Contents lists available at ScienceDirect Geoderma journal homepage: www.elsevier.com/locate/geoderma Laboratory analysis of soil respiration using oxygen-sensitive microplates A. Criado-Fornelio ⁎ , C. Verdú-Expósito, T. Martín-Pérez, C. Moracho-Vilrriales, I. Heredero-Bermejo, J.L. Copa-Patiño, J. Soliveri, J. Pérez-Serrano Departamento de Biomedicina y Biotecnología, Facultad de Farmacia, Universidad de Alcalá, 28871, Alcalá de Henares, (Madrid), Spain ARTICLE INFO Handling Editor: Bernd Marschner Keywords: Soil Microcosm Extracts Respiration Chemicals Amendments ABSTRACT Soil respiration is usually monitored by measuring CO 2 efflux. Most techniques available for such kind of analyses are inconvenient and difficult to adapt to micro-method format. The present study evaluates a new microplate-based method for studying soil respiration in the laboratory. Second-generation oxygen-sensitive microplates (OSM - containing a fluorescent probe attached to the bottom of the well which provides time- resolved fluorescence data) were used to measure soil respiration either in microcosm assays or in soil water extracts. The latter procedure (water extracts) was the least cumbersome, hence it was selected for further experiments. Soil respiration curves generally showed no lag phase, starting with an exponential oxygen consumption phase, followed by a period where respiration became stable after 8–10 h of incubation at 25 °C. Once the procedure for measurement of oxygen consumption in soil was established, the acute toxic effect of diverse chemicals on soil was analysed with OSM. Streptomycin and penicillin failed to reduce soil respiration. Kanamycin plus neomycin, trimethoprim and 5-fluorocytosine exhibited limited inhibitory effects. In contrast, some chemicals (copper sulphate and amphotericin B) and fungicides (such as dodine and fosetyl) noticeably reduced fluorescence readings, showing concentrations to give half-maximal inhibition of respiration (ICR 50 ) ranging from 0.0064 to 0.082 g/L. Finally, some insecticides and soil amendments assayed were either neutral or increased respiration. It is concluded that OSM are reliable, convenient, and yield quantitative results. Moreover, the system is relatively inexpensive and amenable to automation. However, results obtained using soil water extracts may be different from those derived from undisturbed soil aggregates, clods or slurries studied under field conditions. 1. Introduction Inadequate use of pesticides and fertilizers causes worldwide concern. Monitoring environmental changes in terrestrial ecosystems caused by chemical pollution requires a better understanding of soil respiration. The latter is defined as the production of carbon dioxide by heterotrophic soil microorganisms (mainly bacteria and fungi - Sandor, 2010). However, the CO 2 efflux is not always an accurate measurement of the respiration rate, since under field conditions carbon dioxide may migrate with soil water, be part in a carbonate dissolution reaction or in plant root uptake (Angert et al., 2015). In addition, CO 2 efflux is often measured with equipment obtaining data in situ, which show great differences in accuracy, spatial and temporal resolution and applic- ability (Janssens et al., 2000). On the other hand, in a closed environmental system, CO 2 measurement is hampered in alkaline (calcareous) soils for artefacts due to CaCO 3 –CO 2 –H 2 O equilibria (Oren and Steinberg, 2008). One of the best options available for laboratory studies on soil CO 2 efflux are Microresp® microplates, an indirect system, amenable to automation, which measures respiration as a halochromic indicator changes colour when CO 2 reacts with bicarbonate (Campbell et al., 2003). The CO 2 production/O 2 consumption ratio is another parameter that can be used in respiratory studies, but it varies with the kind of substrate consumed and may be biased when [O 2 ] drops below 0.5% due to partial anaerobiosis. Therefore, the best alternative to CO 2 determination under aerobic conditions is the analysis of oxygen consumption. There are diverse methods for measuring dissolved oxygen. Jorge et al. (2004) described an approach based on a device using fluorophores and optical fibres to determine [O 2 ] in water. In edaphology, a number of studies based on the use of electrodes and microelectrodes for oxygen detection have been published (Reddy et al., 1980; Sexstone et al., 1985; Pang et al., 2007; Fan et al., 2014; Angert et al., 2015). Unfortunately, the methods used by these authors showed low sensitivity or proved uneconomic or cumbersome. In contrast, Garland et al. (2003) performed their soil respiration analyses choosing a different approach, based on the BD oxygen microsensor http://dx.doi.org/10.1016/j.geoderma.2017.05.034 Received 19 October 2016; Received in revised form 16 May 2017; Accepted 19 May 2017 ⁎ Corresponding author. E-mail address: angel.criado@uah.es (A. Criado-Fornelio). Geoderma 305 (2017) 12–20 0016-7061/ © 2017 Elsevier B.V. All rights reserved. MARK