Based on the d15N data and streamwater NH4 concentrations we can calculate uptake length and uptake rates of NH4 according to the following methods.

A. NH4 Uptake Length There are three methods for calculating NH4 uptake length using the 15N data: (1) using the water (d15N= delta 15N) d15N-NH4 data corrected for downstream dilution (increases in flow), (2) using the water 15N-NH4 mass flux data (determined from d15N-NH4 data, NH4 concentrations, and flow), and (3) using the organism (or biomass compartment) d15N data corrected for downstream dilution. Each of these methods involves the same calculation: a regression of the natural log of the 15N value (corrected for background, and corrected for dilution if d15N data are used) against distance below the 15N dripper (in meters). The slope of this regression is the distance-normalized NH4 uptake rate and the inverse of the slope is the NH4 uptake length. It is also a good idea to compute the 95% confidence interval for the uptake length using the regression statistics for the slope. See below for details for each method.

The uptake length obtained using the water 15N data (methods 1 and 2) is an instantaneous measure for the time the water samples were collected. The water 15N mass flux method (method 2) should be the most accurate approach; however, it requires good measures of NH4-N concentration at each station where water samples for 15N are collected. This may be difficult in streams with very low (or undetectable) concentrations of NH4. The water d15N-NH4 data can be used directly to calculate uptake length (method 1) assuming that there are minimal changes in streamwater NH4 concentration over distance within the experimental reach (i. e., assumes that regeneration of NH4 plus lateral inputs approximately balance uptake). This may be the situation in many N limited streams (e. g., streams with very low or undetectable NH4-N concentrations; it was the case in Walker Branch and Upper Ball Creek). If so, then the two water 15N data methods should give similar uptake length values. Finally, the two water 15N data methods may overestimate NH4 uptake length on days 20 and 41 slightly due to regeneration of some 15N back to water within the study reach (the uptake length calculation assumes that the tracer flux is changed only by uptake as a parcel of water moves downstream). Likely, there will be some regeneration of 15N back to water at least by day 20. We can correct for this on day 41 by subtracting the background-corrected 15N flux or d15N values determined from the post 12 hour water samples at each station from the day 41 values prior to doing the regressions of ln( 15N flux or d15N) vs. distance. We can also apply this correction to the day 20 water data, although this is likely to be an over-correction because the amount of 15N in the reach will be lower than at post 12 hours.

The uptake length calculated using the organism or biomass compartment 15N data (method 3) is a cumulative uptake length over the period from the beginning of the 15N addition to the time when samples were collected (it is not a time-linear average but rather is weighted somewhat toward more recent periods). The organism/ biomass compartment method also may be an overestimate of NH4 uptake length if there is substantial regeneration back to water of 15N taken up for the same reason the water methods overestimate in this situation (i. e., the water d15N values at the downstream stations are somewhat greater than they would be if only NH4 uptake were occurring). This may be a problem in the latter stages of the 15N addition period; thus, we expect this method to give good results probably only for day 7. The organism/ biomass compartment method may also give poor results if there is considerable movement of organisms/ materials within the stream such that a sample from any one location may not be representative of materials that have remained at that location. This would probably be evident in a poor r2 for the regression. It is likely to be more of a problem for highly mobile organisms or highly transportable materials (e. g., FBOM, leaves). Further, the organism/ biomass compartment method may give poor results for organisms that are feeding on several different food sources that take up 15N at different rates. We would expect more accurate results for primary consumers with a narrow feeding strategy than for omnivores or organisms at higher trophic levels. In summary, we expect that the organism/ biomass method for calculating uptake length will give results similar to the water 15N data methods only early in the 15N addition period (within the first week or so) as long as the stream flow and NH4 concentrations remain roughly constant within that period. In such cases, we expect uptake lengths computed using water 15N data on day 0 and using organism/ biomass 15N data on day 7 to be similar.

Method 1 -Uptake length calculation using streamwater d15N-NH4 data. To calculate uptake length using the streamwater d15N-NH4 data, the d15N-NH4 values at each station below the dripper are corrected for background (by subtracting the water d15N-NH4 value at the station above the dripper) and corrected for dilution (by multiplying the background-corrected d15N-NH4 values by the ratio of flow at that station to flow at the uppermost station below the 15N dripper). Uptake length is then computed as the inverse of the slope of the regression of Ln (background-and dilution-corrected d15N-NH4 values vs. distance downstream from the dripper).

Method 2 -Calculation of uptake length using streamwater 15N mass flux data. To calculate NH4 uptake length using the 15N mass flux data a few more computations are involved. The mass concentration of 15N-NH4 tracer in water at each station is calculated from the water d15N-NH4 values and NH4-N concentrations ([ NH4], in µgN/ L) at station I as follows:

([ NH4] i * 0.003663) * (d15Nb-c/ I /1000) (1)

where d15Nb-c/ I is the background-corrected value in water at station I (note that the dilution correction to d15Ni is not necessary here). The 15N mass flux at each site I is then computed by multiplying by the streamflow at site I (Qi) as follows:

([ NH4] i * 0.003663) * (d15Nb-c/ I /1000) * Qi (2) Uptake length is then calculated as the inverse of the slope of the regression of Ln (mass 15N flux at station I, as given by equation 2) vs. distance downstream from the dripper.

Method 3 -Calculation of uptake length using organism/ biomass d15N values. Calculation of NH4 uptake length using the organism/ biomass d15N values at each station below the dripper is essentially the same as using the water d15N values. It involves first correcting for the background 15N by subtracting the d15N values for that compartment at the station upstream from the dripper and then correcting for dilution by multiplying by the ratio of flow at that station to flow at the uppermost stations below the dripper. Then, uptake length is calculated as the inverse of the slope of the regression of Ln (background-and dilution-corrected d15N value at station I) vs. distance downstream from the dripper.

B. NH4 Uptake Rates We can compute two types of NH4 uptake rates from the 15N data: (1) whole-stream uptake rate, and (2) compartment-specific uptake rate (for each of the primary uptake compartments, i. e., epilithon, filamentous algae, bryophytes, CBOM-leaves, CBOM-wood, FBOM, etc.). Both the whole-stream and compartment-specific uptake rates are computed in terms of N mass area-1 time-1 (e. g., µgN m-2 s-1).

Whole-stream NH4 uptake rate. The whole-stream uptake rate is computed from the NH4 uptake length, streamwater NH4 flux (F, computed as streamflow in L/ s * stream NH4 concentration in µgN/ L), and average stream width (w, wetted width in m) according the following equation from Newbold et al. (1981, Can. J. Fish. Aquat. Sci. 38: 860-863):

NH4 Uptake Rate (µgN m-2 s-1) = (F)/( Uptake Length* w) (3) This is the total NH4 uptake rate (rate of removal of NH4 from stream water) by all stream compartments.

Compartment-specific NH4 uptake rate. Uptake rates of NH4 by individual stream compartments can be computed using the biomass d15N values and the streamwater d15N-NH4 values at one of the stations near the dripper (but one that youíre sure is well-mixed, we used the 10m station in Walker Branch) from the early period of the release (e. g., day 7 biomass samples and day 0 water samples), and

the data on total N standing stock in that compartment per unit streambed area (TN/ m2, in gN/ m2). The compartment TN standing stock values can be computed from the biomass standing stock measurements that were made at the beginning of the experiment (converted to dry mass per m2 using AFDM/ dry mass conversions) and the %N content (% of dry mass) obtained from the C/ N analyses done on subsamples of each compartment from this initial sampling (in most cases done at Univ. of Georgia). The compartment-specific NH4 uptake rates can be computed as the mass rate of 15N uptake (background-corrected) per unit area at site I times 1/( 15N/ 14N) ratio in water (also background-corrected) at site I. Using the compartment d15N values measured on day 7 (background-corrected d15Nbiomass values) and the streamwater d15N-NH4 values (also background-corrected) measured on day 0 (and assuming this is a good average for the period from day 0 to day 7), we can estimate NH4 uptake rates (gN m-2 d-1) into each compartment as (pay close attention to parentheses and brackets):

NH4 Uptake Rate (µgN m-2 d-1) = [((( d15Nbiomass/ 1000)* 0.003663)*( TN/ m2))/ 7] * [1/(( d15Nwater/ 1000)* 0.003663)] (4)

Actually, equation 4 gives only the 14NH4 uptake rate, but this is approximately equal to the total NH4 uptake rate (14N+ 15N) into that compartment because 15N is less than 1% of 14N+ 15N. Also, equation 4 will generally underestimate NH4 uptake rate by each compartment because we must assume that there has been no recycling of 15N (release back to the water) up to day 7 from that compartment. We know that this is not really true, but this is the best we can do. The underestimation will be greater for biomass compartments with rapid N turnover rates (e. g., epilithon), as evidenced by d15N values that approach isotopic steady state (asymptote values) earlier in the experiment, than for biomass compartments with slower N turnover rates (e. g., bryophytes in the Walker Branch study). This is why we can only use this method to estimate compartment-specific NH4 uptake rates early in the experiment.

The compartment-specific mass uptake rates for all major primary uptake compartments can then be summed to give an approximate whole-stream uptake rate that can be compared with the whole-stream uptake rate computed from the uptake length data (method 1, equation 3). Almost certainly the summed compartment-specific uptake rates will be lower than the whole-stream rate from the uptake lengths for two reasons: (1) there may be compartments involved in uptake that were not sampled, and (2) the underestimation of the true compartment-specific uptake rates because of the assumption of no 15N recycling up to day 7 (see above). For some compartments with relatively slow N turnover rates this might be a reasonable approximation in the first few days or so of the 15N addition. This can be verified for different compartments by plots of their d15N values over time. If a particular compartment is near d15N steady state at the time of sampling (flattening of its d15N vs time curve), its NH4 uptake rate is likely

highly underestimated. In Walker Branch, the sum of all compartment-specific NH4 uptake rates was only a little over 1/ 3 the whole-stream NH4 uptake rate computed using the uptake length and NH4 flux (see above). Nonetheless, the biomass-specific calculations give a rough indication of relative importance of different compartments in NH4 uptake (although remember that uptake rates for compartments closer to isotopic steady state are underestimated more than compartments farther from steady state).

There is a second approach for calculating compartment-specific uptake rates that involves determining the N-specific NH4 uptake rate (k, in units of d-1) from the rate of change in background-corrected d15Nbiomass values, assuming first-order tracer kinetics that result in d15Nbiomass values approaching a known asymptote value at a particular station x. Therefore, this approach can only be used when the asymptote d15Nbiomass value for a particular compartment can at a particular station x be determined during the experiment (i. e., the compartment approaches steady state with respect to the 15N tracer). The stream water d15N-NH4 value at a particular station might be used as the estimate of the asymptote d15Nbiomass value for that station if we knew that all the biomass N was actively cycling with the water NH4 pool and if NH4 was the only form of N that was taken up from the water (i. e., no uptake of NO3). But we know that these assumptions are likely not true. Therefore, the asymptote d15Nbiomass value for a particular primary uptake compartment is likely to be considerably lower than the water d15N-NH4 value at that station. However, if we can determine the asymptote for a particular primary uptake compartment from the d15Nbiomass data over time during the experiment at a particular station x (call it (d15Nbiomass) asymptote x), then we can determine the N-specific uptake rate, k (units of d-1), by solving the following equation:

(d15Nbiomass) x, t = (d15Nbiomass) asymptote x * (1 -e-kt) (5) Rearranging, and using the d15Nbiomass data from day 7 gives: k = -(Ln( 1-(( d15Nbiomass) x /( d15Nbiomass) asymptote x)))/ 7 (6)

Then, once we have the N-specific NH4 uptake rate, k, for each compartment we can compute the the compartment specific NH4 uptake rate ( in units of gN m-2 d-1) by multiplying k (in d-1) by the fraction of the compartment that is actively cycling with the water NH4 pool (which is equal to the compartment TN/ m2 value times the ratio (d15Nbiomass) asymptote x / (d15Nwater) x ):

k * (TN/ m2)*(( d15Nbiomass) asymptote x / (d15Nwater) x )) (7) Finally, it should be emphasized that we are calculating only NH4 uptake rate rates, both on a whole-stream basis and for each compartment. If there is appreciable uptake of NO3 as well, then the total N uptake rate will be greater than the uptake rate of NH4.

C. Caveat Concerning d15N values (15NH4 Blank effect) The purpose of this section is to discuss some potential problems with the measurement of d15N-NH4+ and caution against over-interpretation of this data. We intend to run some tests this winter in order to increase our confidence in the d15N-NH4+ values.

The general d15N-NH4+ protocol we follow is as outlined in Holmes et al (in press, Marine Chemistry). In brief, NH4 is converted to NH3 under basic conditions, and the NH3 is collected on an acid trap which is later run on the Mass Spec. One complication with this method is fractionation which we currently correct for with the standard we run with each transect. Another problem with this technique is blanks, and the magnitude of the problem increases with the level of isotopic enrichment, especially at low NH4 concentrations. Therefore, the ìtrueî del value of ammonium is particularly difficult to estimate near the dripper site where the del values are highest. We currently are not able to correct for the blank because the exact amount and del value of the blank are not well known.

In each 4L diffusion, the reagents we add introduce extra nitrogen that is not from stream water. Based on very limited data, the reagents (salt and MgO) introduce something like 0.25 µmol N onto the filter disks for the 4L diffusion sample, even after being ashed (Holmes, pers comm). This is a fairly large amount relative to the total mass of N we recover on the filter in most of these diffusions (range of 0.7 to 2 µmol/ 4L). The d we get from the mass spectrometer is therefore a combination of the blank d and the sample d. If we assume the d of the blank is near 0 (which is likely), then the sample dís we get back from the mass spec are lower than the actual d. The problem is more severe for highly enriched samples because the spread between blank and d15N-NH4+ values will be much greater; samples close to the dripper will be relatively more underestimated (Fig. 1). Therefore, any comparisons between the d15N-NH4+ at a given distance downstream and the biota at that same station will have to be made with this potential problem in mind. Obviously, the greater the percent of mass that is derived from a blank the greater the underestimate will be (Fig. 1). We intend to get a better estimate of the blank mass this winter.

As we have shown, the effect the reagent blank might have on absolute d values can be large. However, the effect on the Sw calculation is relatively minor. Based on calculations using hypothetical conditions, the effect of the blank on Sw is likely to be less than 10% (Table 1). In Table 1, we set up a hypothetical transect with a maximum measured d of 500, an uptake length of 50m, and an N-recovery of 0.7 µmol/ filter (all reasonable estimates from the various experiments run so far). Of this 0.7 µmol, we varied the blank component equal to 0.1, 0.3 and 0.6 µmol/ filter (roughly 17%, 50%, and 86% of the N-recovered). We also varied the d15N of the blank equal to 5, 0, and -5 o/ oo. If the blank is 0, there is no effect on the estimate of

Sw because the slope of the natural log transformed data is changed proportionally along the entire transect. If blank dís are 5 or -5 ( a likely range), the error in the Sw calculation is less than 10% if the mass of the blank is less than 50% of the total mass (Table 1). Even if most of the mass on the filter is derived from the blank (which is unlikely), the effect is relatively small. In short, the blank effect on Sw will be minor.

In summary, (1) interpret d15N-NH4+ data cautiously, being aware that the d values of the highly enriched samples may be significantly underestimated. This could significantly affect compartment specific uptake rates, as discussed in this addendum. After running some experiments this winter, we may revise the d15N-NH4+ values reported thus far.

(2) Problem #1 is only a minor annoyance with regard to calculating Sw.