Presence of pharmaceutical contaminants of emerging concerns in two rivers of western Cuba and their relationship with the extracellular enzymatic activity of microbial communities.

In recent years, the concern derived from the presence of emerging contaminants in the environment and the possible effects on the One Health trilogy has increased. This study determined the concentration of pharmaceutical contaminants of emerging concern and their relationship with the extracellular enzymatic activity of microbial communities from two rivers in western Cuba. Two sampling stations were analyzed; one in the Almendares River (urban) and the other in the San Juan River (rural), taking into account the pollution sources that arrive at these stations and previous physicochemical characterizations. Extracellular protease, acid phosphatase, alkaline phosphatase, lipase, and catalase activities in water and sediments were determined and correlated with contaminants of emerging concern determined by liquid chromatography with mass spectrometry. This study evidenced the presence of different pharmaceutical contaminants found in the categories of antihypertensives, stimulants, anti-inflammatories, and antibiotics in both rivers. Concentrations of contaminants of emerging concern were greater in the Almendares River compared to the San Juan River. In addition, through the canonical redundancy analysis, the influence of these contaminants on the extracellular enzymatic activities of microbial communities was documented, where in most cases they inhibit protease, phosphatase, and lipase activities and enhance catalase activity in response to oxidative stress. The present investigation constitutes the first report in Cuba of the presence of pharmaceutical contaminants of emerging concern and one of the few works that exist in the Latin American region.

Enantioseparation and ecotoxicity evaluation of ibrutinib by Electrokinetic Chromatography using single and dual systems.

In this work, two chiral methods enabling the separation of ibrutinib enantiomers were developed by Electrokinetic Chromatography. A cyclodextrin (CD) or a mixture of the CD and a chiral ionic liquid (CIL) was used as chiral selector. Using the single CD system, seven neutral and six anionic CDs were tested in a formate buffer at pH 3.0 working in positive and negative polarity, respectively. The use of sulfated-γ-CD (S-γ-CD) and negative polarity originated the best results considering analysis time and enantioresolution. The optimization of the experimental conditions allowed obtaining the separation of ibrutinib enantiomers in an analysis time of 4.2 min with an enantioresolution value of 1.5. The effect of the addition of fifteen CILs on the enantioresolution was evaluated showing that both analysis time and enantioresolution were generally increased. A mixture of S-γ-CD and [TMA][L-Lys] was selected which provided the separation of ibrutinib enantiomers in 8.1 min with an enantioresolution value of 3.3 under the same experimental conditions as in the case of using the single CD system. The enantiomeric impurity (S-ibrutinib) was the first-migrating isomer when using the single CD and the combined CD/CIL systems, as corresponds to the most desirable situation. Both chiral methods allowed the detection of the enantiomeric impurity up to a 0.1% as established by the International Council on Harmonization. After establishing the analytical characteristics of both chiral methodologies developed, they were applied to the enantiomeric determination of ibrutinib in a pharmaceutical formulation for hospital use marketed as pure enantiomer (R-ibrutinib) and to evaluate the stability and ecotoxicity of racemic ibrutinib and R-ibrutinib on Daphnia magna. The developed methodologies enabled, for the first time, the rapid chiral quantitation of ibrutinib in abiotic and biotic matrices.

First eco-toxicological evidence of ivabradine effect on the marine bacterium Vibrio fischeri: A chiral view.

Ivabradine (S-ivabradine) is a contemporary antihypertensive drug designed and commercialized for cardiovascular diseases treatment over the world. In this work the enantiomer-specific stability and acute toxicity of ivabradine to the marine bacterium Vibrio fischeri as well as the potential mechanism of action were investigated for the first time. With this aim, real concentrations of ivabradine enantiomers under abiotic and biotic conditions were determined by Capillary Electrophoresis (CE) with cyclodextrins (CDs) as chiral selectors. A moderate chiral stability without enantiomeric interconversion was observed for ivabradine. The bioluminescence inhibition method revealed an enantioselective toxicity of ivabradine to marine bacterium. The order of ecotoxicity was R-ivabradine < racemic ivabradine < S-ivabradine with EC50 (t = 5 min) values about 75.98, 11.11 and 7.93 mg/L, respectively. Confocal Live/Dead stained images showed that bacterial envelops cells were seriously damaged after exposure to S-ivabradine. S-ivabradine also disturbed the esterase activity and significantly increased the ROS level compared with the control. Thus, oxidative stress originating membrane cells damage and enzymatic activity changes was shown to be the primary mechanism of S-ivabradine toxicity to marine bacterium. Our results highlight the need for more eco-toxicological evaluations of the cardiovascular drug S-ivabradine on other aquatic organisms to establish the risk on the environment.

Effect of Carbamazepine, Ibuprofen, Triclosan and Sulfamethoxazole on Anaerobic Bioreactor Performance: Combining Cell Damage, Ecotoxicity and Chemical Information.

Pharmaceuticals and personal care products (PPCPs) are partially degraded in wastewater treatment plants (WWTPs), thereby leading to the formation of more toxic metabolites. Bacterial populations in bioreactors operated in WWTPs are sensitive to different toxics such as heavy metals and aromatic compounds, but there is still little information on the effect that pharmaceuticals exert on their metabolism, especially under anaerobic conditions. This work evaluated the effect of selected pharmaceuticals that remain in solution and attached to biosolids on the metabolism of anaerobic biomass. Batch reactors operated in parallel under the pressure of four individual and mixed PPCPs (carbamazepine, ibuprofen, triclosan and sulfametoxazole) allowed us to obtain relevant information on anaerobic digestion performance, toxicological effects and alterations to key enzymes involved in the biodegradation process. Cell viability was quantitatively evaluated using an automatic analysis of confocal microscopy images, and showed that triclosan and mixed pollutants caused higher toxicity and cell death than the other individual compounds. Both individual pollutants and their mixture had a considerable impact on the anaerobic digestion process, favoring carbon dioxide production, lowering organic matter removal and methane production, which also produced microbial stress and irreversible cell damage.

Microbial Electrochemical Fluidized Bed Reactor: A Promising Solution for Removing Pollutants From Pharmaceutical Industrial Wastewater.

The capacity of electroactive bacteria to exchange electrons with electroconductive materials has been explored during the last two decades as part of a new field called electromicrobiology. Such microbial metabolism has been validated to enhance the bioremediation of wastewater pollutants. In contrast with standard materials like rods, plates, or felts made of graphite, we have explored the use of an alternative strategy using a fluid-like electrode as part of a microbial electrochemical fluidized bed reactor (ME-FBR). After verifying the low adsorption capacity of the pharmaceutical pollutants on the fluid-bed electrode [7.92 ± 0.05% carbamazepine (CBZ) and 9.42 ± 0.09% sulfamethoxazole (SMX)], our system showed a remarkable capacity to outperform classical solutions for removing pollutants (more than 80%) from the pharmaceutical industry like CBZ and SMX. Moreover, the ME-FBR performance revealed the impact of selecting an anode potential by efficiently removing both pollutants at + 200 mV. The high TOC removal efficiency also demonstrated that electrostimulation of electroactive bacteria in ME-FBR could overcome the expected microbial inhibition due to the presence of CBZ and SMX. Cyclic voltammograms revealed the successful electron transfer between microbial biofilm and the fluid-like electrode bed throughout the polarization tests. Finally, Vibrio fischeri-based ecotoxicity showed a 70% reduction after treating wastewater with a fluid-like anode (+ 400 mV), revealing the promising performance of this bioelectrochemical approach.

Stereoselective separation of sulfoxaflor by electrokinetic chromatography and applications to stability and ecotoxicological studies.

An Electrokinetic Chromatography method was developed for the stereoselective analysis of sulfoxaflor, a novel sulfoximine agrochemical with two chiral centers. A screening with fourteen negatively charged CDs was performed and Succinyl-ß-CD (Succ-ß-CD) was selected. A 15 mM concentration of this CD in a 100 mM borate buffer (pH 9.0), using an applied voltage of 20 kV and a temperature of 15 °C made possible the baseline separation of the four stereoisomers of sulfoxaflor in 13.8 min. The evaluation of the linearity, accuracy, precision, LODs and LOQs of the method developed showed its performance to be applied to the analysis of commercial agrochemical formulations, the evaluation of the stability of sulfoxaflor stereoisomers under biotic and abiotic conditions, and to predict, for the first time, sulfoxaflor toxicity (using real concentrations instead of nominal concentrations), on two non-target aquatic organisms, the freshwater plant, Spirodela polyrhiza, and the marine bacterium, Vibrio fischeri.

Enantiomeric separation of panthenol by Capillary Electrophoresis. Analysis of commercial formulations and toxicity evaluation on non-target organisms.

The first CE methodology enabling the enantiomeric separation of panthenol was developed in this work. Electrokinetic chromatography with cyclodextrins (CD-EKC) was the CE mode employed for this purpose. The effect of different experimental variables such as the nature and concentration of the cyclodextrin, the temperature and the separation voltage was investigated. The best enantiomeric separation was obtained with 25 mM (2-carboxyethyl)-ß-CD (CE-ß-CD) in 100 mM borate buffer (pH 9.0), with a separation voltage of 30 kV and a temperature of 30 °C. Under these conditions, an enantiomeric resolution of 2.0 in an analysis time of 4.2 min was obtained, being the biologically active enantiomer d-panthenol (dexpanthenol) the second-migrating enantiomer. The analytical characteristics of the method were evaluated in terms of precision, accuracy, selectivity, linearity, LOD, and LOQ, showing a good performance for the quantitation of dexpanthenol in cosmetic and pharmaceutical formulations. The enantiomeric impurity (L-panthenol) could be detected at a 0.1% level with respect to the majority enantiomer, allowing to accomplish the requirements of the ICH guidelines. The method was also successfully applied to study the stability of panthenol under abiotic and biotic conditions and its toxicity on non-target organisms (the aquatic plant Spirodela polyrhiza).

Enzyme response of activated sludge to a mixture of emerging contaminants in continuous exposure.

The relevant information about the impacts caused by presence of emerging pollutants in mixtures on the ecological environment, especially on the more vulnerable compartments such as activated sludge (AS) is relatively limited. This study investigated the effect of ibuprofen (IBU) and triclosan (TCS), alone and in combination to the performance and enzymatic activity of AS bacterial community. The assays were carried out in a pilot AS reactor operating for two-weeks under continuous dosage of pollutants. The microbial activity was tracked by measuring oxygen uptake rate, esterase activity, oxidative stress and antioxidant enzyme activities. It was found that IBU and TCS had no acute toxic effects on reactor biomass concentration. TCS led to significant decrease of COD removal efficiency, which dropped from 90% to 35%. Continuous exposure to IBU, TCS and their mixtures increased the activities of glutathione s-transferase (GST) and esterase as a response to oxidative damage. A high increase in GST activity was associated with non-reversible toxic damage while peaks of esterase activity combined with moderate GST increase were attributed to an adaptive response.

Stability and toxicity studies for duloxetine and econazole on Spirodela polyrhiza using chiral capillary electrophoresis.

Stability and toxicity studies for duloxetine and econazole were achieved using individual solutions and their mixtures. Stability of drugs racemates and enantiomers was investigated under abiotic and biotic conditions. Toxicity was evaluated for the first time on Spirodela polyrhiza. EC50 values were calculated for each individual drug and for their binary mixture. Real (not nominal) concentrations determined by Capillary Electrophoresis were employed in the calculations of toxicity parameters. The use of a 25 mM phosphate buffer (pH 3.0) with 1.5% S-ß-CD as chiral selector at a temperature of 30 °C and a separation voltage of -20 kV enabled the simultaneous enantiomeric separation of duloxetine and econazole in 7.5 min with enantiomeric resolutions of 7.9 and 6.5, respectively. For individual solutions, decay percentages under abiotic conditions were higher for duloxetine (80%) than for econazole (60%), while in presence of Spirodela polyrhiza they increased for duloxetine but not for econazole. Econazole showed the highest decay percentages under abiotic or biotic conditions (100%) in binary mixtures. EC50 values for duloxetine and econazole enabled to include both drugs within the group of very toxic compounds although econazole showed a higher toxicity than duloxetine and the binary mixture.

Enantiomer stability and combined toxicity of duloxetine and econazole on Daphnia magna using real concentrations determined by capillary electrophoresis.

Enantiomer stability was investigated in this work for the first time for duloxetine and econazole in individual solutions and their mixtures under the standardized ecotoxicity test experimental conditions for Daphnia magna and abiotic conditions. Real (and not nominal) enantiomer concentrations were employed for calculations since their determination was achieved by Capillary Electrophoresis. Relevant differences were found in stability profiles for both drugs in any case. Toxicity was evaluated for the first time in this work for mixtures of duloxetine and econazole on Daphnia magna. Dose-effect parameters were calculated at different exposure times (24, 48, and 72 h) showing a significant inhibition of daphnids mobility when increasing the incubation time. Combination index values enabled to obtain the type and level of interaction of drugs with the organism. A strong synergism was observed at 48 h exposure time and any effect level, which demonstrated the high toxicity of the drug mixture compared with the individual drug solutions. These results were corroborated when evaluating the oxidative stress using fluorescence images.

Biocompatible antimicrobial electrospun nanofibers functionalized with ε-poly-l-lysine.

The antimicrobial polypeptide ε-poly(l-lysine) (ε-PL) was electrostatically incorporated to poly(acrylic acid) (PAA)/poly(vinyl alcohol) (PVA) electrospun nanofibers. ε-PL loading and distribution was assessed by infrared spectra, ζ-potential measurements and the primary amino reactive dye fluorescamine. Functionalized fibers with 485 ±â€¯140 nm diameter, could be loaded with 0.57-0.74 g ε-PL (g dressing)-1 that released at a constant rate of 5.4 ±â€¯2.8 mg ε-PL (g dressing day)-1. Such a dressings resulted in two orders of magnitude lower bacterial colonization than non-functionalized PAA-PVA after 14 days of incubation. Bacterial impairment was attributed to the damage of cell membranes and the formation of intracellular reactive oxygen species. ε-PL functionalized nanofibers did not display cytotoxicity to human corneal epithelial cells, HCEpC, in 24 h MTT assays. However, the viability of rapidly growing tumoral HeLa cells decreased >50% under the same conditions. The prepared biocompatible nanofibrous dressings with durable antibacterial activity show potential application as wound dressings and other biomedical uses.

Incorporation of antimicrobial peptides on electrospun nanofibres for biomedical applications.

The aim of this work was to immobilize antimicrobial peptides onto a fibrous scaffold to create functional wound dressings. The scaffold was produced by electrospinning from a mixture of the water soluble polymers poly(acrylic acid) and poly(vinyl alcohol) and subsequently heat cured at 140 °C to produce a stable material with fibre diameter below micron size. The peptides were incorporated into the negatively charged scaffold by electrostatic interaction. The best results were obtained for lysozyme impregnated at pH 7, which rendered a loading of up to 3.0 × 10-4 mmol mg-1. The dressings were characterized using SEM, ATR-FTIR, elemental analysis, ζ-potential and confocal microscopy using fluorescamine as an amine-reactive probe. The dressings preserved their fibrous structure after impregnation and peptides were distributed homogeneously throughout the fibrous network. The antibacterial activity was assessed by solid agar diffusion tests and growth inhibition in liquid cultures using Staphylococcus aureus, a pathogenic strain generally found in infected wounds. The antibacterial activity caused clear halo inhibition zones for lysozyme-loaded dressings and a 4-fold decrease in S. aureus viable colonies after two weeks of contact of dressings with bacterial liquid cultures. The release profile in different media showed sustained release in acidic environments, and a rapid discharge at high pH values. The incorporation of lysozyme resulted in dressing surfaces essentially free of microbial growth after 14 days of contact with bacteria at pH 7.4 attributed to the peptide that remained attached to the dressing surface.

Dendrimer-functionalized electrospun nanofibres as dual-action water treatment membranes.

This work reports the preparation of composite electrospun membranes combining antimicrobial action with the capacity of retaining low-molecular weight non-polar pollutants. The membranes were electrospun blends of polyvinyl alcohol (PVA) and poly(acrylic acid) (PAA) stabilized using heat curing. The membranes were functionalized by grafting amino-terminated poly(amidoamine) (PAMAM) G3 dendrimers. The antimicrobial effect was assessed using strains of Escherichia coli and Staphylococcus aureus by tracking their capacity to form new colonies and their metabolic impairment upon contact with membranes. The antimicrobial activity was particularly high to the gram-positive bacterium S. aureus with a 3-log reduction in their capacity to colonize dendrimer-functionalized membranes with respect to neat PVA/PAA fibers. The effect to gram-positive bacteria was attributed to the interaction of dendrimers with the negatively charged bacterial membranes and resulted in membranes essentially free of bacterial colonization after 20h in contact with cultures at 36°C. The adsorption of toluene on PAA/PVA fibers and on dendrimer-functionalized membranes was assayed using toluene over a broad concentration range. The host-guest encapsulation of toluene inside dendrimer molecules was computed through docking studies, which allowed calculating a maximum capacity of 14 molecules of toluene per molecule of PAMAM G3. The theoretical prediction was in good agreement with the experimental capacity at the higher concentrations assayed.

A capillary micellar electrokinetic chromatography method for the stereoselective quantitation of bioallethrin in biotic and abiotic samples.

A capillary micellar electrokinetic chromatography (MEKC) method was developed enabling the stereoselective separation of the insecticide bioallethrin. The use of sodium deoxycholate bile salt and acetyl-ß-cyclodextrin (acetyl-ß-CD) made possible the separation of bioallethrin stereoisomers with a high enantioresolution (7.4) in a short analysis time (6.5min). The analytical characteristics of the developed method were evaluated in terms of linearity, accuracy, precision, and limits of detection (LOD) and quantitation (LOQ) showing a good performance for the quantitation of bioallethrin stereoisomers with LODs of 0.2 and 0.3mg/L. The developed method was applied to the stereoselective analysis of a commercial bioallethrin pediculicide formulation and to evaluate the toxicity of bioallethrin stereoisomers on the growth of the unicellular freshwater green alga Pseudokirchneriella subcapitata and on the germination of the higher plant Sorghum bicolor (non-target organisms). The analysis of the commercial pediculicide showed a good agreement between the contents determined for the two stereoisomers and those labelled in the commercial samples. Different toxic responses and biodegradation profiles were found for each stereoisomer in ecotoxicity assays. The mixture of S/R stereoisomers of bioallethrin resulted more toxic than S-bioallethrin for green algae, with EC50 values of 1.10±0.06 for the mixture and of 1.73±0.05mg/L for the pure S-biallethrin (esbiol). Germination of plants seeds was also affected.

Toxicological interactions of ibuprofen and triclosan on biological activity of activated sludge.

The growing use of pharmaceutical and personal care products increases their concentrations in the wastewater entering treatment plants and their levels into biological reactors. The most extended biological wastewater treatment is the activated sludge process. The toxicity of ibuprofen and triclosan, individually and combined, was studied by tracking the biological activity of the activated sludge measuring oxygen uptake rate and the inhibition of the esterase activity. Short-term exposure produced significant inhibition in oxygen uptake, with lower damage to enzymatic activity. Median effect values for oxygen uptake inhibition were 64±13mgL-1 and 0.32±0.07mgL-1 for ibuprofen and triclosan respectively using 125mgL-1 activated sludge. For the inhibition of enzymatic activity values were 633±63mgL-1 for ibuprofen and 1.94±0.32mgL-1 for triclosan. Results indicated that oxygen uptake, related to primary activity of microorganisms, was more strongly affected than the enzymatic activity associated to energy consumption. Toxicity interactions were determined using the Combination Index-isobologram method. Results showed antagonism at lower values of affected population, after which the mixtures tended to additivity and synergism. For the case of enzymatic activity, the antagonism was less marked and the additivity range was higher.

Poly(amidoamine) dendrimers grafted on electrospun poly(acrylic acid)/poly(vinyl alcohol) membranes for host-guest encapsulation of antioxidant thymol.

Amino-terminated fifth generation poly(amidoamine) (PAMAM G5-NH2) dendrimers were grafted onto the surface of poly(acrylic acid)/poly(vinyl alcohol) (PAA/PVA) electrospun fibres with the purpose of creating a host-guest architecture for the controlled delivery of a natural antioxidant, thymol. The nanofibers were stabilized by esterification crosslinking to produce a water insoluble non-woven membrane. The functionalization with PAMAM G5-NH2 led to dendrimer loadings in the 7.4 × 10-7-2.25 × 10-6 mol dendrimer per g membrane range. The resulting materials were characterized using SEM, ATR-FTIR and surface ζ-potential measurements. The loading capacity for thymol reached 2.5 × 10-4 mol thymol per g membrane. The membranes were tested for thymol release in different aqueous and non-aqueous food simulants. Computational modelling was used to get a further insight into the host-guest association of thymol and PAMAM G5-NH2 molecules through docking studies. For this purpose, we examined the molecular level details of the dendrimer-guest complex, calculated the number of included or attached molecules, the exact location of thymol in host-guest complexes and the local environment around the thymol molecules. Docking studies showed that PAMAM-G5-NH2 dendrimers can encapsulate thymol molecules through hydrophobic interactions and hydrogen bonding. The maximum amount of thymol molecules theoretically encapsulated was 16, while another 25 could be hosted at the dendrimer surface through interaction with the outer part or the dendritic branches. The experimental value was 37 ± 5, in agreement with theoretical predictions.

Cleaning-up atrazine-polluted soil by using Microbial Electroremediating Cells.

Biodegradation of pollutants in soil is greatly limited by the availability of terminal electron acceptors required for supporting microbial respiration. Such limitation can be overcome if soil-buried electrodes accept the electrons released in the microbial metabolism. We propose the term bioelectroventing for such a environmental treatment. The process would be performed in a device so-called Microbial Electroremediating Cell. Indeed, our studies demonstrate that the presence of electrodes as electron acceptors effectively stimulated by 5-fold the biodegradation rate of the herbicide atrazine (2-chloro-4-ethylamino-6-isopropyl amino-1,3,5-triazine) in comparison with soil natural attenuation. Furthermore, a different set of toxicological test using Pseudokirchneriella subcapitata green alga e, Salmonella typhimorium bacteria and Sorghum saccharatum plant seeds respectively, confirm that atrazine-polluted soil can be effectively cleaned-up in short time by the use of MERCs.

Electrospun cellulose acetate composites containing supported metal nanoparticles for antifungal membranes.

Electrospun cellulose acetate composites containing silver and copper nanoparticles supported in sepiolite and mesoporous silica were prepared and tested as fungistatic membranes against the fungus Aspergillus niger. The nanoparticles were in the 3-50nm range for sepiolite supported materials and limited by the size of mesopores (5-8nm) in the case of mesoporous silica. Sepiolite and silica were well dispersed within the fibers, with larger aggregates in the micrometer range, and allowed a controlled release of metals to create a fungistatic environment. The effect was assessed using digital image analysis to evaluate fungal growth rate and fluorescence readings using a viability stain. The results showed that silver and copper nanomaterials significantly impaired the growth of fungi when the spores were incubated either in direct contact with particles or included in cellulose acetate composite membranes. The fungistatic effect took place on germinating spores before hyphae growth conidiophore formation. After 24h the cultures were separated from fungistatic materials and showed growth impairment only due to the prior exposure. Growth reduction was important for all the particles and membranes with respect to non-exposed controls. The effect of copper and silver loaded materials was not significantly different from each other with average reductions around 70% for bare particles and 50% for membranes. Copper on sepiolite was particularly efficient with a decrease of metabolic activity of up to 80% with respect to controls. Copper materials induced rapid maturation and conidiation with fungi splitting in sets of subcolonies. Metal-loaded nanomaterials acted as reservoirs for the controlled release of metals. The amount of silver or copper released daily by composite membranes represented roughly 1% of their total load of metals. Supported nanomaterials encapsulated in nanofibers allow formulating active membranes with high antifungal performance at the same time minimizing the risk of nanoparticle release into the environment.

Antimicrobial electrospun silver-, copper- and zinc-doped polyvinylpyrrolidone nanofibers.

The use of electrospun polyvinylpyrrolidone (PVP) nanofibers containing silver, copper, and zinc nanoparticles was studied to prepare antimicrobial mats using silver and copper nitrates and zinc acetate as precursors. Silver became reduced during electrospinning and formed nanoparticles of several tens of nanometers. Silver nanoparticles and the insoluble forms of copper and zinc were dispersed using low molecular weight PVP as capping agent. High molecular weight PVP formed uniform fibers with a narrow distribution of diameters around 500 nm. The fibers were converted into an insoluble network using ultraviolet irradiation crosslinking. The efficiency of metal-loaded mats against the bacteria Escherichia coli and Staphylococcus aureus was tested for different metal loadings by measuring the inhibition of colony forming units and the staining with fluorescent probes for metabolic viability and compromised membranes. The assays included the culture in contact with mats and the direct staining of surface attached microorganisms. The results indicated a strong inhibition for silver-loaded fibers and the absence of significant amounts of viable but non-culturable microorganisms. Copper and zinc-loaded mats also decreased the metabolic activity and cell viability, although in a lesser extent. Metal-loaded fibers allowed the slow release of the soluble forms of the three metals.

Environmental risk of combined emerging pollutants in terrestrial environments: chlorophyll a fluorescence analysis.

The risk assessment in terrestrial environments has been scarcely studied for mixtures of organic contaminants. To estimate toxicity due to these compounds, an ecotoxicological test may be done with the appropriate organism and biomarker. Photosynthesis is principally performed at photosystem II, and its efficiency is affected by any environmental stress. Consequently, the measure of this efficiency may be a good indicator of toxicity if different parameters are employed, e.g., the quantum efficiency of photosystem II and the photochemical quenching coefficient. We did a series of assays to determine the toxicity of two organic contaminants, ibuprofen and perfluorooctanoic acid, using a higher plant (Sorghum bicolor). The results showed more toxicity for the perfluorinated compound and greater sensibility for the quantum efficiency of photosystem II. Regarding the binary combination, three methods were applied to calculate EC50: combination index, concentration addition, and independent action. Synergistic behavior is the principal toxicological profile for this mix. Therefore, the combination index, which considers interactions among chemicals, gave the best estimation to determine risk indices. We conclude that the inhibition of photosynthesis efficiency can be a useful tool to determine the toxicity of the mixtures of organic pollutants and to estimate ecological risks in terrestrial environments.