Joint effects and mechanisms of luteolin and kaempferol on toxigenic Microcystis growth-Comprehensive analysis on two isomers interaction in binary mixture

Allelochemicals are widely accepted as promising algaecide to mitigate Microcystis-dominated cyanobacterial blooms (MCBs). Allelopathic algicidal effect of single luteolin or kaempferol against Microcystis had been confirmed, but their joint effect against Microcystis was unclear. This study comprehensively explored time-dependent joint effect and mechanisms of luteolin and kaempferol on Microcystis growth during 14 day-test. The 50%-inhibitory threshold of their mixture (IC_{50 mix}) was verified as 4.872 and 5.211 mg/L at equitoxic ratio, and 5.167 and 4.487 mg/L at equivalent ratio, respectively, on day 8 and 14. Using toxicity unit, isobologram and predictive models, results revealed that luteolin and kaempferol at equivalent ratio interacted additively at lower, median and higher dosages, while at equitoxic ratio interacted additively at lower dosage but synergistically at median and higher dosages in Microcystis on day 8 and 14, implying that their equitoxic mixture posed better algicidal effect against Microcystis. Various dosages of equitoxic mixture concurrently decreased aqueous and total microcystins (MCs) contents along test. Thus, luteolin and kaempferol could be jointly applied as high-efficacy and eco-safe algaecide with declined MCs pollution risks. As mixture dosage elevated, more strongly weakened cellular MCs retention and inhibited cellular photosynthetic pigments content during late stage, as well as decreased aqueous MCs content long test, jointly explained increasing growth inhibition ratio with rising mixture dosage. Yet, cell damage was gradually repaired due to early stimulated antioxidant defense at each mixture dosage, thus cell damage might not be a major reason for inhibited growth under mixture stress. This study provided novel insights and guidance to coupled application of luteolin and kamepferol for mitigating MCBs and decreasing MCs pollution risks.