Agricultural use of organic wastes as source of nitrogen and phosphorus: risks and opportunities

El nitrógeno (N) y el fósforo (P) son nutrientes esenciales en la producción de cultivos. El desarrollo de los fertilizantes de síntesis durante el siglo XX permitió una intensificación de la agricultura y un aumento de las producciones pero a su vez el gran input de nutrientes ha resultado en algunos casos en sistemas poco eficientes incrementando las pérdidas de estos nutrientes al medio ambiente. En el caso del P, este problema se agrava debido a la escasez de reservas de roca fosfórica necesaria para la fabricación de fertilizantes fosfatados. La utilización de residuos orgánicos en agricultura como fuente de N y P es una buena opción de manejo que permite valorizar la gran cantidad de residuos que se generan. Sin embargo, es importante conocer los procesos que se producen en el suelo tras la aplicación de los mismos, ya que influyen en la disponibilidad de nutrientes que pueden ser utilizados por el cultivo así como en las pérdidas de nutrientes de los agrosistemas que pueden ocasionar problemas de contaminación. Aunque la dinámica del N en el suelo ha sido más estudiada que la del P, los problemas importantes de contaminación por nitratos en zonas vulnerables hacen necesaria la evaluación de aquellas prácticas de manejo que pudieran agravar esta situación, y en el caso de los residuos orgánicos, la evaluación de la respuesta agronómica y medioambiental de la aplicación de materiales con un alto contenido en N (como los residuos procedentes de la industria vinícola y alcoholera). En cuanto al P, debido a la mayor complejidad de su ciclo y de las reacciones que ocurren en el suelo, hay un mayor desconocimiento de los factores que influyen en su dinámica en los sistemas suelo-planta, lo que supone nuevas oportunidades de estudio en la evaluación del uso agrícola de los residuos orgánicos. Teniendo en cuenta los conocimientos previos sobre cada nutriente así como las necesidades específicas en el estudio de los mismos, en esta Tesis se han evaluado: (1) el efecto de la aplicación de residuos procedentes de la industria vinícola y alcoholera en la dinámica del N desde el punto de vista agronómico y medioambiental en una zona vulnerable a la contaminación por nitratos; y (2) los factores que influyen en la disponibilidad de P en el suelo tras la aplicación de residuos orgánicos. Para ello se han llevado a cabo incubaciones de laboratorio así como ensayos de campo que permitieran evaluar la dinámica de estos nutrientes en condiciones reales. Las incubaciones de suelo en condiciones controladas de humedad y temperatura para determinar el N mineralizado se utilizan habitualmente para estimar la disponibilidad de N para el cultivo así como el riesgo medioambiental. Por ello se llevó a cabo una incubación en laboratorio para conocer la velocidad de mineralización de N de un compost obtenido a partir de residuos de la industria vinícola y alcoholera, ampliamente distribuida en Castilla-La Mancha, región con problemas importantes de contaminación de acuíferos por nitratos. Se probaron tres dosis crecientes de compost correspondientes a 230, 460 y 690 kg de N total por hectárea que se mezclaron con un suelo franco arcillo arenoso de la zona. La evolución del N mineral en el suelo a lo largo del tiempo se ajustó a un modelo de regresión no lineal, obteniendo valores bajos de N potencialmente mineralizable y bajas contantes de mineralización, lo que indica que se trata de un material resistente a la mineralización y con una lenta liberación de N en el suelo, mineralizándose tan solo 1.61, 1.33 y 1.21% del N total aplicado con cada dosis creciente de compost (para un periodo de seis meses). Por otra parte, la mineralización de N tras la aplicación de este material también se evaluó en condiciones de campo, mediante la elaboración de un balance de N durante dos ciclos de cultivo (2011 y 2012) de melón bajo riego por goteo, cultivo y manejo agrícola muy característicos de la zona de estudio. Las constantes de mineralización obtenidas en el laboratorio se ajustaron a las temperaturas reales en campo para predecir el N mineralizado en campo durante el ciclo de cultivo del melón, sin embargo este modelo generalmente sobreestimaba el N mineralizado observado en campo, por la influencia de otros factores no tenidos en cuenta para obtener esta predicción, como el N acumulado en el suelo, el efecto de la planta o las fluctuaciones de temperatura y humedad. Tanto el ajuste de los datos del laboratorio al modelo de mineralización como las predicciones del mismo fueron mejores cuando se consideraba el efecto de la mezcla suelo-compost que cuando se aislaba el N mineralizado del compost, mostrando la importancia del efecto del suelo en la mineralización del N procedente de residuos orgánicos. Dado que esta zona de estudio ha sido declarada vulnerable a la contaminación por nitratos y cuenta con diferentes unidades hidrológicas protegidas, en el mismo ensayo de campo con melón bajo riego por goteo se evaluó el riesgo de contaminación por nitratos tras la aplicación de diferentes dosis de compost bajo dos regímenes de riego, riego ajustado a las necesidades del cultivo (90 ó 100% de la evapotranspiración del cultivo (ETc)) o riego excedentario (120% ETc). A lo largo del ciclo de cultivo se estimó semanalmente el drenaje mediante la realización de un balance hídrico, así como se tomaron muestras de la solución de suelo y se determinó su concentración de nitratos. Para evaluar el riesgo de contaminación de las aguas subterráneas asociado con estas prácticas, se utilizaron algunos índices medioambientales para determinar la variación en la calidad del agua potable (Índice de Impacto (II)) y en la concentración de nitratos del acuífero (Índice de Impacto Ambiental (EII)). Para combinar parámetros medioambientales con parámetros de producción, se calculó la eficiencia de manejo. Se observó que la aplicación de compost bajo un régimen de riego ajustado no aumentaba el riesgo de contaminación de las aguas subterráneas incluso con la aplicación de la dosis más alta. Sin embargo, la aplicación de grandes cantidades de compost combinada con un riego excedentario supuso un incremento en el N lixiviado a lo largo del ciclo de cultivo, mientras que no se obtuvieron mayores producciones con respecto al riego ajustado. La aplicación de residuos de la industria vinícola y alcoholera como fuente de P fue evaluada en suelos calizos caracterizados por una alta capacidad de retención de P, lo cual en algunos casos limita la disponibilidad de este nutriente. Para ello se llevó a cabo otro ensayo de incubación con dos suelos de diferente textura, con diferente contenido de carbonato cálcico, hierro y con dos niveles de P disponible; a los que se aplicaron diferentes materiales procedentes de estas industrias (con y sin compostaje previo) aportando diferentes cantidades de P. A lo largo del tiempo se analizó el P disponible del suelo (P Olsen) así como el pH y el carbono orgánico disuelto. Al final de la incubación, con el fin de estudiar los cambios producidos por los diferentes residuos en el estado del P del suelo se llevó a cabo un fraccionamiento del P inorgánico del suelo, el cual se separó en P soluble y débilmente enlazado (NaOH-NaCl-P), P soluble en reductores u ocluido en los óxidos de Fe (CBD-P) y P poco soluble precipitado como Ca-P (HCl-P); y se determinó la capacidad de retención de P así como el grado de saturación de este elemento en el suelo. En este ensayo se observó que, dada la naturaleza caliza de los suelos, la influencia de la cantidad de P aplicado con los residuos en el P disponible sólo se producía al comienzo del periodo de incubación, mientras que al final del ensayo el incremento en el P disponible del suelo se igualaba independientemente del P aplicado con cada residuo, aumentando el P retenido en la fracción menos soluble con el aumento del P aplicado. Por el contrario, la aplicación de materiales orgánicos menos estabilizados y con un menor contenido en P, produjo un aumento en las formas de P más lábiles debido a una disolución del P retenido en la fracción menos lábil, lo cual demostró la influencia de la materia orgánica en los procesos que controlan el P disponible en el suelo. La aplicación de residuos aumentó el grado de saturación de P de los suelos, sin embargo los valores obtenidos no superaron los límites establecidos que indican un riesgo de contaminación de las aguas. La influencia de la aplicación de residuos orgánicos en las formas de P inorgánico y orgánico del suelo se estudió además en un suelo ácido de textura areno francosa tras la aplicación en campo a largo plazo de estiércol vacuno y de compost obtenido a partir de biorresiduos, así como la aplicación combinada de compost y un fertilizante mineral (superfosfato tripe), en una rotación de cultivos. En muestras de suelo recogidas 14 años después del establecimiento del experimento en campo, se determinó el P soluble y disponible, la capacidad de adsorción de P, el grado de saturación de P así como diferentes actividades enzimáticas (actividad deshidrogenasa, fosfatasa ácida y fosfatasa alcalina). Las diferentes formas de P orgánico en el suelo se estudiaron mediante una técnica de adición de enzimas con diferentes substratos específicos a extractos de suelo de NaOH-EDTA, midiendo el P hidrolizado durante un periodo de incubación por colorimetría. Las enzimas utilizadas fueron la fosfatasa ácida, la nucleasa y la fitasa las cuales permitieron identificar monoésteres hidrolizables (monoester-like P), diésteres (DNA-like P) e inositol hexaquifosfato (Ins6P-like P). La aplicación a largo plazo de residuos orgánicos aumentó el P disponible del suelo proporcionalmente al P aplicado con cada tipo de fertilización, suponiendo un mayor riesgo de pérdidas de P dado el alto grado de saturación de este suelo. La aplicación de residuos orgánicos aumentó el P orgánico del suelo resistente a la hidrólisis enzimática, sin embargo no influyó en las diferentes formas de P hidrolizable por las enzimas en comparación con las observadas en el suelo sin enmendar. Además, las diferentes formas de P orgánico aplicadas con los residuos orgánicos no se correspondieron con las analizadas en el suelo lo cual demostró que éstas son el resultado de diferentes procesos en el suelo mediados por las plantas, los microorganismos u otros procesos abióticos. En este estudio se encontró una correlación entre el Ins6P-like P y la actividad microbiana (actividad deshidrogenasa) del suelo, lo cual refuerza esta afirmación. Por último, la aplicación de residuos orgánicos como fuente de N y P en la agricultura se evaluó agronómicamente en un escenario real. Se estableció un experimento de campo para evaluar el compost procedente de residuos de bodegas y alcoholeras en el mismo cultivo de melón utilizado en el estudio de la mineralización y lixiviación de N. En este experimento se estudió la aplicación de tres dosis de compost: 1, 2 y 3 kg de compost por metro lineal de plantación correspondientes a 7, 13 y 20 t de compost por hectárea respectivamente; y se estudió el efecto sobre el crecimiento de las plantas, la acumulación de N y P en la planta, así como la producción y calidad del cultivo. La aplicación del compost produjo un ligero incremento en la biomasa vegetal acompañado por una mejora significativa de la producción con respecto a las parcelas no enmendadas, obteniéndose la máxima producción con la aplicación de 2 kg de compost por metro lineal. Aunque los efectos potenciales del N y P fueron parcialmente enmascarados por otras entradas de estos nutrientes en el sistema (alta concentración de nitratos en el agua de riego y ácido fosfórico suministrado por fertirrigación), se observó una mayor acumulación de P uno de los años de estudio que resultó en un aumento en el número de frutos en las parcelas enmendadas. Además, la mayor acumulación de N y P disponible en el suelo al final del ciclo de cultivo indicó el potencial uso de estos materiales como fuente de estos nutrientes. ABSTRACT Nitrogen (N) and phosphorus (P) are essential nutrients in crop production. The development of synthetic fertilizers during the 20th century allowed an intensification of the agriculture increasing crop yields but in turn the great input of nutrients has resulted in some cases in inefficient systems with higher losses to the environment. Regarding P, the scarcity of phosphate rock reserves necessary for the production of phosphate fertilizers aggravates this problem. The use of organic wastes in agriculture as a source of N and P is a good option of management that allows to value the large amount of wastes generated. However, it is important to understand the processes occurring in the soil after application of these materials, as they affect the availability of nutrients that can be used by the crop and the nutrient losses from agricultural systems that can cause problems of contamination. Although soil N dynamic has been more studied than P, the important concern of nitrate pollution in Nitrate Vulnerable Zones requires the evaluation of those management practices that could aggravate this situation, and in the case of organic wastes, the evaluation of the agronomic and environmental response after application of materials with a high N content (such as wastes from winery and distillery industries). On the other hand, due to the complexity of soil P cycle and the reactions that occur in soil, there is less knowledge about the factors that can influence its dynamics in the soil-plant system, which means new opportunities of study regarding the evaluation of the agricultural use of organic wastes. Taking into account the previous knowledge of each nutrient and the specific needs of study, in this Thesis we have evaluated: (1) the effect of the application of wastes from the winery and distillery industries on N dynamics from the agronomic and environmental viewpoint in a vulnerable zone; and (2) the factors that influence P availability in soils after the application of organic wastes. With this purposes, incubations were carried out in laboratory conditions as well as field trials that allow to assess the dynamic of these nutrients in real conditions. Soil incubations under controlled moisture and temperature conditions to determine N mineralization are commonly used to estimate N availability for crops together with the environmental risk. Therefore, a laboratory incubation was conducted in order to determine the N mineralization rate of a compost made from wastes generated in the winery and distillery industries, widely distributed in Castilla-La Mancha, a region with significant problems of aquifers contamination by nitrates. Three increasing doses of compost corresponding to 230, 460 and 690 kg of total N per hectare were mixed with a sandy clay loam soil collected in this area. The evolution of mineral N in soil over time was adjusted to a nonlinear regression model, obtaining low values of potentially mineralizable N and low constants of mineralization, indicating that it is a material resistant to mineralization with a slow release of N, with only 1.61, 1.33 and 1.21% of total N applied being mineralized with each increasing dose of compost (for a period of six months). Furthermore, N mineralization after the application of this material was also evaluated in field conditions by carrying out a N balance during two growing seasons (2011 and 2012) of a melon crop under drip irrigation, a crop and management very characteristic of the area of study. The mineralization constants obtained in the laboratory were adjusted to the actual temperatures observed in the field to predict N mineralized during each growing season, however, this model generally overestimated the N mineralization observed in the field, because of the influence of other factors not taken into account for this prediction, as N accumulated in soil, the plant effect or the fluctuations of temperature and moisture. The fitting of the laboratory data to the model as well as the predictions of N mineralized in the field were better when considering N mineralized from the soil-compost mixture rather than when N mineralized from compost was isolated, underlining the important role of the soil on N mineralization from organic wastes. Since the area of study was declared vulnerable to nitrate pollution and is situated between different protected hydrological units, the risk of nitrate pollution after application of different doses compost was evaluated in the same field trial with melon under two irrigation regimes, irrigation adjusted to the crop needs (90 or 100% of the crop evapotranspiration (ETc)) or excedentary irrigation (120% ETc). Drainage was estimated weekly throughout the growing season by conducting a water balance, samples of the soil solution were taken and the concentration of nitrates was determined. To assess the risk of groundwater contamination associated with these practices, some environmental indices were used to determine the variation in the quality of drinking water (Impact Index (II)) and the nitrates concentration in the groundwater (Environmental Impact Index (EII)). To combine environmental parameters together with yield parameters, the Management Efficiency was calculated. It was observed that the application of compost under irrigation adjusted to the plant needs did not represent a higher risk of groundwater contamination even with the application of the highest doses. However, the application of large amounts of compost combined with an irrigation surplus represented an increase of N leaching during the growing season compared with the unamended plots, while no additional yield with respect to the adjusted irrigation strategy is obtained. The application of wastes derived from the winery and distillery industry as source of P was evaluated in calcareous soils characterized by a high P retention capacity, which in some cases limits the availability of this nutrient. Another incubation experiment was carried out using two soils with different texture, different calcium carbonate and iron contents and two levels of available P; to which different materials from these industries (with and without composting) were applied providing different amounts of P. Soil available P (Olsen P), pH and dissolved organic carbon were analyzed along time. At the end of the incubation, in order to study the changes in soil P status caused by the different residues, a fractionation of soil inorganic P was carried out, which was separated into soluble and weakly bound P (NaOH-NaCl- P), reductant soluble P or occluded in Fe oxides (CBD-P) and P precipitated as poorly soluble Ca-P (HCl-P); and the P retention capacity and degree of P saturation were determined as well. Given the calcareous nature of the soils, the influence of the amount of P applied with the organic wastes in soil available P only occurred at the beginning of the incubation period, while at the end of the trial the increase in soil available P equalled independently of the amount of P applied with each residue, increasing the P retained in the least soluble fraction when increasing P applied. Conversely, the application of less stabilized materials with a lower content of P resulted in an increase in the most labile P forms due to dissolution of P retained in the less labile fraction, demonstrating the influence of organic matter addition on soil P processes that control P availability in soil. As expected, the application of organic wastes increased the degree of P saturation in the soils, however the values obtained did not exceed the limits considered to pose a risk of water pollution. The influence of the application of organic wastes on inorganic and organic soil P forms was also studied in an acid loamy sand soil after long-term field application of cattle manure and biowaste compost and the combined application of compost and mineral fertilizer (triple superphosphate) in a crop rotation. Soil samples were collected 14 years after the establishment of the field experiment, and analyzed for soluble and available P, P sorption capacity, degree of P saturation and enzymatic activities (dehydrogenase, acid phosphatase and alkaline phosphatase). The different forms of organic P in soil were determined by using an enzyme addition technique, based on adding enzymes with different substrate specificities to NaOH-EDTA soil extracts, measuring the hydrolyzed P colorimetrically after an incubation period. The enzymes used were acid phosphatase, nuclease and phytase which allowed to identify hydrolyzable monoesters (monoester-like P) diesters (DNA-like P) and inositol hexakisphosphate (Ins6P-like P). The long-term application of organic wastes increased soil available P proportionally to the P applied with each type of fertilizer, assuming a higher risk of P losses given the high degree of P saturation of this soil. The application of organic wastes increased soil organic P resistant to enzymatic hydrolysis, but no influence was observed regarding the different forms of enzyme hydrolyzable organic P compared to those observed in the non-amended soil. Furthermore, the different forms of organic P applied with the organic wastes did not correspond to those analyzed in the soil which showed that these forms in soil are a result of multifaceted P turnover processes in soil affected by plants, microorganisms and abiotic factors. In this study, a correlation between Ins6P-like P and the microbial activity (dehydrogenase activity) of soil was found, which reinforces this claim. Finally, the application of organic wastes as a source of N and P in agriculture was evaluated agronomically in a real field scenario. A field experiment was established to evaluate the application of compost made from wine-distillery wastes in the same melon crop used in the experiments of N mineralization and leaching. In this experiment the application of three doses of compost were studied: 1 , 2 and 3 kg of compost per linear meter of plantation corresponding to 7, 13 and 20 tonnes of compost per hectare respectively; and the effect on plant growth, N and P accumulation in the plant as well as crop yield and quality was studied. The application of compost produced a slight increase in plant biomass accompanied by a significant improvement in crop yield with respect to the unamended plots, obtaining the maximum yield with the application of 2 kg of compost per linear meter. Although the potential effects of N and P were partially masked by other inputs of these nutrients in the system (high concentration of nitrates in the irrigation water and phosphoric acid supplied by fertigation), an effect of P was observed the first year of study resulting in a greater plant P accumulation and in an increase in the number of fruits in the amended plots. In addition, the higher accumulation of available N and P in the topsoil at the end of the growing season indicated the potential use of this material as source of these nutrients.

Service capacity reserve under uncertainty by hospital's ER's analogies: a practical model for car services

El objetivo de esta investigación consiste en definir un modelo de reserva de capacidad, por analogías con emergencias hospitalarias, que pueda ser implementado en el sector de servicios. Este está específicamente enfocado a su aplicación en talleres de servicio de automóviles. Nuestra investigación incorpora la incertidumbre de la demanda en un modelo singular diseñado en etapas que agrupa técnicas ARIMA, teoría de colas y simulación Monte Carlo para definir los conceptos de capacidad y ocupación de servicio, que serán utilizados para minimizar el coste implícito de la reserva capacidad necesaria para atender a clientes que carecen de cita previa. Habitualmente, las compañías automovilísticas estiman la capacidad de sus instalaciones de servicio empíricamente, pero los clientes pueden llegar bajo condiciones de incertidumbre que no se tienen en cuenta en dichas estimaciones, por lo que existe una diferencia entre lo que el cliente realmente demanda y la capacidad que ofrece el servicio. Nuestro enfoque define una metodología válida para el sector automovilístico que cubre la ausencia genérica de investigaciones recientes y la habitual falta de aplicación de técnicas estadísticas en el sector. La equivalencia con la gestión de urgencias hospitalarias se ha validado a lo largo de la investigación en la se definen nuevos indicadores de proceso (KPIs) Tal y como hacen los hospitales, aplicamos modelos estocásticos para dimensionar las instalaciones de servicio de acuerdo con la distribución demográfica del área de influencia. El modelo final propuesto integra la predicción del coste implícito en la reserva de capacidad para atender la demanda no prevista. Asimismo, se ha desarrollado un código en Matlab que puede integrarse como un módulo adicional a los sistemas de información (DMS) que se usan actualmente en el sector, con el fin de emplear los nuevos indicadores de proceso definidos en el modelo. Los resultados principales del modelo son nuevos indicadores de servicio, tales como la capacidad, ocupación y coste de reserva de capacidad, que nunca antes han sido objeto de estudio en la industria automovilística, y que están orientados a gestionar la operativa del servicio. ABSTRACT Our aim is to define a Capacity Reserve model to be implemented in the service sector by hospital's emergency room (ER) analogies, with a practical approach to passenger car services. A stochastic model has been implemented using R and a Monte Carlo simulation code written in Matlab and has proved a very useful tool for optimal decision making under uncertainty. The research integrates demand uncertainty in a unique model which is built in stages by implementing ARIMA forecasting, Queuing Theory and a Monte Carlo simulation to define the concepts of service capacity and occupancy, minimizing the implicit cost of the capacity that must be reserved to service unexpected customers. Usually, passenger car companies estimate their service facilities capacity using empirical methods, but customers arrive under uncertain conditions not included in the estimations. Thus, there is a gap between customer’s real demand and the dealer’s capacity. This research sets a valid methodology for the passenger car industry to cover the generic absence of recent researches and the generic lack of statistical techniques implementation. The hospital’s emergency room (ER) equalization has been confirmed to be valid for the passenger car industry and new process indicators have been defined to support the study. As hospitals do, we aim to apply stochastic models to dimension installations according to the demographic distribution of the area to be serviced. The proposed model integrates the prediction of the cost implicit in the reserve capacity to serve unexpected demand. The Matlab code could be implemented as part of the existing information technology systems (ITs) to support the existing service management tools, creating a set of new process indicators. Main model outputs are new indicators, such us Capacity, Occupancy and Cost of Capacity Reserve, never studied in the passenger car service industry before, and intended to manage the service operation.

Amelioration of a degraded acid soil from SW Spain by no-tillage and Ca-amendment

En los suelos, el exceso de acidez lleva asociado deficiencias en ciertos nutrientes y una alta disponibilidad de aluminio, tóxico para los cultivos propios del ambiente mediterráneo. Su laboreo, provoca la pérdida de materia orgánica (MO), deteriora su estructura y reduce la actividad biológica, provocando en última instancia una menor calidad del suelo. Es de esperar pues que cuando se labran suelos ácidos, sus problemáticas particulares tiendan a agravarse. En nuestra zona de estudio, la “raña” de Cañamero (Extremadura, España), predominan los suelos muy ácidos y degradados por un laboreo inadecuado. Las rañas constituyen amplias plataformas casi horizontales, con unos suelos muy viejos (Palexerults), que se caracterizan por tener el complejo de cambio dominado por el aluminio, y un pH ácido que decrece en profundidad. Poseen un potente horizonte Bt rico en arcillas caoliníticas, que propicia que en periodos con exceso de lluvia, se generen capas colgadas de agua cercanas a la superficie. En torno a los años 1940’s estos suelos, que previamente sostenían un alcornocal, o su matorral de sustitución, se pusieron en cultivo. El laboreo aceleró la mineralización de la materia orgánica, agravó los problemas derivados del exceso de acidez y condujo al abandono de los campos cultivados por falta de productividad. Para recuperar la calidad de estos suelos degradados y obtener unos rendimientos compatibles con su uso agrícola es necesario, por un lado, aplicar enmiendas que eleven el pH y reduzcan la toxicidad del aluminio y, por otro, favorecer el incremento en el contenido en MO. En 2005 se implantó en esta raña un ensayo de campo para estudiar la influencia del no laboreo y de la utilización de una enmienda cálcica en parámetros relacionados con la calidad del suelo en un cultivo forrajero. El diseño experimental fue en parcelas divididas con cuatro repeticiones donde el factor principal fue el tipo de laboreo, no laboreo (NL) frente a laboreo convencional (LC), y el factor secundario el uso o no de una enmienda cálcica. La enmienda consistió básicamente en una mezcla de espuma de azucarería y yeso rojo y se incorporó al comienzo del ensayo hasta los 7 cm de profundidad. Desde el comienzo del ensayo el NL influyó positivamente en el contenido de carbono orgánico total (COT) y particulado (COP), mientras que la enmienda tuvo una ligera influencia al principio del ensayo en ambos pero su efecto positivo se desvaneció con el paso del tiempo. Los mayores contenidos en COT y POC se observaron cuando se combinó el NL con la enmienda. La enmienda incrementó con rapidez el pH, y el Ca, y disminuyó el contenido en aluminio hasta una profundidad de 50 cm, incluso en NL, y mejoró ligeramente la agregación del suelo. El NL por sí solo, gracias al aumento en POC, TOC y las proteínas del suelo relacionadas con la glomalina (PSRG), que son capaces de formar compuestos estables no tóxicos con el aluminio, también contribuyó a la reducción de la toxicidad de aluminio en la capa más superficial. Cuando en las campañas con exceso de precipitaciones se generaron capas colgadas de agua próximas a la superficie, el NL generó unas condiciones más favorables para la germinación y desarrollo del cultivo, resultando en una producción más alta que el LC. A ello contribuyó la mayor capacidad de almacenamiento de agua y la mayor transmisividad de esta hacia abajo, en la capa más superficial (0-5 cm) que propició una menor saturación por agua que el LC. Respecto a los parámetros relacionados con la agregación, el NL aumentó los macroagregados hasta los 10 cm de profundidad y favoreció la acumulación de CO y N en todas las fracciones de tamaño de agregados. Sin embargo, la recuperación del grado de macroagregación tras el cese del laboreo resulta lenta en comparación con otros suelos, posiblemente debido al bajo contenido en arcilla en el horizonte Ap. En comparación con el NL, la enmienda mostró también un efecto positivo, aunque muy ligero, en la agregación del suelo. En contradicción con otros estudios en suelos ácidos, nuestros resultados indican la existencia de una jerarquía de agregados, y destacan el papel importante de la MO en la mejora de la agregación. Tanto el NL como la enmienda favorecieron por separado varias propiedades químicas, físicas y biológicas del suelo, pero, en general, encontramos los mayores beneficios con su uso combinado. Además, a largo plazo el efecto positivo de NL en las propiedades del suelo fue en aumento, mientras que el efecto beneficioso de la enmienda se limitó básicamente a las propiedades químicas y se desvaneció en pocos años. Destacamos que las condiciones meteorológicas a lo largo del ensayo beneficiaron la producción de biomasa en NL, y en consecuencia las propiedades relacionadas con la materia orgánica, por lo que son un factor a tener en cuenta a la hora de evaluar los efectos de la enmienda y el laboreo sobre las propiedades del suelo, especialmente en zonas donde esas condiciones son muy variables entre una campaña y otra. Los resultados de este estudio han puesto de manifiesto que el NL no ha mermado la eficacia de la enmienda caliza, posiblemente gracias a la alta solubilidad de la enmienda aplicada, es más, el manejo con NL y enmienda es el que ha favorecido en mayor medida ciertos parámetros de calidad del suelo. Por el contrario el LC sí parece anular los beneficios de la enmienda en relación con las propiedades relacionadas con la MO. Por tanto, cabe concluir que la combinación de NL y la enmienda es una práctica adecuada para mejorar las propiedades químicas y físicas de suelos ácidos degradados por el laboreo. ABSTRACT Excessive acidity in soils is associated with deficiencies in certain nutrients and high concentrations of available aluminum, which is toxic for most Mediterranean crops. Tilling these soils results in the loss of soil organic matter (SOM), damages soil structure and reduces biological activity, ultimately degrading soil quality. It is expected, therefore, that when acid soils are tilled, their particular problems will tend to get worse. In our study area, the "Cañamero’s Raña” (Extremadura, Spain), acid soils degraded by an inappropriate tillage prevail. Rañas are large and flat platforms with very old soils (Palexerults), which are characterized by an exchange complex dominated by aluminum and an acid pH which decreases with depth. These soils have a strong Bt horizon rich in kaolinite clays, which encourages the formation of perched water-tables near the soil surface during periods of excessive rain. During the first third of the 20th century, these soils, that previously supported cork oak or its scrub replacement, were cultivated. Tillage accelerated the mineralization of the SOM, aggravating the problems of excessive acidity, which finally led to the abandonment of the land due to low productivity. To recover the quality of these degraded soils and to obtain consistent yields it is necessary, first, to apply amendments to raise the pH and reduce aluminum toxicity, and second to encourage the accumulation of SOM. In 2005 a field trial was established in the Raña to study the influence of no-tillage and the use of a Ca-amendment on soil quality related parameters in a forage crop agrosystem. The experimental design was a split-plot with four replicates where the main factor was tillage type, no-tillage (NT) versus traditional tillage (TT) and the secondary factor was the use or not of a Ca-amendment. The Ca-amendment was a mixture of sugar foam and red gypsum that was incorporated into the top 7 cm of the soil. Since the beginning of the experiment, NT had a positive influence on total and particulate organic carbon (TOC and POC, respectively), while the Ca-amendment had a small positive influence at the beginning of the study but its effect diminished with time. The highest TOC and POC contents were observed when NT and the Ca-amendment were combined. The Ca-amendment, even under NT, rapidly increased pH and Ca, and decreased the aluminum content to a depth of 50 cm, as well as improving soil aggregation slightly. NT, due to the increased POC, TOC and Glomalin-related soil proteins (GRSP), which can form stable non-toxic compounds with aluminum, also contributed to the reduction of aluminum toxicity in the upper layer. When perched water-tables near the soil surface were formed in campaigns with excessive rainfall, NT provided more favorable conditions for germination and crop development, resulting in higher yields compared with TT. This was directly related to the higher water storage capacity and the greater transmissivity of the water downwards from the upper layers, which led to lower water saturation under NT compared with TT. With regards to the aggregation-related parameters, NT increased macroaggregation to a depth of 10 cm and favored the accumulation of OC and N in all aggregate size fractions. However, the degree of recovery of macroaggregation after tillage ceased was slow compared with other soils, possibly due to the low clay content in the Ap horizon. Compared with NT, the Ca-amendment had a slight positive effect on soil aggregation. In contrast to other studies in acid soils, our results indicate the existence of an aggregate hierarchy, and highlight the important role of SOM in improving aggregation. Both NT and the Ca-amendment separately favored various chemical, physical and biological soil properties, but in general we found the greatest benefits when the two treatments were combined. In addition, the positive effect of NT on soil properties increased with time, while the beneficial effect of the Ca-amendment, which was limited to the chemical properties, vanished after a few years. It is important to note that the meteorological conditions throughout the experiment benefited biomass production under NT and, as a consequence, organic matter related properties. This suggests that meteorological conditions are a factor to consider when evaluating the effects of Ca-amendments and tillage on soil properties, especially in areas where such conditions vary significantly from one campaign to another. The results of this study show that NT did not diminish the effectiveness of the Ca-amendment, possibly due to the high solubility of the selected amendment. Moreover, the combination of NT and the Ca-amendment was actually the management that favored certain soil quality parameters the most. By contrast, TT seemed to nullify the benefits of the Ca-amendment with regards to the OM related properties. In conclusion, the combination of NT and the application of a Ca-amendment is an advisable practice for improving the chemical and physical properties of acid soils degraded by tillage.

Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice

This study investigated whether endothelin-1 (ET-1), a potent vasoconstrictor, which also stimulates cell proliferation, contributes to endothelial dysfunction and atherosclerosis. Apolipoprotein E (apoE)-deficient mice and C57BL/6 control mice were treated with a Western-type diet to accelerate atherosclerosis with or without ETA receptor antagonist LU135252 (50 mg/kg/d) for 30 wk. Systolic blood pressure, plasma lipid profile, and plasma nitrate levels were determined. In the aorta, NO-mediated endothelium-dependent relaxation, atheroma formation, ET receptor-binding capacity, and vascular ET-1 protein content were assessed. In apoE-deficient but not C57BL/6 mice, severe atherosclerosis developed within 30 wk. Aortic ET-1 protein content (P < 0.0001) and binding capacity for ETA receptors was increased as compared with C57BL/6 mice. In contrast, NO-mediated, endothelium-dependent relaxation to acetylcholine (56 ± 3 vs. 99 ± 2%, P < 0.0001) and plasma nitrate were reduced (57.9 ± 4 vs. 93 ± 10 μmol/liter, P < 0.01). Treatment with the ETA receptor antagonist LU135252 for 30 wk had no effect on the lipid profile or systolic blood pressure in apoE-deficient mice, but increased NO-mediated endothelium-dependent relaxation (from 56 ± 3 to 93 ± 2%, P < 0.0001 vs. untreated) as well as circulating nitrate levels (from 57.9 ± 4 to 80 ± 8.3 μmol/liter, P < 0.05). Chronic ETA receptor blockade reduced elevated tissue ET-1 levels comparable with those found in C57BL/6 mice and inhibited atherosclerosis in the aorta by 31% without affecting plaque morphology or ET receptor-binding capacity. Thus, chronic ETA receptor blockade normalizes NO-mediated endothelial dysfunction and reduces atheroma formation independent of plasma cholesterol and blood pressure in a mouse model of human atherosclerosis. ETA receptor blockade may have therapeutic potential in patients with atherosclerosis.

Effect of age on in vivo rates of mitochondrial protein synthesis in human skeletal muscle

A progressive decline in muscle performance in the rapidly expanding aging population is causing a dramatic increase in disability and health care costs. A decrease in muscle endurance capacity due to mitochondrial decay likely contributes to this decline in muscle performance. We developed a novel stable isotope technique to measure in vivo rates of mitochondrial protein synthesis in human skeletal muscle using needle biopsy samples and applied this technique to elucidate a potential mechanism for the age-related decline in the mitochondrial content and function of skeletal muscle. The fractional rate of muscle mitochondrial protein synthesis in young humans (24 ± 1 year) was 0.081 ± 0.004%·h−1, and this rate declined to 0.047 ± 0.005%·h−1 by middle age (54 ± 1 year; P < 0.01). No further decline in the rate of mitochondrial protein synthesis (0.051 ± 0.004%·h−1) occurred with advancing age (73 ± 2 years). The mitochondrial synthesis rate was about 95% higher than that of mixed protein in the young, whereas it was approximately 35% higher in the middle-aged and elderly subjects. In addition, decreasing activities of mitochondrial enzymes were observed in muscle homogenates (cytochrome c oxidase and citrate synthase) and in isolated mitochondria (citrate synthase) with increasing age, indicating declines in muscle oxidative capacity and mitochondrial function, respectively. The decrease in the rates of mitochondrial protein synthesis is likely to be responsible for this decline in muscle oxidative capacity and mitochondrial function. These changes in muscle mitochondrial protein metabolism may contribute to the age-related decline in aerobic capacity and muscle performance.

ZK200775: A phosphonate quinoxalinedione AMPA antagonist for neuroprotection in stroke and trauma

Stroke and head trauma are worldwide public health problems and leading causes of death and disability in humans, yet, no adequate neuroprotective treatment is available for therapy. Glutamate antagonists are considered major drug candidates for neuroprotection in stroke and trauma. However, N-methyl-d-aspartate antagonists failed clinical trials because of unacceptable side effects and short therapeutic time window. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) antagonists derived from the quinoxalinedione scaffold cannot be used in humans because of their insolubility and resulting renal toxicity. Therefore, achieving water solubility of quinoxalinediones without loss of selectivity and potency profiles becomes a major challenge for medicinal chemistry. One of the major tenets in the chemistry of glutamate antagonists is that the incorporation of phosphonate into the glutamate framework results in preferential N-methyl-d-aspartate antagonism. Therefore, synthesis of phosphonate derivatives of quinoxalinediones was not pursued because of a predicted loss of their selectivity toward AMPA. Here, we report that introduction of a methylphosphonate group into the quinoxalinedione skeleton leaves potency as AMPA antagonists and selectivity for the AMPA receptor unchanged and dramatically improves solubility. One such novel phosphonate quinoxalinedione derivative and competitive AMPA antagonist ZK200775 exhibited a surprisingly long therapeutic time window of >4 h after permanent occlusion of the middle cerebral artery in rats and was devoid of renal toxicity. Furthermore, delayed treatment with ZK200775 commencing 2 h after onset of reperfusion in transient middle cerebral artery occlusion resulted in a dramatic reduction of the infarct size. ZK200775 alleviated also both cortical and hippocampal damage induced by head trauma in the rat. These observations suggest that phosphonate quinoxalinedione-based AMPA antagonists may offer new prospects for treatment of stroke and trauma in humans.

Acclimation of Arabidopsis Leaves Developing at Low Temperatures. Increasing Cytoplasmic Volume Accompanies Increased Activities of Enzymes in the Calvin Cycle and in the Sucrose-Biosynthesis Pathway1

Photosynthetic and metabolic acclimation to low growth temperatures were studied in Arabidopsis (Heynh.). Plants were grown at 23°C and then shifted to 5°C. We compared the leaves shifted to 5°C for 10 d and the new leaves developed at 5°C with the control leaves on plants that had been left at 23°C. Leaf development at 5°C resulted in the recovery of photosynthesis to rates comparable with those achieved by control leaves at 23°C. There was a shift in the partitioning of carbon from starch and toward sucrose (Suc) in leaves that developed at 5°C. The recovery of photosynthetic capacity and the redirection of carbon to Suc in these leaves were associated with coordinated increases in the activity of several Calvin-cycle enzymes, even larger increases in the activity of key enzymes for Suc biosynthesis, and an increase in the phosphate available for metabolism. Development of leaves at 5°C also led to an increase in cytoplasmic volume and a decrease in vacuolar volume, which may provide an important mechanism for increasing the enzymes and metabolites in cold-acclimated leaves. Understanding the mechanisms underlying such structural changes during leaf development in the cold could result in novel approaches to increasing plant yield.

Acclimation of Photosynthesis to Elevated CO2 under Low-Nitrogen Nutrition Is Affected by the Capacity for Assimilate Utilization. Perennial Ryegrass under Free-Air CO2 Enrichment1

Acclimation of photosynthesis to elevated CO2 has previously been shown to be more pronounced when N supply is poor. Is this a direct effect of N or an indirect effect of N by limiting the development of sinks for photoassimilate? This question was tested by growing a perennial ryegrass (Lolium perenne) in the field under elevated (60 Pa) and current (36 Pa) partial pressures of CO2 (pCO2) at low and high levels of N fertilization. Cutting of this herbage crop at 4- to 8-week intervals removed about 80% of the canopy, therefore decreasing the ratio of photosynthetic area to sinks for photoassimilate. Leaf photosynthesis, in vivo carboxylation capacity, carbohydrate, N, ribulose-1,5-bisphosphate carboxylase/oxygenase, sedoheptulose-1,7-bisphosphatase, and chloroplastic fructose-1,6-bisphosphatase levels were determined for mature lamina during two consecutive summers. Just before the cut, when the canopy was relatively large, growth at elevated pCO2 and low N resulted in significant decreases in carboxylation capacity and the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase protein. In high N there were no significant decreases in carboxylation capacity or proteins, but chloroplastic fructose-1,6-bisphosphatase protein levels increased significantly. Elevated pCO2 resulted in a marked and significant increase in leaf carbohydrate content at low N, but had no effect at high N. This acclimation at low N was absent after the harvest, when the canopy size was small. These results suggest that acclimation under low N is caused by limitation of sink development rather than being a direct effect of N supply on photosynthesis.

Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice

We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2−/− mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2−/− mice, we used suppressive subtractive hybridization between livers from Usf2−/− and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2−/− hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages.

Capacidade funcional e saúde bucal relacionada à qualidade de vida de idosos

É de extrema importância estudar a relação entre capacidade funcional e saúde bucal relacionado à qualidade vida, para auxiliar nos fatores intervenientes tanto no cuidado odontológico como em outras áreas de atenção à saúde do idoso, além de adequar planos de tratamento as reais necessidades de cada indivíduo, proporcionando melhor qualidade de vida. O objetivo desse estudo foi investigar a capacidade funcional e saúde bucal relacionada à qualidade de vida do idoso. Foram incluídos na pesquisa idosos acima de 65 anos que residem em área de cobertura da Estratégia Saúde da Família de Bauru. Foram investigadas, através de entrevistas, as variáveis capacidade funcional (Índice de Katz), saúde bucal relacionada à qualidade de vida (OHIP-14), condições socioeconômicas e comorbidades. Foram excluídos da pesquisa idosos que apresentavam alterações de compreensão e expressão da comunicação, Para a realização da análise estatística foi utilizado o Teste de Pearson ao nível de significância de 0,05 e a Regressão Linear Multivariada, ao nível de significância de p 0,01. Participaram da pesquisa 238 idosos com 65 anos ou mais, sendo que 56,3% tinham entre 65 e 74 anos e 43,7% tinham 75 anos ou mais, a média geral de idade foi 74,5 anos. Quanto ao sexo, 55,5% eram mulheres, houve predominância da raça branca (65,1%). Quanto a variável capacidade funcional, 8,8% apresentaram incapacidade funcional intermediária ou severa. Sobre os aspectos socioeconômicos, 61,3% dos idosos apresentaram renda mensal de até dois salários mínimos, 88,2% eram aposentados ou pensionistas, 68,9% tinham algum grau de escolaridade e 57,1% eram casados ou mantinham uma união estável. Quanto a saúde bucal relacionada à qualidade de vida, 20,6% dos entrevistados se sentiram desconfortáveis para mastigar alguns tipos de alimentos. A hipertensão arterial foi a comorbidade mais relatada pelos entrevistados (75,6%). As dimensões dificuldade para mastigar (0,21; p<0,05) e necessidade de parar as refeições (0,21; p<0,05) do instrumento OHIP-14 apresentaram correlação estatisticamente significante com o Índice de Katz. Através da análise multivariada, foi observado que quanto maior a incapacidade menos o idoso se preocupa com a saúde bucal. A alimentação é um aspecto importante da vida diária de qualquer pessoa, especialmente o idoso que apresenta doenças crônicas em maior quantidade e severidade conforme o envelhecimento acontece. Além disso, a incapacidade apresentou tendência de aumento com a idade, no entanto a literatura não permite concluir que o envelhecimento em si seja causa da incapacidade funcional. A maior limitação do estudo se relaciona ao próprio desenho epidemiológico, pois o estudo transversal não permite inferências de causalidade, no entanto a amplitude do grupo etário nesta pesquisa permite observar tendências sobre a capacidade funcional e a saúde bucal relacionada à qualidade de vida em idosos. A incapacidade funcional aumentou com a idade e apresentou tendência de maior impacto negativo na saúde bucal relacionada a qualidade de vida dificultando a realização de refeições.

The Relationship Between Social Support and Self-Advocacy in College Students With Disabilities

This study explored the connection between social support and self-advocacy in college students with disabilities. The College Students with Disabilities Campus Climate Survey (Lombardi, Gerdes, & Murray, 2011) was used to gather data from undergraduate students at a midsize western private university. Social support was found to be a significant predictor of self-advocacy in college students with disabilities. Peer support, family support, and faculty teaching practices made up the construct of social support. Peer support and faculty teaching practices were found to be significant predictors of student self-advocacy. Family support was not found to be significant. The data was examined for group differences between genders, disability types, and disability status (high incidence disabilities versus low incidence disabilities). No significant group differences were found. These findings suggest helping students build social support will increase their level of self-advocacy, which in turn may increase academic success.

Anaerobic Digestion Project Working Guide: A Case Study Analysis and Procedural Summary of Anaerobic Digester Projects for the United States Agricultural Sector

Renewable energy such as biomass has given markets, including dairy farms, an effective approach to reducing the costs of sustaining a profitable business. Anaerobic digestion systems offer dairy farms a very effective way to reduce manure odor, comply with soil and water pollution regulations, manufacture compost for general market sales, produce irrigation capacity and generate on-site electricity as well as the ability to sell excess electricity back to the local utilities. This project defines anaerobic digestion technologies and practices, analyzes case studies and presents a step-by-step anaerobic digestion project startup checklist. The result is an anaerobic digestion project working guide that acts as a tool to aid dairy farmers in their own potential anaerobic digestion project.

Ammonia removal using activated carbons: effect of the surface chemistry in dry and moist conditions

The effect of surface chemistry (nature and amount of oxygen groups) in the removal of ammonia was studied using a modified resin-based activated carbon. NH3 breakthrough column experiments show that the modification of the original activated carbon with nitric acid, that is, the incorporation of oxygen surface groups, highly improves the adsorption behavior at room temperature. Apparently, there is a linear relationship between the total adsorption capacity and the amount of the more acidic and less stable oxygen surface groups. Similar experiments using moist air clearly show that the effect of humidity highly depends on the surface chemistry of the carbon used. Moisture highly improves the adsorption behavior for samples with a low concentration of oxygen functionalities, probably due to the preferential adsorption of ammonia via dissolution into water. On the contrary, moisture exhibits a small effect on samples with a rich surface chemistry due to the preferential adsorption pathway via Brønsted and Lewis acid centers from the carbon surface. FTIR analyses of the exhausted oxidized samples confirm both the formation of NH4+ species interacting with the Brønsted acid sites, together with the presence of NH3 species coordinated, through the lone pair electron, to Lewis acid sites on the graphene layers.

An improved procedure for obtaining and maintaining well characterized partial water saturation states on concrete samples to be used for mass transport tests

A conditioning procedure is proposed allowing to install into the concrete specimens any selected value of water saturation degree with homogeneous moisture distribution. This is achieved within the least time and the minimum alteration of the concrete specimens. The protocol has the following steps: obtaining basic drying data at 50 °C (water absorption capacity and drying curves); unidirectional drying of the specimens at 50 °C until reaching the target saturation degree values; redistribution phase in closed containers at 50 °C (with measurement of the quasi-equilibrium relative humidities); storage into controlled environment chambers until and during mass transport tests, if necessary. A water transport model is used to derive transport parameters of the tested materials from the drying data, i.e., relative permeabilities and apparent water diffusion coefficients. The model also allows calculating moisture profiles during isothermal drying and redistribution phases, thus allowing optimization of the redistribution times for obtaining homogeneous moisture distributions.

Self-rated health and mortality: a follow-up study of a Spanish population

Objectives: Self-rated health (SRH) is known to be a valid indicator for the prediction of health outcomes. The aims of this study were to describe and analyse the associations between SRH and health status, socio-economic and demographic characteristics; and between SRH and mortality in a Spanish population. Study design: Longitudinal study. Methods: A sample of 5275 adults (age ≥21 years) residing in the Valencian Community (Spanish Mediterranean region) was surveyed in 2005 and followed for four years. SRH was categorized into good and poor health. The response variable was mortality (dead/alive), obtained from the local mortality register. Logistic regression models were adjusted in order to analyse the associations between SRH and health status, socio-economic and demographic characteristics; odds ratios were calculated to measure the associations. Poisson regression models were adjusted in order to analyse the associations between mortality and explanatory variables; the relative risk of death was calculated to measure the associations. Results: Poor SRH was reported by 25.9% of respondents, and the mortality rate after four years of follow-up was 3.6%. An association was found between SRH and the presence of chronic disease and disability in men and women. A perception of poor health vs good health led to a mortality risk of 3.0 in men and 2.7 in women. SRH was predictive of mortality, even after adjusting for all other variables. In men and women, the presence of disability provided additional predictive ability. Conclusions: SRH was predictive of mortality in both men and women, and acted as a mediator between socio-economic, demographic and health conditions and mortality.