Application of the cellular oxidation biosensor to Toxicity Identification Evaluations for high-throughput toxicity assessment of river water

Toxicity Identification Evaluation (TIE) is a useful method for the classification and identification of toxicants in a composite environment water sample. However, its extension to a larger sample size has been restrained owing to the limited throughput of toxicity bioassays. Here we reported the development of a high-throughput method of TIE Phase I. This newly developed method was assisted by the fluorescence-based cellular oxidation (CO) biosensor fabricated with roGFP2-expressing bacterial cells in 96-well microplate format. The assessment of four river water samples from Langat river basin by this new method demonstrated that the contaminant composition of the four samples can be classified into two distinct groups. The entire toxicity assay consisted of 2338 tests was completed within 12 h with a fluorescence microplate reader. Concurrently, the sample volume for each assay was reduced to 50 μL, which is 600 to 4700 times lesser to compare with conventional bioassays. These imply that the throughput of the CO biosensor-assisted TIE Phase I is now feasible for constructing a large-scale toxicity monitoring system, which would cover a whole watershed scale

Cr(VI) sorption/desorption on untreated and mussel-shell-treated soil materials: fractionation and effects of pH and chromium concentration

We used batch-type experiments to study Cr(VI) sorption/desorption on granitic material, forest soil, pyritic material, mussel shell, and on forest soil and granitic material amended with 12 t ha−1 (1.2 kg m −2) shell, considering the effects of varying Cr(VI) concentration and pH. Sequential extractions were carried out to fractionate adsorbed Cr(VI) and to determine the stability of Cr(VI) retention. The pyritic material had the highest Cr(VI) retention capacity, whereas the granitic material showed the lowest retention potential. When high Cr concentrations were added, some saturation of the adsorbent surfaces became apparent, but Cr release remained low. The highest Cr retention was achieved at a very acid pH value, with release progressively increasing as a function of increasing pH. The amendment with 12 t ha−1 mussel shell did not cause marked changes in Cr(VI) retention. Sorption data were satisfactory adjusted to the Freundlich model. Regarding Cr(VI) fractionation, the soluble fraction (weakly bound) was dominant in mussel shell and in the unamended and amended granitic material, whereas more stable fractions dominated in the pyritic material (residual fraction) and in the forest soil (oxidizable fraction). In conclusion, the pyritic material presented the highest Cr(VI) retention capacity, while the retention was low and weak on the granitic material; mussel shell was not characterized by a marked Cr(VI) retention potential, and it did not cause remarkable increase in Cr(VI) retention when used to amend the granitic material or the forest soil.This study was funded by the Ministerio de Economía y Competitividad (Government of Spain), grant numbers CGL2012-36805-C02-01-02S

Metais pesados em solos de pastagem da A Pastoriza (NO Espanha) tratados com chorume de vaca e adubos NPK

In Galicia (NW Spain), pasturelands cover a broad extension and are mainly used to feed cattle. Farms are managed in an intensive manner, using cattle slurry and inorganic fertilizers to increase pasture production, but also increasing risks of heavy metal pollution. In this work we studied the influence of fertilization practices on total concentrations and in-depth distribution of heavy metals and related elements (As, Cd, Cr, Cu, Mn, Ni, Pb and Zn) in two forest soils (SN1, SN2) and five pastureland soils (P1-P5) fertilized with cattle slurry and NPK, in a broadly exploded farmland area (A Pastoriza, Lugo). Soils SN2 and P4 were developed over slate, whereas soils SN1, P1, P2, P3 and P5 evolved on Candana quartzite. Forest soils presented acid pH (4.58-4.68), high Al saturation (75-90%), and low available P concentration (4.78-11.96 mg kg-1), whereas those parameters exhibited better scores in the pastureland soils, due to previous amendment and fertilization practices, thus giving pH 5.17-7.02, Al saturation 0.58-59.24%, and available P 5.24-42.07 mg kg-1. Regarding heavy metals, soil depth did not affect significantly to total concentrations, contrary to that happening with parent material, with higher As, Cu, Fe, and Ni concentrations found in soils over slate (possibly due to the presence of pyritic materials). In most cases, heavy metal total concentrations were lower than that considered as reference background levels for soils developed over each of the parent materials, and were always lower than that considered phyto-toxic. In this study, natural soils usually presented heavy metal total contents similar or even higher than that of the fertilized soils (unless Zn in the P4 pastureland), thus indicating that the spread doses of fertilizers did not influence significantly their concentration levels.En Galicia las praderas ocupan una gran extensión, siendo utilizadas principalmente para la alimentación del ganado vacuno destinado tanto a la producción de leche como de carne. Las granjas se manejan de manera intensiva, utilizando fertilizantes inorgánicos y purín de vacuno para aumentar la producción de pastos. Esta práctica puede aumentar el contenido de metales pesados en el suelo. En este trabajo se estudian las concentraciones totales y la distribución en profundidad de metales pesados y elementos afines (As, Cd, Cr, Cu, Mn, Ni, Pb y Zn) en dos suelos forestales (SN1, SN2) y en cinco praderas (P1-P5) fertilizadas con purín de vacuno y NPK en una zona con una gran vocación ganadera (A Pastoriza, Lugo). Se pretende conocer el grado de influencia de la fertilización sobre los niveles de estos elementos. Los suelos elegidos se desarrollaron a partir de pizarras (SN2, P4) y de cuarcitas de la Serie de Cándana (SN1, P1, P2, P3, P5). Los suelos forestales presentaron pH ácido (4,58-4,68), elevada saturación del complejo de cambio por Al (75-90%) y baja concentración de P disponible (4,78-11,96 mg kg-1); estos parámetros mejoran en los suelos de pradera como consecuencia de las enmiendas y fertilizantes aplicados, presentando un pH entre 5,17 y 7,02, una saturación de Al que varía entre 0,58-59,24% y P disponible entre 5,24 y 42,07 mg kg-1. En relación con los metales pesados, la profundidad de la muestra no afecta de modo significativo a su concentración total, pero sí el material de partida, presentando concentraciones más elevadas de Fe, As, Cu y Ni los suelos desarrollados sobre pizarras, posiblemente debido a la presencia de materiales piríticos. En la mayoría de los casos, los metales pesados estudiados presentan concentraciones totales inferiores a las establecidas como genéricas para suelos desarrollados sobre los respectivos materiales geológicos, y siempre son menores que los límites considerados fitotóxicos. La fertilización aplicada no parece tener una influencia significativa sobre los contenidos totales de estos elementos, con la excepción del Zn en la pradera P4, ya que los suelos naturales en muchos casos presentan valores similares e incluso superiores a los obtenidos en las parcelas fertilizadas.As pastagens ocupam uma grande área na região da Galicia (NO Espanha) destnando-se sobretudo à alimentação de bovinos para produção de leite e carne. As explorações são geridas de forma intensiva, aplicando-se adubos minerais e chorumes com o objetivo de aumentar a produção de pastos. Contudo, esta prática pode aumentar o teor de metais pesados no solo. Neste trabalho, a concentração total e a distribuição em profundidade de metais pesados e elementos associados (As, Cd, Cr, Cu, Mn, Ni, Pb e Zn) foram estudados em dois solos florestais (SN1, SN2) em cinco talhões (P1-P5) adubados com NPK e chorume de bovino, numa área com uma grande vocação para pecuária (A Pastoriza, Lugo). Teve-se por objetivo principal determinar o grau de influência da fertilização nos teores destes elementos. Os solos selecionados foram desenvolvidos a partir de ardósias (SN2, P4) e quartzitos da Série de Candana (SN1, P1, P2, P3, P5). Os solos florestais apresentavam pH ácido (4,58-4,68), alta saturação de Al (75-90%) no complexo de troca de e baixa concentração de P disponível (4,78-11,96 mg kg-1); estes parâmetros melhoram em solos de pastagem, como resultado da calagem e fertilizantes aplicados, apresentando um pH entre 5,17 e 7,02; saturação por Al variando de 0,58 a 59,24% e P disponível entre 5,24 e 42,07 mg kg-1. Em relação aos metais pesados, a profundidade da amostra não afetou significativamente a sua concentração total, contrariamente ao que se verificou com o material parental, que apresentou as concentrações mais elevadas de Fe, As, Cu e Ni nos solos desenvolvidos a partir de ardósias possivelmente devido à presença de materiais piríticos. Na maioria dos casos, os metais pesados estudados apresentaram concentrações totais inferiores aos valores geralmente considerados como referência para solos desenvolvidos a partir dos respetivos materiais parentais, sendo sempre inferiores aos valores limite considerados fitotóxicos. Neste estudo, os solos naturais apresentaram valores de metais pesados semelhantes ou mesmo superiores aos dos solos fertilizados (exceto para o Zn na pastagem P4), o que indica que as doses aplicadas de fertilizantes não influenciaram de forma significativa os seus níveis de concentração.This work was funded by the Spanish Ministerio de Economía y Competitividad (Grant Reference: CGL2012-36805-C02-01)S

Enmienda de un suelo de mina con subproductos : efecto sobre la biomasa microbiana determinada mediante el uso de los ácidos grasos de los fosfolípidos

In the present work, the effect of two by-products (pine bark and crushed mussel shell) on microbial biomass and community structure was studied in a soil from a mine tailing located in a copper mine. In a laboratory experiment, different doses (0, 12, 24, 48, 96 and 192 Mg ha-1) of pine bark, crushed mussel shell or mixtures of both by-products were added to the soil. The amended soil samples were incubated for one year at 60% of water holding capacity, and then 33 phospholipid fatty acids (PLFAs) were extracted from these samples and quantified. The PLFAs concentrations were used for different microbial biomass estimations: total biomass, bacterial biomass, fungal biomass, gram-positive (G+) biomass and gram-negative (G-) biomass. The addition of crushed mussel had no significant effects on the total soil microbial biomass, either bacterial of fungal biomass. However, the addition of pine bark increased the total microbial biomass in the soil (up to 40%), mainly due to increases in the fungal biomass (it increased 1600%). No synergistic effects were observed when the soil was amended with both, pine bark and crushed mussel shell. The main community structure changes were due to the addition of pine bark to the soil, and were also due to modifications in fungal communities. Our results suggest that the microbial biomass was mainly limited in the mine soil by low organic matter concentrations, and therefore, practices increasing the amount of soil organic matter should be priorities for soil reclamation.En el presente trabajo se estudió el efecto de dos subproductos (corteza de pino y concha de mejillón triturada) sobre la biomasa y estructura microbiana de un suelo procedente de una escombrera localizada en una mina de cobre. En un experimento realizado en laboratorio fueron añadidas al suelo diferentes dosis (0, 12, 24, 48, 96 and 192 Mg ha-1) de corteza de pino, concha de mejillón triturada y mezclas de ambos subproductos. Las muestras de suelo enmendado fueron incubadas durante un año al 60% de la capacidad de campo, y posteriormente 33 ácidos grasos de los fosfolípidos (PLFAs) fueron extraídos de estas muestras y cuantificados. La concentración de PLFAs fue utilizada para realizar distintas estimaciones de la biomasa microbiana: biomasa total, biomasa bacteriana, biomasa fúngica, biomasa de bacterias gram + y biomasa de bacterias gram -. La adición de concha de mejillón triturada no tuvo efectos significativos sobre la biomasa total ni sobre la biomasa bacteriana o fúngica. Sin embargo, la adición de corteza de pino al suelo incrementó la biomasa total del suelo (hasta un 40%), debido mayormente al incremento de la biomasa fúngica (se incrementó un 1600%). Tampoco se observaron efectos sinérgicos cuando el suelo fue enmendado con una mezcla de corteza de pino y concha de mejillón triturada. Los mayores cambios en la estructura de las comunidades microbianas fueron debidos a la adición de corteza de pino al suelo, y fueron además debidas a cambios en las comunidades fúngicas. Nuestros resultados sugieren que la biomasa microbiana del suelo de mina está mayormente limitada por la concentración de materia orgánica y, por tanto, deben ser priorizadas prácticas de manejo que contribuyan a incrementarla para la rehabilitación de este tipo de suelos.Neste trabalho foi estudado o efeito de dois subprodutos (casca de pinheiro e concha de mexilhão triturada) na biomassa e estrutura microbiana de um solo procedente de uma escombreira localizada numa mina de cobre. Numa experiência de laboratório, doses diferentes (0, 12, 24, 48, 96 e 192 Mg ha-1) de casca de pinheiro, concha de mexilhão triturada e misturas de ambos os subprodutos foram adicionados ao solo. Amostras de solo corrigido com os resíduos foram incubadas durante um ano a 60% da sua capacidade de retenção de água, e subsequentemente, 33 ácidos gordos dos fosfolípidos (PLFAs) foram extraídos a partir destas amostras e quantificados. A concentração de PLFAs foi usada para estimar vários tipos de biomassa microbiana: biomassa total, biomassa bacteriana, biomassa fúngica, biomassa de bactérias gram positivas e biomassa de bactérias gram negativas. A adição de concha de mexilhão triturada não teve nenhum efeito significativo na biomassa total ou na biomassa bacteriana ou fúngica. Porém, a adição de casca de pinheiro aumentou a biomassa microbiana do solo (até 40%), principalmente devido ao aumento da biomassa fúngica (a qual aumentou de 1600%). Não foi observado nenhum efeito de sinergismo quando o solo foi corrigido com uma mistura de casca de pinheiro e concha de mexilhão triturada. As maiores alterações na estrutura das comunidades microbianas foram produzidas pela adição de casca de pinheiro ao solo, as quais resultaram em alterações nas comunidades fúngicas. Os resultados sugerem que a biomassa microbiana do solo de mina é, maioritariamente limitada pela concentração de matéria orgânica. Assim, práticas que contribuam para o seu aumento devem ser prioritárias para a reabilitação deste tipo de solos

Correção de um solo de mina com subprodutos: efeito sobre a biomassa microbiana determinada através da utilização de ácidos gordos dos fosfolípidos

In the present work, the effect of two by-products (pine bark and crushed mussel shell) on microbial biomass and community structure was studied in a soil from a mine tailing located in a copper mine. In a laboratory experiment, different doses (0, 12, 24, 48, 96 and 192 Mg ha-1) of pine bark, crushed mussel shell or mixtures of both by-products were added to the soil. The amended soil samples were incubated for one year at 60% of water holding capacity, and then 33 phospholipid fatty acids (PLFAs) were extracted from these samples and quantified. The PLFAs concentrations were used for different microbial biomass estimations: total biomass, bacterial biomass, fungal biomass, gram-positive (G+) biomass and gram-negative (G-) biomass. The addition of crushed mussel had no significant effects on the total soil microbial biomass, either bacterial of fungal biomass. However, the addition of pine bark increased the total microbial biomass in the soil (up to 40%), mainly due to increases in the fungal biomass (it increased 1600%). No synergistic effects were observed when the soil was amended with both, pine bark and crushed mussel shell. The main community structure changes were due to the addition of pine bark to the soil, and were also due to modifications in fungal communities. Our results suggest that the microbial biomass was mainly limited in the mine soil by low organic matter concentrations, and therefore, practices increasing the amount of soil organic matter should be priorities for soil reclamation.En el presente trabajo se estudió el efecto de dos subproductos (corteza de pino y concha de mejillón triturada) sobre la biomasa y estructura microbiana de un suelo procedente de una escombrera localizada en una mina de cobre. En un experimento realizado en laboratorio fueron añadidas al suelo diferentes dosis (0, 12, 24, 48, 96 and 192 Mg ha-1) de corteza de pino, concha de mejillón triturada y mezclas de ambos subproductos. Las muestras de suelo enmendado fueron incubadas durante un año al 60% de la capacidad de campo, y posteriormente 33 ácidos grasos de los fosfolípidos (PLFAs) fueron extraídos de estas muestras y cuantificados. La concentración de PLFAs fue utilizada para realizar distintas estimaciones de la biomasa microbiana: biomasa total, biomasa bacteriana, biomasa fúngica, biomasa de bacterias gram + y biomasa de bacterias gram -. La adición de concha de mejillón triturada no tuvo efectos significativos sobre la biomasa total ni sobre la biomasa bacteriana o fúngica. Sin embargo, la adición de corteza de pino al suelo incrementó la biomasa total del suelo (hasta un 40%), debido mayormente al incremento de la biomasa fúngica (se incrementó un 1600%). Tampoco se observaron efectos sinérgicos cuando el suelo fue enmendado con una mezcla de corteza de pino y concha de mejillón triturada. Los mayores cambios en la estructura de las comunidades microbianas fueron debidos a la adición de corteza de pino al suelo, y fueron además debidas a cambios en las comunidades fúngicas. Nuestros resultados sugieren que la biomasa microbiana del suelo de mina está mayormente limitada por la concentración de materia orgánica y, por tanto, deben ser priorizadas prácticas de manejo que contribuyan a incrementarla para la rehabilitación de este tipo de suelos.Neste trabalho foi estudado o efeito de dois subprodutos (casca de pinheiro e concha de mexilhão triturada) na biomassa e estrutura microbiana de um solo procedente de uma escombreira localizada numa mina de cobre. Numa experiência de laboratório, doses diferentes (0, 12, 24, 48, 96 e 192 Mg ha-1) de casca de pinheiro, concha de mexilhão triturada e misturas de ambos os subprodutos foram adicionados ao solo. Amostras de solo corrigido com os resíduos foram incubadas durante um ano a 60% da sua capacidade de retenção de água, e subsequentemente, 33 ácidos gordos dos fosfolípidos (PLFAs) foram extraídos a partir destas amostras e quantificados. A concentração de PLFAs foi usada para estimar vários tipos de biomassa microbiana: biomassa total, biomassa bacteriana, biomassa fúngica, biomassa de bactérias gram positivas e biomassa de bactérias gram negativas. A adição de concha de mexilhão triturada não teve nenhum efeito significativo na biomassa total ou na biomassa bacteriana ou fúngica. Porém, a adição de casca de pinheiro aumentou a biomassa microbiana do solo (até 40%), principalmente devido ao aumento da biomassa fúngica (a qual aumentou de 1600%). Não foi observado nenhum efeito de sinergismo quando o solo foi corrigido com uma mistura de casca de pinheiro e concha de mexilhão triturada. As maiores alterações na estrutura das comunidades microbianas foram produzidas pela adição de casca de pinheiro ao solo, as quais resultaram em alterações nas comunidades fúngicas. Os resultados sugerem que a biomassa microbiana do solo de mina é, maioritariamente limitada pela concentração de matéria orgânica. Assim, práticas que contribuam para o seu aumento devem ser prioritárias para a reabilitação deste tipo de solos.S

Toxicity of tetramethylammonium hydroxide to aquatic organisms and its synergistic action with potassium iodide

The aquatic ecotoxicity of chemicals involved in the manufacturing process of thin film transistor liquid crystal displays was assessed with a battery of four selected acute toxicity bioassays. We focused on tetramethylammonium hydroxide (TMAH, CAS No. 75-59-2), a widely utilized etchant. The toxicity of TMAH was low when tested in the 72 h-algal growth inhibition test (Pseudokirchneriellia subcapitata, EC50 = 360 mg L−1) and the Microtox® test (Vibrio fischeri, IC50 = 6.4 g L−1). In contrast, the 24 h-microcrustacean immobilization and the 96 h-fish mortality tests showed relatively higher toxicity (Daphnia magna, EC50 = 32 mg L−1 and Oryzias latipes, LC50 = 154 mg L−1). Isobologram and mixture toxicity index analyses revealed apparent synergism of the mixture of TMAH and potassium iodide when examined with the D. magna immobilization test. The synergistic action was unique to iodide over other halide salts i.e. fluoride, chloride and bromide. Quaternary ammonium ions with longer alkyl chains such as tetraethylammonium and tetrabutylammonium were more toxic than TMAH in the D. magna immobilization test

The new multi-frequency instrument (MFI2) for the QUIJOTE facility in Tenerife

The QUIJOTE (Q-U-I joint Tenerife) experiment combines the operation of two radio-telescopes and three instruments working in the microwave bands 10?20 GHz, 26-36 GHz and 35-47 GHz at the Teide Observatory, Tenerife, and has already been presented in previous SPIE meetings (Hoyland, R. J. et al, 2012; Rubiño-Martín et al., 2012). The Cosmology group at the IAC have designed a new upgrade to the MFI instrument in the band 10-20 GHz. The aim of the QUIJOTE telescopes is to characterise the polarised emission of the cosmic microwave background (CMB), as well as galactic and extra-galactic sources, at medium and large angular scales. This MFI2 will continue the survey at even higher sensitivity levels. The MFI2 project led by the Instituto de Astrofísica de Canarias (IAC) consists of five polarimeters, three of them operating in the sub-band 10?15 GHz, and two in the sub-band 15-20 GHz. The MFI2 instrument is expected to be a full two-three times more sensitive than the former MFI. The microwave complex correlator design has been replaced by a simple correlator design with a digital back-end based on the latest Xilinx FPGAs (ZCU111). During the first half of 2019 the manufacture of the new cryostat was completed and since then the opto-mechanical components have been designed and manufactured. It is expected that the cryogenic front-end will be completed by the end of 2022 along with the FPGA acquisition and observing system. This digital system has been employed to be more robust against stray ground-based and satellite interference, having a frequency resolution of 1 MHz.The MFI2 instrument is being developed by the Instituto de Astrofisica de Canarias (IAC), with an instrumental participation from the Universidad Politecnica de Cartagena (UPCT). Partial financial support is provided by the Spanish Ministry of Science and Innovation (MICINN), under the projects AYA2017-84185-P, IACA15-BE-3707, EQC2018-004918-P and the FEDER Agreement INSIDE-OOCC (ICTS-2019-03-IAC-12). We also acknowledge financial support of the Severo Ochoa Programs SEV-2015-0548 and CEX2019-000920-S

A Bacterial Biosensor for Oxidative Stress Using the Constitutively Expressed Redox-Sensitive Protein roGFP2

A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2). Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd2+, Cu2+, Pb2+, Zn2+ and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm) were determined as 1.0 × 10−7 (arsenite), 1.0 × 10−4 (naphthalene), 1.0 × 10−4 (Cu2+), 3.8 × 10−4 (H2O2), 1.0 × 10−3 (Cd2+), 1.0 × 10−3 (Zn2+), 1.0 × 10−2 (menadione), 1.0 (triphenyltin), 1.56 (zinc pyrithione), 3.1 (selenite) and 6.3 (Pb2+), respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation

Distribution and genotype-phenotype correlation of GDAP1 mutations in Spain

Mutations in the GDAP1 gene can cause Charcot-Marie-Tooth disease. These mutations are quite rare in most Western countries but not so in certain regions of Spain or other Mediterranean countries. This cross-sectional retrospective multicenter study analyzed the clinical and genetic characteristics of patients with GDAP1 mutations across Spain. 99 patients were identified, which were distributed across most of Spain, but especially in the Northwest and Mediterranean regions. The most common genotypes were p.R120W (in 81% of patients with autosomal dominant inheritance) and p.Q163X (in 73% of autosomal recessive patients). Patients with recessively inherited mutations had a more severe phenotype, and certain clinical features, like dysphonia or respiratory dysfunction, were exclusively detected in this group. Dominantly inherited mutations had prominent clinical variability regarding severity, including 29% of patients who were asymptomatic. There were minor clinical differences between patients harboring specific mutations but not when grouped according to localization or type of mutation. This is the largest clinical series to date of patients with GDAP1 mutations, and it contributes to define the genetic distribution and genotype-phenotype correlation in this rare form of CMT