european journal of soil biology 45 (2009) 176–183 available at www.sciencedirect.com journal homepage: http://www.elsevier.com/locate/ejsobi Original article Earthworm collection from agricultural fields: Comparisons of selected expellants in presence/absence of hand-sorting Ce´line Pelosia,*, Michel Bertrandb, Yvan Capowiezc, Hubert Boizardd, Jean Roger-Estradea aAgroParisTech, UMR 211 Agronomie, F-78850 Thiverval-Grignon, France bINRA, UMR 211 Agronomie, INRA/AgroParisTech, F-78850 Thiverval-Grignon, France cINRA, UR 1114, Plantes et Syste`mes Horticoles, F-84914 Avignon cedex 9, France dINRA, UR 1158 Agronomie, F-80200 Estre´es-Mons, France article info abstract Article history: The role earthworms play in soil fertility is under increasing scientific scrutiny, especially Received 8 June 2008 in light of the fact that farmers are seeking to reduce soil tillage. However, there are many Received in revised form discrepancies in earthworm sampling methods. The aim of this study is to compare the 22 September 2008 efficiency of three chemical expellants (formaldehyde, commercial ‘hot’ mustard and allyl Accepted 29 September 2008 isothiocyanate, abbreviated AITC), with two sampling methods: (i) a simple method that Published online 26 October 2008 consisted in spreading an expellant solution on the soil and retrieving earthworms that emerged at the soil surface, and (ii) a combined method that consisted in applying Keywords: a chemical to expel earthworms and then hand-sorting the remaining earthworms from Earthworms the block of soil. Sampling efficiency was measured in terms of earthworm density and Hand-sorting biomass, for endogeic and anecic earthworms. Formaldehyde With the simple method, a higher density of earthworms was sampled using formaldehyde Allyl isothiocyanate and AITC than with mustard. Formaldehyde, AITC and mustard expelled not significantly Commercial ‘hot’ different biomasses of 47.7, 31.9 and 20.5 g mÀ2, respectively, on average over the three Mustard plots. The combined method did not yield a significantly different density or biomass with Sampling method the different chemicals. Agricultural fields Formaldehyde is toxic and commercial ‘hot’ mustard is difficult to standardise and ineffi- cient when used without hand-sorting. Accounting for the accuracy of the sampling methods as well as the toxicity of the chemicals to users and soil organisms, AITC appears to be a reliable and promising chemical expellant whether or not in combination with hand- sorting. Its use would be a step towards standardizing earthworm sampling methods. ª 2008 Elsevier Masson SAS. All rights reserved. 1. Introduction structure [37], and they are central to the ecological func- tioning of agroecosystems [9]. Earthworm sampling methods The role earthworms play in soil fertility is under increasing are the means to obtain accurate data on the number and scientific scrutiny, especially in light of the fact that farmers biomass of individuals mÀ2 in addition to the species and are seeking to reduce soil tillage. Earthworms are known to ecological group composition of a given community. However, positively influence organic matter dynamics and soil there are discrepancies in earthworm sampling methods. * Corresponding author. þ33 1 30815347; fax: þ33 1 30815425. E-mail address: [email protected] (C. Pelosi). 1164-5563/$ – see front matter ª 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2008.09.013 european journal of soil biology 45 (2009) 176–183 177 Authors reviewing earthworm sampling methods have (FAO classification), sampling was carried out in a yearly distinguished between ethological methods and hand-sorting ploughed plot (30 cm depth), labelled plot 1, and in a plot that [1,6,25]. Ethological methods, the efficiency of which depends on had been superficially tilled since 2001, labelled plot 2 (Table 1). earthworm activity, include the use of an electrical, i.e., octet In April 2008 sampling was carried out in another yearly method, or chemical methods to expel earthworms from the ploughed plot (30 cm depth), labelled plot 3, at the Thiverval- soil. Octet method efficiency has been discussed [7,18,35] and Grignon INRA experimental centre (4851’ N, 155’E, elevation different chemicals have been evaluated. Hand-sorting, which is 125 m), near Paris, which has orthic luvisol soils (FAO classifi- very time-consuming and labour intensive [8,38], entails a direct cation) (Table 1). The three plots were chosen in order to assess sampling of earthworms by excavating blocks of soil. Several sampling efficiency in cropping systems under different soil authors have reported the efficiency of a method combining the tillage regimes and thus with differing infiltration rates and application of a chemical expellant with hand-sorting [2,6]. depths [39]. The cropping systems are ‘‘intensive’’ ones, in Generally speaking, they found that hand-sorting is more suit- terms of high yield and the amount of pesticides and fertilisers. able than an expellant solution for small earthworms [26] such as endogeics and juvenile anecics. However, when this method 2.2. Experimental design and earthworm is used alone, the number of larger earthworms such as anecics sampling methods is often under-estimated because these earthworms can easily move into deeper soil. Similarly, expellants may give better Earthworms were collected in May 2006 during their results for large anecic earthworms [8,10,42] but smaller earth- maximum period of activity under temperate climatic condi- worms may be under-estimated because their low mobility tions [4,16] to limit the effect of dormancy. In April 2008, might prevent them reaching the surface. earthworms were collected during a cold week (mean air The chemical expellants commonly used in experimental temperature was near 7 C). Soil water content was 17.9%, sampling are formaldehyde [31], commercial hot mustard 14.8% and 21.0% in plots 1, 2 and 3, respectively. Selected [19,27] and, more recently, allyl isothiocyanate (AITC) [43]. weather conditions during the four weeks before the 2006 and Formaldehyde is the most commonly used one [8,33,34] but it 2008 samplings are presented in Fig. 1. has been recently recognised as a cancer-producing chemical Sampling was undertaken using a metal frame (40  40 cm) [23]. It is also toxic to earthworms, plants and their environ- which was placed on the soil and driven into the ground to ment [17,20]. Despite these findings, it is still recommended by a depth of 1 cm to retain the chemical on the sampling site. the ISO/DIS 23611-1, 2007-02 [14]. The expellant efficiency of Chemicals were applied twice at 10-min intervals. Three litres commercially available mustard paste or mustard powder has of expellant were applied for each experiment. Emerging been questioned [2]. Moreover, because its composition is earthworms were retrieved during a 20-min period after the generally a well-kept manufacturing secret [43], protocols first expellant application. Then, a 40 cm  40 cm  30 cm- cannot be standardised. Allyl isothiocyanate, or AITC, is deep block of soil was excavated from the same place and a natural breakdown product of glucosinolates formed in many remaining earthworms were extracted by hand in the labo- Cruciferae; it is the irritant molecule in commercial ‘hot’ ratory to obtain the results for the combined method. In each mustard. It is not noxious to humans [22] and is easily degraded plot, four replicates were made for each of the three chemicals in soil [3]. The isopropanol, used to dilute AITC in water, is also in a randomised design. Earthworms, preserved in 4% not harmful to human health [12] and rapidly biodegrades in formalin solution, were fresh weighed (without emptying gut water [41]. Only two studies have tested AITC as an earthworm contents), counted and identified at the species level accord- expellant [11,43] and it is still rarely used in field samplings. ing to the identification key established by Sims and Gerard Given these discrepancies in earthworm sampling methods, [36]. The development stage of each earthworm was noted. there is a need to move towards standardization. This study Earthworms are considered sub-adult if they have a full compares the efficiency of three chemical expellants, which have tubercula pubertatis but no clitellum and adult if they are never been evaluated in a single study (formaldehyde, commer- clitellate [36]. They are considered juvenile if they have cial ‘hot’ mustard and AITC) with two sampling methods: (i) neither tubercula pubertatis nor clitellum. a simple method that consisted in spreading an expellant solu- tion on the soil and retrieving earthworms that emerged at the soil surface, and (ii) a combined method that consisted in 2.3. Chemicals applying a chemical to expel earthworms and then hand-sorting the remaining earthworms from the block of soil. Sampling effi- Formaldehyde solution (VWR, France; 36%; density 1.09) was ciency was measured in terms of earthworm density and diluted with water to 2 g lÀ1, according to Bouche´ and Aliaga biomass, for endogeic and anecic earthworms. [5] and Gunn [20]. Following Zaborski [43], AITC was first diluted with isopropanol (propan-2-ol) to give a 5 g lÀ1 solu- tion. Then this solution was diluted with water to reach 2. Materials and methods a concentration of 0.1 g lÀ1, which is considered optimal [43]. Commercial ‘hot’ mustard (store-brand mustard) was thor- 2.1. Sites and cropping systems oughly mixed with water to obtain a solution at a concentra- tion of 15 g lÀ1 [10,20]. Both the mustard solution and AITC Field data were collected in two locations in northern France. In stock solution with isopropanol were prepared in the labora- May 2006, at the Estre´es-Mons INRA experimental centre tory shortly before the experiment because the molecule loses (4952’ N, 300’ E, elevation 85 m), which has haplic luvisol soils its irritating activity after a certain time in water [27] due to its 178 european journal of soil biology 45 (2009) 176–183 Table 1 – Sampling dates, site locations and soil characteristics of the three plots used to compare earthworm sampling methods.