# Evaluating Earthworms' Potential for Remediating Soils Contaminated with Olive Mill Waste Sediments
## 3 Results and Discussion
### Microcosm: Impact of OMW on Earthworm Biomarkers
Glutathione \(S\)-transferase activity was induced in the muscle tissue of earthworms from all OMW treatments, and in the gastrointestinal tissue of the earthworms exposed to 10% and 20% OMW. It is well known that GST activity plays an important role in the metabolism of xenobiotics by conjugating glutathione with electrophilic metabolites [26; 51]. Figure 3: Response of glutathione-dependent antioxidant mechanisms in the body wall muscle and gastrointestinal tract of earthworms after 30 d of exposure to olive mill waste (OMW)-amended soils. Horizontal solid lines in the univariate scatterplots are the mean values. Different letters denote statistical differences (\(p<0.05\), Games–Howell post-hoc test). GSH = reduced glutathione, GSSG = oxidized glutathione, GST = glutathione \(S\)-transferase, GR = glutathione reductase. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & \multicolumn{4}{c}{**OMW Dose (\%, _uv/uv_)**} \\
**Digestive Enzymes 1** & **0** & **10** & **20** & **40** & _p-Value 2_ \\ \hline _Gastrointestinal tissue_ & & & & & \\ Esterase & 74.2 \(\pm\) 38.5 & 76.8 \(\pm\) 36.4 & 92.3 \(\pm\) 30.7 & 68.0 \(\pm\) 46.7 & 0.407 \\ Lipase & 3.01 \(\pm\) 0.85 & 2.83 \(\pm\) 0.80 ab & 2.56 \(\pm\) 1.20 ab & 1.84 \(\pm\) 0.80 b & 0.038 \\ Glucosidase & 5.76 \(\pm\) 1.06 & 6.78 \(\pm\) 1.78 a & 7.10 \(\pm\) 3.45 ab & 4.90 \(\pm\) 1.22 b & 0.039 \\ Acid phosphatase & 6.30 \(\pm\) 1.16 a & 8.14 \(\pm\) 1.87 b & 7.82 \(\pm\) 4.15 ab & 7.26 \(\pm\) 1.84 ab & 0.028 \\ Alkaline phosphatase & 6.87 \(\pm\) 1.24 a & 8.58 \(\pm\) 2.50 ab & 10.5 \(\pm\) 5.64 b & 9.00 \(\pm\) 4.67 b & 0.034 \\ _Gastrointestinal content_ & & & & & \\ Glucosidase & 1.09 \(\pm\) 0.39 a & 0.85 \(\pm\) 0.25 ab & 0.77 \(\pm\) 0.25 b & -3 & 0.049 \\ Acid phosphatase & 1.55 \(\pm\) 0.70 & 1.65 \(\pm\) 0.63 & 1.21 \(\pm\) 0.47 & - & 0.086 \\ Alkaline phosphatase & 1.50 \(\pm\) 0.46 a & 1.34 \(\pm\) 0.51 ab & 1.01 \(\pm\) 0.49 b & - & 0.029 \\ \hline \hline \end{tabular} \({}^{1}\) Enzyme activity expressed as nmol min\({}^{-1}\) mg\({}^{-1}\) total protein (tissue), and nmol h\({}^{-1}\) g\({}^{-1}\) content (gastrointestinal content). \({}^{2}\) One-way ANOVA test (Weich) homogeneity correction). Different letters denote within-effect differences for each enzyme’s activity (_p_ < 0.05, [PERSON]–[PERSON] post-hoc test). \({}^{3}\) Not measured (insufficient sample amount).  
\end{table} 
Table 2: Digestive enzyme activities (mean \(\pm\) SD, n = 15) in the gastrointestinal tissue and content of _Lumbricus terrestris_ exposed to olive mill waste (OMW)-amended soils. Figure 4: Sunray plots of the deviation index (\(A_{i}\) scores) of biomarker measured in body wall muscle and gastrointestinal tissue of _Lumbricus terrestris_ exposed after 30 d of exposure to olive mill waste (OMW)-amended soils. Dotted lines in sunray plots indicate the reference values (see Materials and Methods for details). Bar plot shows the integrated biomarker response index (IBRv2) values calculated for each treatment and tissue. AChE = acetylcholinesterase, GSH = reduced glutathione, GSSG = oxidized glutathione, GST = glutathione S-transferase, GR = glutathione reductase, AcP = acid phosphatase, AIP = alkaline phosphatase, Gluc = \(\beta\)-glucosidase, Est Est = esterase. Furthermore, GST activity also removes lipid peroxides, thus contributing to reduce cellular oxidative damage [52]. Its enhanced activity has been suggested as a biomarker of oxidative stress in earthworms, the body wall and foregut being the tissues expressing the highest levels of GST activity [53]. Therefore, the response of this antioxidant enzyme in our earthworms indicated oxidative stress induced by chemicals present in the OMW sediments. Biomarker responses of control earthworms measured in this study were comparable to other studies that used the same species [18]. Therefore, we can assume that these are baseline values for these biomarkers, so they can be used as reference values in IBRv2 index calculations [38].