Effects of Nitrogen and Phosphorus 

Additions on Phytoplankton Productivity 

and Chlorophyll a in a Subtropical Estuary, 

Charlotte Harbor, Florida

By Ralph T. Montgomery, Environmental Quality Laboratory, Inc.,

Port Charlotte, Florida;

Benjamin F. McPherson, U.S. Geological Survey; and 

Edward E. Emmons, Environmental Quality Laboratory, Inc.,

Port Charlotte, Florida

U.S. GEOLOGICAL SURVEY

Water-Resources Investigations Report 91-4077

Prepared in cooperation with the

FLORIDA DEPARTMENT OF ENVIRONMENTAL REGULATION

Tallahassee, Florida 

1991
U.S. DEPARTMENT OF THE INTERIOR 

MANUEL LUJAN, JR., Secretary

U.S. GEOLOGICAL SURVEY 

Dallas L. Peck, Director

For additional information, Copies of this report may be

write to: purchased from:

District Chief U.S. Geological Survey

U.S. Geological Survey Books and Open-File Reports

Suite 3015 Federal Center

227 North Bronough Street Box 25425

Tallahassee, Florida 32301 Denver, Colorado 80225
Contents

Abstract 1 

Introduction 1 

Background 1 

Purpose and scope 3 

Description of study area 3 

Methods 3

In situ C-14 incubations 3 

Determination of photosynthetic C-14 uptake 3 

Determination of chlorophyll a 5 

Chemical analyses 6 

Data analyses 6

Design of nutrient addition experiments 6 

Time series 6

Single and multiple nutrient additions 9 

Concentration response 13 

Results of nutrient addition experiments 15 

Tidal Myakka River fixed station 15 

High flow 15 

Low flow 15 

Second high flow 18

Peace River and Charlotte Harbor salinity stations 18 

Low flow 18 

High flow 18 

Effects of nitrogen and phosphorus additions on phytoplankton productivity and

chlorophyll a 24 

Summary and conclusions 29 

Selected references 30

Figures

1. Map showing location of Charlotte Harbor study area and in situ study sites,

1984-85 4 

2-27. Graphs showing:

2. Total gaged flow of the Peace River, 1980-86, and dates of in situ 

flow-related studies 5

3. Relation between carbon uptake and period of in situ incubation at site 1 

(approximately 12-parts-per-thousand salinity), May 10, 1984 9

4. Relation between chlorophyll-^ concentration of amended samples as

percent of chlorophyll a in control samples and period of in situ incubation 

at site 1, May 10, 1984 9

5. Relation between carbon uptake and period of in situ incubation at site 2 at 

a salinity of approximately 12 parts per thousand on May 17, 1984 10

6. Relation between chlorophyll-^ concentration of amended samples as

percent of chlorophyll a in control samples and period of in situ incubation 

at site 2, May 17, 1984 10

7. Phytoplankton carbon uptake for control and nutrient-amended samples for 

10-hour in situ incubations at salinities of approximately 12 (site 3) and 20 

(site 4) parts per thousand, May 22,1984 12

Contents III
8. Phytoplankton chlorophyll-a concentrations for control and nutrient-amended 

samples for 10-hour in situ incubations at salinities of approximately 

12 (site 3) and 20 (site 4) parts per thousand, May 22, 1984 13

9. Phytoplankton carbon uptake for control and nutrient-amended samples for 

10-hour in situ incubations at site 5, May 31, 1984 14

10. Phytoplankton chlorophyll-a concentrations for control and nutrient-amended 

samples for 10-hour in situ incubations at site 5, May 31, 1984 14

11. Phytoplankton carbon uptake for control and nutrient-amended samples 

for 10-hour in situ incubations at a salinity of approximately 20 parts per 

thousand at site 6, June 1, 1984 16

12. Phytoplankton chlorophyll-a concentrations for control and nutrient-amended 

samples for 10-hour in situ incubations at a salinity of approximately 20 

parts per thousand at site 6, June 1, 1984 16

13. Phytoplankton carbon uptake for control and nutrient-amended samples 

for 10-hour in situ incubations at site 7 on the tidal Myakka River, 

September 27, 1984 17

14. Phytoplankton chlorophyll-a concentrations for control and nutrient-amended 

samples for 10-hour in situ incubations at site 7 on the tidal Myakka River, 

September 27, 1984 17

15. Phytoplankton carbon uptake for control and nutrient-amended samples 

for 10-hour in situ incubations at at site 7 on the tidal Myakka River, 

May 23, 1985 19

16. Phytoplankton chlorophyll-^ concentrations for control and nutrient-amended 

samples for 10-hour in situ incubations at site 7 on the tidal Myakka River, 

September 16, 1985 19

17. Phytoplankton carbon uptake for control and nutrient-amended samples 

for 10-hour in situ incubations at site 7 on the tidal Myakka River, 

September 16, 1985 20

18. Phytoplankton chlorophyll-^ concentrations for control and nutrient-amended 

samples for 10-hour in situ incubations at site 7 on the tidal Myakka River, 

May 23, 1985 20

19. Phytoplankton carbon uptake for control and nutrient-amended samples 

for 10-hour in situ incubations at a salinity of approximately 6 parts per 

thousand at site 8, May 8, 1985 21

20. Phytoplankton chlorophyll-a concentrations for control and nutrient-amended 

samples for 10-hour in situ incubations at a salinity of approximately 

6 parts per thousand at site 8, May 8, 1985 21

21. Phytoplankton carbon uptake for control and nutrient-amended samples 

for 10-hour in situ incubations at a salinity of approximately 20 parts per 

thousand at site 9, May 10, 1985 22

22. Phytoplankton chlorophyll-a concentrations for control and nutrient-amended 

samples for 10-hour in situ incubations at a salinity of approximately 

20 parts per thousand at site 9, May 10, 1985 22

23. Phytoplankton carbon uptake for control and nutrient-amended samples 

for 10-hour in situ incubations at a salinity of approximately 6 parts per 

thousand at site 10, September 17, 1985 23

24. Phytoplankton chlorophyll-a concentrations for control and nutrient-amended 

samples for 10-hour in situ incubations at a salinity of approximately 

6 parts per thousand at site 10, September 17, 1985 23

25. Phytoplankton carbon uptake for control and nutrient-amended samples 

for 10-hour in situ incubations at a salinity of approximately 20 parts per 

thousand at site 11, September 24, 1985 25

Contents IV
26. Phytoplankton chlorophyll-a concentrations for control and nutrient-amended 

samples for 10-hour in situ incubations at a salinity of approximately 

20 parts per thousand at site 11, September 24, 1985 25

27. Mean values for selected macronutrients and measurements of phytoplankton 

production collected monthly from June 1983 through June 1988 at 6 and 

20 parts per thousand along salinity transects in the lower Peace River and 

upper Charlotte Harbor 26

Tables

1. Methods used for chemical analyses 6

2. Summaries by experiment of linear, quadratic, and cubic determinations from 

general linear model procedures for changes in carbon uptake and chlorophyll a 

in response to nutrient additions 7

3. Ambient water-quality data and preceding Peace River flows for each 

experiment 11

4. Dates and locations where there were significant changes in relative percentage 

of either carbon uptake or chlorophyll a for three measured size fractions of 

.phytoplankton in response to nutrient additions 28

CONVERSION FACTORS AND ABBREVIATIONS

Multiply By To obtain

millimeter (mm) 0.0394 inch

centimeter (cm) 0.3937 inch

meter (m) 3.281 foot

kilometer (km) 0.6214 mile

square kilometer (km2 ) 0.3861 square mile

cubic meter per second (m3/s) 35.31 cubic foot per second

kilogram per day (kg/d) 2.205 pound per day

Additional abbreviations used in report:

microcuries (|lCi) 

micrometer (|im) 

milligram per liter (mg/L) 

milliliter (mL) 

parts per thousand (%o)

Contents
Effects of Nitrogen and Phosphorus Additions on 

Phytoplankton Productivity and Chlorophyll a in a 

Subtropical Estuary, Charlotte Harbor, Florida

By Ralph T. Montgomery, Benjamin F. McPherson, and Edward E. Emmons

ABSTRACT

The response of natural phytoplankton assemblages in 

a subtropical coastal plain estuary, Charlotte Harbor, 

Florida, to inorganic nitrogen and phosphorus additions was 

determined from measurements of relative changes in both 

the uptake of carbon-14 and concentrations of chlorophyll a. The effects of nitrate plus nitrite nitrogen, ammonia 

nitrogen, and orthophosphorus additions over a series of 

concentrations were evaluated through in situ experiments 

conducted during periods of seasonally low and high river 

inflows. The responses to nutrient additions were evaluated 

for three different size fractions of phytoplankton. Relative 

changes of phytoplankton carbon uptake and chlorophyll-a 

concentrations were highly variable with regard to season, 

location, nutrient, and size fractions.

Within areas of the estuary characterized by lower 

salinities, phytoplankton exhibited a distinct seasonal 

pattern to additions of inorganic nitrogen. Under seasonally 

high freshwater inflow, phytoplankton showed little 

response to inorganic nitrogen additions, whereas under 

seasonally low inflow, phytoplankton responded to the 

inorganic nitrogen additions. The seasonally high freshwater 

inflow increased ambient inorganic nitrogen concentrations 

and water color. The high water color greatly reduced light 

penetration in the water column and limited phytoplankton 

productivity. The effect of nutrient additions in the higher 

salinity reaches of the estuary indicates that, under normal 

conditions, these areas are continually nitrogen limited. 

During periods of high freshwater inflow during the summer 

months, the estuary can be divided conceptually into a lowsalinity zone where phytoplankton production is mediated 

by light availability, as determined by high water color, and 

a high-salinity zone where phytoplankton production is 

nitrogen limited. Seasonal nutrient concentration data and 

comparisons among inorganic nitrogen inputs and observed 

phytoplankton productivity support the experimental

bioassay results. Each of these lines of evidence indicates 

that, exclusive of seasonal riverine influences that affect 

light penetration of the water column, nitrogen availability 

normally limits phytoplankton production within the 

Charlotte Harbor estuarine system.

INTRODUCTION 

Background

Redfield (1958) suggested that phosphorus might 

ultimately be responsible for limiting net organic production 

in both freshwater and marine systems. Evidence that has 

become available since then, however, has led to a widely 

accepted generalization that marine environments are, for the 

most part, nitrogen limited (Ryther and Dunstan, 1971), 

whereas phosphorus predominantly limits production in 

freshwater systems (Jones and Lee, 1982; Sakshaug and 

Olsen, 1986). Common exceptions to this would include 

those aquatic systems influenced by either phosphate-rich 

deposits or nutrient pollution, although other exceptions have 

been reported. Myers and Iverson (1981) found evidence of 

phosphorus-limiting primary production in an estuary in the 

northeastern Gulf of Mexico, whereas McComb and others 

(1981) found evidence of seasonal shifts between nitrogen 

and phosphorus limitation in an estuary in western Australia. 

Davies and Sleep (1981) reported that phytoplankton 

production in the English Channel at times could be simultaneously and additively stimulated by additions of ammonia, 

nitrate, and phosphate, whereas Agins and Jaccarini (1982) 

found phosphorus to be the limiting nutrient in parts of the 

Mediterranean Sea. Wurtsbaugh and others (1985) observed 

nitrogen stimulation of phytoplankton carbon-14 uptake in a 

tropical alpine lake.

Introduction
Although light, silica, iron, trace elements, and grazing 

have all been observed under certain conditions to limit 

phytoplankton production, macronutrient availability, 

especially nitrogen and phosphorus, has generally been 

demonstrated to be the key factor regulating primary productivity in estuarine and coastal waters (Ketchum, 1967; 

Boynton and others, 1982). Nitrogen and phosphorus exist in 

a variety of dissolved and particulate forms that may be 

directly or indirectly used for phytoplankton production. The 

distribution and availability of these various nutrient forms 

are dependent on the complex interactions of physical, 

chemical, and biological processes. It has been suggested 

that, in estuaries, nutrient recycling sustains much of the 

shorter term seasonal phytoplankton productivity (Nixon, 

1981), whereas the input of new nutrients increases total 

availability, thus supporting longer term production. Anthropogenic development in coastal basins has often resulted in 

increased nutrient loadings and has been implicated in estuarine enrichment and increased phytoplankton productivity 

(Jaworski, 1981).

Measurements of a wide variety of growth or 

photosynthetic characteristics have been utilized to evaluate 

the response of phytoplankton communities to potential 

nutrient limiting conditions. Some of the more commonly 

used measurements of phytoplankton responses include 

changes in biomass (Graneli and others, 1986); assays of 

physiological and enzymatic properties (Smith and Kalff, 

1981; Kana and others, 1985; Elser and Kimmel, 1986); 

measurements of ambient-nutrient ratios (Sklar and Turner, 

1981) and use of these ratios in kinetic models (Kunikane 

and Kaneko, 1984); culture and chemostat studies of nutrient 

uptake rate and growth (Caperon and Myer, 1972a, 1972b; 

Collos, 1984; Kunikane and others, 1984; Sommer, 1985; 

Sanders and others, 1987); and field measurements of 

nutrient uptake or carbon fixation (Goldman, 1960; Davies 

and Sleep, 1981; Nalewajko and Garside, 1983; Wurtsbaugh 

and others, 1985).

This report is one of several published by the U.S. 

Geological Survey, in cooperation with the Florida Department of Environmental Regulation, as part of the Survey's 

environmental assessment of Charlotte Harbor. The report 

discusses the effects of nutrient additions on phytoplankton 

populations in the harbor. Phytoplankton constitute a major 

primary producer in the harbor. Increased population growth 

in the basin is projected to increase nutrient loading to the 

harbor (Hammett, 1988) and could stimulate phytoplankton 

growth and cause changes in water quality.

In this study, changes in the incorporation of carbon-14 

and chlorophyll-a concentrations were used as measures of 

the response of phytoplankton assemblages to a series of 

added nutrient concentrations. Changes in the phytoplankton 

in response to nutrient additions were evaluated for the 

whole and each of three different size fractions: net- 

(>20|um), nano- (between 5 and 20 |um), and picophytoplankton <5 |um). The importance of segmenting

phytoplankton production and biomass measurements, such 

as chlorophyll a, by size fractions has been emphasized in 

freshwater (Munawar and others, 1978; Lane and Goldman, 

1984), estuarine (Bruno and others, 1983; Furnas, 1983; 

Furuya and Marumo, 1983; Sellner, 1983), and marine 

(Malone, 1971; Marshall, 1985; Probyn, 1985) habitats. 

Although a variety of terms and size ranges have been 

applied (Sicko-Goad and Stoermer, 1984), the value of 

partitioning phytoplankton into functional groups based on 

size has been shown in assessing food-chain value (Janicki 

and DeCosta, 1984; Stockner, 1988) and in determining 

phytoplankton community responses to environmental 

variables (Malone, 1980). Differential responses of sizebased assemblages to nutrient levels and ratios (Lane and 

Goldman, 1984; Probyn, 1985), as well as pollutants 

(Munawar and others, 1983), have been demonstrated.

Microscopic surveys of the taxonomic composition of 

the phytoplankton assemblages within each of the three 

measured size fractions have indicated the following generalized patterns with regard to salinity ranges within Charlotte 

Harbor. The smallest size fraction (<5 |im), at both intermediate and higher salinities, is characteristically dominated by 

Cryptophyceae (Chroomonas spp. and Cryptomonas spp.), 

with small Bacillariphyceae (Thalossiosira spp., Nitzschia 

spp. Naviculla spp.) also common nano-plankton components. In high salinity waters, the net-plankton size 

fraction is often dominated by chain-forming and larger 

diatoms. Skeletonema costatum, Asterionella glacialis, 

Odentella sinensis, Corethron criphilum, Coscinodiscus 

centralis, and C. eccentricus, as well as species of 

Chaetoceros and Rizosolenia, are seasonally important 

diatoms. Dinophyceae (Ceratium spp. and Peridinum spp.) 

are seasonally common in the largest size fraction during the 

summer months.

At intermediate salinities, blooms of Skeletonema 

costatum are commonly associated with relative increases in 

carbon uptake and chlorophyll-a concentration within the 

largest size fraction. Occasionally, however, dinoflagellates 

(Prorocentrum micans, P. minimum, Gymnodinium spp., and 

Gyrodinium spp.) comprise major components of the largest 

size fractions. Specifically, blooms of Gyrodinium 

resplendens seasonally dominate the larger size fractions at 

the mouth of the Peace River.

The pico-plankton size fraction at the low salinities 

often contained significant numbers of nonflagellated, 

smooth, circular to ovoid, green cells. Taxonomically, such 

cells probably include both Cyanophyceae (Synechoccus 

spp., Chroococcus spp., and Anacystis spp.) as well as 

Chlorophyceae (Nannochloris spp. and Chlorella spp.). 

Small phytoflagellates (Chlamydomans spp., Carteria spp., 

Chroomonas spp., and Cryptomonas spp.) are also common 

components of the pico-plankton at the lowest harbor salinities. The larger size fractions in the riverine areas of the 

estuary generally are characterized by mixtures of both 

Chlorophyceae (Ankistrodesmus spp., Coelastrum spp.,

Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
Crucigenia spp., Pediastrum spp., Scenedesmus spp., and 

Tetraedron spp.), Bacillariophyceae (Cyclotella spp., 

Nitschia spp., Navicula spp., and Fragillaria spp.), and 

Cyanophyceae (Anabaena spp., Afttfcysto spp.).

Purpose and Scope

This report presents the results of a study to evaluate 

the effects of inorganic nitrogen and phosphorus additions on 

phytoplankton productivity and chlorophyll-a concentrations 

in a subtropical Florida coastal plain estuary and assesses the 

potential effects of increased nutrient concentrations on 

future phytoplankton production within the Charlotte Harbor 

system. The results of this estuarine phytoplankton productivity study are discussed in terms of the seasonal and areal 

variations in ambient-nutrient concentrations and hydrologic 

conditions. The potential implications that increased 

anthropogenic-nutrient inputs may have on primary productivity within specific hydrologically defined areas of the 

harbor also are examined.

DESCRIPTION OF STUDY AREA

Charlotte Harbor in southwest Florida (fig. 1) is a 

large, relatively shallow, subtropical, coastal plain estuary 

enclosed by a series of barrier islands. Water exchange with 

the Gulf of Mexico occurs primarily through Boca Grande 

Pass at the southern end of the harbor. The estuarine system 

covers an area of approximately 300 km2 and has an average 

depth of only about 3 m. The estuary is vertically well mixed 

because of tidal flow, wind, and the shallow water depth. 

However, strong vertical-density stratification does occur in 

the northern parts of the harbor, predominantly during 

periods of high freshwater inflow (Fraser and Wilcox, 1981; 

Fraser, 1986).

Two rivers flow into the upper estuary: the Peace 

River, which has an average, total gaged, long-term inflow of 

approximately 49.5 m3/s; and the Myakka River, which has 

an average inflow of about 18 m3/s. Seasonal variations of 

inflow to the estuary are reflected in the hydrograph for the 

Peace River (fig. 2).

Charlotte Harbor is one of the least disturbed estuarine 

complexes in Florida (Taylor, 1974; Fraser and Wilcox, 

1981; Froelich and others, 1985). During the past decade, 

however, there has been concern over the long-term effects 

of rapid population growth in the area and the effects of 

nonpoint sources of runoff on the water quality of the estuary. 

Parts of the northern Charlotte Harbor drainage basin contain 

rich phosphate deposits in the surficial and intermediate 

aquifers. Phosphate mining and processing have been active 

in the upper Peace River basin since the late 1800's, first 

beginning with direct dredging of river sediments. These 

deposits are natural sources of phosphorus to the Peace and 

Myakka Rivers and, ultimately, to Charlotte Harbor.

METHODS

The effects of nutrient additions were evaluated 

through a series of in situ experiments conducted during 

seasonally low and high periods of river inflows. In each 

experiment, natural phytoplankton populations were exposed 

to increasing concentrations of added nitrate plus nitrite 

(NO3-N), ammonia (NH4-N), and orthophosphorus (PO4-P). 

The patterns of the responses of the natural phytoplankton 

assemblages to increased concentrations of each of these 

nutrients were determined from measurements of relative 

changes in the uptake of carbon-14 and chlorophyll-a 

concentrations. Estimates of phytoplankton production and 

biomass were made using the procedures described below.

In Situ C-14 Incubations

To reduce the chances of trace inhibitory or stimulatory 

contamination (Marra and Heinemann, 1984), all incubation 

bottles and lined caps were washed in 6 normal hydrochloric 

acid (6N HCL) prior to use, rinsed, triple rinsed with doubledistilled water, and rinsed again with sample water 

immediately prior to filling. At each station, 450-mL 

subsurface-water samples were filtered through 500-micron 

cloth screening to remove particulate material and larger 

zooplankton. All in situ incubations were conducted using 

473-mL, clear, flint glass, narrow mouth, round bottles. A 

predetermined number of replicate treatment bottles were 

then prepared by the addition of 0.5 mL of stock nutrient 

solutions to obtain the desired final added concentrations. An 

identical volume of the deionized, double distilled water 

used to prepare the stock nutrient solutions was added to an 

equal number of replicate controls. Dark bottles were used to 

determine blank background uptake. All incubation bottles 

were then inoculated with 10 fiCi of trace-metal-free carbon-14 

(Fitzwater and others, 1982) and quickly suspended at the 

predetermined 50-percent light depth and incubated in situ. 

To reduce potential diel variability (Harding and others, 

1982a, 1982b; Malone, 1982; Brown and Field, 1985; Kana 

and others, 1985), samples at all stations were incubated for 

a predetermined period of time around apparent noon. After 

incubation, the samples were stored in the dark on ice and 

quickly returned to the laboratory where they were filtered.

Determination of Photosynthetic C-14 Uptake

To determine the total carbon-14 uptake of the whole 

sample and that attributable to the net-, nano- and picoplankton size fractions, three separate 50-mL subsamples 

(Arthur and Rigler, 1967) were taken from each of the 

replicate-sample bottles. The initial subsample was filtered 

directly onto a 2.4-cm glass fiber filter using a low vacuum 

(<120 (im) of mercury (Hg). Two subsequent subsamples

Methods
82' 82°00'

27°00'

26°45'

CHARLOTTE 

HARBOR

10 MILES

EXPLANATION 

lA TIME SERIES STUDY SITE, MAY 1984

3 NUTRIENT COMBINATION, 12 PARTS PER THOUSAND 

STUDY SITE, MAY 1984

4 NUTRIENT COMBINATION, 20 PARTS PER THOUSAND 

STUDY SITE, MAY 1984

5 NUTRIENT CONCENTRATION, 12 PARTS PER THOUSAND 

STUDY SITE, MAY 1984

6 NUTRIENT COMBINATION, 20 PARTS PER THOUSAND 

STUDY SITE, JUNE 1984

7 FIXED TIDAL MYAKKA SITE

8 SALINITY BASED SITES---- 6 PARTS PER THOUSAND, MAY 1985

99 20 PARTS PER THOUSAND, MAY 1985

100 6 PARTS PER THOUSAND, SEPTEMBER 1985 

11 * 20 PARTS PER THOUSAND, SEPTEMBER 1985

Figure 1 . Location of Charlotte Harbor study area and in situ sites, 1984-85.

Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
o 

o

LU 

CO

CC 

LLJ 

Q_

CO

CC 

LUhLJJ

g 

m

o

z

g

LlCC 

LU

E

350

300

250

200

150

100

50

0

I I I I

SAMPLING PERIODS

A= LOW-FLOW EXPERIMENTAL DESIGN, MAY-JUNE 1984

B= HIGH FLOW, SEPTEMBER 1984

C= LOW FLOW, MAY 1985

D= HIGH FLOW, SEPTEMBER 1985

A B

1980 1981 1982 1983 1984 1985 1986

Figure 2. Total gaged flow of the Peace River, 1980-86, and dates of in situ flow-related studies.

were likewise filtered after having been respectively filtered 

through 20- and 5-micron screening. Three parallel filter 

blanks for each station were prepared by adding 10 LiCi of 

carbon-14 to a corresponding 450-mL ice-stored water 

sample, taking 50-mL subsamples, and filtering and treating 

the blanks in the same manner as the initial subsample 

described above. The wet filters were then placed in scintillation vials. Any residual inorganic carbon-14 was removed by 

adding 0.5 mL of 0.1 N HCL (Lean and Bumison, 1979) and 

by allowing the vials to stand for 3 hours before the addition 

of 10.0 mL of scintillation fluor. The scintillation vials were 

stored in the dark for at least 24 hours before being counted. 

Each sample was counted for 30 minutes using a liquid 

scintillation system to provide counts per minute (CPM) in 

spectral region A with standard deviations of less than 0.2 

percent. CPM were then converted to disintegrations per 

minute (DPM) using the channels ratio method. The counting 

efficiency was determined using a series of filters quenched 

by increasing amounts of phytoplankton onto which a known

amount of carbon-14 labeled sucrose (Pugh, 1970) had been 

dried. The specific activity of the stock carbon-14 bicarbonate added to the samples was checked (Iverson and others, 

1976) after each experiment. DPM were converted to milligrams carbon per cubic meter fixed, based on equations of 

Strickland and Parsons (1972).

Determination of Chlorophyll a

Chlorophyll-a concentrations for the >20 |im, 5 to 

20 |im, and <5 |im size phytoplankton fractions were 

determined fluorimetrically (Strickland and Parsons, 1972) 

using 90-percent acetone extracted from unground filters 

prepared using the same filtering procedures used for the 

determination of carbon-14 incorporation. All reported 

chlorophyll-a concentrations represent single determinations 

obtained by compositing equal volumes from each of the 

replicate incubation bottles.

Methods
Chemical Analyses

Corresponding water samples at each station were 

collected in new, clean polyethylene containers. The containers were rinsed with sample water, filled, and immediately 

placed on ice in the dark. Analyses were conducted within 

recommended holding times for unpreserved samples using 

either U.S. Environmental Protection Agency (1979) or 

American Public Health Association (1989) standard 

methods (table 1).

Table 1. Methods used for chemical analyses

[ERA, U.S. Environmental Protection Agency, 1979; 

APHA, American Public Health Association, 1989]

Constituent or property Method and reference

Chloride

Color

NO2+NO3 nitrogen

NHLt nitrogen

Silica

Inorganic carbon

Orthophosphorus

Total kjeldahl nitrogen

Total phosphorus

Chlorophyll a (fluorometric)

4500B

110.2

353.2

350.1

370.1

415.1

365.1

351.3

365.2

10200H,3

(APHA)

(EPA)

(EPA)

(EPA)

(EPA)

(EPA)

(EPA)

(EPA)

(EPA)

(APHA)

Data Analyses

Graphical and statistical analyses of results were 

conducted utilizing procedures within PC SAS (Statistical 

Analysis System Institute, Inc., 1987). In a number of 

experiments, carbon uptake and chlorophyll-a responses to 

nutrient additions were tested over a series of nitrogen and 

phosphorus concentrations. In these instances, general linear 

model procedures (polynomial regression or curve-fitting 

analysis) were employed to determine if the coefficients of 

the resulting phytoplankton responses over the range of 

nutrient concentrations tested were significantly different 

from zero and to describe the shape of the response curves. 

This methodology explicitly differs from that of the more 

often applied multiple-comparative procedures, such as 

Duncan's Multiple Range of Analysis of Variance (Duncan, 

1957; Waller and Duncan, 1969). Multiple-comparative 

analysis would treat the series of nutrient concentrations as 

qualitatively different variables and test and contrast means 

for individual concentrations.

In testing responses to treatments with increasing 

concentrations of a single quantitative variable, such as a 

nutrient, polynomial regression or curve-fitting analysis 

provides the most effective and statistically valid method of 

estimating the relation of the observed response with the 

applied treatment (Chew, 1971; 1980; Peterson, 1977; Little,

1981). In utilizing polynomial regression to estimate the 

relation between the dependent response (phytoplankton 

carbon or chlorophyll a) and independent treatment variables 

(nitrogen forms and phosphorus), it is usually sufficient to 

partition the resultant treatment sum of squares into its linear, 

cubic, and quadratic components (table 2) while testing the 

significance of each (Chew, 1971). The advantages of this 

methodology over standard analysis of variance procedures 

are evident when applied to actual field data where progressive patterns in the data may be obscured by the variability 

between discrete-measurement intervals.

Nutrient concentrations were log transformed before 

applying polynomial regression procedures because phytoplankton responses were tested over nutrient concentration 

ranges of up to three orders of magnitude. The relative 

shapes of the resulting response curves were further analyzed 

using nonlinear, least squares, and fitting procedures to solve 

and plot the estimated resultant curves.

DESIGN OF NUTRIENT ADDITION 

EXPERIMENTS

A series of experiments was initially conducted to 

develop an overall experimental design. The purpose of the 

design was to evaluate adequately the potential phytoplanktoncommunity responses to nutrient additions under high and 

low freshwater inflow conditions.

Time Series

Collos and Slawyk (1984) noted the importance of 

conducting an incubation time series experiment as part of 

any interpretation of the response of C-14 uptake to nitrogen 

additions. Their results indicated that, during the early 

periods of a time-course experiment, the addition of nitrogen 

could result in inhibition of C-14 uptake, whereas longer 

incubations resulted in marked stimulation. In addition, 

experimental incubation periods should be of sufficient 

length to observe a response, if present, while at the same 

time be short enough to reduce the possible "bottle-induced" 

effects often associated with extended incubations.

To define an adequate incubation period, two independent 

time-series experiments were conducted at estuarine salinities of approximately 12%o during a period when ambient 

NO3-N and NtLt-N concentrations were at detection limits 

(table 3). Each time-series experiment was conducted by 

incubating sets of control and treated bottles for periods 

ranging from 2 to 12 hours centered around apparent noon 

(eastern standard time). The longest incubation period 

samples were collected first. A drogue was used to mark the 

initial body of water sampled and sequential samplings were 

conducted hourly up to the 2-hour incubation period, which 

was collected 1 hour prior to apparent noon.

Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
Table 2. Summaries by experiment of linear, quadratic, and cubic determinations from general linear model procedures 

for changes in carbon uptake and chlorophyll a in response to nutrient additions

[Student t value for testing null hypothesis; significance level, probability, underlined equals statistical significance; mg/m 3 , milligrams per 

cubic meter]

c . . Parameter estimate

Linear

Nutrient concentration

\2%o 

NO3-N 167.279 

NH4-N -89.478

20%o 

NO3-N 297.327 

NH4-N 318.211

Flow-related studies 

Myakka fixed station 

September 1984 

PO4 -P - 2.774 

N03-N 76.137 

NH4-N 14.071

May 1985 

PO4-P -38.526

NO3-N 55.627 

NH4-N 3.906

September 1985 

PO4-P - 1.459 

N03-N - .059 

NH4-N .906

Peace River salinity zones 

May 1985 

6%o 

PO4 -P -36.438

N03-N -17.823 

NH4-N 20.202

20%o 

PO4-P 13.916 

NO3-N 82.569 

NH4-N 82.122

September 1985 

6%o 

PO4-P 2.485 

NO3-N 3.031 

NH4-N 3.411

20%o 

PO4 -P -16.847 

N03-N 230.491 

NH4-N 309.663

Nutrient concentration

12%o 

N03-N 14.565 

NH4-N 10.002

20%o 

NO3-N -8.7419 

NH4-N -7.6802

Quadratic

11.450 

-6.848

9.159 

9.050

.598 

-1.682 

-1.583

- .402 

2.020 

4.693

.092 

- .022 

- .046

.931

.255 

.429

- .243 

2.041 

8.590

- .074 

.262 

.177

.414 

2.134 

-5.945

.278 

.349

.897 

.746

Cubic

-1.479 

1.464

-3.367

-3.575

- .066 

-1.072

- .237

.439 

- .366 

.619

.024 

- .003 

- .019

.516 

.304 

- .197

- .246 

- .631 

- .058

- .037 

- .019 

- .037

.358 

-2.682 

-4.477

- .185 

- .114

.217 

.192

Linear

2.61 

1.50

3.85 

3.39

- .24 

2.62 

.53

-3.20

3.22 

.21

-1.52 

- .05 

.89

-2.29 

-1.63 

2.11

1.92 

2.96 

3.96

.93 

1.18 

1.06

- .65 

2.48 

4.16

1.88 

1.60

-18.88 

- 2.79

Student T

Quadratic Cubic

Carbon uptake (mg/m

6.51 -1.67 

-5.26 1.62

4.91 -2.70 

3.91 -2.35

1.23 -' .36 

-1.38 -2.31 

-1.42 - .56

- .80 2.29 

2.79 -1.33 

6.02 2.10

2.29 1.57 

- .46 - .17 

-1.07 -1.16

1.40 2.04 

.56 1.75 

1.07 -1.30

- .80 -2.14 

1.75 -1.42 

9.90 - .18

- .67 - .89 

2.45 - .48 

1.32 - .74

.38 .87 

.55 -1.82 

-1.91 -3.79

Sig

Linear

3 ) 

0.0139

.1421 

.0005

.0018

.8135 

.0148

.6007 

.0036

.0035

.8355

.1401 

.9593

.3827

.0302

.1145 

.0445

.0660 

.0065

.0005

.3589 

.2471 

.2970

.5208 

.0198

.0003

Chlorophyll a (mg/m3 )

1.31 -1.72 .1569 

2.60 -1.21 .1705

80.16 29.01 .0001

11.20 4.30 .0495

;nificance level

Quadratic

0.0001

.0001

.0001

.0004

.2309 

.1796 

.1662

.4329 

.0099

.0001

.0304

.6474 

.2939

.1734 

.5813 

.2937

.4306 

.0921 

.0001

.5098 

.0212

.1976

.7056 

.5876 

.0672

.2824 

.0485

.0001

.0004

Cubic

0.1056 

.1124

.0105

.0249

.7226 

.0291

.5787 

.0302

.1955 

.0460

.1275 

.8664

.2574

.0131

.0912 

.2054

.0421 

.1661 

.8618

.3842 

.6375 

.4687

.3912 

.0806 

.0008

.1831 

.2793

.0001

.0127

Standard error

Linear

64.168

59.743

77.191 

93.782

11.638 

29.086 

26.554

12.047 

17.272 

18.622

.958 

1.135 

1.021

15.895 

10.913 

9.569

7.252 

27.886 

20.738

2.661 

2.560 

3.206

25.880 

92.866

74.370

7.753 

6.252

.463

2.757

Quadratic

1.759 

1.301

1.865 

2.314

.487 

1.218 

1.111

.504

.724 

.779

.040 

.047 

.043

.665

.457 

.400

.031 

1.167 

.868

.111 

.107 

.134

1.083 

3.886 

3.112

.213 

.135

.011 

.066

Cubic

0.888 

.901

1.248 

1.522

.185 

.463

.422

.191

.275 

.296

.015 

.018 

.016

.252 

.173 

.152

.115

.443 

.329

.042 

.041 

.051

.411 

1.475 

1.181

.107 

.094

.007 

.045

Design of Nutrient Addition Experiments
Table 2. Summaries by experiment of linear, quadratic, and cubic determinations from general linear model procedures 

for changes in carbon uptake and chlorophyll a in response to nutrient additions Continued

[Student t value for testing null hypothesis; significance level, probability, underlined equals statistical significance; mg/m3 , milligrams per 

cubic meter]

Experiment -

Parameter estimate

Linear Quadratic Cubic

Flow-related studies 

Myakka fixed station 

September 1984

2%c

PO4 -P

NO3-N

NH4-N

May 1985

23%e

PO4 -P

NO3 -N

NH4-N

September 1985

0%0

PO4-P

NO3-N

NH4-N

.298

2.508

1.610

- .810

.961

- .472

- .054

.218

- .025

- .20

.015

.073

.008

.152

.296

- .004

- .013

- .003

- .007

- .027

- .011

.011

.004

.037

.001

.004

.001

Linear

Student T Significance level

Quadratic Cubic Linear Quadratic Cubic

Standard error

Linear Quadratic Cubic

Chlorophyll a (mg/m3 ) Continued

1.08

2.08

2.35

-1.92

1.09

- .37

- .62

3.14

- .48

-1.72

.30

2.55

.46

4.13

3.68

- .97

4.40

-1.19

-1.69

-1.44

- .97

1.57

.30

1.22

.58

-3.49

.35

.3216

.0827

.0572

.1033

.3182

.8142

.5611

.0201

.6453

.1364

.7758

.0438

.6643

.0062

.0103

.3718

.0045

.2785

.1423

.2005

.3694

.1679

.7714

.2697

.5857

.0130

.7398

.276

1.206

.686

.422

.883

1.920

.087

.069

.051

.012 .004

.050 .019

.029 .011

.018 .007

.037 .014

.080 .031

.004 .001

.003 .001

.002 .001

Peace River salinity zones

May 1985

6%e

PO4 -P

NO3-N

NH4-N

20%o

PO4-P

NO3 -N

NH4-N

September 1985

6%e

PO4 -P

NO3-N

NH4-N

20%o

PO4-P

NO3 -N

NH4-N

- .304

1.072

.598

- .673

1.573

.193

.082

.247

.244

.061

- .920

2.490

.007

.038

.750

.045

.311

.496

- .001

.013

.017

.017

.889

.297

.011

- .007

.005

.012

.016

.048

- .001

- .003

- .003

- .002

.102

- .001

- .51

1.80

1.88

-1.56

.47

.09

.62

1.24

2.10

.16

- .18

.55

.30

1.55

5.64

2.49

2.20

5.53

- .26

1.62

3.43

1.05

4.18

1.57

1.20

- .78

1.07

1.76

.30

1.42

- .61

-1.00

-1.68

- .37

1.26

- .01

.6251

.1212

.1086

.1706

.6578

.9312

.5606

.2608

.0801

.8791

.8622

.6024

.7767

.1728

.0013

.0472

.0897

.0015

.8033

.1556

.0139

.3354

.0058

.1679

.2739

.4631

.3247

.1295

.7763

.2059

.5627

.3556

.1430

.7212

.2552

.9897

.591

.594

.315

.432

3.376

2.143

.134

.199

.116

.385

5.081

4.530

.025 .009

.249 .009

.013 .005

.018 .007

.141 .054

.090 .034

.006 .002

.008 .003

.005 .002

.016 .006

.212 .081

.190 .071

The first time-series experiment investigated responses to 

the addition of 0.5 mg/L of NO3-N. The results indicated a lag 

of 8 to 10 hours was required before a significant increase in the 

rate of carbon uptake was observed (fig. 3). A slightly shorter 

period of time was needed to observe increases in concentrations of chlorophyll a above control levels (fig. 4). No significant differences were observed among the measured phytoplankton size fractions in their responses to nitrogen addition.

A second experiment was conducted to further investigate 

phytoplankton reactions to nitrogen additions with respect to 

incubation length. Additions of 0.5 mg/L of NO3-N and 

NH4-N were compared with controls (figs. 5 and 6). Stimulation patterns of carbon uptake and chlorophyll a were similar 

for both nitrogen forms. The incubation period required to

observe an increase in concentrations of chlorophyll a was 

again slightly less than that required for a corresponding 

significant increase in carbon uptake.

Time lags, as observed in these experiments between 

nutrient additions and measurable stimulation, should be 

expected to vary both with phytoplankton community 

composition and physiological condition. In each of the 

preceding experiments, incubation periods of 6 to 8 hours 

were sufficient to observe increases in phytoplankton carbon 

uptake and concentrations of chlorophyll a. However, to 

assure adequate detection of phytoplankton responses to 

experimental nutrient additions, standardized 10-hour 

incubations, centered at 1200 hours eastern standard time, 

were utilized in all subsequent investigations.

8 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
Single and Multiple Nutrient Additions

An experiment was conducted to investigate potential 

differences in the responses of phytoplankton communities at 

two salinity zones within the estuary to different concentrations and combinations of potentially limiting nutrients.

Phytoplankton were collected from two salinity zones 

(approximately 12 and 20%o) at a time when both areas of the 

estuary were characterized by low ambient-nitrogen levels 

(table 3). Figures 7 and 8 depict the effects of nutrient 

additions. Differences in carbon uptake were analyzed using 

analysis of variance and contrast methods (alpha = 0.05) to 

compare mean uptake rates for various treatments.

tr

- UJ

UJ

UJ

°- 9

3 CD

2 ^

O O

< 1

UJ rr

2.0

1.5

1.0

0-5

I I I I I I 1 I I I I I I I \ \ 1 I I I T

CONTROL SAMPLES (VALUES ARE MEANS 

FOR FIVE REPLICATE SAMPLES)

SAMPLES AMENDED WITH 0.5 MILLIGRAM PER LITER 

N03 -N. (VALUES ARE MEANS FOR FIVE REPLICATE 

SAMPLES)

INDICATES MEANS FOR AMENDED AND CONTROL

SAMPLES ARE SIGNIFICANTLY DIFFERENT AT

A 5-PERCENT SIGNIFICANCE LEVEL /

+ -+ - +

r~~*"

/

t I I I I I I I I I I I I I I I I I I I i I I I I 

6 8

HOURS

10 12

Figure 3. Relation between carbon uptake and period of in situ incubation at site 1 (approximately 12-parts-per-thousand 

salinity), May 10, 1984.

2 400

UJ

O

tr

UJ 

0.

2

. 300 

2 

O

DCh2 2 00 

O

2 

O 

O

ce I

100

X 

CL

O tr 

O

x 

o

i i i i i i i i i i i i i i i i i i i i y i

P P P CONTROL SAMPLES

4. 4. + SAMPLES AMENDED WITH 0.5

MILLIGRAM PER LITER NOg-N

t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ! 1 1 1 1

1 1 1

~"

-

II 1

10 12

HOURS

Figure 4. Relation between chlorophyli-a concentration of amended samples as percent of chlorophyll a in control samples 

and period of in situ incubation at site 1, May 10, 1984.

Design of Nutrient Addition Experiments 9
LU 1.5

LU

-J CO

gs

CD CC

CC LU

< CL

° CO 

Z 5

LU CC 

5 O

1.0

0.5

CONTROL SAMPLES (VALUES ARE MEANS FOR 

FIVE REPLICATE SAMPLES)

+- + .+ SAMPLES AMENDED WITH 0.5 MILLIGRAM PER LITER NO3 -N. 

(VALUES ARE MEANS FOR FIVE REPLICATE SAMPLES)

^_._^-._^ SAMPLES AMENDED WITH 0.5 MILLIGRAM PER LITER NH4 -N. 

(VALUES ARE MEANS FOR FIVE REPLICATE SAMPLES)

INDICATES MEANS FOR AMENDED AND CONTROL 

SAMPLES ARE SIGNIFICANTLY DIFFERENT AT A 

5-PERCENT SIGNIFICANCE LEVEL

/>

10

HOURS

12

Figure 5. Relation between carbon uptake and period of in situ incubation at site 2 at a salinity of approximately 

12 parts per thousand on May 1 7, 1984. (No significant differences between means of amended samples.)

IZ 300

UJ

O

CC

ULJ

0.

 200

z

ULJ 

O

z 

o 

o

«i 100

I 

Q. 

O 

CC

g

i 

o

I

-

T

2

i i i i I I i i i I I I I i I I I I I I I I I I r,'( I I 

0 0 a CONTROL SAMPLES

+ + + SAMPLES AMENDED WITH 0.5 ,''',^

MILLIGRAM PER LITER NO,-N jr--^

n^s

£.,---{* & SAMPLES AMENDED WITH 0.5 ^ ''^'--^' 

MILLIGRAM PER LITER NH4 -N ^^,^-^T.---"

t i i i 

46 

i I i i i i I i i I I I I I i i i I I I ill

8 10 12

HOURS

I 1

_

-

-

1 1

1

Figure 6. Relation between chlorophyll-a concentration of amended samples as percent of chlorophyll a in control 

samples and period of in situ incubation at site 2, May 1 7, 1984.

10 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
Table 3. Ambient water-quality data and preceding Peace River flows for each experiment

[NO3 = nitrite plus nitrate; NH4 = annomia nitrogen; OP = orthophosphate; IOC = inorganic carbon; TKN = total kjeldahl nitrogen; 

TP = total phosphorus; < = less than; %o = parts per thousand. All values are in milligrams per liter except color, which is in 

platinum-cobalt units]

Experiment

Preliminary design

experiments

Time series no. 1

2 hours

4 hours

6 hours

8 hours

10 hours

Time series no. 2

2 hours

4 hours

6 hours

8 hours

10 hours

12 hours

14 hours

Nutrient combinations

12%o

20%o

Nutrient concentrations

12%o

20%0

Date

5/10/84

5/10/84

5/10/84

5/10/84

5/10/84

5/17/84

5/17/84

5/17/84

5/17/84

5/17/84

5/17/84

5/17/84

5/22/84

5/22/84

5/31/84

6/01/84

Chloride

7,110

7,260

7,270

7,130

7,270

6,480

6,480

6,610

6,280

6,160

6,460

6,470

7,500

10,800

6,670

10,900

Color

49.0

50.0

50.5

52.0

54.0

50.0

50.0

51.0

51.5

53.0

51.0

52.0

50.0

33.0

50.0

28.0

NO3

<0.001

< .001

< .001

< .001

< .001

.002

.001

.001

.001

.002

.002

.004

< .003

< .003

.003

< .001

NH4

<0.013

< .013

< .013

< .013

< .013

< .004

< .004

< .004

< .004

< .004

< .004

.004

< .018

< .018

< .010

< .001

OP

0.363

.360

.363

.378

.368

.396

.404

.380

.397

.401

.389

.400

.399

.247

.471

.251

Silica

1.34

1.37

1.41

1.44

1.47

.40

.40

.40

.39

.40

.38

.40

1.16

.78

1.17

.42

IOC

18.1

18.1

18.6

18.2

18.3

17.8

17.4

18.1

17.8

17.9

17.9

18.2

19.2

22.1

17.2

19.9

TKN

1.01

.96

.99

1.26

.99

.14

.02

.95

.02

.02

.02

.10

1.15

.78

.93

.78

TP

0.393

.395

.398

.403

.395

.689

.486

.423

.469

.441

.442

.469

.454

.272

.543

.280

Average total gaged 

Peace River flow,

in meters per

second for the

preceding indicated

number of days

7 14 28

days days days

5.7 12.5 26.8

16.1 5.7 18.3

5.3 4.8 15.1

9.3 4.1 5.7

9.1 3.6 5.2

Flow-related studies

Myakka River fixed 

station 

September 1984

2%0 9/27/84 1,300 122 .017 .019 .227 2.25 16.8 .90 .267 23.8 38.8 64.9

May 1985

23%0

September 1985

0%0

Peace River salinity

zones

May 1985

6%o

20%o

September 1985

6%o

20%o

5/22/85

9/16/85

5/08/85

5/10/85

9/17/85

9/24/85

12,500

190

4,220

11,200

3,720

10,700

33.0

390

52

43

275

70

< .001

.483

.077

< .001

.003

.001

< .001

.185

.052

< .001

.221

< .001

.397

.518

.830

.313

.631

.183

.32

3.18

1.29

.04

3.68

1.08

19.6

6.72

2.5

25.4

12.6

18.5

1.35

1.47

.96

1.10

1.39

.76

.412 2.4 1.3

.559 122.2 68.2

.886 .9 1.8

.330 1.9 1.7

.674 111.8 77.0

.204 36.4 111.8

1.8

43.9

2.4

2.3

40.5

39.6

Design of Nutrient Addition Experiments 11
NO- O.I NO, 0.5 NH 4 0.5 NH4 +N03 0.5 NH 4+N03 P04 0.5 

+ P04 0.5

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

(0

2 PHYTOPLANKTON

cc cc r~7

0 UJ U

ztL 1.0 - 

-o ^ O 1

Raw f? o-*-^

<5 jum

5-20 Aim

>20 jum

SIZE FRACTION

cc 

o

1.13 = TOTAL CARBON UPTAKE, IN GRAMS 

PER CUBIC METER, FOR THE THREh 

FRACTIONS

SIZE

Figure 7. Phytoplankton carbon uptake for control and nutrient-amended samples for 10-hour 

in situ incubations at salinities of approximately 12 (site 3) and 20 (site 4) parts per thousand, 

May 22, 1984. (Each bar represents mean of five replicates.)

Comparisons of phytoplankton carbon uptake at 12%o 

indicated:

1. No significant difference between controls and replicates 

treated with PCVP;

2. Both forms of nitrogen stimulated production, with the 

effect of NH4-N at 0.1 mg/L being significantly 

greater;

3. Increasing the concentration of NOa-N resulted in 

increased uptake, whereas increasing NH4-N from 0.1 

to 0.5 mg/L had the opposite effect;

4. NOa-N + NH4-N had no greater effect than either of the 

two nutrients individually, whereas the addition of 

PO4-P in combination with the two forms of nitrogen 

resulted in a significantly higher level of carbon 

uptake; and

5. NOa-N at 0.5 mg/L and both concentrations of NH4-N 

significantly increased the relative amount of carbon 

taken up by the largest phytoplankton size fraction 

(>20 jim).

The effects on carbon uptake to similar nutrient additions at 

20%o salinity showed slightly different patterns:

1. No significant difference was noted between controls 

and replicates treated with PO4-P;

2. Both forms of nitrogen significantly increased carbon 

uptake. Stimulation by NH4-N was significantly 

greater than that for NOa-N;

3. Increasing the concentration of either of the forms of 

nitrogen did not significantly increase carbon uptake;

4. Neither the combination of nitrogen forms nor the 

further addition of PO4-P increased carbon uptake;

5. Both forms of nitrogen at 0.5 mg/L resulted in a significant 

increase in relative percent of production by the 5- to 

20-jim size fraction.

Within each salinity zone, chlorophyll-a concentrations generally 

exhibited stimulation patterns similar to those observed for 

carbon uptake.

12 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
NO O.I NOjO.5 NH 4 O.I NH 4 0.5 NH 4 +N03 0.5 NH^+NOj

+ P04 0.5

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

<5 jjm

5-20 urn

>20jum

50 = TOTAL CHLOROPHYLL - a. CONCENTRATION, IN 

MILLIGRAMS PER CUBIC METER

Figure 8. Phytoplankton chlorophyll-a concentrations for control and nutrient-amended samples for 

10-hour in situ incubations at salinities of approximately 12 (site 3) and 20 (site 4) parts per thousand, 

May 22, 1984.

Concentration Response

Based on the results of the preceding experiments, the 

effects of nutrient additions over a wider range of nitrogen 

concentrations were measured at the same two salinities. The 

purpose of this experiment was to determine a standardized 

range of added-nutrient concentrations adequate to statistically evaluate phytoplankton responses within different 

estuarine salinity regimes. At 12%o, the effects of nitrogen on 

phytoplankton carbon uptake and concentrations of 

chlorophyll a were investigated using nutrient additions that 

resulted in final concentrations for NO3-N ranging from 0.0 

to 1.0 mg/L and from 0.0 to 0.8 mg/L for NH4-N. At 20%o 

salinity, phytoplankton responses to nutrient additions were 

tested over final concentrations ranging from 0.0 to 0.4 mg/L 

for both NO3-N and NH4-N.

Observed changes in phytoplankton carbon uptake and 

chlorophyll a over ranges of nitrogen additions at the 12%o 

salinity zone are shown in figures 9 and 10. Concentrations 

of the additions are presented in ascending order and are not 

linear. Changes in carbon uptake and chlorophyll a are indicated for the whole (total block height) and each of the three

measured size fractions. In this (and following concentration 

experiments), polynomial regression procedures were used 

to test for changes in the carbon uptake and chlorophyll-a 

concentrations. Total phytoplankton responses were tested 

for significant linear, cubic, and quadratic treatment sums for 

models with coefficients significantly different from zero. 

Additions of NOs-N significantly increased carbon uptake, 

which steadily increased and then flattened at the highest 

concentrations. Analysis indicated that the overall carbon 

uptake response to NOs-N could best be described by linear 

and quadratic functions (table 2). Carbon uptake also 

increased with NFLj-N additions. The pattern of the treatment 

response, however, was significantly different from that for 

NO3-N. Carbon uptake was greatest at the lowest levels of 

NH4-N addition and then slowly declined with increasing 

concentrations. Analysis of this pattern indicated a significant quadratic component (table 2). Corresponding changes 

in chlorophyll-a concentrations that resulted from increasing 

additions of the two nitrogen forms (fig. 10) were similar to 

one another and characteristically different from the patterns 

of increase in carbon uptake. Maximum stimulation of chlorophyll a took place at much lower NOs-N concentrations

Design of Nutrient Addition Experiments 13
NO

CONTROL 0.05 O.I OTT 0.4 6.8"

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

0.01 0.025 O.05 O.075 0.10 0.20 0.40

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

1.35 = TOTAL CARBON UPTAKE, IN GRAMS

PER CUBIC METER, FOR THE THREE SIZE 

FRACTIONS

Figure 9. Phytoplankton carbon uptake for control and nutrient-amended samples for 10-hour in situ incubations at site 5, 

May 31,1984. (Each bar represents mean of five replicates.)

CONTROL 0.05 O.I 0 2 0.4 0.8

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

001 0025 0.05 0.075 010 020 0.40

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

<5 jjm

5-20 jjm

>20,um

39 = TOTAL CHLOROPHYLL-^ CONCENTRATION, 

IN MILLIGRAMS PER CUBIC METER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

<5 jjm 

5-20 jjm

>20 jjm

44 = TOTAL CHLOROPHYLL-a. CONCENTRATION, 

IN MILLIGRAMS PER CUBIC METER

Figure 10. Phytoplankton chlorophyll-a concentrations for control and nutrient-amended samples for 10-hour in situ 

incubations at site 5, May 31, 1984.

14 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
than that for carbon uptake. By comparison, the maximum 

stimulation of chlorophyll a occurred at high levels of NH4-N, 

unlike the carbon uptake response.

At 20%o salinity, both carbon uptake and chlorophyll-a 

concentrations (figs. 11 and 12) significantly increased with 

additions of NO3-N and NI-Lj-N (table 2). Solution of carbon 

uptake models, using nonlinear least squares fitting procedures, indicated that the general shape of the resulting curves 

were similar, with the asymptote of the response curve being 

slightly higher for NH4-N than for NO3-N. Corresponding 

analysis of chlorophyll-a increases in response to increasing 

NO3-N and NH4-N additions indicated the resulting curves 

were similar.

Analysis of relative changes in carbon uptake and 

chlorophyll-a concentrations within the three measured 

phytoplankton size fractions in response to nutrient additions 

at 12 and 20%c salinity generally failed to indicate any 

systematic or progressive differences. An exception, 

however, was the relative "changes in chlorophyll-a concentrations among size fractions in response to increasing 

NC>3-N levels at 20%c. Although chlorophyll-a concentrations within all size fractions rose with increasing NO3-N 

concentrations, there was a progressive increase in the 

relative percent of chlorophyll a in the >20-jim size fraction 

and a corresponding decrease in the <5-|im fraction.

RESULTS OF NUTRIENT ADDITION 

EXPERIMENTS

Based on the preceding series of experimental 

findings, a methodology was developed to investigate the 

response of phytoplankton communities within the Charlotte 

Harbor estuarine system to nutrient additions under differing 

conditions of freshwater inflow. Changes in carbon uptake 

and chlorophyll-a concentrations that resulted from additions 

of NC»3-N, NH4-N, and PC»4-P, each over a series of 10 final 

concentrations (0.0, 0.001, 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 

and 1.0 mg/L), were investigated. Nutrient addition experiments were conducted at a fixed location in the tidal part of 

the Myakka River (fig. 1) during two periods of high and one 

period of low freshwater inflow and at two salinity zones 

(6 and 20%o) within the lower tidal Peace River-upper 

Charlotte Harbor part of the estuary during corresponding 

periods of low and high Peace River inflow.

In each experiment polynomial models of the form

carbon uptake = bo + bi(Conc.) + b2(Conc.) 2 + b3(Conc.) 3

were constructed for each nutrient. Similar models also were 

constructed using chlorophyll-a concentrations as a response 

variable.

Significance determinations of the models were done 

by testing the coefficient for each term of the regression (bi, 

b2, b3 not equal to 0). Significance of these coefficients

dictated shape of the response curve. A significant linear (bi) 

response was characterized by monotonic changes in carbon 

uptake with increasing nutrient concentrations; significant 

quadratic (b2) responses by polynomial curves with one 

inflection point; and significant cubic responses by polynomial curves with two inflection points. Different response 

curves were generated for each experiment (location, date), 

nutrient, and response variable (carbon uptake, chlorophyll-a 

concentrations) and are summarized in table 2.

Tidal Myakka River Fixed Station 

High Flow

The first experiment at the fixed Myakka River site 

was conducted in September 1984 during a period of 

relatively high river flow (fig. 2). Ambient water conditions 

were characterized by low salinity, high color, and relatively 

high macronutrient concentrations (table 3). Phytoplankton 

carbon uptake and chlorophyll-a concentrations were highest 

in the smallest size fraction (<5 Jim), as indicated by the 

controls (figs. 13 and 14).

Carbon uptake and chlorophyil-a concentrations 

significantly increased in response to nitrogen additions 

(table 2). Uptake and concentrations initially increased and 

then reached a point where the effects of stimulation from 

increasing NC»3-N additions remained relatively constant 

(figs. 13 and 14). The observed changes in carbon uptake in 

response to increasing NH4-N additions, by comparison, 

were greatest at lower concentrations and then declined 

steadily with increasing NH4-N concentrations. Changes in 

chlorophyll-a concentrations in response to increasing NH4-N 

and NC»3-N additions initially increased and then remained 

relatively constant with further additions. There were no 

systematic changes or differences apparent in the responses 

of the three measured phytoplankton size fractions resulting 

from stimulation by nitrogen additions.

Graphical analysis appeared to indicate that both 

carbon uptake and chlorophyll-a concentrations declined 

slightly with increasing concentrations of PC»4-P (figs. 13 and 

14). Statistical analysis, however, failed to identify any 

significant decline (table 2).

Low Flow

A second experiment was conducted at the Myakka 

River site in May 1985 during a period of low river flow. 

Ambient water conditions were characterized by high salinity, reduced water color, and inorganic nitrogen concentrations at detection limits. Phytoplankton carbon uptake and 

chlorophyll-a concentrations were divided nearly equally 

between the largest (>20 Jim) and smallest (<5 Jim) size 

fractions, as indicated by the untreated controls.

Results of Nutrient Addition Experiments 15
.01 0.02 0.04 0 06 O.O8 O.IO

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

1.0 = TOTAL CARBON UPTAKE, IN GRAMS

PER CUBIC METER, FOR THE THREE SIZE 

FRACTIONS

Figure 11. Phytoplankton carbon uptake for control and nutrient-amended 

samples for 10-hour in situ incubations at a salinity of approximately 20 

parts per thousand at site 6, June 1, 1984. (Each bar represents mean of

001 002 O.04 O 06 0.08 0.10

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

<5 Aim

<20 jjm

50 = TOTAL CHLOROPHYLL-_a_ CONCENTRATION, 

IN MILLIGRAMS PER CUBIC METER

Figure 12. Phytoplankton chlorophyll-a concentrations for control and 

nutrient-amended samples for 10-hour in situ incubations at a salinity 

of approximately 20 parts per thousand at site 6, June 1, 1984.

16 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
CONTROL 0001 0.01 005 O.I 02 0.4 0.6 0.8 

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

w EXPLANATION

3 PHYTOPLANKTON SIZE FRACTION

<5.um

0.80 = TOTAL CARBON UPTAKE, IN GRAMS

PER CUBIC METER, FOR THE THREE SIZE 

FRACTIONS

Figure 13. Phytoplankton carbon uptake for control and nutrient-amended 

samples for 10-hour in situ incubations at site 7 on the tidal Myakka River, 

September 27, 1 984. (Each bar represents mean of three replicates.)

CONTROL 0.001 0.01 0.05 O.I 0.2 04 06 0.8 

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

20 __

<5 jjm

5-20 um 

>20 jjm

20 = TOTAL CHLOROPHYLL-^ CONCENTRATION, 

IN MILLIGRAMS PER CUBIC METER

Figure 14. Phytoplankton chlorophyll-a concentrations for control and 

nutrient-amended samples for 10-hour in situ incubations at site 7 on the 

tidal Myakka River, September 27, 1984.

Results of Nutrient Addition Experiments 17
The additions of both forms of nitrogen significantly 

(table 2) increased carbon uptake and chlorophyll a (figs. 15 

and 16). The patterns of changes in carbon uptake in 

response to increasing additions of NOs-N and NH4-N were 

similar. In each instance, carbon uptake increased with 

increasing nitrogen additions and then remained relatively 

constant at concentrations greater than 0.2 mg/L. Solutions 

of these models using nonlinear-fitting procedures indicated 

that the response curve for NO3-N initially increased at a 

slightly greater rate than that for NH4-N. The asymptote of the 

NH4-N response curve, however, was higher than that for 

NO3-N. Analysis of relative changes among the size fractions indicated the smallest phytoplankton size fraction 

(<5 jim) accounted for most of the observed increase in 

carbon uptake and chlorophyll-a concentration in response to 

NOs-N and NH4-N additions.

In contrast to the stimulation by nitrogen, carbon 

uptake significantly (table 2) decreased with increasing PCVP 

additions. Graphically, chlorophyll-a concentrations also 

seemed to decrease slightly with increasing PO4-P additions. 

This conclusion was supported by the results of the general 

linear-models analysis. Further examination indicated that 

the largest phytoplankton size fraction (>20 jim) was 

responsible for the observed decrease in carbon uptake and 

chlorophyll-a concentration.

Second High Flow

The final experiment at the Myakka River site was 

conducted in September 1985, again during a period of high 

river flow (fig. 2). Ambient water conditions were characterized by low salinity, extremely high water color, and 

elevated concentrations of macronutrients (table 3). The 

controls indicated that, although the phytoplankton chlorophyll-a concentration was slightly less than that of the 

preceding experiments at this location, the relative rate of 

carbon uptake was extremely low (less than 15 percent) 

when compared to previous observed rates (figs. 17 and 18). 

Neither phytoplankton carbon uptake nor chlorophyll-a 

concentrations indicated any biologically significant 

response to the additions of nutrients.

Peace River and Charlotte Harbor Salinity 

Stations

Low Flow

Experiments were conducted at 6%o salinity in the 

lower Peace River and at approximately 20%c in upper 

Charlotte Harbor in May 1985, a period of extremely low 

river inflow (fig. 2). Ambient water conditions at the lower 

salinity zone were characterized by low color and relatively 

high concentrations of NO3 -N, NH4-N, and PO4-P (table 3). 

The controls indicated that approximately 75 percent of

phytoplankton chlorophyll a and carbon uptake within the 

phytoplankton community occurred in the <5-{im size 

fraction (figs. 19 and 20). Ambient conditions in the higher 

salinity zone, by comparison, were characterized by low 

color and NOs-N and NH4-N concentrations at or below 

detection limits. Phytoplankton carbon uptake and 

chlorophyll-a concentrations within the controls at 20%o 

were divided approximately equally between the largest and 

smallest size fractions (figs. 21 and 22).

Carbon uptake at 6%o (fig. 19) salinity was slightly 

depressed by the addition of PO4-P, slightly stimulated by 

NH4-N, and showed no significant response to additions of 

NO3-N (table 2). Graphical analysis (fig. 20) indicated that 

the concentration of chlorophyll a at 6%o salinity was 

initially relatively high and moderately (approximately 

30-50 percent) stimulated by additions of each of the three 

macronutrients (table 2). None of the observed changes in 

carbon uptake or chlorophyll a in response to increasing 

nutrient additions resulted from systematic differences in 

stimulation among the three measured phytoplankton size 

fractions.

At 20%o salinity, additions of PO4-P caused no 

biologically meaningful changes in either phytoplankton 

carbon uptake or chlorophyll-a concentrations (figs. 21 and 

22). Additions of both nitrogen forms, by comparison, 

resulted in highly significant increases. In general, the 

patterns of the responses to NOs-N and NH4-N were similar. 

Solutions of these curves using nonlinear fitting procedures 

indicated that the curve for NOs-N initially increased at a 

rate greater than that of NH4-N, whereas the asymptote of the 

NH4-N response curve was significantly higher. The 

responses of chlorophyll a followed patterns similar to those 

for carbon uptake (table 2). Both nitrogen forms 

caused threefold increases in phytoplankton chlorophyll-a 

concentration.

Analysis by size fraction of changes in carbon uptake 

in response to nitrogen additions indicated that, as concentrations of both forms increased, there were significant small 

increases in the relative percentages attributable to the two 

smallest (<5 (im and <20 and >5 jim) size fractions. A similar 

pattern occurred with respect to changes in concentration of 

chlorophyll a with increasing NH4-N additions.

High Flow

A second series of experiments was conducted in the 

Charlotte Harbor Estuary at approximately 6 and 20%o 

salinity in September 1985, a period of relatively high river 

inflow (fig. 2). Water color and the ambient concentration of 

NH4-N at the more riverine-influenced lower salinity were 

extremely high, whereas the ambient level of NC^N was 

relatively low (table 3). Phytoplankton chlorophyll-a 

concentrations and carbon uptake at 6%o salinity were both 

quite low (figs. 23 and 24 controls). Ambient water-quality 

conditions at 20%o salinity, by comparison, were

18 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
CONTROL 0 001 001 005 01 02 04 06 08

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

co EXPLANATION

§£ 1-D

z cc.

PHYTOPLANKTON SIZE FRACTION

<5 .um

5-20 jjm 

>20.xim

1.0 = TOTAL CARBON UPTAKE, IN GRAMS

PER CUBIC METER, FOR THE I HREt SIZE 

FRACTIONS

Figure 15. Phytoplankton carbon uptake for control and nutrient-amended 

samples for 10-hour in situ incubations at site 7 on the tidal Myakka River, 

May 23, 1985. (Each bar represents mean of three replicates.)

0 001 001 005 01 02 04 06 08

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

<5 jj

5-20 jum 

>20.um

24 = TOTAL CHLOROPHYLL-a_ CONCENTRATION, 

IN MILLIGRAMS PER CUBIC METER

Figure 16. Phytoplankton chlorophyll-a concentrations for control and 

nutrient-amended samples for 10-hour in situ incubations at site 7 on the 

tidal Myakka River, May 23, 1985.

Results of Nutrient Addition Experiments 19
0.07 / 0.06 / 0.06 / 0.06 / 0.06 / 0.06 / 007 / 0.06 / 0.06 / 0.07

CONTROL 0.001 0.01 005 O.I 0.2 0.4 0.6 O.S 1.0 

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION 

0.6

_ <5 ,um 

0.4

5-20 ,um

>20jum 

0.0 "

0.06 = TOTAL CARBON UPTAKE IN GRAMS PER CUBIC 

METER FOR THE THREE SIZE FRACTIONS

Figure 17. Phytoplankton carbon uptake for control and nutrient-amended 

samples for 10-hour in situ incubations at site 7 on the tidal Myakka River, 

September 16, 1985. (Each bar represents mean of three replicates.)

CONTROL 0 001 0.01 0 05 06 o a 10

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

5 = TOTAL CHLOROPHYLL-^ CONCENTRATION, 

IN MILLIGRAMS PER CUBIC METER

Figure 18. Phytoplankton chlorophyll-a concentrations for control and 

nutrient-amended samples for 10-hour in situ incubations at site 7 on the 

tidal Myakka River, September 16, 1985.

20 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
CONTROL o 001 001 005 01 02 04 oe 08 

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

5 EXPLANATION

ir PHYTOPLANKTON SIZE FRACTION

uJS 0.6

So

J- CD 0.4

&; O

1° 0.2

og

CD Q. 0.0

<5 .urn

5-20 jum 

>20 jjm

0.80 = TOTAL CARBON UPTAKE, IN GRAMS

PER CUBIC METER, FOR THE THREE SIZE 

FRACTIONS

Figure 19. Phytoplankton carbon uptake for control and nutrient-amended 

samples for 10-hour in situ incubations at a salinity of approximately 6 parts 

per thousand at site 8, May 8, 1985. (Each bar represents mean of three 

replicates.)

6 001 O.OI 005 Ol 02 04 06 08 

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

~ 30

5-20 Jim 

>20 jjm

30 = TOTAL CHLOROPHYLL-a_ CONCENTRATION, 

IN MILLIGRAMS PER CUBIC METER

Figure 20. Phytoplankton chlorophyll-a concentrations for control and 

nutrient-amended samples for 10-hour in situ incubations at a salinity of 

approximately 6 parts per thousand at site 8, May 8, 1985.

Results of Nutrient Addition Experiments 21
CONTROL 0001 001 0 05 01 02 0.4 0.6 08 

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

<5.um

1.5 = TOTAL CARBON UPTAKE, IN GRAMS

PER CUBIC METER, FOR THE I HREE SIZE 

FRACTIONS

Figure 21 . Phytoplankton carbon uptake for control and nutrient-amended 

samples for 10-hour in situ incubations at a salinity of approximately 20 

parts per thousand at site 9, May 10, 1985. (Each bar represents mean of 

three replicates.)

10 / 14 / 12 / 13 / 26 / 45 / 33 / 40 / 43

CONTROL 0 001 001 005 01 02 04 06 0.8

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

S|§ 2o

piB

-JH o

<5 jum

5-20 jum 

>20 jum

40 = TOTAL CHLOROPHYLL-a^ CONCENTRATION, 

IN MILLIGRAMS PER CUBIC METER

Figure 22. Phytoplankton chlorophyll-a concentrations for control and 

nutrient-amended samples for 10-hour in situ incubations at a salinity of 

approximately 20 parts per thousand at site 9, May 10, 1985.

22 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

5 EXPLANATION

0.1

£ a O.os

ii

PHYTOPLANKTON SIZE FRACTION

<5 jjm

5-20 jjm 

>20 jjm

0.1 = TOTAL CARBON UPTAKE, IN GRAMS

PER CUBIC METER, FOR THE THREE SIZE 

FRACTIONS

Figure 23. Phytoplankton carbon uptake for control and nutrient-amended 

samples for 10-hour in situ incubations at a salinity of approximately 6 parts 

per thousand at site 10, September 1 7, 1985. (Each bar represents mean of 

five replicates.)

CONTROL O.OOI 001 005 O.I 0.2 0.4 0.6 0.8

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

5-20 .um 

>20>im

6 = TOTAL CHLOROPHYLL-^. CONCENTRATION, 

IN MULLIGRAMS PER CUBIC METER

Figure 24. Phytoplankton chlorophyll-a concentrations for control and 

nutrient-amended samples for 10-hour in situ incubations at a salinity of 

approximately 6 parts per thousand at site 10, September 1 7', 1985.

Results of Nutrient Addition Experiments 23
characterized by moderate color and by NO3-N and NH4-N 

concentrations near detection limits (table 3). Initially, chlorophyll-a concentrations and carbon uptake rates at 20%o 

salinity (figs. 25 and 26 controls) were higher than those at 

6%o salinity.

Experimental nutrient additions at 6%o salinity had no 

biologically significant effects on either carbon uptake or 

chlorophyll-a concentration (figs. 23 and 24). This was in 

sharp contrast to the effects of corresponding additions at the 

higher salinity.

Additions of NO3 -N and NH4-N at 20%o salinity 

significantly increased (table 2) carbon uptake by more than 

100 percent (fig. 25). The carbon uptake responses for 

nitrogen initially increased with increasing concentrations, 

reached maximum levels, and then significantly declined 

with further additions. Solutions of the response curves, 

using nonlinear fitting procedures, indicated no significant 

differences between the asymptotes of the two nitrogen 

response curves. The shape of the NH4-N curve, however, 

indicated greater stimulation at lower concentrations.

Chlorophyll-a concentrations significantly increased 

(table 2) as much as fivefold in response to experimental 

nitrogen additions at 20%o salinity (fig. 26). The shapes of the 

responses of phytoplankton chlorophyll-a concentration to 

the two forms of nitrogen (fig. 26) increased to maximum 

levels and then declined slightly at the highest additions. The 

magnitude of chlorophyll-a stimulation was greater than that 

of carbon uptake, whereas the decline of chlorophyll-a 

concentrations at the highest nitrogen concentrations was 

less dramatic.

. Analysis by size fraction indicated that, over the ranges 

of NO3-N additions, where carbon uptake and chlorophyll a 

were stimulated, the relative percentage attributable to the 

5- to 20-(im size fraction increased with increasing concentration of NO3-N. By comparison, the relative percentage of 

chlorophyll a within the largest size fraction (>20 (im) 

increased in response to NI-Lj-N stimulation.

EFFECTS OF NITROGEN AND PHOSPHORUS 

ADDITIONS ON PHYTOPLANKTON 

PRODUCTIVITY AND CHLOROPHYLL a

The experiments conducted within the 6%o salinity part 

of the Charlotte Harbor Estuary showed distinct seasonal 

changes in phytoplankton carbon uptake and chlorophyll a 

that resulted from inorganic nitrogen additions. Other investigators, such as Myers and Iverson (1981) working in the 

fresh and brackish estuarine waters of Apalachicola Bay in 

northwest Florida and D'Elia and others (1986) working in 

Chesapeake Bay, have also observed seasonal differences in 

the response of phytoplankton assemblages to nitrogen and 

phosphorus additions. The seasonal changes in phytoplankton responses in Charlotte Harbor were directly attributable to seasonal periods of low and high freshwater inflow.

Nutrient additions at the end of the wet season, when water 

color and ambient macronutrient concentrations were high 

(September 1985), did not stimulate phytoplankton carbon 

uptake or chlorophyll-a production at either the riverine 

Myakka River (0%o salinity) or Peace River (6%o salinity) 

locations. The experimental results, by comparison, from the 

Myakka River site in May 1985 (23%o salinity) and the 20%o 

salinity zone in upper Charlotte Harbor in May and September 1985 indicate that phytoplankton communities within the 

more saline parts of the estuary do not exhibit seasonality in 

their response to nitrogen additions and are continually nitrogen limited, except possibly under very high freshwater 

inflow conditions.

The results from the nutrient addition experiments 

indicate that, during low freshwater inflow, the availability of 

nitrogen may limit phytoplankton productivity over a wide 

range of salinities throughout much of the Charlotte Harbor 

system. During seasonal high freshwater inflow, however, 

nitrogen availability probably does not limit phytoplankton 

production in these lower salinity areas of the estuary. 

Freshwater runoff from the Peace and Myakka River basins 

contains high concentrations of dissolved humic compounds 

that color the water and greatly reduce light penetration 

(McPherson and Miller, 1987; Fraser, 1991; McPherson and 

others, in press). In such highly colored waters, light availability probably is the dominant factor controlling phytoplankton production. In regions of the estuary characterized 

by higher salinities, dilution and photooxidation of humic 

substances reduce water color and increase light penetration. 

Thus, during periods of high freshwater inflow, the estuary 

conceptually can be divided into two regions based on salinity. In the lower salinity region, phytoplankton production is 

limited by light availability mediated by elevated water 

color, whereas in the higher salinity regions, phytoplankton 

communities are nutrient (nitrogen) limited.

Ambient conditions are often useful in determining 

potential limiting factors controlling phytoplankton productivity within estuarine systems. The results from Charlotte 

Harbor indicate a general pattern of increasing phytoplankton response to nitrogen additions with decreasing 

ambient nutrient concentrations. Other investigators (Davies 

and Sleep, 1981; Myers and Iverson, 1981; D'Elia and 

others, 1986) have reported similar patterns.

Mean monthly ambient values for selected macronutrients, 

color, phytoplankton production, and chlorophyll-a concentrations in the Charlotte Harbor Estuary from June 1983 

through June 1988 at 6 and 20%o salinity are shown in 

figure 27 (Montgomery and Emmons, 1989). Several of 

these measurements exhibit distinct patterns related to 

seasonal river inflow. Basin rainfall and runoff are characterized by a spring dry season and a summer wet period. 

Orthophosphorus concentrations within the more riverineinfluenced upper harbor increase significantly in the spring 

dry season as the concentration of phosphorus in the rivers 

increases with declining river flow and then declines through

24 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
0.001 001 005 01 02 04 06 08 

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

to EXPLANATION

PHYTOPLANKTON SIZE FRACTION

2.0 = TOTAL CARBON UPTAKE, IN GRAMS

PER CUBIC METER, FOR THE THREE SIZE 

FRACTIONS

Figure 25. Phytoplankton carbon uptake for control and nutrient-amended 

samples for 10-hour in situ incubations at a salinity of approximately 20 

parts per thousand at site 11, September 24, 1 985. (Each bar represents a 

mean of three replicates.)

15 / 13 14 / IS / 2S / 47 / 78 / 79 / 74 / 61

CONTROL 0001 001 005 01 0.2 O4 0.6 0.8 

NUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER

EXPLANATION

PHYTOPLANKTON SIZE FRACTION

44 = TOTAL CHLOROPHYLL - a. CONCENTRATION, 

IN MILLIGRAMS PER CUBIC METER

Figure 26. Phytoplankton chlorophyll-a concentrations for control and 

nutrient-amended samples for 10-hour in situ incubations at a salinity of 

approximately 20 parts per thousand at site 11, September 24, 1 985.

Effects of Nitrogen and Phosphorus Additions 25
CO

h;

Z

.

CC OQ 

O O

200

150

100

50

 i i i i i i 

6-PARTS-PERrHOUSAND SALINITY

-<r' \ j THOUSAND 

" V-' SALINITY

2.0

N D

MONTH MONTH

LLJ 

O

O DC 

CC LU

LU

<

§

LU 5

0.4

0.3

0.2

= 0

MONTH

CC

LU

<0 |5 0.

§£

I CL 0.6 - 

CO «

I < 0.4 - 

Q. CC 

O O

X H p 

I- _J 0.2 -

? 0

ND

I I 

J J

MONTH

CO

i c

< 3

CC O

O I

li CC

^ LU

5 CL

Z CC LU

LU }-,

Q. O

^ OQ

-, D

O °

OQ CC

CC LU

< CL

250

20 °

'50

100

50

1 i I 1 1 1

I I 

J J

MONTH

LU

(B|ffi

do

>tr

ft S 

gco

I 1

I CC

o o

50

40

O4 o

fO o

1 i i i i y i i i i

i i r

J J A 

MONTH

Figure 27. Mean values for selected macronutrients and measurements of phytoplankton production 

collected monthly from June 1983 through June 1988 at 6 and 20 parts per thousand along salinity transects 

in the lower Peace River and upper Charlotte Harbor. (From Montgomery and Emmons, 1989.)

the summer as PO4-P concentrations in river inflow decrease. 

Ambient nitrite plus nitrate concentrations in the highly 

riverine-influenced areas of the estuary (6%o salinity) are 

highest in winter and then decline sharply throughout the 

spring as phytoplankton production increases. Nitrate 

concentrations in this salinity zone increase again during the 

wet season as river inflow increases and high water color 

results in decreased phytoplankton production. Concentrations of NOs-N in the more saline parts of the harbor (20%o

salinity) remain near detection limits through all but the 

winter months. These generalized macronutrient patterns are 

similar to those reported by Fraser (1986; 1991) from longterm seasonal studies of water-quality characteristics of the 

lower Peace River.

Low ambient nutrient levels by themselves, however, 

cannot be taken as definitive indications of potential nutrient 

limitation. Smith (1984) proposed further evaluation of 

potential limiting nutrient conditions in estuarine systems by

26 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
analyzing inorganic atomic N:P ratios and comparisons of 

concentrations of nutrients and conservative constituents 

along natural gradients. The N:P ratios of natural phytoplankton assemblages generally average near the Redfield (1958) 

value of 16. Waters with ambient N:P ratios of less than 10 

usually indicate potential conditions in which phytoplankton 

production is nitrogen limited, whereas values of greater than 

21 are commonly indicative of phosphorus limitation (Ram 

and Plotkin, 1983; Sakshaug and Olsen, 1986). Determinations of limiting nutrient conditions based on ambient 

inorganic N:P ratios and corresponding results from bioassay 

determinations generally have been found to be in good 

agreement (Schanz and Juon, 1983). Extensive naturalphosphate deposits within the Charlotte Harbor drainage 

basin result in unusually high ambient-orthophosphorus 

concentrations and unusually low inorganic N:P ratios 

throughout the entire estuarine system. The nitrogen and 

phosphorus ratios in 6%c and 20%o salinity zones are shown 

in figure 27.

Another method commonly used to evaluate potential 

limiting nutrient conditions for phytoplankton growth is the 

comparison of concentrations of nutrients and conservative 

constituents along natural gradients (Smith, 1984). Fraser 

and Wilcox (1981), Froelich and others (1985), and 

McPherson and Miller (1990) evaluated nutrient sources and 

distributions in Charlotte Harbor using nutrient dilution 

curve models constructed by plotting nutrient concentrations 

against salinity. Nitrite-nitrate concentrations always 

deviated from conservative behavior, declining rapidly with 

increasing salinity, whereas orthophosphorus levels 

exhibited conservative behavior reflecting simply straight 

dilution. Such nutrient dilution curve observations, 

combined with determinations of atomic N:P ratios and 

studies of seasonal ambient-nutrient concentration patterns 

(fig. 27), support the overall findings of the nutrient-addition 

experiments. The results of these experiments stress the 

importance of nitrogen in limiting phytoplankton production, 

except in those estuarine areas that are seasonally influenced 

by high water color.

Population growth in the Charlotte Harbor basin is 

expected to increase nitrogen loading to the estuary by more 

than 2,700 kg/d by the year 2020 (Hammett, 1988). Such 

additional nitrogen loading would be equivalent to 18 percent 

of the current gaged-basin nitrogen input (McPherson and 

Miller, 1990). New inorganic nitrogen would be immediately 

available for phytoplankton uptake, whereas mineralization 

by bacteria and other microorganisms would make other 

forms available over time. Additional nitrogen loadings 

could be expected to result in increased seasonal phytoplankton production in those areas of the harbor where water color 

is low enough to allow for sufficient light availability.

Total freshwater inorganic nitrogen inputs in Charlotte 

Harbor (Hammett, 1988; Fraser, 1991) are more than an 

order of magnitude below that required to support observed 

levels of phytoplankton production (Montgomery and

Emmons, 1989; McPherson and others, in press). Therefore, 

some mechanism, such as the rapid recycling of nitrogen by 

processes such as remineralization by microheterotrophs 

(Furnas and others, 1986), is required to support observed 

photosynthesis rates. Pennock (1987) suggested that, in the 

Delaware Estuary system, more than 80 percent of the annual 

nitrogen demand is supplied by recycling of NH4-N, even 

though NO3-N is the dominant ambient-nitrogen form in 

estuarine freshwater inflow (Fisher and others, 1982). Other 

than during ephemeral events associated with extremely 

large inputs of freshwater (as may occur during tropical 

depressions), the inorganic nitrogen entering the estuary is 

rapidly taken up by phytoplankton and joins a larger organic 

pool of fixed nitrogen. Such a concept is consistent with a 

number of findings from other estuarine systems.

Phytoplankton responses to stimulation by nitrogen in 

the presented bioassay experiments exhibited a number of 

well-defined patterns. Distinct differences existed in the 

response time and the absolute magnitudes of the two 

productivity measurements, carbon uptake and chlorophyll a. Other investigators (Vince and Valiel, 1973; Davies 

and Sleep, 1981; Wurtsbaugh and others, 1985; Graneli and 

others, 1986) have noted that nitrogen stimulation of carbon 

uptake is normally associated with corresponding increases 

in chlorophyll-^ concentrations. Results from the presented 

time-series experiments indicated that chlorophyll a 

responded more quickly to nitrogen stimulation than carbon 

uptake. More apparent, however, were the differences 

observed in the absolute magnitudes of the maximum 

responses of carbon uptake and chlorophyll a. Nitrogen 

additions resulted in maximum increases in carbon uptake 

ranging from 40 to 150 percent, whereas corresponding 

increases in chlorophyll a were always far greater, increasing 

from 150 to 400 percent.

Another characteristic distinction of the response of 

the phytoplankton assemblages to nitrogen additions was the 

observed difference in carbon uptake stimulation between 

the two forms of nitrogen, NO3-N and NH4-N. Nitrogen 

uptake, under limiting conditions, generally has been found 

to be both extremely rapid and nearly light independent 

(Goldman and others, 1981; Fisher and others, 1982; Whalen 

and Alexander, 1984; Morel, 1987; Pennock, 1987; Priscu, 

1987). Researchers have almost universally reported that 

phytoplankton preferentially take up more reduced forms of 

nitrogen, with uptake rates generally being greater for the 

smaller phytoplankton size fractions (Malone, 1971; Malone 

and Neale, 1981). Carpenter and Dunham (1985) and 

Pennock (1987), working on the seasonal estuarine uptake of 

nitrogen, found strong preferences for NH4-N over NO3-N. 

They, and others (Takahashi and Saijo, 1981), also observed 

that NH4-N inhibited NO3-N uptake. Such repressive inhibition was not apparent at high concentrations of NO3-N or low 

concentrations of NH4-N. Dortch and others (1982) found 

that, under nitrogen limitation, the ability to take up NO3-N 

may actually decrease, whereas the potential to take up NH4-N

Effects of Nitrogen and Phosphorus Additions 27
Table 4. Dates and locations where there were significant changes 

in relative percentage of either carbon uptake or chlorophyll a 

for three measured size fractions of phytoplankton in response 

to nutrient additions

[A (+) denotes a significant increase (a = 0.05) in the percent contribution 

of a size fraction over the controls, and a ( ) indicates a corresponding 

significant decline. < = less than; > = greater than; (J,m = micrometer]

Carbon uptake 

for indicated size fraction

<5 5-20 |^m >20

Chlorophyll a

for indicated size fraction

<5 urn 5-20 (j,m <20 (j,r

Myakka River 

May 1985

23%o 

PO4-N

NO3-N 

NH4-N

Peace River 

June 1984 

20%o 

NO3-N

NH4-N

May 1985 

20%o 

PO4-N 

NO3-N

NH4-N

September 1985

PO4 -N

NH4-N

increases. Under such conditions, phytoplankton, even at low 

ambient NKLj-N levels, may be able to satisfy cellular 

demands quickly. Such observations support the findings of 

the current study in which the maximum response of carbon 

uptake was often greater for Nlfy-N than for NO3-N.

Nitrogen stimulation during several of the experiments 

also caused relative changes in the distribution of carbon 

uptake and chlorophyll-a concentrations among phytoplankton size fractions. The patterns of such responses often 

differed between nitrogen forms. Within an estuary, such as 

Charlotte Harbor, natural phytoplankton communities are 

exposed continuously to both short-term patchy and longterm seasonally ephemeral physical and chemical gradients 

that may differentially limit the growth of various taxonomic 

groups. Differences in nutrient ratios also may be an important factor in determining the relative dominance of specific 

taxonomic groups. Sommer (1985) found that neither pulsed 

nutrient additions, such as those that occur naturally during 

periods of high river input, nor steady-state nutrient conditions uniformly provided a competitive advantage to any 

particular size class of phytoplankton; rather, stimulation was 

taxa specific.

Sanders and others (1987) investigated the effects of 

seasonal additions of ammonium, nitrate, and phosphorus to 

large cultures of natural phytoplankton assemblages from the 

Pautuxent River Estuary. Their results indicated that nitrogen 

enrichment during the summer and fall quickly changed the 

species composition of the phytoplankton community from a 

predominance of flagellates to one dominated by small 

centric diatoms. Such changes in dominance were observed 

to result from increased growth rates of specific taxa. Malone 

(1971; 1982) observed that smaller phytoplankton size fractions are often stimulated at far lower concentrations than 

larger size fractions. He proposed that smaller size fractions 

might be expected to respond more quickly because such 

cells, with their greater surface to volume ratio, generally 

have greater uptake rates and lower half-saturation constants 

for macronutrients.

Findings of those experiments where nutrient addition 

over a range of concentrations not only stimulated either 

phytoplankton carbon uptake or chlorophyll-a concentrations but resulted in significant changes in the relative 

percentages among the three measured size fractions are 

summarized in table 4. As indicated, nutrient additions at

28 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida
lower salinities either did not stimulate phytoplankton carbon 

uptake or chlorophyll a (high-flow conditions), or resulted in 

an equal increase among all three size fractions. Similar 

nutrient additions at higher salinities, by comparison, often 

resulted in significant differences in percentages of carbon 

uptake and chlorophyll a among size fractions of the phytoplankton. Although the overall results of this study tend to 

support the view that smaller size fractions generally exhibit 

a greater response to nutrient additions, the data indicated 

that the relative responses of the size fractions were highly 

variable with regard to season, location, and nitrogen form.

Additions of orthophosphorus over a series of 

concentrations did not stimulate carbon uptake during any of 

the bioassay experiments. However, such additions did 

inhibit carbon uptake slightly in May 1985 at both the 

Myakka River site and at 6%o salinity along the Peace River. 

Ambient conditions at these sites included high 

orthophosphorus concentrations (0.397 and 0.830 mg/L, 

respectively) and low atomic N:P ratios (<0.5).

A possible explanation for the inhibition of carbon 

uptake might be the differences in availability between the 

measured ambient and added forms of orthophosphorus, 

combined with the widely observed phenomenon of luxuryphosphorus consumption by phytoplankton. Procedures for 

determining ambient-orthophosphorus concentrations 

measure "reactive forms" of the nutrient, large percentages 

of which can exist as "lightly bound" forms. In much of the 

Charlotte Harbor basin, large percentages of the measured 

orthophosphorus may be in the form of trimetaphosphate 

associated with calcium ions. Although this form is chemically measurable as "reactive" orthophosphorus, its direct 

availability to phytoplankton across cellular membranes 

could be more limited (Kuhl, 1974). The orthophosphorus 

experimentally added, by comparison, was in a chemically 

"pure" form (NaH2PC«4), readily available for phytoplankton 

uptake. Orthophosphorus uptake, unlike that of nitrogen, 

generally has been found to be stimulated by light and dependent on adenosine triphosphate (ATP) (Nalewajko and Lee, 

1983). Thus, luxury-phosphorus consumption has the potential of competing with other metabolic-intensive processes, 

such as photosynthesis, for ATP and may explain the 

observed slight depression of carbon uptake at the two sites 

in May 1985.

SUMMARY AND CONCLUSIONS

1. In regions of Charlotte Harbor characterized by low 

salinities, phytoplankton carbon uptake and chlorophyll-tf concentrations were stimulated by additions of 

inorganic nitrogen during seasonal periods of low 

freshwater inflow. Neither phytoplankton measurement, by comparison, responded to nitrogen additions 

at low salinities during periods of high freshwater

inflows. This could be directly attributed to the effects 

of increased water color, reduced light availability, and 

high ambient nitrogen concentrations.

2. Under normal conditions, in the more saline reaches of 

the estuary, phytoplankton carbon uptake and 

chlorophyll a may be continually nitrogen limited.

3. During periods of high summer freshwater inflow, the 

estuary can be divided conceptually into a low salinity 

zone where phytoplankton production is mediated by 

light availability, as determined by high water color, 

and a high salinity zone, where production is nitrogen 

limited.

4. Results from seasonal and dilution curve studies of 

ambient-nutrient concentration patterns, as well as 

comparative calculations of inorganic nitrogen inputs 

and observed phytoplankton productivity, support the 

observations of the bioassay investigations. Each of 

these lines of evidence indicates that, exclusive of 

seasonal riverine influences affecting light penetration 

of the water column, nitrogen availability normally 

limits phytoplankton production within the Charlotte 

Harbor estuarine system.

5. Although the overall results of this study tend to support 

the view that smaller size fractions generally exhibit a 

greater response to nitrogen additions, the data 

indicated that the relative responses of the size 

fractions were highly variable with regard to season, 

location, and nutrient form.

The reported experimental results indicate that 

seasonal freshwater inflows influence the occurrence and 

distribution of the nutrient limitation of phytoplankton 

production in the Charlotte Harbor estuarine system. Care 

must be taken, however, in directly transferring the results of 

such short-term macronutrient-bioassay determinations to 

the ecosystem level. The scope of the present study was 

limited to specific salinities and flows and may have 

neglected other nutrients and biological and physical factors 

that could have significantly influenced the potential 

responses of the phytoplankton assemblages. Factors that 

limit bottle-held phytoplankton growth and productivity or 

both may differ from those that limit production over an 

entire system. Understanding the components controlling 

phytoplankton production in a shallow, subtropical estuarine 

system such as Charlotte Harbor is further confounded by the 

complex interactions of physical factors such as salinity, 

temperature, light (as influenced by both annual cycles and 

seasonal increases in water color), and nutrient additions 

through inputs and regeneration.

Conceptually, phytoplankton populations can seldom 

be classified as either strictly light or nutrient limited. First, 

the potential exists for distinct spatial and temporal differences among the factors controlling phytoplankton 

production. Further, various algal taxonomic groups, influenced by distinctive uptake and growth rates, can exhibit 

marked variations in their responses to limiting conditions.

Summary and Conclusions 29
The view that potentially limiting nutrients are available for 

phytoplankton uptake at homogeneous low concentrations is, 

furthermore, restrictive. Phytoplankton assemblages within 

estuarine systems encounter a wide variety of ambient 

nutrient concentrations, including discrete nutrient pulses, 

deterministic of a patchy environment, as well as ephemeral 

and seasonal influences.

Population growth in the Charlotte Harbor basin is 

expected to cause an increase in nitrogen loading to the 

estuary by more than 2,700 kg/d by the year 2020. Such 

additional nitrogen loading would be equivalent to 18 

percent of the current gaged-basin nitrogen input. New 

inorganic nitrogen would be immediately available for 

phytoplankton uptake, whereas mineralization by bacteria 

and other microorganisms would make other forms available 

over time. Additional nitrogen loadings could be expected to 

result in increased seasonal phytoplankton production in 

those areas of the harbor where water color is low enough to 

allow for sufficient light availability.

Incorporation of the results of nutrient-limiting studies, 

such as presented here, as well as seasonal patterns of phytoplankton production into generalized hydrodynamic models 

would aid the development of predictive management tools. 

The present study indicates that management strategies 

developed to maintain existing water quality need to 

emphasize point and nonpoint loadings of nitrogen into the 

Charlotte Harbor system, seasonal and locational factors, and 

rates of nitrogen recycling.

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