# Evaluating Earthworms' Potential for Remediating Soils Contaminated with Olive Mill Waste Sediments
## 2 Materials and Methods
### Experimental Design  
The experimental design was identical to that in previously conducted research into biochar-spiked soils by [PERSON] et al. [18]; therefore, it is only briefly described in the current paper. OMW toxicity was assessed by two successive bioassays with earthworms. The first one followed the ISO 2007 guidelines [19] for testing the avoidance behaviour response (ABR) of earthworms. The ABR assay consisted of quantifying the ability of earthworms to avoid OMW-amended soil in a two-chamber system (\(265\times 162\times 100\) mm) holding the test soil in a compartment and the control (non-contaminated) soil in the opposite side. The doses of OMW sediment were: 0%, 10%, 20%, 40% and 80% OMW _w/w_ dry mass (each treatment was run in triplicate). OMW sediment was mixed in soil using a spatula, and then moistened to 15% _v/w_ with distilled water. Test boxes containing the OMW-amended and control soils were placed in an acclimatization chamber at 15 \({}^{\circ}\)C and continued dark for acclimatization (24 h). Next, five earthworms were released in each treatment and the containers were kept again for 48 h in the acclimatization chamber to allow earthworms to freely distribute between both soil sections. After this incubation time, individuals were counted in both compartments following the criteria by [PERSON] and [PERSON] [20]. \begin{table}
\begin{tabular}{c c c} \hline
**Parameter** & **OMW Sediments** & **Soil** \\ \hline pH & 9.38 \(\pm\) 0.02 & 8.76 \(\pm\) 0.06 \\ Electrical conductivity (dS/cm) & 1.40 \(\pm\) 0.06 & 0.08 \(\pm\) 0.01 \\ Organic matter (\%) & 3.06 \(\pm\) 0.09 & 0.36 \(\pm\) 0.01 \\ Total organic carbon (\%) & 3.08 \(\pm\) 0.05 & 0.27 \(\pm\) 0.02 \\ Total nitrogen (\%) & 0.26 \(\pm\) 0.05 & 0.03 \(\pm\) 0.01 \\ Total soluble polyphenols (mg/kg) & 873 \(\pm\) 55 & 85 \(\pm\) 6.5 \\ CEC (meq/100 g OM) & 244 \(\pm\) 31 & 381 \(\pm\) 17 \\ Total P (g/kg) & 3.2 \(\pm\) 0.09 & 0.067 \(\pm\) 0.005 \\ K (g/kg) & 11.9 \(\pm\) 0.7 & 3.9 \(\pm\) 0.35 \\ Na (g/kg) & 1.9 \(\pm\) 0.01 & 0.6 \(\pm\) 0.05 \\ PO\({}_{4}\)\({}^{-3}\) (mg/kg) & 32 \(\pm\) 0.6 & 8 \(\pm\) 0.4 \\ SO\({}_{4}\)\({}^{-2}\) (mg/kg) & 0.8 \(\pm\) 0.09 & 0.4 \(\pm\) 0.03 \\ \hline \end{tabular}
\end{table} 
Table 1: Selected physicochemical properties (mean \(\pm\) SD, n = 3) of OMW sediments and soil used in toxicity testing with _Lumbricus terrestris_. Figure 1: Study area at the locality of Mora (Toledo, Spain). Disposal of olive-mill waste (OMW) in open-air evaporation ponds. The second bioassay consisted of a microcosm study whereby earthworms were incubated in OMW-amended soils for 30 d. Treatments (0%, 10%, 20%, 40% and 80% _w/w_ dry mass) were prepared in triplicate as described above, and 2.0 kg (wet mass) of each spiked soil was placed in a plastic container with a holed plastic lid for reducing water loss during incubation. Containers (n = 15) were placed in the thermostatized chamber for 24 h for soil acclimatization, and five earthworms were released in each container after recording body weight. Individuals were maintained in Petri dishes for 24 h to allow gut voiding, and then body weight was recorded. Before adding the worms, we took one soil sample from each replicate, which served to measure the soil enzyme activity at \(t\) = 0 d. Containers were incubated for 30 d at 15 \({}^{\circ}\)C. During this period, earthworms received approx. 5 g of _Morus alba_ leaves per container and week as food, which were put on soil surface. At the end of microcosm experiment, we recovered earthworms, and collected soil samples from the burrow walls and casts (exceta) from the soil surfaces. Soil and cast samples were frozen at -20 \({}^{\circ}\)C until measurement of enzyme activities. Earthworms were put again on placed in Petri dishes (15 \({}^{\circ}\)C for 24 h) to record post-incubation body weight.