# 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 1991U.S. DEPARTMENT OFTHEINTERIOR MANUEL LUJAN, JR., Secretary

U.S. GEOLOGICAL SURVEY Dallas L. Peck, Director

For additional information, Copies ofthis 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 ofstudy area 3 Methods 3 In situ C-14 incubations 3 Determination ofphotosynthetic C-14 uptake 3 Determinationofchlorophylla 5 Chemical analyses 6 Data analyses 6 Design ofnutrient addition experiments 6 Time series 6 Single and multiple nutrient additions 9 Concentration response 13 Results ofnutrient addition experiments 15 Tidal Myakka River fixed station 15 High flow 15 Low flow 15 Second high flow 18 PeaceRiver and Charlotte Harbor salinity stations 18 Low flow 18 High flow 18 Effects ofnitrogen andphosphorus additions onphytoplankton productivity and chlorophylla 24 Summary and conclusions 29 Selectedreferences 30

Figures 

### 1. Map showing location ofCharlotte Harbor study area and insitu study sites, 1984-85 4 2-27. Graphs showing: 2. Total gaged flow ofthe PeaceRiver, 1980-86, and dates ofin situ flow-related studies 5 3. Relationbetween carbon uptake andperiod ofin situ incubation at site 1 (approximately 12-parts-per-thousand salinity), May 10, 1984 9 4. Relationbetweenchlorophyll-^ concentration ofamended samples as percentofchlorophyll a in control samples andperiod ofinsitu incubation at site 1, May 10, 1984 9 5. Relationbetween carbonuptake andperiodofinsitu incubation at site 2 at
a salinity ofapproximately 12partsperthousand on May 17, 1984 10 6. Relationbetween chlorophyll-^ concentration ofamended samples as percentofchlorophylla in control samples andperiod ofinsitu incubation at site 2, May 17, 1984 10 7. Phytoplankton carbon uptake forcontrol andnutrient-amended samples for 10-hour insitu incubations at salinities ofapproximately 12 (site 3) and 20 (site 4)parts perthousand, May 22,1984 12

Contents III

### 8. Phytoplanktonchlorophyll-aconcentrationsforcontrol andnutrient-amended samples for 10-hourinsitu incubations at salinities ofapproximately 12 (site 3) and 20 (site 4) parts perthousand, May 22, 1984 13 9. Phytoplankton carbon uptake forcontrol and nutrient-amended samples for 10-hour insitu incubations at site 5, May 31, 1984 14 10. Phytoplankton chlorophyll-aconcentrationsforcontrol andnutrient-amended samples for 10-hour in situ incubations at site 5, May 31, 1984 14 11. Phytoplankton carbon uptake forcontrol and nutrient-amended samples for 10-hour in situ incubations ata salinity ofapproximately 20parts per thousand at site 6,June 1, 1984 16 12. Phytoplankton chlorophyll-aconcentrationsforcontrol andnutrient-amended samples for 10-hourinsitu incubations ata salinity ofapproximately 20 partsperthousand at site 6,June 1, 1984 16 13. Phytoplankton carbon uptake forcontrol and nutrient-amended samples for 10-hour in situ incubations at site 7 on the tidal MyakkaRiver, September27, 1984 17 14. Phytoplankton chlorophyll-aconcentrationsforcontrol andnutrient-amended samples for 10-hour insitu incubations at site 7 on the tidal Myakka River, September27, 1984 17 15. Phytoplankton carbon uptake forcontrol and nutrient-amended samples for 10-hour in situ incubations at at site 7 on the tidal MyakkaRiver, May 23, 1985 19 16. Phytoplankton chlorophyll-^concentrationsforcontrol andnutrient-amended samples for 10-hour insitu incubations at site 7 on the tidal MyakkaRiver, September 16, 1985 19 17. Phytoplankton carbon uptake forcontrol and nutrient-amended samples for 10-hour in situ incubations at site 7 on the tidal MyakkaRiver, September 16, 1985 20 18. Phytoplankton chlorophyll-^concentrationsforcontrol andnutrient-amended samples for 10-hour insitu incubations at site 7 on the tidal MyakkaRiver, May 23, 1985 20 19. Phytoplankton carbon uptake forcontrol andnutrient-amended samples for 10-hour in situ incubations ata salinity ofapproximately 6 parts per thousand at site 8, May 8, 1985 21 20. Phytoplanktonchlorophyll-aconcentrationsforcontrol andnutrient-amended samples for 10-hour insitu incubations at a salinity ofapproximately 6parts perthousand at site 8, May 8, 1985 21 21. Phytoplankton carbon uptake forcontrol andnutrient-amended samples for 10-hour in situ incubations ata salinity ofapproximately 20parts per thousand at site 9, May 10, 1985 22 22. Phytoplankton chlorophyll-a concentrationsforcontrol andnutrient-amended samples for 10-hour in situ incubations at a salinity ofapproximately 20parts perthousand at site 9, May 10, 1985 22 23. Phytoplankton carbon uptake forcontrol andnutrient-amended samples for 10-hour insitu incubations ata salinity ofapproximately 6partsper thousand at site 10, September 17, 1985 23 24. Phytoplanktonchlorophyll-aconcentrationsforcontrol andnutrient-amended samples for 10-hour insitu incubations at a salinity ofapproximately 6parts perthousand at site 10, September 17, 1985 23 25. Phytoplankton carbon uptake forcontrol andnutrient-amended samples for 10-hour in situ incubations ata salinity ofapproximately 20parts per thousand at site 11, September24, 1985 25

Contents IV

### 26. Phytoplanktonchlorophyll-aconcentrationsforcontrol andnutrient-amended samples for 10-hour in situ incubations at a salinity ofapproximately 20parts perthousand at site 11, September 24, 1985 25 27. Meanvaluesfor selectedmacronutrients andmeasurements ofphytoplankton production collectedmonthly fromJune 1983 throughJune 1988 at6 and 20parts perthousand along salinitytransects in the lowerPeaceRiver and upper Charlotte Harbor 26

Tables 

### 1. Methods used forchemical analyses 6 2. Summaries by experimentoflinear, quadratic, andcubic determinations from general linearmodelprocedures forchangesincarbonuptake andchlorophylla inresponse to nutrient additions 7 3. Ambient water-quality data andpreceding Peace Riverflows foreach experiment 11 4. Dates and locations wherethere were significantchanges inrelativepercentage ofeithercarbon uptake orchlorophyll a forthree measured size fractions of .phytoplankton inresponse 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 squarekilometer (km2) 0.3861 squaremile cubic meterper second (m3/s) 35.31 cubic footper second kilogramperday (kg/d) 2.205 poundperday

Additional abbreviations used in report: 

### microcuries (|lCi) micrometer (|im) milligramper liter (mg/L) milliliter(mL) partsperthousand (%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 ultimatelybe 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 acceptedgeneralizationthatmarineenvironments are,forthe 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 depositsornutrientpollution, althoughotherexceptionshave been reported. Myers and Iverson (1981) found evidence of phosphorus-limiting primary production in an estuary in the northeastern Gulf ofMexico, whereas McComb and others (1981) found evidence of seasonal shifts between nitrogen andphosphorus limitation in anestuaryinwesternAustralia. Davies and Sleep (1981) reported that phytoplankton production intheEnglish Channel attimes could be simultaneously and additively stimulated by additions ofammonia, nitrate, and phosphate, whereas Agins and Jaccarini (1982) found phosphorus to be the limiting nutrient in parts ofthe Mediterranean Sea. Wurtsbaugh and others (1985) observed nitrogen stimulation ofphytoplankton carbon-14 uptake in a tropical alpine lake.

IntroductionAlthough 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 demonstratedto be the key factorregulating primary productivity in estuarine and coastal waters (Ketchum, 1967; Boynton and others, 1982). Nitrogen andphosphorus existin
a variety of dissolved and particulate forms that may be directly or indirectly usedforphytoplanktonproduction. The distribution and availability ofthese 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 longerterm production. Anthropogenic development in coastal basins has often resulted in increased nutrient loadings andhas 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 andchemostat studies ofnutrient 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 ofnutrient 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 waterquality. In this study, changes in the incorporationofcarbon-14 and chlorophyll-a concentrations were used as measures of the response of phytoplankton assemblages to a series of addednutrientconcentrations. 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 ofthe taxonomic composition of the phytoplankton assemblages within each of the three measured size fractions have indicated the following generalizedpatterns with regardto salinityranges withinCharlotte Harbor. The smallest size fraction (<5 |im), at both intermediate and higher salinities, is characteristically dominatedby 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 ofthe largest size fractions. Specifically, blooms of Gyrodinium resplendens seasonally dominate the larger size fractions at the mouth ofthe 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 ofthe 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 ofa study to evaluate theeffects ofinorganicnitrogen andphosphorus additionson phytoplanktonproductivityandchlorophyll-aconcentrations in a subtropicalFloridacoastalplainestuary and assesses the potential effects of increased nutrient concentrations on futurephytoplankton production withinthe Charlotte Harbor system. The results ofthis estuarine phytoplankton productivity study are discussed in terms ofthe seasonal and areal variations in ambient-nutrient concentrations andhydrologic 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 ofbarrier islands. Water exchange with the Gulf of Mexico occurs primarily through Boca Grande Pass at the southern end ofthe harbor. The estuarine system covers an areaofapproximately 300km2 andhas an average depthofonly about 3 m. The estuaryis vertically wellmixed 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 ofhigh freshwaterinflow (Fraser and Wilcox, 1981; Fraser, 1986). Two rivers flow into the upper estuary: the Peace River, whichhas an average, total gaged, long-terminflowof 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 PeaceRiver (fig. 2). Charlotte Harboris one ofthe leastdisturbed 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 nonpointsourcesofrunoffonthe waterqualityoftheestuary. Parts ofthenorthernCharlotteHarbordrainagebasincontain 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 ofphosphorus to the Peace and MyakkaRivers 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, naturalphytoplanktonpopulations wereexposed to increasing concentrations of added nitrate plus nitrite (NO3-N), ammonia (NH4-N), and orthophosphorus (PO4-P). The patterns ofthe 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 madeusing theprocedures describedbelow.



### In SituC-14 Incubations

Toreducethe chancesoftraceinhibitoryor stimulatory contamination (Marra and Heinemann, 1984), all incubation bottles and lined caps were washed in 6 normalhydrochloric acid (6NHCL)priortouse,rinsed,triplerinsed withdoubledistilled 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 solutionsto obtainthedesiredfinal addedconcentrations.An identical volume of the deionized, double distilled water used to prepare the stock nutrient solutions was added to an equalnumberofreplicate controls. Darkbottles wereusedto determine blank background uptake. All incubation bottles
weretheninoculatedwith 10 fiCioftrace-metal-freecarbon-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
apredetermined period oftime around apparentnoon. After incubation, the samples were stored in the dark on ice and quicklyreturned tothe laboratory where they were filtered.

Determination of Photosynthetic C-14 Uptake



### To determine the total carbon-14 uptake ofthe 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

Methods82' 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



### gLlCC 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 describedabove.The wetfilterswerethenplacedin scintillationvials. Anyresidual inorganiccarbon-14 was removedby adding 0.5 mLof0.1 N HCL (Lean and Bumison, 1979) and by allowing the vials to stand for 3 hours before the addition of 10.0 mL ofscintillation fluor. The scintillation vials were stored in the darkfor 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)usingthechannelsratiomethod.Thecounting efficiency was determined using a series offilters quenched by increasing amounts ofphytoplanktononto which aknown amount ofcarbon-14 labeled sucrose (Pugh, 1970) had been dried. The specific activity ofthe 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 ofChlorophyll 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 concentrationsrepresent single determinations obtained by compositing equal volumes from each of the replicate incubationbottles.

Methods

### Chemical Analyses

Corresponding water samples at each station were collectedinnew, cleanpolyethylenecontainers.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 NHLtnitrogen 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)



### DataAnalyses 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 ofnitrogen and phosphorus concentrations. In these instances, general linear model procedures (polynomial regression or curve-fitting analysis) were employed to determine ifthe coefficients of the resulting phytoplankton responses over the range of nutrient concentrations tested were significantly different from zero and to describe the shape ofthe response curves. This methodology explicitly differs from that of the more often applied multiple-comparative procedures, such as Duncan's Multiple Range ofAnalysis ofVariance (Duncan, 1957; Waller and Duncan, 1969). Multiple-comparative analysis would treat the series ofnutrient concentrations as qualitatively different variables and test and contrast means forindividual 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 appliedtreatment (Chew, 1971; 1980; Peterson, 1977; Little, 1981). In utilizing polynomial regression to estimate the relation between the dependent response (phytoplankton carbon orchlorophylla) andindependenttreatmentvariables (nitrogen forms and phosphorus), it is usually sufficient to partitiontheresultanttreatmentsumofsquaresintoitslinear, cubic, and quadratic components (table 2) while testing the significance of each (Chew, 1971). The advantages ofthis methodology over standard analysis ofvariance procedures
are evident when applied to actual field data where progressive patterns in the data may be obscured by the variability between discrete-measurementintervals. 

### 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 shapesoftheresultingresponsecurveswerefurtheranalyzed usingnonlinear, least squares, andfittingproceduresto solve andplottheestimatedresultantcurves.

DESIGN OF NUTRIENT ADDITION EXPERIMENTS

A series of experiments was initially conducted to develop an overall experimental design. The purpose ofthe design was to evaluate adequatelythepotentialphytoplanktoncommunity responses to nutrient additions under high and low freshwaterinflow conditions.

Time Series

Collos and Slawyk (1984) noted the importance of conducting an incubation time series experiment as part of any interpretation ofthe response ofC-14 uptake tonitrogen additions. Their results indicated that, during the early periods ofatime-course experiment, the addition ofnitrogen 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, ifpresent, while at the same time be short enoughtoreduce thepossible "bottle-induced" effects often associated with extended incubations. Todefine an adequateincubationperiod,twoindependent 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. Adrogue was usedto mark the initialbody ofwater sampled and sequential samplings were conducted hourly up to the 2-hour incubation period, which
was collected 1 hourpriorto apparentnoon.

Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, FloridaTable 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/m3, milligrams per cubic meter]

c

. . Parameter estimate

Linear

Nutrientconcentration \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 PeaceRiver 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 ExperimentsTable 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

Thefirsttime-seriesexperimentinvestigatedresponsesto the addition of0.5 mg/LofNO3-N. The results indicated a lag of8 to 10hourswasrequiredbeforeasignificantincreaseinthe rate ofcarbon uptake was observed (fig. 3). A slightly shorter period oftime was needed to observe increases in concentrations ofchlorophyll a above control levels (fig. 4). No significant differences were observed among the measured phytoplankton sizefractions intheirresponses to nitrogen addition. A second experiment was conducted tofurtherinvestigate phytoplanktonreactions to nitrogen additions withrespect to incubation length. Additions of 0.5 mg/L of NO3-N and NH4-Nwere comparedwith controls (figs. 5 and 6). Stimulationpatterns ofcarbon 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 inphytoplankton 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 differencesintheresponses ofphytoplanktoncommunities 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) atatime whenboth areas ofthe 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 analyzedusing analysis ofvariance and contrast methods (alpha = 0.05) to compare mean uptake rates forvarious 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 200 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 ofNutrientAddition Experiments 9LU 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 i I i i i i I i i I I I I I i i i I I I ill

46 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, FloridaTable 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 seriesno. 1 2 hours 4 hours 6 hours 8 hours 10hours
Time series no. 2 2 hours 4 hours 6 hours 8 hours 10 hours 12 hours 14 hours
Nutrientcombinations 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 ofdays 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
zonesMay 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 11NO- O.I NO, 0.5 NH 4 0.5 NH4 +N03 0.5 NH4+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 1 ^ O Raw 

## f? o-*-^ <5jum

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 ofphytoplankton carbon uptake at 12%o indicated: 1. No significantdifferencebetweencontrolsandreplicates treated withPCVP; 2. Both forms ofnitrogen 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/Lhad the opposite effect; 4. NOa-N + NH4-N had no greatereffect than either ofthe two nutrients individually, whereas the addition of PO4-P in combination with the two forms ofnitrogen resulted in a significantly higher level of carbon uptake; and 5. NOa-N at 0.5 mg/L and both concentrations ofNH4-N significantly increased the relative amount of carbon taken up by the largest phytoplankton size fraction (>20jim). The effects on carbon uptake to similar nutrient additions at 20%o salinity showed slightly differentpatterns: 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 greaterthan that forNOa-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 ofPO4-Pincreased carbon uptake; 5. Bothforms ofnitrogen at0.5 mg/Lresultedin a significant increase in relative percent ofproduction by the 5- to 20-jim size fraction. Withineachsalinityzone,chlorophyll-aconcentrations generally exhibited stimulation patterns similar to those observed for carbon uptake.

12 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, FloridaNO 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

<5jjm

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 ofthe preceding experiments, the effects ofnutrient additions over a wider range ofnitrogen concentrations were measured atthe sametwo salinities. The purpose ofthis experiment was to determine a standardized range of added-nutrient concentrations adequate to statistically evaluate phytoplankton responses within different estuarine salinityregimes. At 12%o, the effects ofnitrogen on phytoplankton carbon uptake and concentrations of chlorophyll a were investigated using nutrient additions that resulted in final concentrations forNO3-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 overfinal concentrationsrangingfrom0.0to0.4mg/L forboth NO3-N andNH4-N. Observed changes inphytoplanktoncarbonuptake and chlorophyll a over ranges ofnitrogen additions at the 12%o salinity zone are shown in figures 9 and 10. Concentrations ofthe additions are presented in ascending order and are not linear. Changes in carbon uptake and chlorophyll a are indicated forthe whole (total blockheight) and each ofthe 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 significantlinear, cubic, andquadratictreatment sumsfor models with coefficients significantly different from zero. Additions ofNOs-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 withNFLj-N additions. Thepattern ofthetreatment 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 thatresulted from increasing additions ofthe two nitrogen forms (fig. 10) were similar to
one another and characteristically different from the patterns ofincrease in carbon uptake. Maximum stimulation ofchlorophyll a took place at much lower NOs-N concentrations

Design of Nutrient Addition Experiments 13NO

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 ofchlorophyll a occurred athigh levels ofNH4-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 ofNO3-N andNI-Lj-N (table 2). Solution ofcarbon uptake models, using nonlinear least squares fitting procedures, indicated thatthe general shape oftheresulting curves were similar, with the asymptote ofthe response curvebeing slightly higher for NH4-N than for NO3-N. Corresponding analysis ofchlorophyll-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 inresponse 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 ofchlorophyll a in the >20-jim size fraction and acorresponding 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 ofphytoplankton communities within the Charlotte Harborestuarine system to nutrient additions underdiffering conditions of freshwater inflow. Changes in carbon uptake andchlorophyll-aconcentrationsthatresultedfrom additions ofNC»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 MyakkaRiver (fig. 1) duringtwoperiods ofhigh andone 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 oflow andhigh Peace Riverinflow. In each experimentpolynomial models ofthe form
carbon uptake = bo + bi(Conc.) + b2(Conc.)2 + b3(Conc.)3



### were constructedfor each nutrient. Similarmodels also were constructedusing chlorophyll-aconcentrations as aresponse variable. Significance determinations ofthe models were done by testing the coefficient for each term ofthe regression (bi, b2, b3 not equal to 0). Significance of these coefficients dictated shape oftheresponse curve. Asignificantlinear (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, andresponse variable (carbon uptake, chlorophyll-a concentrations) and are summarizedin 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, andrelatively high macronutrient concentrations (table 3). Phytoplankton carbonuptake andchlorophyll-aconcentrations werehighest 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 inresponse toincreasing 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 ofthe 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 increasingconcentrations ofPC»4-P(figs. 13 and 14). Statistical analysis, however, failed to identify any significantdecline (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 indicatedby the untreatedcontrols.

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, FloridaCONTROL 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 ofboth 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 ofNOs-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 ofthese models using nonlinear-fitting procedures indicated that the response curve for NO3-N initially increased at a slightly greaterratethanthatforNH4-N. The asymptote ofthe 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 (<5jim) accounted for most of the observed increase in carbonuptake andchlorophyll-a concentrationinresponseto NOs-N andNH4-N additions. In contrast to the stimulation by nitrogen, carbon uptake significantly (table 2)decreased with increasing PCVP additions. Graphically, chlorophyll-a concentrations also seemedto decrease slightly with increasing PO4-Padditions. This conclusion was supported by the results ofthe 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 aperiod ofhigh 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 comparedto previous observedrates (figs. 17 and 18). Neither phytoplankton carbon uptake nor chlorophyll-a concentrations indicated any biologically significant response to the additions ofnutrients.

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 ofNO3-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 ofeach ofthe 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 ofthe responses to NOs-N andNH4-N were similar. Solutions ofthese curves using nonlinear fitting procedures indicated that the curve for NOs-N initially increased at a rate greaterthanthatofNH4-N, whereasthe asymptoteofthe NH4-N response curve was significantly higher. The responses ofchlorophylla followed patterns similarto those for carbon uptake (table 2). Both nitrogen forms caused threefold increases in phytoplankton chlorophyll-a concentration. Analysis by size fraction ofchanges in carbon uptake inresponsetonitrogen additions indicatedthat, asconcentrations ofboth forms increased, there were significant small increases in the relative percentages attributable to the two smallest (<5 (im and<20 and >5jim) sizefractions. Asimilar pattern occurred with respect to changes in concentration of chlorophylla with increasing NH4-N additions.
High Flow



### A second series ofexperiments was conducted in the Charlotte Harbor Estuary at approximately 6 and 20%o salinity in September 1985, a period ofrelatively high river inflow (fig. 2). Water colorand 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, FloridaCONTROL 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

5-20jum >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 190.07 / 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, FloridaCONTROL 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 21CONTROL 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, FloridaNUTRIENT ADDITIONS, IN MILLIGRAMS PER LITER 5 EXPLANATION

0.1

£ a O.os 

# ii PHYTOPLANKTON SIZE FRACTION

<5jjm

5-20jjm >20jjm
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 contrastto the effects ofcorresponding additions atthe 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 ofthe NH4-N curve, however, indicated greater stimulation atlowerconcentrations. Chlorophyll-a concentrations significantly increased (table 2) as much as fivefold in response to experimental nitrogen additions at20%osalinity (fig. 26). The shapesofthe responses of phytoplankton chlorophyll-a concentration to the two forms of nitrogen (fig. 26) increased to maximum levels andthendeclined slightly atthehighest additions. The magnitude ofchlorophyll-a stimulation was greaterthanthat of carbon uptake, whereas the decline of chlorophyll-a concentrations at the highest nitrogen concentrations was less dramatic.

. Analysisby sizefractionindicatedthat,overtheranges ofNO3-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 ofNO3-N. By comparison, the relativepercentage of chlorophyll a within the largest size fraction (>20 (im) increased in response toNI-Lj-N stimulation.

EFFECTS OF NITROGEN AND PHOSPHORUS ADDITIONS ON PHYTOPLANKTON PRODUCTIVITY AND CHLOROPHYLL a


### The experiments conducted withinthe 6%osalinitypart of the Charlotte Harbor Estuary showed distinct seasonal changes in phytoplankton carbon uptake and chlorophyll a that resulted from inorganic nitrogen additions. Otherinvestigators, 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 ofphytoplankton assemblages to nitrogen and phosphorus additions. The seasonal changes in phytoplankton responses in Charlotte Harbor were directly attributable to seasonalperiods oflow andhighfreshwaterinflow. Nutrient additions at the end ofthe 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 experimentalresults, by comparison, from the MyakkaRiver site inMay 1985 (23%o salinity) and the 20%o salinity zone in upper Charlotte Harbor in May and September 1985 indicate thatphytoplanktoncommunities withinthe
more saline parts ofthe estuary do not exhibit seasonality in theirresponsetonitrogen additions and arecontinuallynitrogen limited, except possibly under very high freshwater inflow conditions. The results from the nutrient addition experiments indicatethat,during lowfreshwaterinflow,the availabilityof nitrogen may limit phytoplankton productivity over a wide range ofsalinities throughout much ofthe 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 runofffrom the Peace and Myakka River basins contains high concentrations ofdissolvedhumic 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 ofthe estuary characterized by higher salinities, dilution and photooxidation of humic substancesreduce watercolorand increase lightpenetration. Thus, during periods ofhigh freshwater inflow, the estuary conceptually canbe dividedintotworegionsbasedon salinity. In the lower salinity region, phytoplanktonproduction 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 ambientnutrient concentrations. Otherinvestigators (Davies and Sleep, 1981; Myers and Iverson, 1981; D'Elia and others, 1986) have reported similarpatterns. Meanmonthlyambientvaluesforselectedmacronutrients, 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 seasonalriverinflow. Basinrainfall andrunoffare 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 ofphosphorus in the rivers increases withdecliningriverflow andthendeclinesthrough

24 Effects of Nitrogen and Phosphorus Additions on Phytoplankton, Charlotte Harbor, Florida0.001 001 005 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 25CO h; Z

. CC OQ O O 200

150

100

50 i i i i i i6-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

O4o

fOo 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.)


### thesummerasPO4-Pconcentrationsinriverinflowdecrease. 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 ofNOs-N in the more saline parts ofthe harbor (20%o salinity) remain near detection limits through all but the wintermonths. Thesegeneralized macronutrientpatterns are similar to those reported by Fraser (1986; 1991) from longterm seasonal studies of water-quality characteristics ofthe lowerPeace River. Low ambient nutrient levels by themselves, however, cannotbe taken as definitive indications ofpotential nutrient limitation. Smith (1984) proposed further evaluation of potential limiting nutrient conditions inestuarine 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 alongnaturalgradients. TheN:Pratiosofnaturalphytoplankton assemblages generally average near the Redfield (1958) value of 16. Waters with ambient N:P ratios ofless than 10 usually indicate potential conditions in whichphytoplankton production isnitrogenlimited, whereas valuesofgreaterthan 21 are commonly indicative ofphosphorus limitation (Ram and Plotkin, 1983; Sakshaug and Olsen, 1986). Determinations of limiting nutrient conditions based on ambient inorganicN:Pratios and correspondingresults frombioassay 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 ofconcentrations ofnutrients and conservative constituents along natural gradients (Smith, 1984). Fraser and Wilcox (1981), Froelich and others (1985), and McPherson andMiller (1990) evaluated nutrient sources and distributions in Charlotte Harbor using nutrient dilution
curve models constructedbyplottingnutrientconcentrations 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), supportthe overall findings ofthe nutrient-addition experiments. The results of these experiments stress the importanceofnitrogeninlimitingphytoplanktonproduction, except in thoseestuarine areas that are seasonally influenced by high watercolor. 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 additionalnitrogen loading wouldbeequivalentto 18percent of the current gaged-basin nitrogen input (McPherson and Miller, 1990). New inorganicnitrogenwouldbeimmediately available for phytoplankton uptake, whereas mineralization by bacteria and other microorganisms would make other forms available over time. Additional nitrogen loadings couldbe expectedtoresult in increased seasonalphytoplanktonproduction in those areas ofthe harbor where watercolor is low enough to allow for sufficient light availability. Total freshwaterinorganic nitrogen inputs in Charlotte Harbor (Hammett, 1988; Fraser, 1991) are more than an order ofmagnitude below that required to support observed levels of phytoplankton production (Montgomery and Emmons, 1989; McPherson and others, inpress). Therefore,
some mechanism, such as the rapid recycling ofnitrogen by processes such as remineralization by microheterotrophs (Furnas and others, 1986), is required to support observed photosynthesis rates. Pennock (1987) suggested that, in the DelawareEstuary system, morethan 80percentofthe 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 upby phytoplankton andjoins a largerorganic pool of fixed nitrogen. Such a concept is consistent with a number offindings from otherestuarine 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 ofcarbon 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 alwaysfargreater, increasing from 150to400percent. Another characteristic distinction of the response of the phytoplankton assemblages to nitrogen additions wasthe 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 andothers, 1981; Fisherandothers, 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 smallerphytoplankton size fractions (Malone, 1971; Malone and Neale, 1981). Carpenter and Dunham (1985) and Pennock (1987), working on the seasonal estuarineuptakeof nitrogen, found strong preferences for NH4-N over NO3-N. They, and others (Takahashi and Saijo, 1981), also observed that NH4-N inhibitedNO3-Nuptake. Such repressive inhibitionwasnotapparent athighconcentrations ofNO3-Norlow concentrations of NH4-N. Dortch and others (1982) found that, undernitrogen limitation, the ability to take up NO3-N may actually decrease, whereasthepotentialtotakeupNH4-N

Effects of Nitrogen and Phosphorus Additions 27Table 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. Undersuchconditions,phytoplankton,even atlow 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 ofcarbon uptake wasoften greaterforNlfy-Nthan forNO3-N. Nitrogen stimulation during several ofthe 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 thatmay differentially limitthegrowthofvarioustaxonomic groups. Differences in nutrient ratios also may be an important factor in determining the relative dominance ofspecific taxonomic groups. Sommer (1985) found thatneitherpulsed nutrient additions, such as those that occur naturally during periods ofhigh river input, nor steady-state nutrient conditions uniformly provided a competitive advantage to any particularsizeclassofphytoplankton; rather, stimulation was taxa specific. Sanders and others (1987) investigated the effects of seasonal additions ofammonium, nitrate, and phosphorus to largeculturesofnaturalphytoplanktonassemblagesfromthe PautuxentRiverEstuary. Theirresultsindicatedthatnitrogen enrichment during the summer and fall quickly changed the species composition ofthephytoplankton community from a predominance of flagellates to one dominated by small centric diatoms. Such changes in dominance were observed toresultfromincreasedgrowthratesofspecifictaxa. 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 greateruptake rates and lowerhalf-saturationconstants formacronutrients. Findings ofthose 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 salinitieseitherdidnot stimulatephytoplanktoncarbon

uptake orchlorophyll a (high-flow conditions), orresulted 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 ofthe 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 ofthe size fractions were highly variable with regard to season, location, andnitrogen form. Additions of orthophosphorus over a series of concentrations didnot 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:Pratios (<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 ofluxuryphosphorus consumption by phytoplankton. Procedures for determining ambient-orthophosphorus concentrations
measure "reactive forms" of the nutrient, large percentages ofwhich can exist as "lightly bound" forms. In much ofthe 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 forphytoplankton uptake. Orthophosphorus uptake, unlike that of nitrogen, generallyhasbeenfoundtobe stimulatedbylightanddependent 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 ofcarbon 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-tfconcentrations were stimulatedby additionsof 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 couldbe directly attributedto the effects ofincreased watercolor,reducedlightavailability, and high ambientnitrogenconcentrations. 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 canbe dividedconceptually into alow 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 affectinglightpenetration of the water column, nitrogen availability normally limits phytoplankton production within the Charlotte Harbor estuarine system. 5. Although the overall results ofthis 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 mustbe taken, however, in directlytransferringthe 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 asCharlotte Harborisfurtherconfoundedbythe 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 andregeneration. 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 29The view that potentially limiting nutrients are available for phytoplankton uptake athomogeneous lowconcentrations is, furthermore, restrictive. Phytoplankton assemblages within estuarine systems encounter a wide variety of ambient nutrient concentrations, including discrete nutrient pulses, deterministic ofa 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 othermicroorganisms wouldmake otherforms available
over time. Additional nitrogen loadings couldbe expectedto result in increased seasonal phytoplankton production in those areas ofthe harborwhere water color is low enough to allow for sufficient light availability. Incorporation ofthe results ofnutrient-limiting studies, such as presentedhere, as well as seasonal patterns ofphytoplankton production into generalized hydrodynamic models would aid the development ofpredictive management tools. The present study indicates that management strategies developed to maintain existing water quality need to emphasize point and nonpoint loadings ofnitrogen into the Charlotte Harbor system, seasonal andlocationalfactors, and rates ofnitrogen recycling.

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