Supplementary Materialsijerph-16-00883-s001. types are native to mainly freshwaters of the northern hemisphere. are often found under rocks, in sandy or coarse substrates, and under leaves, where they predominantly occur in high abundance and, thus, often dominate macroinvertebrate communities . As benthic organisms, gammarids spend significant periods of their life cycle in close contact with the sediment and the water column above . Consequently, gammarids are steadily exposed to not only water-soluble substances in the water column but also to hydrophobic substances that are adsorbed to the sediment and their diet . These hydrophobic compounds are taken up by the gammarids predominantly via food and accumulate in their tissues . Due to the long lifespans of gammarids in combination with low environmental concentrations of substances that elicit sublethal effects, the accumulation of such compounds is possible over quite a while . Therefore, biota become some sort of unaggressive sampler for bioavailable substances  and will be utilized as time-integrative device BLR1 in contamination evaluation. The removal of gammarid tissue and lipids with following evaluation has an sign of the inner focus of substances, which is a more suitable HMN-214 surrogate for biologically effective concentration than external concentrations . A encouraging analytical tool is the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) multi-residue method, which was originally developed to extract and recover residues of pesticides from food matrices, such as fruits or vegetables [37,38]. With QuEChERS, it is possible to effectively examine a wide range of compounds, including highly polar as well as highly acidic and basic ones . The aim of the present study was to bioanalytically evaluate the ecotoxicological state of the River Wurm and to determine the ecotoxicological impact of treated wastewater released predominantly by the WWTP Aachen-Soers and the smaller WWTP Eilendorf into the receiving streams. Therefore, in this study, an in situ feeding inhibition experiment was combined with the quantification of micropollutants in whole-body extracts of gammarids that were HMN-214 collected at sites along the analyzed rivers. 2. Material and Methods 2.1. Study Area and Design The River Wurm has a length of 53 km and has its source in forests south-west of Aachen, Germany. According to a recent statement by the Ministry for Environment, Agriculture, Conservation, and Consumer Protection of the state of North Rhine-Westphalia, the catchment area of the River Wurm is usually dominated by agriculture (42.3%) and settlements or industry (30.6%), while only a small fraction is covered by grassland (13.6%) and forests (9.7%) . Consequently, the Wurm is HMN-214 usually predominantly influenced by agriculture as well as by urban run-off, traffic, industry, and treated wastewater . The River Haarbach is a tributary of the River Wurm, has a length of 13.5 km, and flows into the River Wurm approximately 2 km upstream of the WWTP Aachen-Soers. The WWTPs Aachen-Soers and the Eilendorf have populace equivalents of 458,000 and 87,000, which translate into a total volume of sewage of 98,000,000 L/day and 13,000 L/day, respectively. As a consequence, the Haarbach consists of at least 50% treated effluent, while the proportion of treated wastewater is usually more than 70% of the River Wurm downstream of the WWTP Aachen-Soers; HMN-214 thus, sewage dominates the water program and with regards to product tons  quantitatively. Because of their located area of the WWTPs upstream, the sampling sites W1 and H1 had been considered as guide sites to look for the influence from the WWTPs Aachen-Soers and Eilendorf. The scholarly study area using the seven sampling sites is shown in Figure.