Free Metal Concentrations Research Articles

PyChelator: a Python-based Colab and web application for metal chelator calculations

BackgroundMetal ions play vital roles in regulating various biological systems, making it essential to control the concentration of free metal ions in solutions during experimental procedures. Several software applications exist for estimating the concentration of free metals in the presence of chelators, with MaxChelator being the easily accessible choice in this domain. This work aimed at developing a Python version of the software with arbitrary precision calculations, extensive new features, and a user-friendly interface to calculate the free metal ions.ResultsWe introduce the open-source PyChelator web application and the Python-based Google Colaboratory notebook, PyChelator Colab. Key features aim to improve the user experience of metal chelator calculations including input in smaller units, selection among stability constants, input of user-defined constants, and convenient download of all results in Excel format. These features were implemented in Python language by employing Google Colab, facilitating the incorporation of the calculator into other Python-based pipelines and inviting the contributions from the community of Python-using scientists for further enhancements. Arbitrary-precision arithmetic was employed by using the built-in Decimal module to obtain the most accurate results and to avoid rounding errors. No notable differences were observed compared to the results obtained from the PyChelator web application. However, comparison of different sources of stability constants showed substantial differences among them.ConclusionsPyChelator is a user-friendly metal and chelator calculator that provides a platform for further development. It is provided as an interactive web application, freely available for use at https://amrutelab.github.io/PyChelator, and as a Python-based Google Colaboratory notebook at https://colab.research.google.com/github/AmruteLab/PyChelator/blob/main/PyChelator_Colab.ipynb.

Glutathionylation of a glycolytic enzyme promotes cell death and vigor loss during aging of elm seeds.

Seed deterioration during storage is a major problem in agricultural and forestry production and for germplasm conservation. Our previous studies have shown that a mitochondrial outer membrane protein VOLTAGE-DEPENDENT ANION CHANNEL (VDAC) is involved in programmed cell death-like viability loss during the controlled deterioration treatment (CDT) of elm (Ulmus pumila L.) seeds, but its underlying mechanism remains unclear. In this study, we demonstrate that the oxidative modification of GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) is functioned in the gate regulation of VDAC during the CDT of elm seeds. Through biochemical and cytological methods and observations of transgenic material [Arabidopsis (Arabidopsis thaliana), Nicotiana benthamiana, and yeast (Saccharomyces cerevisiae)], we demonstrate that cysteine S-glutathionylated UpGAPDH1 interacts with UpVDAC3 during seed aging, which leads to a mitochondrial permeability transition and aggravation of cell death, as indicated by the leakage of the mitochondrial proapoptotic factor cytochrome c and the emergence of apoptotic nucleus. Physiological assays and inductively coupled plasma mass spectrometry analysis revealed that GAPDH glutathionylation is mediated by increased glutathione, which might be caused by increases in the concentrations of free metals, especially Zn. Introduction of the Zn-specific chelator TPEN [(N,N,N',N'-Tetrakis (2-pyridylmethyl)ethylenediamine)] significantly delayed seed aging. We conclude that glutathionylated UpGAPDH1 interacts with UpVDAC3 and serves as a proapoptotic protein for VDAC-gating regulation and cell death initiation during seed aging.

Abstract 5571: FoxP3 absolute quantification to build an effective risk model for cancer patient survival

Abstract Background: Forkhead box protein P3 (FOXP3), belonging to the forkhead/winged-helix family, is of great importance in modulating the differentiation and development of T-regulatory cells (Tregs). The numbers of FOXP3+ Tregs as well as exhaustive subsets of CD4+ and CD8+ T cells have been observed increased in solid tumor tissues of patients with colorectal and breast cancers, which helps to create an immunosuppressive environment and promotes tumor progression. However, the correlation between the presence of FOXP3+ Tregs and patients’ survival has been extensively studied in many cancer types, which remains conflicting. The approach presented here aims to absolutely quantify the level of FOXP3 in single cells and constructed effective immune risk models to predict tumor prognosis. Method: Imaging mass cytometry (IMC) was used to quantify FOXP3 inside each single cell. The indirect quantification used a free-metal titration curve in order to match the level of intensity of the metal-tagged antibodies to the level of expression of the antigen. Deep learning (StarDist QuPath extension) was used for cell segmentation and expression quantification. FOXP3 quantification in tissue or at the single cell level was validated against ELISA assay that measure FOXP3 in tissue lysate. Results: Linearity of the metal signals with an average %CV of 17% was observed emphasizing the linear correlation between free metal concentration and intensity. With the IMC technique, the minimum amount of FoxP3 detectable is 0.09 femtogram and its absolute quantification in the different cells in tumor tissues was confronted to disease prognosis in order to start building an effective immune risk model. Conclusion: With the introduction of new biologics in cancer therapy, it is imperative to follow real quantitative change in the expression of biological marker that are indicator of tumor prognosis. Indeed, although the FOXP3 distribution was already seen to be heterogenous in tumors, its absolute level of expression indicative of disease progression can be directly correlated to gain better understanding of the patient response stratification to biologics. Citation Format: Adele Ponzoni, Sandra Delebecq, Melodie Boute, Corinne Ramos, Jonathan Stauber. FoxP3 absolute quantification to build an effective risk model for cancer patient survival. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5571.

ISIDORE, a Probe for In Situ Trace Metal Speciation Based on the Donnan Membrane Technique and Electrochemical Detection Part 2: Cd and Pb Measurements during the Accumulation Time of the Donnan Membrane Technique

The Donnan membrane technique (DMT), in which a synthetic or natural solution (the "donor") is separated from a ligand-free solution (the "acceptor") by a cation-exchange membrane, is a recognized technique for measuring the concentration of a free metal ion in situ, with coupling to electrochemical detection allowing for the quantification of the free metal ion directly on site. However, the use of the DMT requires waiting for the free metal ion equilibrium between the donor and the acceptor solution. In this paper, we investigated the possibility of using the kinetic information and showed that non-equilibrium experimental calibrations of Cd and Pb with the ISIDORE probe could be used to measure free metal concentrations under conditions of membrane-controlled diffusion transport. The application of this dynamic approach made it possible to successfully determine the concentration of free Cd in synthetic and natural river samples. Furthermore, it was found that the determination of free Cd from the slope was not affected by the Ca concentration ratio between the acceptor and donor solution, as opposed to the traditional approach based on Donnan equilibrium. This ISIDORE probe appears to be a promising tool for determining free metal ions in natural samples.

Direct determination of free Zn concentration in samples of biological interest

The speciation of essential metal ions in biological fluids, such as blood plasma and serum, is of fundamental importance to understand the homeostasis of these elements. The activity of metal ions such as Zn2+ in extracellular media is thought to affect their interaction with membrane-bound transporters, and thus is critical for their cellular uptake. Previous approaches to determine “free” Zn2+ (i.e. the hexa-aquo ion) are based on separation by either chromatography or ultrafiltration, or on metallochromic dyes. However, both types of approach are prone to affect the relevant equilibria. These drawbacks can be circumvented with the electroanalytical technique AGNES (Absence of Gradients and Nernstian Equilibrium Stripping), since it can measure free zinc concentration without perturbing the sample speciation. Here, a Bovine Serum Albumin (BSA) + Zn synthetic mixture and Fetal Bovine Serum (FBS) are analyzed as proof of concept. Adsorption of BSA on the surface of the Hanging Mercury Drop Electrode (HMDE), despite the advantage of its renewal, is so intense that it blocks appropriate attainment of the required equilibrium, and only estimations of [Zn2+] can be derived. In contrast, a rotating disc electrode with a thin mercury film deposited on it (TMF-RDE) is advantageous because of its small volume and enhanced mass transfer. Protein adsorption can be prevented by covering the TMF-RDE with Nafion. A free Zn concentration [Zn2+] = 2.7 nmol L−1 was found at pH 7.0, total Zn 20 μμmol L−1 and BSA 600 μμmol L−1. A sample of FBS with fixed pH 7.2 (MOPS 0.08 mol L−1) yielded [Zn2+] = 0.25 nmol L−1. This methodology opens the way to free metal concentration determinations in biological fluids.

Combining a Standardized Batch Test with the Biotic Ligand Model to Predict Copper and Zinc Ecotoxicity in Soils.

Extraction of soil samples with dilute CaCl2 solution in a routinely performed batch test has potential to be used in site‐specific assessment of ecotoxicological risks at metal‐contaminated sites. Soil extracts could potentially give a measure of the concentration of bioavailable metals in the soil solution, thereby including effects of soil properties and contaminant “aging.” We explored the possibility of using a 0.001 M CaCl2 batch test combined with biotic ligand models (BLMs) for assessment of ecotoxicity in soils. Concentrations of Cu2+ and Zn2+ in soil extracts were linked to responses in ecotoxicity tests (microbial processes, plants, and invertebrates) previously performed on metal‐spiked soils. The batch test data for soils were obtained by spiking archived soil materials using the same protocol as in the original studies. Effective concentration values based on free metal concentrations in soil extracts were related to pH by linear regressions. Finally, field‐contaminated soils were used to validate model performance. Our results indicate a strong pH‐dependent toxicity of the free metal ions in the soil extracts, with R 2 values ranging from 0.54 to 0.93 (median 0.84), among tests and metals. Using pH‐adjusted Cu2+ and Zn2+ concentrations in soil extracts, the toxic responses in spiked soils and field‐contaminated soils were similar, indicating a potential for the calibrated models to assess toxic effects in field‐contaminated soils, accounting for differences in soil properties and effects of contaminant “aging.” Consequently, evaluation of a standardized 0.001 M CaCl2 batch test with a simplified BLM can provide the basis for an easy‐to‐use tool for site‐specific risk assessment of metal toxicity to soil organisms. Environ Toxicol Chem 2022;41:1540–1554. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Determination of affinities of lanthanide-binding proteins using chelator-buffered titrations.

The recent discoveries of the first proteins that bind lanthanides as part of their biological function not only are relevant to the emerging field of lanthanide-dependent biology, but also hold promise to revolutionize the technologically critical rare earths industry. Although protocols to assess the thermodynamics of metal-protein interactions are well established for "traditional" metal ions in biology, the characterization of lanthanide-binding proteins presents a challenge to biochemists due to the lanthanides' Lewis acidity, propensity for hydrolysis, and high-affinity complexes with biological ligands. These properties necessitate the preparation of metal stock solutions with very low buffered "free" metal concentrations (e.g., femtomolar to nanomolar) for such determinations. Herein we describe several protocols to overcome these challenges. First, we present standardization methods for the preparation of chelator-buffered solutions of lanthanide ions with easily calculated free metal concentrations. We also describe how these solutions can be used in concert with analytical methods including UV-visible spectrophotometry, circular dichroism spectroscopy, Förster resonance energy transfer (FRET), and sensitized terbium luminescence, in order to accurately determine dissociation constants (Kds) of lanthanide-protein complexes. Finally, we highlight how application of these methods to lanthanide-binding proteins, such as lanmodulin, has yielded insights into selective recognition of lanthanides in biology. We anticipate that these protocols will facilitate discovery and characterization of additional native lanthanide-binding proteins, will motivate the understanding of their biological context, and will prompt their applications in biotechnology.

Investigating the Binding Heterogeneity of Trace Metal Cations With SiO2 Nanoparticles Using Full Wave Analysis of Stripping Chronopotentiometry at Scanned Deposition Potential

Silica oxides nano- and microparticles, as well as silica-based materials, are very abundant in nature and industrial processes. Trace metal cation binding with these bulk materials is generally not considered significant in speciation studies in environmental systems. Nonetheless, this might change for nanoparticulate systems as observed in a previous study of Pb(II) with a very small SiO2 particle (7.5 nm diameter). Besides, metal binding by those nanoparticles is surprisingly characterized by a heterogeneity that increases with the decrease of metal-to-particle ratio. Therefore, it is interesting to extend this study to investigate different trace metals and the influence of the nanoparticle size on the cation binding heterogeneity. Consequently, the Cd(II), Pb(II), and Zn(II) binding by two different sized SiO2 nanoparticles (Ludox LS30 and TM40) in aqueous dispersion was studied for a range of pH and ionic strength conditions, using the combination of the electroanalytical techniques Scanned Stripping ChronoPotentiometry and Absence of Gradients and Nernstian Equilibrium Stripping. The coupling of these techniques provides the free metal concentration in the bulk (AGNES) and information of the free and complex concentration at the electrode surface for each Stripping Chronopotentiometry at Scanned deposition Potential (SSCP). A recent mathematical treatment allows the reconstruction of a portion of the metal to ligand binding isotherm with the included heterogeneity information using the full SSCP wave analysis. In this work, we observed that the Zn(II) binding is homogeneous, Cd(II) is slightly heterogeneous, and Pb(II) is moderately heterogeneous, whereas the results obtained with the 7.5 nm diameter nanoparticle are slightly more heterogeneous than those obtained with the one of 17 nm. These findings suggest that the Zn(II) binding is electrostatic in nature, and for both Cd(II) and Pb(II), there should be a significant chemical binding contribution.

Full wave analysis of stripping chronopotentiometry at scanned deposition potential (SSCP): Obtaining binding curves in labile heterogeneous macromolecular systems for any metal-to-ligand ratio

Abstract The different deposition potentials applied in Scanned Stripping ChronoPotentiometry (SSCP) probe different values of the free metal and complex concentrations at the electrode surface. The knowledge of these concentrations gives access to a relevant window of the binding curve of a metal to a homogeneous or heterogeneous ligand. Here, a suitable mathematical treatment for the determination of these surface concentrations, when using a mercury thin film rotating disk electrode, is reported. The proposed procedure does not require ligand excess conditions and takes advantage of the knowledge of the free metal ion concentration in the bulk solution provided by the technique AGNES (Absence of Gradients and Nernstian Equilibrium Stripping). It is experimentally shown that the information derived from the SSCP points (i.e. at different deposition potentials in a unique solution) is consistent with the speciation results yielded by the technique AGNES in pertinent solutions prepared within a range of metal-to-ligand ratios. Successful examples with polystyrene sulfonate, Laurentian Fulvic acid and a peat humic acid are reported. It is concluded that, by running consecutively SSCP and AGNES in a single solution, speciation information corresponding to different compositions (i.e. those locally generated at the electrode surface by the end of the deposition step) can be retrieved, this enabling an easy determination of the binding curve.

A novel Cyphos IL 104-based polymer inclusion membrane (PIM) probe to mimic biofilm zinc accumulation.

The presence of Zn in surface waters from abandoned mining zones is a critical issue since excess Zn concentrations may affect aquatic life and whole ecosystems. We present, for the first time, a simple tool based on a polymer inclusion membrane (PIM) intended to monitor Zn in river water by mimicking metal accumulation in the biofilm. The PIM-based probe contains a polymeric membrane prepared using cellulose triacetate (CTA, 50% w/w) as the base polymer, nitrophenyloctyl ether (NPOE) as the plasticizer (20% w/w), and the ionic liquid (IL) Cyphos 104 as the extractant (30% w/w). The accumulation of Zn in the acceptor phase (0.01 M HNO3) was evaluated for different free metal concentrations at 4 h accumulation time resulting in a good correlation between the free metal concentration and the accumulated one. We also found that the metal accumulated agrees with the free metal fraction upon addition of EDTA in the donor solution. The results for Zn accumulation with the PIM-based probe were found to be comparable to those obtained for a biofilm that was grown in a stream from an abandoned mine area and subsequently translocated to the laboratory and put in contact with Zn polluted stream water, so confirming the effectiveness of this new probe in mimicking Zn accumulation in the biofilm.

A review of chemical speciation techniques used for predicting dissolved copper bioavailability in seawater

Environmental contextCopper (Cu) is a metal of ecotoxicological concern in contaminated coastal areas. Cu present as the free ion is considered the most bioavailable fraction of the metal, and there is a need for the correct measurement or estimation of free Cu-ion concentrations in seawater. I review studies that have combined a biological measure of Cu bioavailability with chemical speciation measurements, and critically assess the ability of current methods to predict Cu bioavailability in contaminated coastal ecosystems. AbstractCopper (Cu) is an essential metal, but it is also toxic at concentrations reached in polluted coastal areas. In seawater, the speciation of this metal is largely controlled by the presence of dissolved organic matter (DOM), which binds Cu ions decreasing the concentration of inorganic and free forms of the metal. This is important to aquatic life, given that the bioavailability of dissolved metals is generally expected to be determined by the free ion concentration according to bioavailability models such as the free ion activity model and biotic ligand model (FIAM/BLM). The analytical determination of free metal concentrations in seawater is a challenging task that is needed (in combination with toxicity tests or other means of testing bioavailability) in order to test the applicability of the FIAM/BLM in particular systems and also for its application in monitoring and risk assessment of metals. This review summarises the studies that combine a biological measure of Cu bioavailability with the use of a chemical speciation technique for the measurement of Cu speciation in seawater, and it presents a critical examination of the results of those studies in order to determine which techniques are more suitable for the prediction of Cu bioavailability in seawater and to highlight research needs in the field. The technique showing the highest level of agreement with bioavailability data is anodic stripping voltammetry (ASV). Cathodic stripping voltammetry (CSV), aluminium hydroxide coated exchange resin (ALSA), and diffusion gradients in thin films (DGT) are also promising in this regard, although DGT slightly overestimates bioavailable Cu. More research is needed comparing the performance of different chemical speciation techniques with Cu bioavailability in seawater, especially at environmentally relevant concentrations of Cu.

A retrospective review on extended carboplatin infusion and incidence of hypersensitivity reaction in gynecologic malignancies in a single institution.

e17102 Background: Carboplatin has been the standard treatment for advanced gynecologic malignancies in the first line setting as well as treatment of relapsed disease. Carboplatin induced hypersensitivity reaction (HSR) is observed more frequently in a second line setting and symptoms can range from rash to life threatening symptoms such as anaphylaxis, which could preclude further treatment. A retrospective study by O’Cearbhaill showed HSR rate with extended infusion protocol to be 3.4%. However, two subsequent prospective studies by Lax and O’Cearbhaill did not find that extended infusion reduced the risk or severity of HSR. We reviewed the incidence of HSR with three hour extended-infusion carboplatin at our institution. Methods: Our practice at the Monter Cancer Center changed from standard 1 hour carboplatin retreatment infusion to extended-infusion in 2011. We reviewed charts of 162 patients from 2011 to present who received a three hour extended infusion of carboplatin. All patients received prophylactic H1 and H2 blockers along with steroids. HSR was determined in two ways: 1) Identifying patients who reported clinical symptoms of HSR. 2) Identifying which patients had received diphenhydramine, hydrocortisone and/or epinephrine as indicated for an infusion reaction. Results: 162 patients were identified: 83 with ovarian cancer, 44 with uterine cancer, and 16 with fallopian tube cancer, 10 with primary peritoneal cancer, and 9 with cervical/vulvar cancer. Most had relapsed disease and were previously treated for stage IV (35%), or stage III (47%) with platinum doublet chemotherapy at least six months prior. Only 1 out of 162 (0.6%) had a grade 1 reaction with hives and was treated with dexamethasone and diphenhydramine. She was previously treated with carboplatin 17 months prior. Her current HSR occurred during her fifth cycle of retreatment. She was not re challenged with the drug and elected not to undergo desensitization. Conclusions: Hypersensitivity reactions develop in about 12-33% of women retreated with carboplatin peaking at eight cycles (Lax et al.). The mechanism behind HSR is unclear but considered to be from repeated exposure to free platinum metal concentration. Incidence of HSR at our institution was lowest ever-reported at 0.6%, suggesting that our extended infusion protocol with the appropriate premedication is appropriate for carboplatin-retreat patients. Further prospective studies to evaluate this regimen is warranted.

Assessment of labilities of metal complexes with the dynamic ion exchange technique

Environmental contextIn natural waters, the impact of metals on biota is modulated by their binding with ligands. Ion-exchange techniques can provide information about metal-ligand complexes in solution, which can be linked to metal bioavailability in natural waters. We investigate modelling approaches to interpreting data from ion-exchange experiments to help elucidate the contribution of a particular complex to the overall metal uptake. AbstractThe dynamic ion exchange technique (DIET) is proposed to provide speciation information, which can be used to establish links with metal bioavailability in natural waters. The experimental setup consists of a few milligrams of a sulfonic acid type ion exchange resin packed in a plastic microcolumn that is coupled to a peristaltic pump for a sample to interact with the resin which is subsequently eluted. The evolution of both the accumulated number of moles in the resin and the concentration of the effluent can provide information on the dissociation of different metal-ligand complexes when compared with the transport properties. This information can be converted into the lability degree of a given complex or the DIET concentration cDIET, which accounts for the labile fraction contributing to the metal accumulation by the resin column at the operation conditions. cDIET can be extended to columns containing chelating resins (such as those with Chelex) or to chromatography. A comprehensive modelling of the involved phenomena (such as diffusion, advection, reaction kinetics and electrostatic partitioning) leads to the quantitative interpretation of the accumulation time series (accumulation curves) or effluent evolution (breakthrough curves). Particularly simple analytical expressions can be used for short exposure times, when a (quasi) steady-state is attained. These models have been checked against the results from complexes of Cu and Ni with ligands, such as ethylenediamine, and ethylenediaminetetraacetic, iminodiacetic, glutamic, salicylic, malonic and malic acids, which yield complexes with contrasting charges. Caution is advised when estimating the free metal fraction from DIET measurements, as cDIET and the free metal concentration can be considered to be equal only in the case of extremely inert complexes.

Characterization of the Polysaccharides Released by the Toxic Marine Dinoflagellate Alexandrium catenella Under Metal Stress

A laboratory study was conducted in order to characterize the polysaccharides exudated by the marine toxic dinoflagellate Alexandrium catenella under metal stress conditions. Cultures were exposed to increasing free metal concentrations of copper ([Cu2+] = 2.51 × 10−9–1.27 × 10−8 mol/l), zinc ([Zn2+] = 2.80 × 10−7–1.00 × 10−6 mol/l), cadmium ([Cd2+] = 2.59 × 10−8–7.80 × 10−8 mol/l) and lead ([Pb2+] = 1.14 × 10−8–4.37 × 10−8 mol/l). The composition in aldoses of the polysaccharides exudated in the culture media was analyzed by gas chromatography. In comparison with the controls, the exudation of polysaccharides was greatly increased under metal stress, to reach its maximal values expressed as specific amounts in total aldoses at the highest levels of metal contamination (copper: 1.1 × 10−3 μg Cell−1 at 7.06 × 10−9 mol/l and 4.7 × 10−3 μg Cell−1 at 1.27 × 10−8 mol/l; lead: 1.15 × 10−3 μg Cell−1 at 2.6 × 10−8 mol/l). Composition in aldoses of the polysaccharides exudated was different according to the metal studied and to the contamination levels. Results showed that glucose represented the major aldose composing the polysaccharides released and being able to take part in metal speciation. The authors conclude that high levels of glucose released by A. catenella in its extracellular medium could take a part in some survival strategies setup by the dinoflagellate to adapt to metal stress.

Characterization of the dissociation kinetics of Cd and Ni in soils based on diffusive gradients in thin films technique

A new theoretical method was established for the combinatorial calculation of the dissociation rate constant (K-1) of the metal-organic complexes (MLs), the concentration of free ionic soil metals (CM), the labile concentration of soil metal-organic complexes (CML) based on diffusive gradients in thin-films (DGT) technique with a range of diffusive layer thicknesses (0.053–0.173 mm) in soils. The fitting results agreed well with the determined values. The values of K-1, CML and CM were calculated without other morphological analysis software and the fitting results agreed well with the determined values with some advantages such as the use of fewer hypothetical parameters, ease of calculation, the full embodiment of the contribution of MLs to the labile content. According to the results of model fitting, cation exchange capacity and soil organic matter were found to be the key environmental factors for K-1 values of Cd and Ni, respectively. The labile contents of Cd and Ni in soil were closely related with pH, soil organic matter and the total contents of heavy metals.

A stress-associated protein, LmSAP, from the halophyte Lobularia maritima provides tolerance to heavy metals in tobacco through increased ROS scavenging and metal detoxification processes

Agricultural soil pollution by heavy metals is a severe global ecological problem. We recently showed that overexpression of LmSAP, a member of the stress-associated protein (SAP) gene family isolated from Lobularia maritima, in transgenic tobacco led to enhanced tolerance to abiotic stress. In this study, we characterised the response of LmSAP transgenic tobacco plants to metal stresses (cadmium (Cd), copper (Cu), manganese (Mn), and zinc (Zn)). In L. maritima, LmSAP expression increased after 12 h of treatment with these metals, suggesting its involvement in the plant response to heavy metal stress. LmSAP transgenic tobacco plants subjected to these stress conditions were healthy, experienced higher seedling survival rates, and had longer roots than non-transgenic plants (NT). However, they exhibited higher tolerance towards cadmium and manganese than towards copper and zinc. LmSAP-overexpressing tobacco seedlings accumulated more cadmium, copper, and manganese compared with NT plants, but displayed markedly decreased hydrogen peroxide (H2O2) and lipid peroxidation levels after metal treatment. Activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were significantly higher in transgenic plants than in NT plants after exposure to metal stress. LmSAP overexpression also enhanced the transcription of several genes encoding metallothioneins (Met1, Met2, Met3, Met4, and Met5), a copper transport protein CCH, a Cys and His-rich domain-containing protein RAR1 (Rar1), and a ubiquitin-like protein 5 (PUB1), which are involved in metal tolerance in tobacco. Our findings indicate that LmSAP overexpression in tobacco enhanced tolerance to heavy metal stress by protecting the plant cells against oxidative stress, scavenging reactive oxygen species (ROS), and decreasing the intracellular concentration of free heavy metals through its effect on metal-binding proteins in the cytosol.

Relation between different metal pollution criteria in sediments and its contribution on assessing toxicity

Several tools have been developed and applied to evaluate the metal pollution status of sediments and predict their potential ecological risk assessment. To date, a comprehensive relationship between the information given by these sediment tools for predicting metal bioavailability and the effective toxicity observed is lacking. In this work, the possible inter-correlations between the data outcoming from using several qualitative evaluation tools of the sediment contamination (contamination factor, CF, the enrichment factor, EF, or the geoaccumulation index, Igeo), metal speciation on sediments (evaluated by the modified BCR sequential extraction procedure) and free metal concentrations in pore waters were studied. It was also our aim to evaluate if these assessment tools could be used for predicting the pore waters toxicity data as toxicity proxy. Principal component analysis and cluster analysis revealed that two quality indices used (CF and EF) were highly correlatable with the more labile fractions from BCR sediment speciation. However, neither of these parameters did correlate with the toxicity of pore waters measured by the chronic toxicity (72 h) in Pseudokirchneriella subcapitata. In contrast, the toxic effects of the given total metal load in sediments were better evaluated by using an additive metal approach using pore water free metal concentrations.

Use of high metal-containing biogas digestates in cereal production – Mobility of chromium and aluminium

Biogas digestate use as organic fertilizer has been widely promoted in recent years as a part of the global agenda on recycling waste and new sustainable energy production. Although many studies have confirmed positive effects of digestates on soil fertility, there is still lack of information on the potential adverse effects of digestates on natural soil heavy metal content, metal leaching and leaching of other pollutants. We have investigated the release of aluminium (Al) and chromium (Cr) from different soils treated with commercial digestates high in mentioned potentially problematic metals in a field experiment, while a greenhouse and a laboratory column experiment were used to address mobility of these metals in two other scenarios. Results obtained from the field experiment showed an increase in total concentrations for both investigated metals on plots treated with digestates as well as a significant increase of water-soluble Al concentrations. Factors that were found to be mostly affecting the metal mobility were dissolved organic carbon (DOC), pH and type of soil. Metal binding and free metal concentrations were modelled using the WHAM 7.0 software. Results indicated that the use of digestates with high metal content are comparable to use of animal manure with respect to metal leaching. Data obtained through chemical modelling for the samples from the field experiment suggested that an environmental risk from higher metal mobility has to be considered for Al. In the greenhouse experiment, measured concentrations of leached Cr at the end of the growing season were low for all treatments, while the concentration of leached Al from digestates was higher. The high irrigation column leaching experiment showed an increased leaching rate of Cr with addition of digestates.

Trace metal speciation in a wastewater wetland and its bioaccumulation in tilapia Oreochromis niloticus

Trace metal species in the water column of a canal system forming a wetland filled with wastewater were analyzed to determine their correlation with metal accumulation in the gills of locally fished Oreochromis niloticus. The metal concentrations in the suspended particles and water dissolved were analyzed. The metal species were calculated using Windermere Humic Aqueous Model version VII showing that the high organic matter and major cation contents were important parameters. Also, the free ion metal concentration was expected to correlate with the organic matter aromaticity; however, organic compounds other than humic susbtances seem to be complexing the metals in the system. Additionally, no clear correlation could be found between metal accumulation in gills and any of the dissolved metal species. Nonetheless, certain trends could be seen between the calculated metal species and metal accumulation in the tilapia from the suspended particles.

Polymer inclusion membrane to access Zn speciation: Comparison with root uptake

Metal speciation studies can be performed with a new technique based on a functionalized membrane. The estimation of not only the total amount of metal, but also the metal available to living organisms is very important. In this context, we have investigated the use of a polymer inclusion membrane (PIM) in a new tool for the determination of free metal ion concentration. In order to check the usefulness of PIM devices in metal speciation studies and metal availability to potato plants (Solanum tuberosum), Zn has been chosen as a case study. The PIM designed for Zn transport uses polyvinyl chloride (PVC) as polymer and di-(2-ethylhexyl) phosphoric acid (D2EHPA) as carrier, with 0.01M nitric acid in the receiving solution. The stability of the PIM has been demonstrated and good linearity of PIM-device fluxes (JPIM) with free metal concentration was observed for total metal concentrations ranging from 3μM up to 70μM. The presence of different ligands, such as ethylenediaminetetraacetic acid (EDTA), humic acid (HA) and citrate, greatly influences the measured JPIM because the formation of metal complexes in the donor phase decreases the free Zn concentration in the sample. Good correlation has been found when comparing PIM fluxes and metal accumulation in potato plants roots in the presence of EDTA. But, the root uptake did not change when adding citrate and HA to the hydroponic medium, so the uptake does not always follows the Free Ion Activity Model (FIAM). These ligands might induce physiological changes in the roots and enhance metal uptake.