Examination of residual chloroform interference in the measurement of microbial biomass C by fumigation-extraction Nativ Rotbart a, b , Mikhail Borisover a , Nadezhda Bukhanovsky a , Alla Nasonova a , Asher Bar-Tal a , Adi Oren a, * a Department of Soil Chemistry, Plant Nutrition and Microbiology, Institute of Soil, Waterand Environmental Sciences, Volcani Center, Agricultural Research Organization (ARO), Rishon LeZion 75359, Israel b Department of Soil and Water Sciences, Faculty of Agriculture, Environment and Food Sciences, The Hebrew University of Jerusalem, Rehovot, Israel article info Article history: Received 13 January 2017 Received in revised form 5 March 2017 Accepted 29 March 2017 Keywords: Chloroform fumigation-extraction Residual chloroform Soil moisture level Procedure validity Soil microbial biomass abstract Using the chloroform fumigation-extraction (CFE) method for determining microbial biomass in soils, a presumption is followed, i.e., that chloroform becomes fully evacuated from the fumigated soil before extraction. Otherwise, the C-containing fumigant may comprise an unknown part of the extractable C flush, resulting in certain overestimation of soil microbial biomass. A quantitative assessment of extractable residual chloroform levels in fumigated soils was performed, testing soils varying in clay and organic matter contents. The tests were performed over a broad range of soil moisture conditions as to facilitate a mechanistic interpretation of possible chloroform interactions in the studied soils (i.e., dissolution, sorption). The results support the validity of the CFE method's application in agricultural soils, demonstrating the insignificance of biomass overestimation arising from residual chloroform. © 2017 Published by Elsevier Ltd. 1. Introduction Chloroform fumigation-extraction (CFE) is by far the most widely used method for determining microbial biomass in soils. The pioneering work of Vance et al. (1987) has been cited 6899 times, third of the citing papers being published since 2013 (Google Scholar; accessed at March 5, 2017). The attractiveness of the method arises from the ability to assess the bulk microbial biomass C (or N) rather than just biomarker compounds such as ATP, phospholipids or nucleic acids (Blagodatskaya and Kuzyakov, 2013). Using the CFE method, a presumption is followed, i.e., that chlo- roform becomes fully evacuated from the fumigated soil before extraction. Otherwise, the C-containing fumigant may comprise an unknown part of the extractable C flush, resulting in certain over- estimation of soil microbial biomass. Yet, to the best of our knowledge, evidence for complete evacuation of the chloroform has never been presented. Moreover, the use of smell as means of residual chloroform detection has been explicitly reported in the publication formally prescribing the chloroform fumigation- incubation method preceding the CFE method (Jenkinson and Powlson, 1976) as well as in the Laboratory Manual of the Soil Microbial Biomass Group at the Rothamsted Experimental Station (Grace et al., 2006). It should be emphasized that solubility of chloroform in water is not negligible (8416 mg l 1 at 21  C; Chen et al., 2012). Hence, fumigation of soil maintained at 40% water holding capacity (WHC) as prescribed by Vance et al. (1987) or at 50e60% WHC as found by Ross (1989) to yield maximum extractable-C flush values, might result in certain levels of chloro- form dissolution in soil water as governed by Henry's law (Ong and Lion, 1991). Alternatively, chloroform vapor may undergo sorption interactions with soil clay and/or organic matter (SOM) constitu- ents (Chen and Dural, 2002), possibly hindering its evacuation as well. To our knowledge, only a single work by Alessi et al. (2011) contended with the risk of residual chloroform in the CFE proced- ure, alarmingly reporting significant chloroform levels remaining in the extracts from two fumigated natural loam soils, as well as from pure clay minerals-particularly in their hydrated state. Attempts to remove the chloroform prior to extraction by increasing the evac- uation time, or to remove chloroform in the extracts by sparging them vigorously with nitrogen gas, both failed. The main goal of the current study was to quantify extractable residual chloroform levels in fumigated soils, testing soils varying * Corresponding author. E-mail address: adior@volcani.agri.gov.il (A. Oren). Contents lists available at ScienceDirect Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio http://dx.doi.org/10.1016/j.soilbio.2017.03.018 0038-0717/© 2017 Published by Elsevier Ltd. Soil Biology & Biochemistry 111 (2017) 60e65