## 2 Materials and Methods
### Sampling and Analysing  
The long-term test lasted for almost two years. Automatic samplers continuously collected flow-propositional samples of the untreated wastewater, the MBR-effluent and the final effluent after the GAC-biofilter and stored them cooled. Each week, composite samples were collected and frozen. During start-up and after some weeks of operation, weekly composite samples were sent for analyses. With the analyses at hand, coming weeks for analyses were planned for or previously collected samples were added in order to cover periods with significant changes. Grab samples for bacteria analyses were collected at the final day of a sampling campaign. Investigated micropollutants include a wide range of relevant pharmaceuticals and other emerging substances, oestrogen effect, bacteria and microplastics (see Table S1 for details about investigated substances). Generally, triplicate analyses were performed on all samples. Only certified laboratories were utilized in the project. Thus, standard analytical methods for all analyses and are not described in detail here. Pharmaceuticals and microplastic particles were analysed at IVL's own certified laboratory using the following methods. Pharmaceuticals were analysed using aliquots of 100 to 200 mL thawed composite samples that were spiked with 50 \(\mu\)L internal standard carbamazepine-13C15N (2000 ng/mL) and ibuprofen-D3 (2000 ng/mL). One millilitre of 0.1 wt% ethylenediaminetetraacetate (EDTA-Na2) dissolved in  
Figure 1: Schematic illustration of the pilot setup including a membrane bioreactor MBR and granulated active carbon (GAC)-biofilter for removal of micropollutants. methanol:water (1:1) was added. Prior to extraction using solid phase extraction (SPE) cartridges (Oasis HLB, 6 mL, Waters), the sample was shaken. Cartridges were conditioned with methanol followed by Milli-Q (MQ) water. Thereafter, the samples were applied to the columns at a flow rate of two drops per second. The substances were eluted from the SPE cartridges using 5 mL methanol followed by 5 mL acetone. The supernatants were transferred to vials for final analysis on a binary liquid chromatography (UFLC) system with auto injection (Shimadzu, Japan). The chromatographic separation was carried out using gradient elution on a C18 reversed phase column (dimensions 50 \(\times\) 3 mm, 2.5-\(\upmu\)m particle size, XBridge, Waters, UK) at a temperature of 35 \({}^{\circ}\)C and a flow rate of 0.3 mL/ min. The mobile phase consists of 10 mM acetic acid in water. In addition to pharmaceuticals and phenolic compounds in water, also the contents in the filter material was analysed at the end of the experiment. Pharmaceuticals residues in the carbon were determined after representative samples were taken, dewatered and freeze-dried. The substances were extracted with acetone: acetic acid (20:1). The eluate was then treated as for the water samples. Even so, replicate analyses have been performed, complex wastewater and filter material matrixes imply challenges during sample preparation and analyses. For example, other organic substances can reduce the recovery during sample preparation and affect the signal during analysis or some substances to be analysed can interact with free ions from the matrix and form chelate complex, which result in reduced recovery and detection. As the test are based on real wastewater including the daily, weekly and seasonal quality variation, analyses uncertainty varies during the 2 years of analyses as also the water matrix varies. Therefore, only average values of replicate analyses are presented in this study. Microplastic particles were analysed by following method [48] commonly used in screenings in Nordic countries as standards for microplastic analyses are not yet established. The water samples were filtered through filters with a mesh size of 20 \(\upmu\)m and the material collected on the filters was analysed with a stereo microscope (50 times magnification).