RESEARCH ARTICLE Sewage sludge toxicity assessment using earthworm Eisenia fetida: can biochemical and histopathological analysis provide fast and accurate insight? S. Babić 1 & J. Barišić 1 & O. Malev 1 & G. Klobučar 2 & N. Topić Popović 1 & I. Strunjak-Perović 1 & N. Krasnići 3 & R. Čož-Rakovac 1 & R. Sauerborn Klobučar 1,4 Received: 3 October 2015 /Accepted: 11 January 2016 # Springer-Verlag Berlin Heidelberg 2016 Abstract Sewage sludge (SS) is a complex organic by- product of wastewater treatment plants. Deposition of large amounts of SS can increase the risk of soil contamination. Therefore, there is an increasing need for fast and accurate assessment of SS toxic potential. Toxic effects of SS were tested on earthworm Eisenia fetida tissue, at the subcellular and biochemical level. Earthworms were exposed to depot sludge (DS) concentration ratio of 30 or 70 %, to undiluted and to 100 and 10 times diluted active sludge (AS). The ex- posure to DS lasted for 24/48 h (acute exposure), 96 h (semi- acute exposure) and 7/14/28 days (sub-chronic exposure) and 48 h for AS. Toxic effects were tested by the measurements of multixenobiotic resistance mechanism (MXR) activity and lipid peroxidation levels, as well as the observation of mor- phological alterations and behavioural changes. Biochemical markers confirmed the presence of MXR inhibitors in the tested AS and DS and highlighted the presence of SS- induced oxidative stress. The MXR inhibition and thiobarbi- turic acid reactive substance (TBARS) concentration in the whole earthworm’ s body were higher after the exposition to lower concentration of the DS. Furthermore, histopathological changes revealed damage to earthworm body wall tissue layers as well as to the epithelial and chloragogen cells in the typhlosole region. These changes were proportional to SS concentration in tested soils and to exposure duration. Ob- tained results may contribute to the understanding of SS- induced toxic effects on terrestrial invertebrates exposed through soil contact and to identify defence mechanisms of earthworms. Keywords Soil toxicity . Earthworm . Oxidative stress . Lipid peroxidation . MXR . Histopathology Introduction Sewage sludge (SS) is an inevitable by-product of wastewater treatment plants (WWTPs), and its large amounts produced in the past decades represent a rising challenge for waste man- agement. After the implementation of the Directive 91/271/ EEC (1991) regarding wastewater treatment, the production of SS is expected to increase continuously (Egiarte et al. 2008). Currently, the annual production of dry SS in the Eu- ropean Union reaches over 10.96 million tons (He et al. 2014). At the same time, estimated SS production for China is 2.97 million tons, USA 6.51 million tons and in Japan 2 million tons (LeBlanc et al. 2008). An economical and sustainable alternative to SS disposal through composting, incineration or landfill is land deposition of SS as fertilizer of agricultural soils (Latare et al. 2014). Generally, SS is rich in organic matter, plant macro- or micro-nutrient contents and therefore can potentially substi- tute fertilizer and increase dry matter yield of many crops (Silveira et al. 2003; Antolin et al. 2005; Singh and Agrawal Responsible editor: Thomas Braunbeck * R. Sauerborn Klobučar rsauer@pathcon.com 1 Division of Materials Chemistry, Ruđer Bošković Institute, Zagreb, Croatia 2 Division of Zoology, Department of Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia 3 Division for Marine and Environmental Research, Ruđer Bošković Institute, Zagreb, Croatia 4 PathCon Laboratories EU, Prilaz Ivana Visina 1-3, 10000 Zagreb, Croatia Environ Sci Pollut Res DOI 10.1007/s11356-016-6097-3