Nitrification inhibitors (NIs) applied to soil reduce nitrogen fertilizer losses from agr o -ecosystems. NIs that are currently registered for use in agriculture appear to selectively inhibit ammonia-oxidizing bacteria (AOB), while their impact on other nitrifiers is limited or unknown. Ethoxyquin (EQ), a fruit preservative shown to inhibit ammonia-oxidizers (AO) in soil, is rapidly transformed to 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (QI), and 2,4-dimethyl-6-ethoxy-quinoline (EQNL). We compared the inhibitory potential of EQ and its derivatives with that of dicyandiamide (DCD), nitrapyrin (NP), and 3,4-dimethylpyrazole-phosphate (DMPP), NIs that have been used in agricultural settings. The effect of each compound on the growth of AOB ( Nitrosomonas europaea, Nitrosospira multiformis ), ammonia-oxidizing archaea (AOA; “ Candidatus Nitrosocosmicus franklandus,” “ Candidatus Nitrosotalea sinensis”), and a nitrite-oxidizing bacterium (NOB; Nitrobacter sp. NHB1), all being soil isolates, were determined in liquid culture over a range of concentrations by measuring nitrite production or consumption and qPCR of amoA and nxrB genes, respectively. The degradation of NIs in the liquid cultures was also determined. In all cultures, EQ was transformed to the short-lived QI (major derivative) and the persistent EQNL (minor derivative). They all showed significantly higher inhibition activity of AOA compared to AOB and NOB isolates. QI was the most potent AOA inhibitor (EC 50 = 0.3–0.7 μM) compared to EQ (EC 50 = 1–1.4 μM) and EQNL (EC 50 = 26.6–129.5 μM). The formation and concentration of QI in EQ-amended cultures correlated with the inhibition patterns for all isolates suggesting that it was primarily responsible for inhibition after application of EQ. DCD and DMPP showed greater inhibition of AOB compared to AOA or NOB, with DMPP being more potent (EC 50 = 221.9–248.7 μM vs EC 50 = 0.6–2.1 μM). NP was the only NI to which both AOA and AOB were equally sensitive with EC 50s of 0.8–2.1 and 1.0–6.7 μM, respectively. Overall, EQ, QI, and NP were the most potent NIs against AOA, NP, and DMPP were the most effective against AOB, while NP, EQ and its derivatives showed the highest activity against the NOB isolate. Our findings benchmark the activity range of known and novel NIs with practical implications for their use in agriculture and the development of NIs with broad or complementary activity against all AO.

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NIs could improve N use efficiency and decelerate N cycling. Still, we know little about their activity on the distinct AOM groups and about their effects on off-target soil microorganisms.

Nitrification inhibitors (NIs) applied to soil reduce nitrogen fertilizer losses 19 from agricultural ecosystems. Currently available NIs appear to selectively inhibit 20 ammonia-oxidizing bacteria (ΑΟΒ), while their impact on other groups of nitrifiers is 21 limited. Ethoxyquin (EQ), a preservative shown to inhibit ammonia-oxidizers (AO) in soil, 22 is rapidly transformed to 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (QI) and 2,4-23 dimethyl-6-ethoxy-quinoline (EQNL). We compared the inhibitory potential of EQ and its 24 derivatives in vitro with other established NIs that have been applied in an agricultural 25 setting (dicyandiamide (DCD), nitrapyrin (NP), 3,4-dimethylpyrazole phosphate (DMPP)) 26 by evaluating their impact on the activity and growth of five soil-derived strains (two AOB 27 (Nitrosomonas europaea, Nitrosospira multiformis), two ammonia-oxidizing archaea 28 (AOA) ("Candidatus Nitrosocosmicus franklandus", "Candidatus Nitrosotalea sinensis"), 29 and one nitrite-oxidizing bacterium (NOB) (Nitrobacter sp.)). NIs degradation was also 30 determined. AOA were more sensitive than AOB or NOB to EQ and its derivatives. Despite 31 its transient character, QI was primarily responsible for AO inhibition by EQ, and the most 32 potent NI against AOA. For AOB, QI was more potent than DCD but less than nitrapyrin 33 and DMPP. AOA and NOB showed higher tolerance to the persistent compounds DCD 34 and DMPP. Our findings benchmark the activity range of known and novel NIs with 35 practical implications for their use, and the development of novel NIs with broad or 36 complementary activity against all AO. 37 DMPP: 3,4-dimethylpyrazole phosphate, AOB: ammonia-oxidizing bacteria, AOA: ammonia-oxidizing archaea, AO: ammonia-oxidizers, NOB: nitrite-oxidizing bacteria, comammox: complete ammonia-oxidizing bacteria; AMO: ammonia monooxygenase 3 38 KEYWORDS nitrification inhibitors, ethoxyquin, quinone imine, ammonia-oxidizing 39 bacteria, ammonia-oxidizing archaea, nitrite-oxidizing bacteria, in vitro assays 40 41Modern agricultural systems depend heavily on large inputs of synthetic N fertilizers to 42 maintain crop productivity and alleviate food crisis for the growing global population (1). 43However, approximately 70% of the annual global input of 100 Tg N fertilizer is lost from 44 agricultural ecosystems due to nitrification and subsequent denitrification processes 45 leading to groundwater and atmospheric pollution through nitrate leaching and nitrogen 46 oxides (NxO) emissions, respectively (2). To reduce N losses and improve nitrogen use 47 efficiency, nitrification inhibitors (NIs) are routinely incorporated into N-stabilized 48 fertilizers to reduce the activities of nitrifying prokaryotes and increase N retention time in 49 soil (3, 4). 50Hundreds of compounds have been identified as potential NIs (5), but only three of 51 them have gained importance for practical use on a global scale: 2-chloro-6-52 (trichloromethyl) pyridine (nitrapyrin) (NP) (6), dicyandiamide (DCD) (7), and 3,4-53 dimethylpyraz...