RESUMO
Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants. However, for a timely transmission of results, sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required. Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis, comparative quality control of the analytical procedures is essential to report reliable and comparable results. In this study, we performed an interlaboratory comparison at laboratories specialized for PCR and high-throughput-sequencing (HTS)-based WBE analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic SARS-CoV-2 variants in graduated concentrations and ratios. Samples were sent to the participating laboratories for analysis using laboratory specific methods and the reported viral genome copy numbers and the detection of viral variants were compared with the expected values. All PCR-laboratories reported SARS-CoV-2 genome copy equivalents (GCE) for all spiked samples with a mean intra- and inter-laboratory variability of 19 % and 104 %, respectively, largely reproducing the spike-in scheme. PCR-based genotyping was, in dependence of the underlying PCR-assay performance, able to predict the relative amount of variant specific substitutions even in samples with low spike-in amount. The identification of variants by HTS, however, required >100 copies/ml wastewater and had limited predictive value when analyzing at a genome coverage below 60 %. This interlaboratory test demonstrates that despite highly heterogeneous isolation and analysis procedures, overall SARS-CoV-2 GCE and mutations were determined accurately. Hence, decentralized SARS-CoV-2 wastewater monitoring is feasible to generate comparable analysis results. However, since not all assays detected the correct variant, prior evaluation of PCR and sequencing workflows as well as sustained quality control such as interlaboratory comparisons are mandatory for correct variant detection.
RESUMO
Micropollutants (MPs), especially endocrine disrupting compounds (EDCs), are mainly released from WWTPs into surface water bodies and can subsequently lead to adverse effects in biota. Treatment with ozone proved to be a suitable method for eliminating such MPs. This method was implemented at the WWTP Aachen-Soers by commissioning the largest full-scale ozonation plant in Europe at the moment. Recently, effect-based methods (EBMs) have been successfully proved for compliance monitoring, e.g. estrogenic compounds. Therefore, the impact of ozone treatment on endocrine potential (agonistic and antagonistic) of treated wastewater was investigated using the ERα- and AR CALUX assays. Additionally, the impact on the receiving stream and a potential preload of the water body was assessed. Therefore, the current study could deal as a case study for small rivers being highly impacted by WWTPs. The estrogenic potential was nearly fully eliminated after ozone treatment. Contrary, the antagonistic (anti-estrogenic and anti-androgenic) potential did not show a clear elimination pattern after ozone treatment independent of the applied ozone dosage and control system. Therefore, further investigations are required regarding the antagonistic potential. Additionally, preloading of the receiving stream was found during the study period. One significant impact is a rain overflow basin (ROB) located upstream of the WWTP effluent. The highest endocrine potential was found after a ROB overflow (2.7 ng EEQ/L, 2.4 µg TMX-EQ/L, 104 µg FLU-EQ/L), suggesting that such runoff events after a heavy rainfall may act as a driver of endocrine loading to the water body. This manuscript contributes significantly to the basic understanding of the efficiency of eliminating the endocrine potential of ozone treatment by, e.g., showing that there is a further need for improving the removal efficiency of antagonistic potential. Moreover, it highlights the need to include other point sources, such as ROBs, to assess polluted surface waters comprehensively.