Introduction

The question of whether earthworms are able to restructure compacted soils has been addressed by several authors (e.g. Capowiez et al. 2009; Joschko et al. 1989; Langmaack et al. 1999; Larink et al. 2001). Investigations have been carried out in field and laboratory experiments using natural soil cores or repacked soil cores. Various designs of repacked soil cores have been used to study the behaviour of earthworms. Many authors used packed soil columns in which the soil was compacted in layers (Bastardie et al. 2003; Capowiez et al. 2001; Jégou et al. 1998). Others constructed experimental pots in which earthworms were offered different degrees of soil compaction in a single soil column (Söchtig and Larink 1992; Stovold et al. 2004). As a general problem, it emerged that earthworms preferred the interface between the cylinder wall and the soil, thus compromising the expected result. To prevent this, some authors roughened the walls, for example using a combination of sharp fine sand and sealing varnish (Capowiez et al. 2011).

A new experimental setup to study earthworm behaviour towards compacted soil is presented in this study. Features of the design include (1) the soil column containing the earthworms is surrounded by repellent soil. This should circumvent the edge effect at the cylinder wall because the earthworms are kept within the soil column by chemical avoidance, (2) a compacted soil block is placed on top of the soil column containing the earthworms. This ensures a closer resemblance to the situation in the field where earthworms may be in their burrows below the compaction zone.

To examine the experimental setup, a laboratory experiment was conducted using two different degrees of compaction to establish which soil bulk densities are accessible to earthworms.

Materials and methods

Soil and earthworms

Different soils were used. “Habitat soil” [52.5 % sand, 43.2 % silt, 4.3 % clay, 3.47 % total organic C, and pH(CaCl2) 7.3] was taken from an agricultural field. “Repellent soil” [45.5 % sand, 49.6 % silt, 4.9 % clay, 5.23 % total organic C, and pH(CaCl2) 3.1] was taken from the topsoil of a spruce forest. Both were dried at 60 °C and sieved to 2 mm. Furthermore, the proctor compaction test (Proctor 1933) was conducted with the habitat soil. In this test, the relationship between water content and dry unit weight (bulk density) of a soil sample at a constant work of compression is determined under controlled conditions. The maximum dry unit weight amounts to 1.7 g cm−3 at an optimum water content (w/w) of 17.8 % (mould, 100 mm diameter; 120 mm height; rammer, 2.5 kg; drop, 300 mm; blows, 25).

Adult individuals of the anecic earthworm Lumbricus terrestris L., obtained from a commercial supplier (Denus Würmer, Stuttgart) were used for the experiment. The worms were adapted to experimental conditions for 15 days in the habitat soil. During the adaptation period, the following parameters, according to Lowe and Butt (2005), were adjusted: 15 cm soil depth, 70 % field capacity, 24 h dark, <1 cm food particle size, and a stocking density of three adults per litre. The earthworms were fed with 1 g rolled oats per individual, applied to the soil surface.

Experimental setup

Figure 1 shows a scheme of the experimental setup. A vessel consists of a polyvinyl chloride (PVC) pipe with a perforated PVC cap at the end, fixed by two polyoxymethylene (POM) plugs. First, a geotextile (20 g m−2) was fitted into the vessel and the repellent soil was artificially compacted to a bulk density of 0.8 g cm−3 (layer 1). A stainless steel pipe (wall thickness 0.6 mm) was then placed in the centre of the vessel with a combination of a ring and three spacers (cf. Fig. 1). Subsequently, the habitat soil was poured into the centre and the repellent soil filled in around the stainless steel pipe. The filling aids were then removed and the soil was compressed (habitat soil, 1.3 g cm−3; repellent soil, 0.8 g cm−3). This process was repeated three times (layers 2–5). Finally, the compacted test soil block was placed on top and surrounded with wetted repellent soil (21.5 % water content, w/w) compressed to a bulk density of 0.8 g cm−3 (layer 6). The test soil block consisted of compacted habitat soil (pH 7.3). Each compaction was induced using a round POM plate and a drop hammer.

Fig. 1
figure 1

The experimental setup (33 cm high and approximately 24 cm internal diameter; lid, 15 cm diameter; core, 10 cm diameter; layers 1–5, 5 cm high; layer 6, 4 cm high; and hole, 2 mm diameter) and the filling aids (stainless steel pipe and a ring with three spacers) in a vessel during the construction of layer 5 (right)

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Pretest for avoidance of acid soil

2D terraria were used to verify that L. terrestris is repelled by the acid soil (repellent soil). 2D terraria consisted of two glass plates (20 × 14.5 cm, distance of 0.9 cm) framed by plastic rails. Habitat soil and acid soil were filled in the left and right half of the terrarium, respectively, resulting in a bulk density of 1.3 g cm−3 (habitat soil) and 0.8 g cm−3 (repellent soil). The soil in the terrarium was wetted by capillary rise from a water bath for 3 days. At the start of the test, one L. terrestris (live weight 3.53–4.21 g) was placed on the soil surface. After 7 days, the traces of earthworm burrows and casts were recorded on transparencies and the trace area calculated for each soil, respectively. The test was performed using five replicates.

Experimental procedure

The study was conducted in a cooling chamber at 13–14 °C. Ten experimental pots (EP) were prepared with different soil blocks and positioned in a randomised order. According to the degree of compaction in forest soils examined by Ampoorter et al. (2010) and Goutal et al. (2012), five pots with test soil blocks of 1.3 g cm−3 were prepared. Furthermore, five pots with test soil blocks of 1.7 g cm−3 bulk density, referring to the maximum dry unit weight of the proctor compaction test, were prepared.

Every soil block had a water content of 17.8 % (w/w). The pots were built up to layer 5 and then moistened by capillary absorption. After moisture saturation of the core, two holes (5 mm diameter; 2 cm deep) were stabbed into the core with a glass rod. Two earthworms with a total fresh biomass (without gut clearance) of 9.3 ± 0.6 g per vessel were then placed in the holes. Once the earthworms had dug themselves in completely, the test soil block was placed on top and surrounded with the repellent soil. Subsequently, 10 straws of Hordeum sp. were placed parallel on the surface as an indicator of earthworm activity (5 cm length; collectively 0.3 g dry weight). To prevent the earthworms from escaping, a perforated film was affixed by rubber band to the top of each vessel. The soil surface was controlled daily for signs of earthworm activity.

X-ray computed tomography

The experimental pots were scanned by X-ray computed tomography (120 kV, 250 mA s−1) using a Toshiba Aquilion 64. Subsequently, the scan slices were examined using the software iQ-Lite Viewer (version 2.1.0 R1). The scan images verify that the experimental setup was constructed in compliance with the scheme in Fig. 1.

Expulsion of the earthworms and handling of the soil blocks

The earthworms were extracted using allyl isothiocyanate (Zaborski 2003). The expellant solution (80 μl allyl isothiocyanate, 1.6 ml methanol, 1 l water) was injected into the burrow holes using a syringe. With the exception of EP10 (no burrow hole), earthworm sampling was realised without the removal of the compacted soil block. It was then possible to remove the soil blocks compacted to 1.7 g cm−3 without destroying them. The adherent soil and earthworm casts were carefully cleaned from them. Although it was not possible to remove the soil blocks with a bulk density of 1.3 g cm−3 without destroying them, traces could be recorded on the top of the soil blocks. To quantify earthworm activity, the traces on the soil blocks were copied on transparency films. The transparency films were scanned (resolution 300 dpi) and the trace areas determined using the software ImageJ (version 1.44p).

Statistical analyses

The measurements were presented by the arithmetic mean and the standard error of the mean. A paired t test (p < 0.05) was used to compare the earthworm traces (cast and burrow areas) of the different soils while the pretest for avoidance.

Results

The pretest for avoidance shows that the areas of cast of habitat (pH 7.3) and repellent (pH 3.1) soils were 35.27 ± 3.31 and 4.08 ± 1.38 cm2, respectively; those for the burrow traces were 9.11 ± 1.66 and 1.35 ± 0.41 cm2, respectively. The cast areas and burrow areas were significantly larger in the left half of the terraria. This means that the acid soil was avoided by L. terrestris to a great extent. This was confirmed by 2D terraria containing only habitat soil (data not shown).

The soil blocks with a bulk density of 1.3 g cm−3 had been penetrated completely within 1 day. In most cases, the burrow holes showed radial cracks. In contrast, only one compacted soil block (EP7, bulk density 1.7 g cm−3) demonstrated a round burrow hole with a depth of 21 mm at the bottom of the soil block (cf. Fig. 2, incomplete 1.7 at day 15). Further burrow holes were observed at the periphery between the soil block and the coat in the repellent soil. In some cases, initial middens were observed on the surface. Figure 2 shows the distribution of the burrow holes over time. There was greater earthworm activity at the lower bulk density of the soil blocks. Each burrow wall in the repellent soil was covered with casts (coloured like the habitat soil).

Fig. 2
figure 2

Development of burrow holes in compacted soil blocks in experimental pots containing two L. terrestris (five replicates). Penetrating = burrows penetrate compacted soil block. Marginal = burrows at margin between compacted soil block and surrounding repellent soil. Incomplete = burrow at bottom of soil block, not penetrated. There were no penetrating holes in soil blocks of 1.7 g cm−3 bulk density

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Earthworm traces appeared as holes (2–10 mm diameter, 1–3 mm deep) on the soil surface. These traces occurred at the top, side and bottom of each heavily compacted soil block, except for the soil block of EP 10 which had only traces at the bottom. The mean area of traces was 4.15 ± 1.22 cm2 at the bottom, 2.91 ± 1.44 cm2 at the side and 10.57 ± 5.69 cm2 at the top of the soil blocks compacted to 1.7 g cm−3. This amounts to around 3.3 % of the total soil block surface. On the surface of the low density soil blocks, the mean area of traces was 16.09 ± 1.77 cm2 at the top. This was twice as much as in the heavily compacted soil blocks. It was not possible to quantify earthworm traces at the side and bottom of the 1.3 g cm−3 soil blocks because the blocks disintegrated during investigation.

All earthworms dug in by directly forcing themselves into the soil after the earthworms were placed in the stabbed holes. There was no indication of soil uptake. It took between 25 and 45 min for the earthworms to disappear into the soil. In some cases, an earthworm left the stabbed hole and had to be placed again. For the expulsion of the earthworms 10–40 ml of the expellant solution was required per experimental pot. In two cases (EP2, EP9), the earthworms remained at the surface. Apart from one earthworm (EP6), all individuals survived (average weight loss 21 %). On a single occasion, an earthworm showed injuries most likely caused by placing the soil block on top of the habitat core at the start of the experiment (EP2).

Discussion

The two ways in which earthworms tunnel through soil can be characterised as “displacement burrowing” and “ingestion burrowing”. Displacement burrowing takes place in soil voids widened by radial pressure exerted by the earthworm (Lee and Foster 1991). The soil around the burrow then becomes compacted. Schrader et al. (2007) demonstrated this for L. terrestris. In our experiment, the radial cracks in soil blocks and the total penetration of 4 cm of 1.3 g cm−3 compacted soil within 24 h are evidence that L. terrestris traversed the soil block by displacement burrowing. Superficial traces in 1.7 g cm−3 soil blocks and a non-penetrating burrow at the bottom side of one soil block suggest that L. terrestris attempted to perform ingestion burrowing in the soil blocks compacted to 1.7 g cm−3. However, the earthworms did not manage to penetrate 4 cm of 1.7 g cm−3 compacted soil within 15 days.

As a chemical barrier, the acid soil (pH 3.1) should prevent the edge effect at the cylinder wall. Nevertheless, L. terrestris tunnelled through the aversive soil to reach the soil surface by creating burrows lined with habitat soil. As a result, the earthworms were able to overcome the chemical avoidance and circumvent the experimental setup.

It was not possible to remove the 1.3 g cm−3 soil blocks for investigation. For this reason, the experimental setup is not suitable for soil blocks with a low degree of compaction. The capillary water rise may have increased the water content in the soil blocks, reducing soil strength. The insertion hole should be made deeper to accelerate the invasion of the earthworms and to avoid injuries when placing the compacted block on top of the habitat core. In this study, two grades of compaction were applied without amendments. It would be possible to conduct investigations using several ingredients (e.g. supplementary food sources). Furthermore, the soil block can be removed for further investigation (e.g. diffusion of gases, development of roots). In addition, the experimental setup allows further research to be undertaken on the behaviour of different ecological types of earthworm and on other earthworm species.

Conclusions

The presented experimental setup is novel in superimposing a compacted soil block on top of the earthworm habitat, thereby more closely resembling the compaction event for the earthworm in the field. In addition, the repellent soil should circumvent the wall problem of the experimental container. However, our results show that L. terrestris could circumvent the experimental setup by creating burrows (lined with earthworm casts) in the acid soil. It would be interesting to compare the edge effect between a habitat and a repellent soil with the edge effect between a cylinder wall and the soil.