J. Aquat. Plant Manage. 41: 2003. 57

J. Aquat. Plant Manage. 41: 57-60

Nutrient Limitation of Periphyton in a

Spring-Fed, Coastal Stream in Florida, USA

SKY K. NOTESTEIN1, T. K. FRAZER1, M. V. HOYER1, AND D. E. CANFIELD, JR.1

ABSTRACT

There is strong evidence to suggest that ground-water

nitrate concentrations have increased in recent years and

further increases are expected along portions of the central

Gulf coast of Florida. Much of the nitrate enriched groundwater is discharged into surface waters through numerous

freshwater springs that are characteristic of the area and the

potential for eutrophication of their receiving waters is a

legitimate concern. To test the potential effects of elevated

nutrient concentrations on the periphyton community an in

situ nutrient addition experiment was conducted in the

spring-fed Chassahowitzka River, FL, USA, during the summer of 1999. Plastic tubes housing arrays of glass microscope

slides were suspended in the stream. Periphyton colonizing

the microscope slides was subjected to artificial increases in

nitrogen, phosphorus or a combination of both. Slides from

each tube were collected at 3- to 4- day intervals and the

periphyton communities were measured for chlorophyll concentration. The addition of approximately 10 μg/L of phosphate above ambient concentrations significantly increased

the amount of periphyton on artificial substrates relative to

controls; the addition of approximately 100 μg/L of nitrate

above ambient concentrations did not. The findings from

this experiment implicated phosphorus, rather than

nitrogen, as the nutrient that potentially limits periphyton

growth in this system.

Key words: Nitrogen, Phosphorus, Bioassay, Eutrophication, Nutrient Enrichment.

INTRODUCTION

Ground-water nitrate enrichment is being documented

throughout much of Florida, particularly in the north central region of the state (Hatzell 1996, Ham and Hatzell 1996,

Jones et al. 1997). Because of the occurrence of large and

productive aquifer systems and highly permeable karst geology, there are numerous locations and multiple pathways by

which nitrate enriched ground-water can permeate and mix

with the surface water environment. For example, freshwater

springs, of which there are greater than 300 in the state, provide a direct conduit for ground-water to move from the

aquifer to surface waters (Katz et al. 1997).

Elevated nitrate concentrations in ground-water can result

from both past and present land use activities, such as commercial or residential fertilizer application (Jones et al.

1997). Because of a presumed lag-time associated with

ground-water movement, it is likely that further increases in

nitrate concentrations in spring discharge will occur because

of past and present human population growth and land use

activities in this region. As nutrient-enriched ground-water

enters the surface water system via spring input there is a

potential for ecological change to occur. In many instances,

increases in nutrient concentrations to streams with low

nutrient concentrations have been linked to changes in

autotrophic community composition, vegetative biomass and

an increase of nuisance species (Wright and McDonnell

1986a, 1986b). Such changes can, in turn, affect shifts in

community structure and alter food web dynamics of a given

system (Hershey et al. 1988, Peterson et al. 1993).

Although increased nutrient concentrations can influence periphyton abundance, the responses to nutrient additions are frequently variable. For example, nitrogen alone

may stimulate periphyton growth (Stelzer and Lamberti

2001) especially when light is not a limiting factor (Lohman

et al. 1991, Mosisch et al. 2001). In other cases, the addition

of phosphorus, both solely (Bothwell 1985, Pan and Lowe

1994) and concurrently with nitrogen (Dodds et al. 1997,

Winter and Duthie 2000), has been shown to increase

periphyton abundance.

The objective of this study was to examine the potential

consequences of additional nutrient inputs on periphyton

abundance as measured as chlorophyll per unit area. To accomplish this, an in situ manipulative field experiment was

conducted to determine if increasing water column nutrient

concentrations of nitrogen and phosphorus would affect the

abundance of periphyton growing on submerged surfaces.

MATERIALS AND METHODS

The Chassahowitzka River located at 28° 42’ 54” North,

82° 34’ 35” West, is a spring-fed coastal river of about 8 km in

length located in southwest Citrus County, FL, USA. The

land surrounding the river typically has elevations of 3 m or

less and is dominated by coastal flatwoods and wetlands that

transition into an extensive salt marsh complex along the

rocky flat coast of the Gulf of Mexico. The climate is subtropical and Wolfe et al. (1990) provides further details on the

ecology of the region.

The Chassahowitzka River is shallow with a mean depth of

about 0.9 m and mean flow rates of less than 0.20 m/s. Average stream discharge calculated from measurements taken

near the main spring between 1998 to 2000, ranged from 2

to 8 m3

/s. The primary substrate is approximately 54% sand,

1

University of Florida, Institute of Food and Agricultural Sciences,

Department of Fisheries and Aquatic Sciences, 7922 NW 71st Street, Gainesville, Florida 32653 USA. skynote@ufl.edu. Received for publication June

11, 2002 and in revised form October 14, 2002
58 J. Aquat. Plant Manage. 41: 2003.

although various mixtures of sand, mud, and rock are common. The river is sufficiently wide that terrestrial vegetation

is not a significant factor in reducing light availability for

photosynthesis, as terrestrial canopy coverage only shades

about 3% of the total river area. Submersed aquatic vegetation is ubiquitous and the light environment is favorable for

the growth of rooted macrophytes, macroalgae and associated periphyton, as the river bottom is visible throughout most

of the river. Light attenuation coefficients (Kd) though variable, are generally less than 1.5/m. Submersed aquatic vegetation occurs throughout most of the river, but declines

gradually with distance downstream. Common aquatic plants

include tape grass, (Vallisneria americana Michx.), Sago pondweed, (Potamogeton pectinatus L.), southern naiad, (Najas

guadalupensis (Spreng.) Magnus), Eurasian water milfoil,

(Myriophyllum spicatum L.), and hydrilla, (Hydrilla verticillata

(L.f.) Royle). Filamentous macroalgae, including Lyngbya sp.

and Chaetomorpha sp., are also abundant.2

In the summer of 1999, an in situ experiment was conducted to test the effects of nutrient enrichment on periphyton

growth. Periphyton sampling was similar to that of Peterson

et al. (1983) and allowed replicate treatments of nitrogen,

phosphorus and nitrogen combined with phosphorus and

control groups to be simultaneously exposed to ambient environmental conditions of stream flow and light. Eight 1.2-m

long by 9.2-cm inside-diameter sections of clear acrylic tubing

were attached to a sheet of Plexiglas (Figure 1) to house the

glass slides. The structure was supported in the water column

by a wood frame and anchored to the bottom so that it was

oriented below the surface and parallel to stream flow. Within each tube, plastic baffles were placed in the upstream end

to facilitate mixing of water passing through the tube. Five

microscope slide holders, each holding six microscope slides,

were fixed inside each tube (Figure 2).

Nutrients were added through tubes connected to remote

supplies of concentrated nutrient solution near the upstream

end of the experimental assembly (Figure 1). Nutrient supply

containers were large enough to last several days, and the entire experiment was monitored at regular time intervals of 2

to 3 days to ensure proper function. Target nutrient enrichment concentrations were 500 μg/L for nitrate and 25 μg/L

for soluble reactive phosphorus, representing an increase of

approximately 100 μg/L of nitrate and 10 μg/L of soluble reactive phosphorus above ambient concentrations measured

at the study location.2 Potassium nitrate and potassium phosphate were the specific salts used in the nutrient addition

and both were certified to meet American Chemical Society

specifications for purity of greater than 99%.

Once the experimental assembly was in place, 6 days were

allowed for colonization. Nutrient additions were initiated on

the 7th day (see Peterson et al. 1983). Four days after nutrient

addition, one slide from each of the five assemblies was randomly removed from each tube. This sampling methodology

was repeated three more times, on the 14th, 18th and 21st day

following the initial set-up. After removal, the microscope

slides were placed in pre-labeled plastic bags containing a

small amount of deionized water. Samples were brought to

the laboratory inside coolers filled with ice for subsequent

processing. The periphyton associated with the microscope

slides was harvested by scraping both sides clean with a razor

blade into a small container with deionized water. These contents were then filtered through a 47-mm Gelman® type A/E

glass-fiber filter. Filters were stored over silica gel desiccant

and frozen prior to analysis. Chlorophyll was subsequently extracted with a hot ethanol method described by Sartory and

Grobbelaar (1984) and chlorophyll concentrations were determined spectrophotometrically (APHA 1989).

Ten samples from the control, nitrogen, phosphorus and

phosphorus plus nitrogen treatment groups for each collection period were pooled and mean chlorophyll values calculated. Mean data were then log10-transformed to

accommodate heteroscedasticity (Sokal and Rohlf 1981).

Log10-transformed data were analyzed with a repeated-measures ANOVA (PROC Mixed, SAS Institute 1996), where

each tube assembly was considered a subject (random factor) and sampling interval and treatments considered as

fixed factors. Various covariance structures were tested to determine which model best fit the data, with a first-order autoregressive structure resulting. Follow-up pair-wise

comparisons were carried out with a least squares means procedure (SAS Institute 1996). Statements of significance

imply P < 0.05, unless noted otherwise.

2 Frazer, T. K., M. V. Hoyer, S. K. Notestein, J. A. Hale and D. E. Canfield, Jr.

2001. Physical, chemical and biological characteristics of five Gulf Coast rivers.

Final Report. Southwest Water Management District. Brooksville, FL 357 pp.

Figure 1. Schematic representation of the experimental set-up (C = control,

N = nitrogen addition, P = phosphorus addition and N + P = combined

nitrogen and phosphorus addition).
J. Aquat. Plant Manage. 41: 2003. 59

RESULTS AND DISCUSSION

The experiment was conducted approximately 200 m

downstream from the main spring, which serves as the origin

of flow at this location. The mean depth was approximately

1 m and the mean stream velocity was about 0.11 m/s, which

represents a discharge of ca. 4 m3

/s. The stream was oriented

east to west and riparian vegetation was not a factor in stream

shading. Submersed plants were not immediately adjacent to

the study site although were prevalent nearby. The ambient

concentrations of nitrate and soluble reactive phosphorus at

the time of sampling were approximately 400 μg/L and

14 μg/L, respectively2

 (Table 1).

Both treatment and time effects were statistically significant (P = 0.055) and the interaction between these two effects

was not significant. A minimum mean periphyton biomass of

1.2 μg chl/cm2 was calculated from the control treatment on

day 11 of the experiment and a maximum mean periphyton

biomass of 5.7 μg chl/cm2 was calculated on day 21 (Figure

3). The nitrogen treatment had a minimum mean periphyton biomass of 1.2 μg chl/cm2 on day 11 of the experiment

and a maximum mean periphyton biomass of 6.8 μg chl/cm2

was calculated on day 21. The phosphorus treatment had a

minimum mean periphyton biomass of 1.4 μg chl/cm2 on

day 11 of the experiment and a maximum mean periphyton

biomass of 7.8 μg chl/cm2 was calculated on day 21. The

nitrogen plus phosphorus treatment had a minimum mean

periphyton biomass of 1.4 μg chl/cm2 on day 11 of the

experiment and a maximum mean periphyton biomass of

8.2 μg chl/cm2

 was calculated day 21 (Figure 3).

The addition of phosphorus and nitrogen plus phosphorus resulted in greater periphyton biomass than that of the

controls (Table 2, Figure 3). Nitrate additions alone did not

result in greater periphyton biomass than that of the controls

(Table 2, Figure 3). Differences among the treatments containing phosphorus, nitrogen with phosphorus and nitrogen

were not significant.

The experiment was terminated 26 days after installation,

before the maximum potential periphyton biomass may have

been achieved, because the microscope slides began to be

colonized by an epiphytic species of Chaetomorpha, a filamentous green alga. Strands of Chaetomorpha sp. began to grow

long enough so as to become entangled with strands attached to neighboring microscope slides. This made quantifying the amount of material attached to any single

Figure 2. Enlarged view of the artificial substrates and their layout in the

experiment.

TABLE 1. CHEMICAL AND PHYSICAL VARIABLES MEASURED JUST BELOW THE

MAIN-SPRING IN THE CHASSAHOWITZKA RIVER. EACH VALUE IS THE MEAN OF 15

SAMPLES EXCEPT SOLUBLE REACTIVE PHOSPHORUS, WHERE N = 12.

Parameter Mean Std Dev

Total Nitrogen (μg/L) 464.0 84.2

Ammonium (μg/L) 15.5 12.5

Nitrate (μg/L) 397.0 71.7

Total Phosphorus (μg/L) 19.5 5.1

Soluble Reactive Phosphorus (μg/L) 13.8 2.7

Chlorophyll (μg/L) 2.5 1.4

Dissolved Oxygen (mg/L) 6.3 2.2

Total Alkalinity (mg/L as CaCO3) 143.7 2.0

Specific Conductivity (mS/cm) 3.2 1.8

Color (Pt-Co units) 3.6 2.1

pH 7.89 0.1

Temperature (C) 23.6 1.1

Salinity (ppt) 1.7 1.0

Depth (m) 0.82 0.09

Flow (m/s) 0.11 0.09

Discharge (m3/s) 4.0 2.4

Figure 3. Mean periphyton biomass in μg chl/cm2 by treatment type and

time of collection (C = control, N = nitrogen addition, P = phosphorus addition and N + P = combined nitrogen and phosphorus addition).
60 J. Aquat. Plant Manage. 41: 2003.

microscope slide uncertain and would have confounded the

determination of the effects of nutrient addition.

In the Chassahowitzka River, we were able to increase periphyton abundance, as measured by the amount of chlorophyll per unit area, by adding phosphorus; suggesting that

phosphorus was the primary limiting nutrient for periphyton

during the course of this experiment. Nitrogen addition

treatments did not exhibit a different response from controls. However, the nitrogen addition treatments were not

different than those treatments where only P was added

either. This latter observation suggests that the addition of

nitrogen might also produce an increase in periphyton in

this system. Nevertheless, phosphorus appears to be the

primary nutrient limiting periphyton growth. The ambient

nitrogen to phosphorus ratio in the stream, which was

approximately 24:1 by weight, further supports the potential

for phosphorus limitation (Goldman et al. 1979).

Urban development within the Chassahowitzka River watershed is expected to continue and as a consequence, further increases in nutrient loading (nitrate in particular) are likely to

occur. However, our findings suggest that increased delivery of

nitrate-nitrogen may not significantly alter the abundance of

periphyton in this river. Concomitant increases in phosphorus, on the other hand, will likely stimulate the growth of

periphyton. Water resource managers are compelled to

consider the implications of these suggestions, particularly as

they relate to nutrient remediation and reduction strategies.

ACKNOWLEDGEMENTS

We thank the staff of Florida Lakewatch at the University

of Florida, Department of Fisheries and Aquatic Sciences, for

providing chemical analysis of water samples. Dr. W. T. Haller

provided a thorough and constructive review of an early

draft of this manuscript that greatly improved its content and

readability. Dr. M. S. Allen provided assistance with statistical

analysis. Funding for this work was provided, in part,

through the Surface Water Improvement and Management

Program and the Coastal Rivers Basin Board of the Southwest Florida Water Management District and also in part by

the Florida Sea Grant College Program (Grant R/LR-B-46

awarded to T. K. Frazer). Journal Series R-08886 of the

Florida Agricultural Experiment Station.

LITERATURE CITED

American Public Health Association. 1989. Standard methods for the examinations of water and wastewater. 17th Ed. American Public Health Assoc.

Washington, DC.

Bothwell, M. L. 1985. Phosphorus limitation of lotic periphyton growth

rates: An intersite comparison using continuous flow troughs (Thompson River System, British Columbia). Limnol. Oceanogr. 30:527-554.

Dodds, W. K., V. H. Smith and B. Zander. 1997. Developing nutrient targets

to control benthic chlorophyll levels in streams: a case study of the Clark

Fork River. Water Res. 31:1738-1750.

Goldman, J. C., J. J. McCarthy and D. G. Peavey. 1979. Growth rate influence

on the chemical composition of phytoplankton in oceanic waters.

Nature 279:210-215.

Hatzell, H. H. 1996. Influence of dairy and poultry operations on nitrate

concentrations in the upper Floridan aquifer in north central Florida.

In: Puckett, L. J. and F. J. Triska (eds.). U.S. Geological Survey nitrogen

cycling workshop: U.S. Geological Survey Open-File Report 96-477.

Ham, L. K. and H. H. Hatzell. 1996. Analysis of nutrients in the surface

waters of the Georgia-Florida coastal plain study unit, 1970-91. U.S. Geological Survey. Water-Resources Investigations Report 96-4037. Tallahassee, Florida.

Hershey, A. E., A. L. Hiltner, M. A. J. Hullar, M. C. Miller, J. R. Vestal, M. A.

Lock, S. Rundle and B. J. Peterson. 1988. Nutrient influence on a stream

grazer: Orthocladius microcommunities respond to nutrient input. Ecology 69:1383-1392.

Jones, G. W., S. B. Upchurch, K. M. Champion and D. J. Dewitt. 1997. Waterquality and hydrology of the Homosassa, Chassahowitzka, Weeki

Wachee, and Aripeka spring complexes, Citrus and Hernando Counties,

Florida (Origin of Increasing Nitrate Concentrations). Ambient

Ground-Water Quality Monitoring Program. Southwest Florida Water

Management District. Brooksville, FL. 167 pp.

Katz, B. G., R. S. DeHan, J. J. Hirten and J. S. Catches. 1997. Interactions

between ground water and surface water in the Suwannee River basin,

Florida. J. Am. Water Res. Assoc. 33:1237-1254. 

Lohman, K., J. R. Jones and C. Baysinger-Daniel. 1991. Experimental evidence for nitrogen limitation in northern Ozark stream. J. North Am.

Benthol. Soc. 10:14-23.

Mosisch, T. D., S. E. Bunn and P. M. Davies. 2001. The relative importance of

shading and nutrients on algal production in subtropical streams. Freshwater Biol. 46:1269-1278.

Pan, Y. and R. L. Lowe. 1994. Independent and interactive effects of nutrients and grazers on benthic algal community structure. Hydrobiologia

291:201-209.

Peterson, B. J., J. E. Hobbie and T. L. Corliss. 1983. A continuous-flow

periphyton bioassay: Tests of nutrient limitation in a tundra stream. Limnol. Oceanogr. 28:583-591.

Peterson, B. J., L. Deegan, J. Helfrich, J. E. Hobbie, M. Hullar, B. Moller, T.

E. Ford, A. Hershey, A. Hiltner, G. Kepphut, M. A. Lock, D. M. Fiebig, V.

McKinley, M. C. Miller, J. R. Vestal, R. Ventullo and G. Volk. 1993. Biological responses of a tundra river to fertilization. Ecol. 74(3):653-672.

Sartory, D. P. and J. U. Grobbelaar. 1984. Extraction of chlorophyll a from

freshwater phytoplankton for spectrophotometric analysis. Hydrobio–

logia. 114:177-187.

SAS Institute. 1996. SAS users guide. SAS Institute, Inc. Cary, NC.

Sokal, R. R. and F. J. Rohlf. 1981. Biometry. 2nd Ed. W.H. Freeman and Co.

San Francisco, CA. pp. 768-774.

Stelzer, R. S. and G. A. Lamberti. 2001. Effects of N:P ratio and total nutrient

concentration on stream periphyton community structure, biomass, and

elemental composition. Limnol. Oceanogr. 46:356-367.

Winter, J. G. and H. C. Duthie. 2000. Epilithic diatoms as indicators of

stream total N and total P concentrations. J. North Am. Benthol. Soc.

19:32-49.

Wolfe, S. H., R. H. Simons, R. E. Noss, J. A. Reidenauer, M. S. Flannery and

M. J. Bland. 1990. An ecological characterization of the Florida Springs

Coast: Pithlachascotee to Waccasassa Rivers. U.S. Fish and Wildlife Service Biological Report. 90(21).

Wright, R. M. and A. J. McDonnell. 1986a. Macrophyte growth in shallow

streams: Field investigations. J. Environ. Eng. 112:953-966.

Wright, R. M. and A. J. McDonnell. 1986b. Macrophyte growth in shallow

streams: Biomass model. J. Environ. Eng. 112:967-981.

TABLE 2. LEAST SQUARES MEANS COMPARISONS FOR THE BIOASSAY PERIPHYTON

EXPERIMENT. TREATMENTS ARE AS FOLLOWS: C = CONTROL, N = NITROGEN

ADDITION, P = PHOSPHORUS ADDITION, N+P = COMBINED NITROGEN AND PHOSPHORUS ADDITION. TIME REFERS TO THE DATE AT WHICH SAMPLES WERE COLLECTED WITH 1 BEING THE FIRST AND 4 BEING THE LAST. SIGNIFICANT

DIFFERENCES ARE IN BOLD.

Treatment Significance Time Significance

C vs. N 0.07 1 vs. 2 0.0001

C vs. N+P 0.02 1 vs. 3 0.0001

C vs. P 0.02 1 vs. 4 0.0001

N vs. N+P 0.26 2 vs. 3 0.0001

N vs. P 0.33 2 vs. 4 0.0001

N+P vs. P 0.85 3 vs. 4 0.0001