# Evaluating Earthworms' Potential for Remediating Soils Contaminated with Olive Mill Waste Sediments
## 3 Results and Discussion
### Microcosm: Impact of OMW on Earthworm Biomarkers  
All individuals (n = 15) from the 80% OMW treatment were found dead during the first two weeks of exposure. Furthermore, the dose of 40% OMW caused a 53.3% mortality after 30 d of incubation. The interaction between OMW treatment and time of exposure had a significant impact of earthworm body weight (\(F_{3,104}\) = 4.2, \(p\) = 0.008) (Figure 2B). While the earthworm weight was similar between treatments at the beginning of the microcosm trial (\(F_{3,56}\) = 2.42, \(p\) = 0.076), it decreased after 30 d of incubation (\(F_{3,48}\) = 15.8, \(p\) < 0.001) in the 20% and 40% treated groups (\(p\) < 0.05, Tukey post-hoc test). We measured a set of biomarkers linked to oxidative stress, neurotoxicity and digestive disruption, using the body wall muscle and gastrointestinal tract as target tissues. We found no significant variation in muscle AChE activity (\(F_{3,20}\) = 1.44, \(p\) = 0.26), which is a common biochemical endpoint of neurotoxicity. However, exposure to OMW-amended soils caused signs of oxidative stress (Figure 3), which were evidenced by alteration of GSH (\(F_{3,17}\) = 9.25, \(p\) < 0.001) and GSSG (\(F_{3,17,5}\) = 4.30, \(p\) = 0.019) concentrations, and GST activity (\(F_{3,184}\) = 6.73, \(p\) = 0.003). The earthworm group exposed to 20% OMW-amended soils had the highest mean concentrations of muscle GSH (13.3 \(\pm\) 4.3 nmol\({}^{-1}\) mg protein, mean \(\pm\) SD, n = 15) and GSSG (3.06 \(\pm\) 1.12 nmol\({}^{-1}\) mg protein, n = 15), and the highest GST activity (310 \(\pm\) 87 nmol\({}^{-1}\) min\({}^{-1}\) mg protein, n = 15) with respect to controls (GSH = 8.20 \(\pm\) 1.35 nmol\({}^{-1}\) mg protein, GSSG = 1.93 \(\pm\) 0.62 nmol\({}^{-1}\) mg protein and GST activity = 202 \(\pm\) 38 nmol\({}^{-1}\) min\({}^{-1}\) mg protein; n = 15). Similar findings were found in the gastrointestinal tract, although the earthworms exposed to 40% OMW-amended soils had also the gastrointestinal GSH and GSSG concentrations significantly higher with respect to the control group (\(p\) < 0.05, Games-Howell post-hoc test) (Figure 3). These data suggest that exposure to OMW-derived toxic chemicals occurred via both the earthworm skin and the gut epithelium, which is in agreement with experimental data of organic contaminant uptake by earthworms [50]. In general, there was a dose-dependent response of digestive enzyme activities to OMW concentrations (Table 2). Lipase activity was the only enzyme with a significant inhibition (39% of controls) response in the gastrointestinal tissue of earthworms exposed to 40% OMW-amended soils (\(p\) = 0.037, Games-Howell post-hoc test). Additionally, there was a slight although significant increase of tissue phosphatase activities of earthworms exposed 10% and 20% OMW sediment. We also measured the activity of these enzymes in the gastrointestinal content, although the amount of sample was sufficient enough for all five enzymes (Table 2). At the dose of 20% OMW sediments, both alkaline phosphatase and \(\beta\)-glucosidase activities of the gastrointestinal content were significantly lower than those of the control group (\(p\) < 0.05, Games-Howell post-hoc test). This finding suggests toxic effects on gut symbionts because the mean activities of these two enzymes did not change (\(\beta\)-glucosidase), or even were higher (alkaline phosphatase), when the activities of gastrointestinal tissue are compared between 0% and 20% OMW treatments. The IBRv2 index was used to assess the biomarker responses. The index clearly showed that the highest OMW concentration induced oxidative stress in earthworms, as evidenced by the high \(A_{\mathrm{i}}\) scores for both GSH and GSSG (Figure 4). Moreover, the \(A_{\mathrm{i}}\) score for the gastrointestinal GSSG concentration of 40% OMW treatment was higher than that of GSH, probably suggesting an oxidation of GSH by free radicals [26].