Comparison of two widely used sampling methods in assessing earthworm community responses to agricultural intensification
Introduction
Earthworms are an important component of soil biodiversity in terms of biomass and function, with strong effects on soil structure and processes (Blouin et al., 2013, Brown et al., 2000, Darwin, 1892, Pulleman et al., 2012) and on plants and other soil biota (Andriuzzi et al., 2015, Andriuzzi et al., 2016, Newington et al., 2004, Nuzzo et al., 2015). It is therefore unsurprising that they are the focus of many studies. What may surprise is the lack of conformity in sampling protocols, even though standardized methods exist (Anderson and Ingram, 1993, ISO/DIS, 2006): field studies with similar aims often differ in the technique used to extract earthworms, the amount of soil sampled, the size and number of sampling units. This complicates comparisons between studies, as has been emphasized recently in the earthworm distribution map for Europe by Rutgers et al. (2016). Rigorous comparisons of sampling techniques across multiple land use types are few, and it is not clear how the spatial extent (e.g. sampling area, simple or composite samples) may affect the results. In this study we sought to compare two sampling methods that are widely used in international research and that contribute to global databases. Specifically, we evaluated whether they provide similar results in terms of earthworm community response to agricultural intensification, and derived recommendations in terms of data quality and practicality for future studies as well as for the integration of data obtained by the different methods.
Both methods are based on a combination of hand-sorting and chemical extraction (Anderson and Ingram, 1993, Van Vliet and de Goede, 2006, Bartlett et al., 2010). Hand-sorting consists of excavating a soil monolith and searching for earthworms manually; chemical extraction consists of applying an irritant, non-lethal solution that infiltrates into the soil and induces earthworms to come to the surface. Other methods are available (reviewed in Bartlett et al., 2010), but hand sorting and chemical extraction techniques have the advantage of not requiring specialised equipment, and used together they ensure reliable sampling (Bartlett et al., 2010). Therefore, the current ISO protocol for earthworm sampling in temperate soils recommends a combination of hand-sorting and chemical extraction (ISO (International Organization for Standardization), 2004, Roembke et al., 2006).
Apart from the sampling technique, there are important variations in sampling protocols that may affect the results. Firstly, whether the chemical is applied on the undisturbed soil surface (e.g. Bouché, 1972), or after excavation of the soil below the sample (e.g. Van Vliet and de Goede, 2006); secondly, how many samples are collected and the size (area, depth) of each sample. There is scarce experimental evidence to assist researchers in making choices on trade-offs between sampling area and number of replicates or sub-replicates − for instance, is extracting earthworms from a composite soil sample of several small sub-replicates equivalent to extracting from fewer sub-replicates but larger size?
The two sampling protocols that we compare in this study differ in operational details, sampling area, and the number of spatially distinct sub-samples. Using an ongoing experiment in a European experimental field site, we assessed (1) whether the two methods provided similar results in terms of earthworm abundance (density, biomass) and community characteristics (diversity, ecological groups, development stage, and body size distribution); and (2) whether they provided similar results for earthworm response to agricultural management practices of different intensity. We also discuss merits and drawbacks of the two methods in terms of feasibility (time and labour needed) and statistical variation. Lastly we discuss whether harmonization of the results obtained by different methods is feasible. Our aim was not to identify the better of two competing approaches, not only because we lack reference data provided by an infallible sampling method, but especially because our interest was to evaluate the potential for linking data obtained through different protocols. Assessing whether soil biological data obtained with distinct sampling methods are comparable is a necessary first step before integration of single studies into larger databases.
Section snippets
Sampling methods and study sites
Two earthworm sampling protocols were compared. Method A consists of excavating and hand-sorting a soil monolith (35 × 35 cm, 20 cm deep), and then applying 0.5 L of a 0.2% formaldehyde solution in the resulting pit to extract deep-dwelling earthworms. This method is widely used (Bartlett et al., 2010, Crittenden et al., 2014, Roembke et al., 2006, Van Vliet and de Goede, 2006) and technically based on ISO protocol 23611-1. Method B consists of first applying formalin to a 100 × 100 cm square (three
Earthworm abundance
The two methods differed in their estimates of absolute earthworm abundance, i.e. density and biomass (Table 2). Earthworm densities were not significantly different in 2012, but in 2013 greater numbers were found using Method A (Fig. 1). Most of the earthworm biomass recovered with Method A was obtained through hand-sorting (formalin 11.4% ± 6.0% in 2012, 6.2% ± 2.6% in 2013), and the same was found for earthworm density (formalin 3.4% ± 0.1% in 2012, 6.9% ± 5.4% in 2013). By contrast, for Method B
The two methods lead to the same conclusions on earthworm community response to land use
Although the methods differed in total sampling area, technical protocol (chemical extraction or hand-sorting first), sample type (composite vs continuous), and the volume of hand-sorted soil, the data they produced led to the same inferences about earthworm community response to land use intensification. Both methods showed an increase in earthworm abundance along the intensification gradient of arable rotation system (Cr) − arable rotation system with a grassland phase (CrGr) − permanent
Acknowledgements
We are thankful to Hoël Hotte, Yannick Benard, Sylvain Busnot, Nassirou Maman, Romain Tourel and Alexis Le Coulteux for contributing to the fieldwork, to Florent Lelu for helping with earthworm identification, and to Xavier Charrier (INRA-Poitiers-Lusignan) for access to the site and his great help with the sampling campaign. This work was supported by the EU SNOWMAN program through project SN03–14, “Soil Functional Biodiversity and Ecosystem Services, a Transdisciplinary Approach (SUSTAIN)”.
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