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Appendix A. Fish nutrient excretion was not a confounding factor.
Under some conditions, nutrient excretion from fish can affect algal communities and
therefore the zooplankton [1]. We use multiple lines of evidence to show that such nutrient
excretion by fish did not confound our results.
We estimated nutrients excreted by fish during the experiment based on the minimal
diet used as well as the fish’s basal metabolic demands. Our experimental protocol was
designed to minimize fish nutrient excretion by starving the fish before placement in the cages.
Fish were rotated into the experiments weekly, which allowed us to feed the fish to ensure their
health while not in the experiment, and to feed them minimally while in the experiment. Bluegill
in our experiments were fed 200 Daphnia twice weekly, each Daphnia containing
approximately 1 µg of N [2] and 0.1 µg of P [3]. Assuming that bluegill assimilate a
minimum of 30% of the N and 37% of the P in their diet [4], we estimate that the three
bluegill in each tank excreted 0.09 µg N l-1 d-1 and 0.01 µg P l-1 d-1 as a direct result of their
consumption of Daphnia. We estimated nutrients excreted through basal metabolic
demands using rates from a previous study of bluegill about the same size as those in our
experiment (mean total length of 50.6 mm and weight of 1.96 g wet mass) that were
subject to a 36 hour starvation period [5]. In this case, nutrient excretion due to the three
fish in each tank was estimated as 1.40 g N l-1 d-1 and 0.15g P l-1 d-1. Summing these two
sources, total excretion rates from fish are estimated as 1.49 g N l-1 d-1 and 0.16g P l-1 d-1.
Similar rates were obtained based on results from another study of bluegill [6] and from
studies of other fish species that examined fish of size and diet used our experiment [7-9]
supporting the robustness of our estimates.
2
We next provide several lines of evidence that indicate that fish nutrient excretion was
negligible compared to other sources of nutrients in the experiment. First, nutrients added
directly to the tank to provide nutrients for phytoplankton growth were added at levels that far
exceeded the predicted influence of fish. Nutrient addition rates of 93.5 g N l-1 d-1, and 7.0 g
P l-1 d-1 were used before the initiation of predator (fish) treatments, and over time reduced to
46.8 g N l-1 d-1, and 3.5 g P l-1 d-1 after fish were added to the tanks (see main text). Even
these lower supply rates at the end of the experiment far exceeded those of fish by 31 and 22 fold
for N and P, respectively.
Second, the nutrient supply from the entire plankton community, including zooplankton
and bacteria, far exceeded that of fish. Hudson et al. [10] and Nowlin et al. [11] demonstrated
that P regeneration by the planktonic community dominates the net supply to plankton, and is
strongly correlated in lakes over a nutrient gradient of 4-80 g/l of total phosphorous. Applying
the strong relationship between P regeneration and total P found by Hudson et al. [10] to our
average total P (40 ug P l-1) value yields a regeneration rate of 5.4 g P l-1 d-1, which far exceed
(by 33 fold based on [7]) that predicted for fish in the experiments. Using the results of Nowlin
et al. [11] yields an even higher estimate. Further, there was additional contribution to the total
nutrient regeneration by snails, hydra, and microorganisms on the sides and bottoms of the tanks,
further reducing the contribution of fish excretion. A similar analysis yields the same results for
N excretion.
Third, a second method of estimating organism nutrient regeneration indicates that it far
exceeded that of fish. There has been much research performed on zooplankton nutrient
regeneration. Urabe et al. [12] examined nutrient regeneration by a natural assemblage of
zooplankton in a natural lake monthly from June to September. In this study two parameters
3
known to affect nutrient excretion rate were similar to that in our tanks; the mean temperature
was 23˚C, and total N:P ratio was 17:1. Further, zooplankton were dominated by copepods in
some months, and by cladocerans in others, and therefore covered a large range of communities
similar to ours. Therefore, this is an ideal study to use to estimate the mass specific nutrient
supply by zooplankton, which averaged a nutrient regeneration rate per unit dry weight of
zooplankton of 2.9 ug P mg-1 DW d-1. Appling this rate to the mean dry weight density of
zooplankton in the fish treatment tanks (0.378 mg/l) leads to an estimate of 1.1 g P l-1d-1. This
value far exceeds that of that estimated from fish by 6.9 fold. This value is also a rather
conservative estimate based on other studies. For example Carrillo et al. [13] and Attayde &
Hansson [14] have reviewed the literature on nutrient excretion rates of zooplankton, and the
median value (15 ug P mg-1 DW d-1) represents even larger predicted nutrient supply rate from
zooplankton. Further, estimated zooplankton excretion using published equations that estimate
excretion as function of zooplankton size and temperature [15,16] yielded equivalently high
estimates nutrient regeneration by zooplankton (both over 10 g P l-1d-1). Therefore, analysis
using published zooplankton excretion rates indicates that the contribution of P excretion from
fish was very much lower than that of zooplankton. As in the previous analysis, the fact that
zooplankton only represent one organism of many organisms regenerating nutrients (e.g.
microplankton, snails) further reduces the predicted negligible contribution from fish. A similar
analysis yields the same results for N excretion.
Fourth, nutrient analyses showed no effect of fish on total phosphorous, total nitrogen, and
the TN:TP ratio. Water samples were collected on September 2 and 3 by collecting 50 ml of
water, 5 cm below the surface in the middle of each tank of four of the six replicates. September
2 was chosen because it was a day nutrients were added to tanks. By measuring nutrients before
4
these manipulations (on Sept 2), and the day after (on Sept 3), we hoped to span the largest range
of nutrient levels in the tanks. Nutrient concentrations were determined using standard automated
colorimetric procedures on a Technicon Auto Analyzer II according to methods detailed in Davis
& Simmons [17]. Total nitrogen (TN) was determined using the persulfate digestion method
4500-N (C) [18] followed by analysis for NO3. Total phosphorus (TP) was determined by
persulfate digestion after the method of Menzel & Corwin [19], followed by analysis for soluble
reactive phosphorus. The effect of fish treatment and day on total phosphorous (TP), total
nitrogen (TP), and TN:TP ratio was analyzed using a repeated-measures ANOVA after
confirming that the data met assumptions of normality and homogenetity of variances, based on
Levene’s tests. Fish treatment did not significantly affect TP (F1,6 = 0.06, p = 0.81), TN (F1,6 =
0.10, p = 0.77), or the TN:TP ratio (F1,6 = 0.11, p = 0.76). The day of sampling was a significant
factor for TP (p=0.001) and TN (p=0.004), and a marginally significant factor for TN:TP (p =
0.054), although these differences were very small. Average values for each response on the two
consecutive days were, respectively, 42 and 49 ug l-1 TP, 850 and 925 ug l-1 TN, and 20.5 and
19.0 TN:TP.
Fifth, the amount of nutrients in an active form that can be utilized by phytoplankton
excreted by fish was estimated to be low compared to the total amount in the tanks. Although we
only measured total N and P, in a cattle tank experiment that used a methodology similar to ours
(containing a natural assemblage of zooplankton and snails), Hall et al. [20] measured soluble
reactive phosphorus (SRP). They added N and P in several ratios including 13:1, which was very
close to our supply ratio. They used two nutrient supply rates, the higher of which yielded
zooplankton density and TP nearly identical to that in the fish treatments in our experiment. The
SRP level in this treatment was 10 g l-1. This level is 62.5 times the estimated daily
5
phosphorous contribution from fish in the experiment, indicating that any contribution from fish
was negligible. An analysis of NH4 yielded similar results for nitrogen.
In summary, the nutrient release from fish was negligible because we supplied much more
nutrients then the fish excreted on a daily basis, and regeneration by other organisms in the tank
far exceeded any contribution from the fish.
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