new attainments, the rapidly increasing used of ENMs raise concerns about their safety for the environment and humans. There is growing evidence showing that if engineered nanomaterials are released into the environment, there is a possibility that they could cause harm to aquatic microorganisms. Among the divers effects triggering their toxicity the ability of ENMs to generate reactive oxygen species (ROS) capable of oxidizing biomolecules is currently considered a central mechanism of toxicity. Therefore, development of sensitive tools for quantification of the ROS generation and oxidative stress are highly sought. After briefly introducing ENMs-induced ROS generation and oxidative stress in the aquatic microorganisms (AMOs), this overview paper focuses on a new optical biosensor allowing sensitive and dynamic measurements of H2O2 in real-time using multiscattering enhanced absorption spectroscopy. Its principle is based on sensitive absorption measurements of the heme protein cytochrome c whose absorption spectrum alters with the oxidation state of constituent ferrous FeII and ferric FeIII. For biological applications cytochrome c was embedded in porous random media resulting in an extended optical path length through multiple scattering of light, which lowers the limit of detection to a few nM of H2O2. The sensor was also integrated in a microfluidic system containing micro-valves and sieves enabling more complex experimental conditions. To demonstrate its performance, abiotic absorption measurements of low concentrations of dye molecules and 10 nm gold particles were carried out achieving limits of detection in the low nM range. Other biologically relevant reactive oxygen species can be measured at sub-µM concentrations, which was shown for glucose and lactate through enzymatic reactions producing H2O2. In ecotoxicological investigations H2O2 excreted by aquatic microorganisms exposed to various stressors were measured. Pro-oxidant effects of nano-TiO2 and nano-CuO towards green alga Chlamydomonas reinhardtii were explored in various exposure media and under different light illuminations. Dynamics of Cd2+ induced effects on photosynthetic activity, sensitisation and recovery of cells of C. reinhardtii was also studied.

Transcriptomic and Physiological Responses of the Green Microalga Chlamydomonas reinhardtii during Short-Term Exposure to Subnanomolar Methylmercury Concentrations.

The effects of short-term exposure to subnanomolar methyl-mercury (MeHg) concentrations, representative of contaminated environments, on the microalga Chlamydomonas reinhardtii were assessed using both physiological end points and gene expression analysis. MeHg bioaccumulated and induced significant increase of the photosynthesis efficiency, while the algal growth, oxidative stress, and chlorophyll fluorescence were unaffected. At the molecular level, MeHg significantly dysregulated the expression of genes involved in motility, energy metabolism, lipid metabolism, metal transport, and antioxidant enzymes. Data suggest that the cells were able to cope with subnanomolar MeHg exposure, but this tolerance resulted in a significant cost to the cell energy and reserve metabolism as well as ample changes in the nutrition and motility of C. reinhardtii. The present results allowed gaining new insights on the effects and uptake mechanisms of MeHg at subnanomolar concentrations in aquatic primary producers.

A Multimethod Approach for Investigating Algal Toxicity of Platinum Nanoparticles.

The ecotoxicity of platinum nanoparticles (PtNPs) widely used in for example automotive catalytic converters, is largely unknown. This study employs various characterization techniques and toxicity end points to investigate PtNP toxicity toward the green microalgae Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii. Growth rate inhibition occurred in standard ISO tests (EC50 values of 15-200 mg Pt/L), but also in a double-vial setup, separating cells from PtNPs, thus demonstrating shading as an important artifact for PtNP toxicity. Negligible membrane damage, but substantial oxidative stress was detected at 0.1-80 mg Pt/L in both algal species using flow cytometry. PtNPs caused growth rate inhibition and oxidative stress in P. subcapitata, beyond what was accounted for by dissolved Pt, indicating NP-specific toxicity of PtNPs. Overall, P. subcapitata was found to be more sensitive toward PtNPs and higher body burdens were measured in this species, possibly due to a favored binding of Pt to the polysaccharide-rich cell wall of this algal species. This study highlights the importance of using multimethod approaches in nanoecotoxicological studies to elucidate toxicity mechanisms, influence of NP-interactions with media/organisms, and ultimately to identify artifacts and appropriate end points for NP-ecotoxicity testing.

Potential of hyperspectral imaging microscopy for semi-quantitative analysis of nanoparticle uptake by protozoa.

Hyperspectral imaging with enhanced darkfield microscopy (HSI-M) possesses unique advantages in its simplicity and non-invasiveness. In consideration of the urgent need for profound knowledge on the behavior and effects of engineered nanoparticles (NPs), here, we determined the capability of HSI-M for examining cellular uptake of different metal-based NPs, including nanosized metals (silver and gold, both citrate stabilized), metal oxides (copper oxide and titanium dioxide), and CdSe/ZnS core/shell quantum dots at subtoxic concentrations. Specifically, we demonstrated that HSI-M can be used to detect and semi-quantify these NPs in the ciliated protozoan Tetrahymena thermophila as a model aquatic organism. Detection and semi-quantification were achieved on the basis of spectral libraries for the NPs suspended in extracellular substances secreted by this single-celled organism, accounting for matrix effects. HSI-M was able to differentiate between NP types, provided that spectral profiles were significantly different from each other. This difference, in turn, depended upon NP type, size, agglomeration status, and position relative to the focal plane. As an exception among the NPs analyzed in this study, titanium dioxide NPs showed spectral similarities compared to cell material of unexposed control cells, leading to false positives. High biological variability resulted in highly variable uptake of NPs in cells of the same sample as well as between different exposures. We therefore encourage the development of techniques able to reduce the currently long analysis times that still hamper the acquisition of statistically strong data sets. Overall, this study demonstrates the potential and challenges of HSI-M in monitoring cellular uptake of synthetic NPs.

Towards Mechanistic Understanding of Mercury Availability and Toxicity to Aquatic Primary Producers.

The present article reviews current knowledge and recent progress on the bioavailability and toxicity of mercury to aquatic primary producers. Mercury is a ubiquitous toxic trace element of global concern. At the base of the food web, primary producers are central for mercury incorporation into the food web. Here, the emphasis is on key, but still poorly understood, processes governing the interactions between mercury species and phytoplankton, and macrophytes, two representatives of primary producers. Mass transfer to biota surface, adsorption to cell wall, internalization and release from cells, as well as underlying toxicity mechanisms of both inorganic mercury and methylmercury are discussed critically. In addition, the intracellular distribution and transformation processes, their importance for mercury toxicity, species-sensitivity differences and trophic transfer are presented. The mini-review is illustrated with examples of our own research.

Exploring the subcellular distribution of mercury in green alga Chlamydomonas reinhardtii and diatom Cyclotella meneghiniana: A comparative study.

Mercury (Hg) is a priority pollutant of global concern because of its toxicity, its ability to bioaccumulate throughout the food web and reach significant concentrations in top predators. Phytoplankton bioconcentrate large amounts of Hg and play a key role in the entry of Hg into the aquatic food web. However, the subcellular distribution of Hg in freshwater phytoplankton, known to affect it toxicity and trophic transfer is understudied. The present study aimed at investigating the accumulation of inorganic Hg (iHg) and its subcellular distribution in freshwater phytoplankton species. To this end green alga Chlamydomonas reinhardtii and diatom Cyclotella meneghiniana were exposed to 10 and 100 nM of iHg for 2 h. The concentrations of Hg in the adsorbed, intracellular and subcellular (granules, debris, organelles, heat-stable peptides (HSP) and heat-denaturable proteins (HDP)) fractions were determined. The results showed that C. meneghiniana accumulated more Hg compared to C. reinhardtii at both iHg exposure concentrations (10 nM: 4.41 ± 0.74 vs. 1.10 ± 0.25 amol cell-1; 100 nM: 79.35 ± 10.78 vs. 38.31 ± 4.15 amol cell-1). The evaluation of the subcellular distribution of Hg, revealed that the majority of Hg was concentrated in the organelles fraction (59.7 % and 74.6 %) in the green algae. In the diatom, Hg was mainly found in the organelles (40.9 % and 33.3%) and in the HSP fractions (26.8 % and 40.1 %). The proportion of Hg in HDP fraction decreased in favor of the organelles fraction in C. reinhardtii when the exposure concentration increased, whereas the proportions in the debris and organelles fractions decreased in favor of HSP fraction in C. meneghiniana. This study provides pioneering information on the subcellular distribution of Hg within in freshwater phytoplankton, a knowledge that is essential to understand the toxicity and trophic transfer of Hg in contaminated aquatic environment.

Exploring the impact of silver-based nanomaterial feed additives on green algae through single-cell techniques.

Silver in its various forms, including dissolved silver ions (Ag+) and silver nanoparticles (AgNPs), is a promising alternative to traditional antibiotics, largely used in livestock as feed additives and could contribute to the decrease and avoidance of the development of antibiotic resistance. The present study aims to assess the potential ecotoxicity of a silver-based nanomaterial (Ag-kaolin), the feed supplemented with the nanomaterial and the faeces since the latter are the ones that finally reach the environment. To this end, green alga Raphidocellis subcapitata was exposed to the extracts of Ag-kaolin, supplemented feed, and pig faeces for 72 h, along with Ag+ and AgNPs as controls for comparison purposes. Given the complexity of the studied materials, single-cell techniques were used to follow the changes in the cell numbers and chlorophyll fluorescence by flow cytometry, and the accumulation of silver in the exposed cells by single cell inductively coupled plasma mass spectrometry (SC-ICP-MS). Changes in cell morphology were observed by cell imaging multimode reader. The results revealed a decrease in chlorophyll fluorescence, even at low concentrations of Ag-kaolin (10 µg L-1) after 48 h of exposure. Additionally, complete growth inhibition was found with this material like the results obtained by exposure to Ag+. For the supplemented feed, a concentration of 50 µg L-1 was necessary to achieve complete growth inhibition. However, the behaviour differed for the leachate of faeces, which released Ag2S and AgCl alongside Ag+ and AgNPs. At 50 µg L-1, inhibition was minimal, primarily due to the predominance of less toxic Ag2S in the leachate. The uptake of silver by the cells was confirmed with all the samples through SC-ICP-MS analysis. These findings demonstrate that the use of Ag-kaolin as a feed supplement will lead to a low environmental impact.