Effectiveness of Lumbricidae Extracting with an Environmentally Friendly Method

Lumbricidae, as representatives of edaphone, are used in ecological research. Over the years, various attempts to improve the ways of earthworm extraction have been made. One of the pro-environmental methods for extracting these animals from the soil is the method involving the application of electric current (octet). The aim of the study was to compare the efficiency of the earthworm extraction using the mixed method (manual segregation along with the use of 0.5% formalin solution) with the method applying the electroshocking. The species composition of earthworms extracted using both methods was the same (seven species of earthworms belonging to three ecomorphological groups were found). A lower number and biomass of earthworms were found in the permanent grassland and arable land with the help of octet than using the mixed method. Further research is needed to improve the existing methods for Lumbricidae extraction and to implement new ones that would be safe for the edaphon representatives.


INTRODUCTION
Due to the specific lifestyle, Lumbricidae play a key role in soil environments as "ecosystem engineers" [Jouquet et al. 2006]. These organisms are subject to ecological worldwide research. In order to determine the qualitative and quantitative composition of earthworms in a given environment, both correct soil sampling and subsequent animal extraction are necessary. There are two groups of methods for obtaining Lumbricidae from the environment, i.e. physical and behavioral [Bartlett et al. 2010, Valckx et al. 2012]. The first group consists of manual sorting of the soil or washing the soil sample with a stream of water and then sieving it on sieves of varying mesh size. In turn, the behavioral methods are based on the extraction of Lumbricidae using, e.g. chemical, electrical or thermal means. A combination of the methods from both groups is also used, i.e. manual segregation and extraction using a chemical agent [EN ISO 23611- 1: 2006]. The advantage of manual soil sorting is the ability to find Lumbricidae in all developmental stages: mature, young individuals and cocoons as well as active forms and those being at rest -diapause [Gutierrez-Lopez et al. 2016]. However, the young and small specimens are often omitted [Jiménez et al. 2006]. In turn, the disadvantages of the screening method include low efficiency and damage to individuals arising under the influence of e.g. a water jet. This makes it difficult to identify the collected material [Valckx et al. 2012].
The behavioral methods usually use a solution of formaldehyde (formalin), mustard suspension and allyl isothiocyanate (AITC) [Butt et al. 2010, Singh et al. 2015. High efficiency and shorter extraction time, e.g. compared to manual screening, have made the chemotactic methods widely used in the studies of Lumbricidae clusters [Valckx et al. 2012]. Despite the high efficiency in earthworm extraction, formalin is a toxic compound for the plant, animal and human organisms [Conolly et al. 2002[Conolly et al. , Čoja et al. 2008]. The irritants contained in mustard (allyl isothiocyanate) are less harmful compounds to the environment. For this purpose, a ready-made commercial mustard emulsion is used [Pelosi et al. 2009] or mustard powder extract is prepared [Eisenhauer et al. 2008]. The disadvantage, however, is that the concentration of AITC in mustard and mustard powder is not exactly known and may vary in the starting material used to make solutions, which hinders the standardization of the method [Valckx et al. 2012]. The work on the application of onion (Allium cepa L.) extracts is also underway; however, implementation of this method requires further research in diverse habitats [Steffen et al. 2013, Singh et al. 2017].
In addition to the use of mustard and onion, the method using low intensity electric current (octet) is characterized by low harmfulness to the soil organisms [Thielemann 1986, Schmidt 2001. In Poland, the use of this pro-environmental method is not widespread.
The aim of the study was to determine the efficiency of Lumbricidae extraction from the soil using low intensity electric current, compared to the mixed method recommended by ISO [EN ISO 23611-1: 2006].

MATERIAL AND METHODS
The research was carried out at two sites -permanent grassland (50 a) and arable land sown with cereals (5 ha) ( Table 1)   direct voltage is supplied from the battery and directed to the inverter, where it is converted into 230 V alternating voltage. From there, i.e. the regulated autotransformer, it is fed to the transformer, where depending on the needs, it is possible to increase the voltage to 450 V. The current with the appropriate voltage is directed from the transformer to the soil probes. The voltage and current parameters at the output to the soil probes are measured by a voltmeter and ammeter ( Figure 1). The time for each sequence is 2.5 min.
The earthworms for analysis were collected from the central part defined by the ring and probe (Figure 1).
In the test sites, apart from the collection of earthworms with the octet method, a mixed method of Lumbricidae extraction proposed by ISO [EN ISO 23611-1: 2006] was used in parallel. For this purpose, the 25×25×25 cm soil blocks were sorted manually. From the deeper layers of the soil profile, the earthworms were extracted by pouring the surface of the well formed after selecting the sample with 10 liters of 0.4% formalin solution (HCHO from CHEM-PUR). Applying Zisci's recommendations [1962], eight test samples were taken each time at the sites (using the current method and the mixed method), determining them randomly by casting a metal frame. The samples were collected annually from May to November 2012 and from April to November 2013. Identification of the found species was carried out using the keys for marking the Polish terrestrial Oligochaeta [Kasprzak 1986].
At the test sites, the soil temperature and humidity were measured each time at a depth of 0-20 cm and beneath. The soil moisture was determined with a drying method at 105°C [PN ISO 11465: 1999]. The soil was also taken from each site for pH analysis in KCl, organic carbon (using the Tiurin method in the Nikitin modification), the content of available phosphorus, potassium and magnesium forms according to Mehlich III [MAFF1981].
In order to characterize the Lumbricidae population, Shannon-Wiener (H) indices were determined: (1) where: pi is the number of specimens of a particular species vs. population size of all organisms.
The data obtained are presented as mean ± standard deviation (SD). The results were statistically analyzed in the STATISTICA ver. 10 software using the Student's t-test or Mann-Whitney U test (if the assumptions for parametric tests were not met).   (Table 2). With the use of electricity, both the permanent grassland and the arable land have lower density of earthworms (22.03 ± 4.12 ind.·m -2 and 18.21 ± 4.15 ind.·m -2 , respectively) compared to mixed extraction method (101.01 ± 18.03 ind.·m -2 and 82.12 ± 18.26 ind.·m -2 ) (p < 0.001) ( Table 3). A similar trend was observed for Lumbricidae biomass (p < 0.001). Shannon-Wiener diversity index on the meadow and arable land was similar for both methods (p > 0.05) ( Table 3).

Seven earthworm species were identified in
The density and biomass of earthworms from individual ecomorphological groups extracted applying the octet method were lower compared to the mixed method (p < 0.05) ( Table 4).

DISCUSSION
The use of electricity to extract the earthworms has been tested, among others, by Tiho and Josens [2000], Schmidt [2001], Čoja et al. [2008] and Butt et al. [2010]. In this method, the voltage is transmitted from the soil probes of the octet device to the soil column, which irritates Lumbricidae and causes them to escape to the soil surface.
In this study, the species structure of Lumbricidae extracted by means of the octet method coincided with that obtained applying the mixed method recommended by ISO [EN ISO 23611-1: 2006]. Seven earthworm species belonging to all three ecomorphological groups were found using both methods (Table 2). It can be assumed that the use of electric current reflects the qualitative structure of earthworms in a given area. This is confirmed by the similar values of the Shannon-Wiener index at the tested sites for the earthworms extracted with electric current and the mixed method ( Table 3). The analysis of the qualitative structure of earthworms extracted with the help of the octet device is recommended in the places where the chemical extraction is prohibited or ineffective. This method can be more effective on slopes, in urban areas or in the areas where minimal environmental disturbances are required, i.e. in protected areas or natural communities [Schmidt 2001]. Staddon et al. [2003] have shown that this method is safe for the plant and animal organisms. The electroshocking did not have any negative effect on the groups of Enchytraeidae, nematodes, Collembola and mites [Blair et al. 1995]. Lower density and biomass of earthworms in the study area obtained with the help ab -statistically significant differences (p < 0.05). aa -statistically insignificant differences (p > 0.05). of octet (Table 3) could be due to low soil moisture in the summer months. Low water content in the soil is one of the factors limiting this method [Schmidt 2001, Szlavecz et al. 2013]. Eisenhauer et al. [2008] verified the improvement of octet efficiency by adding water to the dry soil, from which the samples were previously taken. The addition of water to dry soil did not increase the efficiency of the method. This was probably due to the earlier transition of some species into the state of anabiosis caused by adverse environmental factors. It was also found that the extraction of the earthworms from the endogeic group was more effective with the octet method than using formalin. In this study, with the help of the octet method, in turn, a lower number and Lumbricidae biomass from all three ecomorphological groups were recorded ( Table 4). The effectiveness of the octet method largely depends on the type of soil, vegetation cover and pH of the substrate [Weyers et al. 2008]. More earthworms are extracted from the low pH soil [Čoja et al. 2008].

1.
In the studied ecosystems, the species composition of Lumbricidae found with the help of the octet method coincided with the structure of earthworms extracted by means of the mixed method. The octet method, due to its pro-environmental nature, can be used to identify the qualitative structure of Lumbricidae in the protected areas.

Lower number and biomass of earthworms
were found at the tested sites applying octet compared to the mixed method. Extraction of earthworms using electric current did not reflect the quantitative structure of earthworms.
The effectiveness of the electroshocking method depends largely on the moisture content of the substrate, soil type and type of plant cover. 3. The research on the effectiveness of the Lumbricidae extraction with a help of an octet device requires continuation in diverse ecosystems. ab -statistically significant differences (p < 0.05). aa -statistically insignificant differences (p > 0.05).