J. N. Am. Benthol. Soc., 2007, 26(2):167–177 Ó 2007 by The North American Benthological Society Improving the fluorometric ammonium method: matrix effects, background fluorescence, and standard additions Brad W. Taylor 1 , Christine F. Keep 2 , Robert O. Hall, Jr. 3 , Benjamin J. Koch 4 , AND Lusha M. Tronstad 5 Department of Zoology and Physiology, University of Wyoming, Laramie, Wyoming 82071 USA Alexander S. Flecker 6 AND Amber J. Ulseth 7 Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, New York 15853 USA Abstract. Our understanding of the N cycle is affected by how accurately we can measure NH 4 þ in natural waters. Measuring NH 4 þ concentrations requires accounting for matrix effects (ME) that are caused by substances in the sample that attenuate or intensify the signal of the samples relative to the standards. We show that the ME calculation in the recently published fluorometric NH 4 þ method is mathematically incorrect, producing results that consistently underestimate NH 4 þ concentration as a nonlinear function of the ME. We provide the correct equation and offer an alternative approach that accounts for ME by using sample water rather than deionized water to make the standards, thereby producing a standard curve that contains the same background chemical properties as the samples. In addition, we show that the previous method for measuring a sample’s background fluorescence does not include the background signal of the reagent or its interaction with the matrix constituents of the sample. We provide a new method for measuring a sample’s background fluorescence that includes the background fluorescence of the sample, reagent, and their interaction. The simple changes we suggest produce more accurate and precise NH 4 þ measurements. Key words: fluorometry, nitrogen cycle, quenching, standard additions. NH 4 þ is often difficult to measure in natural waters (Aminot et al. 1997), but it is ecologically important. NH 4 þ is preferred over NO 3 – by autotrophs and heterotrophs (Dodds and Priscu 1989, Kirchman 1994), controls nitrification rates (Schlesinger 1997), is excreted by animals, and has increased globally in fresh and marine waters because of human activities (Howarth et al. 1996). 15 N-enriched NH 4 þ (e.g., 15 N- NH 4 Cl) also is used widely as a tracer to understand the N cycle (e.g., Peterson et al. 2001). Despite widespread attention and modern analytical instru- ments with high sensitivity, quantifying NH 4 þ remains difficult. For example, NH 4 þ can be relatively unstable during sample preservation, and numerous sources of contamination during sample collection, preservation, and analysis hinder measuring low concentrations of NH 4 þ (Eaton and Grant 1979, Aminot et al. 1997). Recently, Ke ´rouel and Aminot (1997) and Holmes et al. (1999) introduced an elegant fluorometric method for measuring low NH 4 þ concentrations that solves many of these problems. The fluorometric method works well because samples can be combined with reagents and analyzed immediately in the field, alleviating problems with the instability of NH 4 þ during storage (Avanzino and Kennedy 1993, Zhang et al. 1997). The method also decreases contamination from laboratory materials and atmospheric NH 3 by using a single working reagent (WR) and a time- 1 Present address: Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755 USA. E-mail: brad.taylor@dartmouth.edu 2 E-mail addresses: chrisfish11@gmail.com 3 bhall@uwyo.edu 4 bkoch@uwyo.edu 5 tronstad@uwyo.edu 6 asf3@cornell.edu 7 Present address: Department of Zoology and Physiol- ogy, University of Wyoming, Laramie, Wyoming 82071 USA. E-mail: aulseth@uwyo.edu 167