Multi-Trophic Biological Responses and Ecological Integrity Diagnosis Along a Water-Quality Pollution Gradient in the Lixiahe Plain River Network, China

Plain river networks are weakly flushed and frequently regulated, which can decouple instantaneous water quality from biological responses. We integrated physicochemical variables, phytoplankton, zooplankton, benthic macroinvertebrates and fish environmental DNA (eDNA) to diagnose ecological condition in the Lixiahe plain river network, China. Regional water quality was assessed at 62 sites, and coupled analyses used 36 sites with complete multi-trophic data. Moderate pollution accounted for 62.90% of classified sites, with total nitrogen as the principal pressure. After Benjamini–Hochberg correction, seven independent associations with primary biological indicators remained significant, mainly linking TOC and TDS with planktonic metrics; the TOC-MEII correlation was treated separately as a non-independent composite association. Mean fish eDNA observed OTU richness declined to 22.33 at the three severely polluted matched sites, although grade-wise contrasts were descriptive because both extreme groups contained only three sites. Adjusted R2 values for the four RDA models ranged from 9.8% to 11.4%; all overall fixed-seed permutation tests were significant (p = 0.0002–0.0298), indicating a detectable but limited water-quality signal. The study-specific MEII ranged from 29.50 to 67.42 (mean 48.04). These results show asynchronous trophic responses and support MEII as a preliminary within-survey screening tool that should be interpreted with its component sub-indices rather than as a calibrated reference-condition index.