Reduction of As(V) to As(III) under anaerobic conditions significantly increases arsenic (As) toxicity and bioavailability, making it a crucial process that drives As contamination. Simultaneously, co-occurring microbial nitrogen (N) transformations may accelerate As(V/III) conversion through complex interactions and competition, yet their competing effects remain insufficiently resolved. To address this, we applied six N-addition treatments with varying total N input and form to anaerobic microcosms established with As-contaminated soil. N treatments receiving NH4+ only (MN(NH4+)) and NO3− only (MN(NO3−)) were included to examine how specific N forms and their associated transformations affect As(V/III) speciation. Additionally, low (LN), medium (MN), and high (HN) N levels were applied as NH4NO3 to increase N availability and intensify competition among N transformations. Results showed that MN(NH4+) increased As(III) in soil (1.4–36.0 mg kg−1) and porewater (0.1–138.7 μg L−1) by enhancing anammox (∼40 %) and promoting DOC and Fe(II) accumulation. Conversely, MN(NO3−) lowered As(III) by stimulating denitrification and restricting DOC and Fe(II) accumulation. Increasing N input from LN to HN decreased denitrification rates by 19.9–51.5 % while enhancing anammox rates by 51.9–199.2 % and the transcriptional activity of the anammox gene hzs (up to 1.5). It also increased the abundance of the As-reducing gene arrB, DOC accumulation, and Fe(III) reduction, ultimately elevating As(III) by 22.3–31.4 mg kg−1 and 35.0–130.8 μg L−1 in soil and porewater, respectively. Structural equation modeling (SEM) and linear mixed-effects models (LMM) identified the largest standardized effect (0.79) and importance (19.1 %) for anammox, highlighting anammox as the dominant driver of As(V/III) speciation. This study provides novel insights into N–As interactions.
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