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The response of microbial communities to releases of hydraulic fracturing flowback and produced water (PW) may influence ecosystem functions. However, knowledge of the effects of PW spills on freshwater microbiota is limited. Here, we conducted two separate experiments: 16S rRNA gene sequencing combined with random forests modelling was used to assess freshwater community changes in simulated PW spills by volume from 0.05% to 50%. In a separate experiment, live/dead cell viability in a freshwater community was tested during exposure to 10% PW by volume. Three distinct patterns of microbial community shifts were identified: (i) indigenous freshwater genera remained dominant in <2.5% PW, (ii) from 2.5% to 5% PW, potential PW organic degraders such as Pseudomonas , Rheinheimera and Brevundimonas became dominant, and (iii) no significant change in the relative abundance of taxa was observed in >5% PW. Microbial taxa including less abundant genera such as Cellvibrio were potential bioindicators for the degree of contamination with PW. Additionally, live cells were quickly damaged by adding 10% PW, but cell counts recovered in the following days. Our study shows that the responses of freshwater microbiota vary by spill size, and these responses show promise as effective fingerprints for PW spills in aquatic environments. [ABSTRACT FROM AUTHOR]