FINAL REPORT 

"High Frequency Monitoring of Wastewater Nutrient Discharges 

and Their Ecological Effects in the Florida Keys National Marine 

Sanctuary" 

Submitted To: 

Water Quality Protection Program 

U.S. Environmental Protection Agency 

Marathon, FL 

Submitted By: 

Brian E. Lapointe and William R Matzie 

Division of Marine Science 

Harbor Branch Oceanographic Institution, Inc. 

3754 Pine Street 

Big Pine Key, FL 3 3 043 

Date: August 28, 1997 
Acknowledgements 

We thank our volunteer staff, including Colleen Murphey, Margaret Vogelsang, Nicole 

Logan, Andy Cannon, and Damon Marunyak for assistance with field sampling, processing 

of samples, data analysis, and graphics. Dr. Clinton Dawes (University of South Florida) 

kindly assisted with identification of macroalgae. Dr. Larry Brand and Arrieta Venizelos 

(University of Miami) are thanked for the analysis of chlorophyll a . Myron Gunsalus, 

Rebecca Elliot, Chandler Griffin, and other staff of the HBOI Environmental Laboratory 

provided low level nutrient analysis. Dr. B. B. MacIntyre (Isotope Analytical Services) 

performed the 615~ analyses and Carl Zimmerrnan (Chesapeake Biological Laboratory, 

University of Maryland) analyzed the tissue C:N:P samples. Finally, we are grateful to G. P. 

Schmahl (Looe Key National Marine Sanctuary) and Bill Kruczynski (USEPA) for their 

assistance in various aspects of the study. Sampling in the Florida Keys National Marine 

Sanctuary was conducted under Permit FKNMS (LR) - 08-95. 
Abstract 

The Florida Keys National Marine Sanctuary (FKNMS) has identified nutrient 

enrichment as a critical management issue. This study assessed how physical forcing 

mechanisms (rainfall, wind, tides) linked land-based wastewater discharges with the initiation 

of algal blooms in shallow (< 10 m) inner-shelf waters between Big Pine Key and hoe Key 

National Marine Sanctuary (LKNMS) from January to October 1996. Three stations were 

selected along the offshore gradient that included: 1) an inshore station (Avenue J, "AJ") 

downgradient of - 2,000 septic tankslcesspits in Spanish Harbor Channel, 2) a nearshore 

station ("PR") near a patch Reef - 0.5 km south of Munson Island, and 3) an offshore station 

("LK") in the back Reef at LKNMS. Monthly sampling included water column 

concentrations of dissolved inorganic nitrogen (DIN = NH4++ N03- + N02-; SRP = soluble 

reactive phosphorus); phytoplankton biomass (chlorophyll a, chl a); biomass, species 

composition, alkaline phosphatase activity, tissue carbon:nitrogen:phosphorus (C:N:P) 

contents, and natural abundance of 615~ in macroalgae; and C:N:P content, blade epiphyte 

loads, and natural abundance of 615~ of the seagrass Thalassia testudinurn. In addition, 

DIN, SRP, and chl a were measured at higher frequencies (daily) prior to, during, and 

following selected physical events to resolve on temporal relationships between the events, 

nutrient enrichment of the water column, and initiation of algal blooms. 

Elevated concentrations of DIN, SRP, and chl a following episodic events (i.e. high 

winds, low tides, and rain) demonstrated the importance of physical forcing to wastewater 

nutrient discharges and eutrophication. During and following a wind event (- 20 knots, NE) 

in mid-February, elevated DIN, SRP, and chl a concentrations were observed at PR and LK 

but not at the more protected inshore station AJ. The highest DIN (mostly NH4+) and SRP 

concentrations of the entire study were measured at AJ during low tide on March 19. With 

the onset of the wet season in late May and early June, DIN concentrations (mostly NH4+) 

reached maximum values at PR and LK, which was followed by maximal chl a 

concentrations at all three stations during the mid-summer period. Chl a concentrations were 

high at all stations throughout the study and averaged 1.86 ug/l at AJ (n = 87), 0.55 ugh at PR 

(n = 86), and 0.60 ugll at LK (n = 83). 

Fleshy macroalgal biomass averaged < 100 g dry wt . m-2 at all three stations in winter 

and early spring but increased to levels of 100 - 300 g dry wt . m-2 at AJ and PR following 

the onset of the rainy season in May. The red macroalga hurencia poiteaui was abundant at 

all three stations and 6 15~ values of this plant were highest at AJ (+ 5.0 oloo), intermediate 

.at PR (+ 3.5 oloo), and lowest at LK ( + 3.0 oloo) -- indicating increasing wastewater N 

xontributions to this alga with increasing proximity to shore. Large-scale blooms of ' Cladophorafuliginosa were first observed at LK in February and this alga became the 

dominant benthic cover by late spring. Transient, short-term increases in 615~ of C. 

fuliginosa from -1 to 5 oloo were associated with increased DIN concen~ations and tissue N 
following the first major rain event in late May. Increased APA of C. fuliginosa at LK was 

also associated with increased DIN in late May and resulted in significant increases in tissue 

P of this alga. The highest algal epiphyte loads on Thlassia testudinum occurred at LK rather 

than the inshore stations, a result of high DIN concentrations combined with high submarine 

irradiance at this station; these high epiphyte loads culminated in a bloom of the phaeophyte 

Cladosiphon occidentalis during May when epiphyte:blade ratios approached 3: 1, among the 

highest values reported in the scientific literature. 

In summary, these results corroborate previous findings that episodic stormwater 

discharges of land-based wastewater nutrients initiate harmful algal blooms in nearshore 

waters of the FKNMS, including the offshore bank reef at LKNMS. Our results also 

underscore the need for high frequency water quality sampling to obtain statistically robust 

annual estimates of contaminant concentrations needed for long-term "status and trends" 

monitoring and also to resolve on the ecologically important short-term effects of wastewater 

nutrient discharges. 
Introduction 

The Water Quality Protection Program (WQPP) of the Florida Keys National Marine 

Sanctuary (FKNMS) has identified water quality as a priority management issue. A major 

concern is the effect of increased nutrient inputs from land-based wastewater discharges on 

the oligotrophic coral reef and seagrass communities. Eutrophication caused by land-based 

human activities is a major mechanism altering coastal ecosystems worldwide (GESAMP 

1990, NAS 1994) and can cause dramatic ecosystem change in tropical and subtropical 

ecosystems. Bell (1992) critically reviewed case studies of eutrophication in coral reef 

regions and noted that enrichment above concentrations of - 1.0 pI14 dissolved inorganic 

nitrogen @IN) and 0.1 pA4 soluble reactive phosphorus (SRP) caused the demise of coral 

reefs in Barbados, Kaneohe Bay, Hawaii, and the Great Barrier Reef Lagoon. Nutrient 

enrichment associated with eutrophication typically causes phase-shifts from slowly growing 

corals to faster growing macroalgae and phytoplankton, as first documented in Kaneohe Bay 

where sewage-driven eutrophication caused blooms of phytoplankton (Caperon et al. 197 1) 

and the green "bubble alga" Dictyosphaeria cavemosa (Banner 1974). Macroalgal blooms 

have correlated tightly with nutrient enrichment on reefs in Jamaica (Lapointe 1997, Lapointe 

et al. 1997), southeast Florida (Lapointe 1997), Belize (Lapointe et al. 1993), and the inner 

Great Barrier Reef lagoon (Bell and Elmetri 1996). Water column nutrient enrichment of 

seagrass meadows in the FKNMS also increases the biomass of fast-gowing blade epiphytes, 

macroalgae, and phytoplankton, resulting in light limitation, seagrass die-off, and habitat loss 

(Tomasko and Lapointe 1991; Lapointe et al. 1994). Based on empirical evidence from the 

many case studies around the world, eutrophication is now considered a leading cause of both 

seagrass aapointe et al. 1994, Duarte 1995) and coral reef (Bell 1992, Ginsberg 1993) 

decline worldwide. 

Because restoration of ecosystems altered by eutrophication is difficult, early diagnosis 

and management of the problem is desirable. Taxonomic or biotic shifts have been used as 

indicators of eutrophication (Schmitt and Osenberg 1995) but because the appearance of 

"indicator species" signals an advanced stage of the problem, considerable environmental 

damage is likely to have already occurred. More desirable is an "early warning" tracer of 

nutrient enrichment, such as that indicating enrichment from wastewater nutrient discharges. 

"McClelland et al. (1997) used 15~114~ ratios (= 815~) asa tracer and found that septic tank 

DIN loads contributed significantly to blooms of the rnacroalgae Cladophora vagabunda, 

Enteromorphu sp., and Gracilaria tikvahiae in the Waquoit Bay estuary, Cape Cod, USA. 
Lapointe (1997), also using 615~ techniques, provided evidence that rainfall during summer 

months increased groundwater discharges of wastewater N from septic tanks or injection 

wells that contributed to blooms of Codium isthmocladum on deep (30 m) reefs offshore 

northern Palm Beach County, Florida. 

There exists considerable potential for wastewater enrichment of coastal waters in the 

FKNMS. There are currently some 30,000 on-site sewage disposal systems (OSDS, 

conventional septic tanks and cesspits) in the Florida Keys. Because of high groundwater 

tables and a transmissive geological substrate, these wastewater discharges cause nutrient 

(Lapointe et al. 1990) and bacterial (Paul et al. 1995 a) contamination of shallow 

groundwaters. Continuous down-gradient flow of nutrient-enriched groundwaters has already 

altered water quality in nearshore waters, evidenced by increased DIN and SRP 

concentrations, phytoplankton biomass, macroalgal blooms, reduced dissolved oxygen, and 

seagrass epiphytization and die-off (Tomasko and Lapointe 1990; Lapointe and Clark 1992; 

Lapointe et al. 1994). Shinn et al. (1994) assessed the fate and pathways of Class V injection 

well effluent using core drilled monitoring wells and found evidence of fecal contamination 

of groundwaters in three offshore wells in the lower Florida Keys (Shinn et al. 1994). Paul et 

al. (1995 b), using viral tracers, found that wastewater transport from septic tanks through 

Key Largo limestone into surface waters occurred in as little as 11 h. Lapointe and Matzie 

(1996) showed that land-based stormwater-driven wastewater nutrient discharges in the 

summer wet season initiate nearshore phytoplankton blooms (along with anoxia and hypoxia) 

and that this phenomenon was observable at the offshore reefs at Looe Key National Marine 

Sanctuary (LKNMS). 

This study used measurement of 615~ to specifically assess the degree to which landbased wastewater DIN contributes to macroalgal blooms in nearshore and offshore waters of 

the FKNMS. Because tidal pumping and rainfall events are two important physical 

mechanisms that result in episodic "pulses" of groundwater-borne nutrients into surface 

waters (Lapointe et al. 1990; Lapointe and Matzie 1996), we assessed how episodic physical 

forcing (rainfall, wind, tides) link nutrient disharges from land with the initiation of blooms 

of phytoplankton, macroalgae, and seagrass epiphytes in coastal waters of the FKNMS. Our 

.approach involved high-frequency sampling at three stations along an offshore eutrophication 

.gradient before and after episodic events for DIN and SRP concentrations; biomass, tissue 9 

C:N:P ratios and alkaline phosphatase activity (APA) of macroalgae; C:N:P ratios and blade 

epiphyte loads of Thalassia testudinum; and phytoplankton biomass (chlorophyll a). We 
also measured 615~ in both T. testudinum and macroalgal tissue as a tracer of wastewater 

DIN enrichment. 

Materials and Methods 

The study was conducted between January and October, 1996, at three shallow (1-2 m) 

seagrass communities along an offshore nutrient gradient from Big Pine Key to Looe Key 

National Marine Sanctuary (LKNMS, Fig. 1). The stations included: 1) an inshore station 

(Avenue J, "AJ", 240 40' 351 N, 810 20' 320 W) downgradient of - 1,000 septic 

tanks/cesspits - 0.25 krn off the east side of Big Pine Key in Spanish Harbor Channel (Fig. 

2), 2) a station inside of Hawk Channel near a patch reef ("PR", 240 36' 800 N, 810 23' 670 

W) - 0.5 krn from Little Palm Island (Fig. 2), and 3) stations in the back reef at LKNMS 

("LK I", 240 32' 564 N, 810 24' 245 W, a shallow seagrass meadow used for sampling all 

parameters except for macroalgal biomass which was sampled at the adjacent "LK 2", 240 33' 

050 W, 810 24' 339 W, Fig. 3) . At each station a 25 m transect was established by installing 

aluminum pins into the sediment; GPS posits for the stations were cross-checked with 

FKNMS staff. The GPS posits and subsurface buoys aided in the relocation and sampling. 

Monthly water samples were collected at each station to determine DIN, SRP, and chl 

a concentrations. Near-bottom (0.15 m above bottom) water samples were collected in 

triplicate at the three stations into clean 250 ml Nalgene bottles, filtered through 0.45 pm 

GFIF filters, and held on ice in the dark until return to the lab where they were frozen. 

Subsequently, they were analyzed for NH4+, N03- plus NO2-, and SRP on a Bran and 

Luebbe TRACS Analytical Console at the HBOI Environmental Laboratory in Ft. Pierce, FL 

(see Appendix I for Quality Assurance Summary). For chl a, three 100 ml replicate water 

samples were filtered (after adding 1 mg of MgC03) through GFIF glass fiber filters and the 

filters were frozen until extraction within a few days (in the laboratory of Dr. Larry Brand, 

University of Miami). The filters were extracted for 30 minutes with 10 rnl of dimethyl 

sulfoxide and then with an added 15 ml of 90% acetone at 5 OC overnight and measured 

fluorometrically before and after acidification for the measurement of chlorophyll and 

phaeopigment concentrations (Burnison 1980; Parsons et al. 1984). Fluorescence 

.measurements were made using a Turner Designs 10-000R fluorometer equipped with a 

jnfrared-sensitive photomultiplier and calibrated using pure chlorophyll a. 3 

In addition to the monthly samples, additional samples were collected at higher 

frequency (daily) prior to, during, and following physical forcing events. For example, 
samples were collected between 14 and 19 February as daily average wind speed increased 

from 5 to 15 knots during a winter northeaster and on July 14 when winds blew from the 

northeast at 15 knots. Samples were collected between 15 and 20 March as a function of tidal 

stage ranging from a high tide on 15 March (+1.0 ft) to low tides on 18 March (-0.33 ft), 19 

March (- 0.43 ft), and 20 March (-0.47 ft). Samples were collected through four different rain 

events on 1 1 March (- 1 .O inch), 15 to 25 May (- 4.0 inches), 1 to 3 July (7.5 inches), and 23 

to 25 September (3.8 inches). Rainfall was continuously monitored (Fig. 4) with a rainfall 

gauge at the Big Pine Key field station and weather and tide data were obtained from the Key 

West National Weather Service Station as recorded in the National Climatic Database. 

Monthly sampling of macroalgae included species composition, biomass, tissue C:N:P 

ratios and alkaline phosphatase activity (APA) of predominant species. Random samples of 

macroalgae (n=10) were collected along the transects with a 0.1 m2 quadrat to estimate 

biomass. The samples were returned to the lab and sorted, cleaned of sediment, epiphytes, 

epizoa, then weighed. When possible, we sampled species common to the three stations (e.g. 

Laurencia, Dictyota) for tissue analysis to allow intraspecific comparisons among the three 

stations. Subsamples of two macroalgae from each station, as well as new clean blade tissue 

of Thalassia testudinum, were dried in a laboratory oven (60 OC) to constant weight and 

analyzed for C:N:P ratios at the University of Maryland's Analytical Services Laboratory in 

Solomons, MD. Blade epiphyte levels were also measured on T. testudinum at the three 

stations by the methods described by Lapointe et al. (1994). APA of macroalgae was 

measured (n = 4) by the spectrophotometric methods detailed in Lapointe et al. (1994). 

We analyzed dried tissue of abundant macroalgae and T. testudinum sampled monthly 

fiom each station for natural abundances of stable nitrogen isotope ratios (615 N =15~/14~). 

Analyses for total N and 15N atom % were performed by Isotope Analytical Services, Inc. 

(Los Alarnos, New Mexico). The samples were processed through a Carlo-Erba NIA 1500 

elemental analyzer using Dumas combustion. The purified nitrogen gas was then measured 

by a VG Isomass mass spectrometer. 

The data were analyzed using one-way and two-way ANOVA. We assessed overall 

variability in water column DIN, SRP, chl a, macroalgae (biomass, C:N:P ratios, alkaline 

phosphatase activity, 615 N), and seagrass (C:N:P ratio, blade epiphytes, 615 N) as a function 

' of location (AJ, PR, LK) and time (month). Additional comparisons were made using 

pairwise t-tests and linear regression. 
Results 

Dissolved Inorganic Nutrients and Chl a in the Water Column 

There was a significant interaction (F = 3.124, P = 0.00008) between location and time 

on the mean DIN concentrations, which decreased with increasing distance from shore. Over 

the entire study, DIN averaged 2.10 + 1.43 pM (n = 102) at AJ, 1.18 + 0.87 pM (n = 102) at 

PR and 0.87 f 0.68 pM (n = 99) at LK. Maximum DIN concentrations were 7.75 pM at AJ , 

5.77 ~LM at PR, and 5.59 pM at LK. NH4+ was the predominant DIN species at AJ, 

accounting for - 70 % of the total DIN pool. NO3- was relatively more important at the more 

offshore PR and LK stations, although the maximum concentrations of NH4+ were - 3-fold 

higher than the maximum concentrations of N03- at these offshore stations. 

Considerable variability in DIN concentrations resulted from physical forcing. 

Elevated DIN concentrations at the inshore AJ station were associated with high winds, rain, 

and low tides. For example, DIN (primarily NH@) increased from - 1.0 pM to 4.0 pM as 

strong northeasterly winds developed between 15 and 19 February. The highest DIN 

concentrations measured during the entire study, 7.75 pM, occurred during the low tide 

sampling on March 19 (Fig. 5). Significant DIN pulses also followed rain events on 11 

March, 15 to 24 May, 1 July, and 23 September. DIN also increased at both PR and LK with 

high northeasterly winds between 15 and 19 February and on July 14 when the maximum 

DIN concentrations occurred at these stations (Figs. 2 and 3). DIN spikes occurred at PR and 

LK following rain events on 24 May, 1 July, and 23 September. In contrast to AJ, the higher 

DIN concentrations at the more offshore PR and LK (Figs. 6 and 7) occurred following the 

onset of the rainy season in mid-May. 

There was a significant interaction (F = 1.82, P = 0.029) between location and 

time on the mean SRP concentrations, which decreased with increasing distance from shore. 

Concentrations of this primary limiting nutrient were relatively lower and displayed less 

variability compared to DIN. Over the entire study, SRP averaged 0.06 f 0.04 pM (n = 102) 

at AJ, 0.04 f 0.03 pM (n = 102) at PR, and 0.03 f 0.03 pM (n = 99) at LK. The highest SRP 

concentrations at all three stations were associated with the high northeast wind event 

*between 15 and 19 February (Figs. 8,9, 10). Annual average DIN:SRP ratios varied from 

50.2 at AJ, 63.4 at PR, to 34.8 at LK. 
There was a significant interaction (F = 3.16, P = 0.00007) between location and time 

on the mean chl a concentrations, which decreased with increasing distance from shore. Chl a 

was significantly (F = 15.7, P < 0.00001) higher during the summer wet season compared to 

the winter and spring dry season. For example, the average chl a concentration at AJ was 

over Pfold higher in the wet season (2.62 ugh) compared to the dry season (0.62 ugh) while 

at PR and LK, chl a concentrations increased by - 50 - 70 % from the dry to the wet seasons. 

Over the entire study, chl a averaged 1.86 f 2.11 ugh (n = 87) at AJ, 0.55 f 0.77 ugh (n = 86) 

at PR, and 0.59 + 0.46 ugll (n = 83) at LK. At AJ, chl a increased to high values (> 5.0 pg/l) 

on 24 May following several days of intense rainfall (> 7 " rain between 15 and 25 May, Fig. 

11). A similar pattern was observed at PR where the highest chl a concentrations occurred on 

July 1 (Fig. 12) following heavy rain (Fig. 4). The seasonal pattern in chl a at LK was similar 

to that at AJ, increasing in late May following the onset of the rainy season (Fig. 13). 

I Biomass and Species Composition of Benthic Macroalgae 

Biomass of frondose, fleshy macroalgae at AJ increased from seasonal low values of - 

25 g dry wt-m-2 during February to peak values of - 90 g dry wt-m-2 in late May following 

the onset of the rainy season (Fig. 14). During February and March, the rhodophytes 

Laurencia poiteaui and Heterosiphonia wurdmanni dominated the fleshy macroalgal biomass 

and blooms of the chlorophytes Cladophora crispula and Chaetomorpha gracilis appeared in 

late April and May. The rhizophytic chlorophyte Caulerpa sertulariodes became abundant in 

June following heavy rainfall and blooms of the chlorophyte Cladophora montagneaena and 

the blue green Lyngbya majescula developed in August and September, respectively. 

Biomass of the calcareous macroalgae (primarily Halimeda opuntia) increased from low 

values of - 150 g dry wt -m-2 in the winter to maximum values of - 1,000 g dry wt am-2 in 

August (Fig. 15). 

Biomass of fleshy macroalgae at PR was higher than at AJ, averaging 40 to 50 g dry wt 

am-2 between February and May and increasing to maximum values of - 300 g dry wt 

in September following the onset of rain in late May (Fig. 16). Between February and May, 

the rhodophytes Laurencia poiteaui and Heterosiphonia wurdmanni dominated the fleshy 

macroalgal biomass and blooms of Ceramium nitens appeared as an epiphyte on Gorgonians 

"in late April. Extensive blooms of the blue-green Lyngbya gracilis (with Lyngbya 

rivularianum as an epiphyte) developed in June on seagrasses, corals, soft corals, and 

macroalgae. In contrast to the fleshy macroalgae, biomass of calcareous macroalgae at PR 
showed a decreasing trend from February to October with maximum values < 140 g dry wt 

m-2 (Fig. 17). 

Biomass of fleshy macroalgae at LK averaged - 40 to 50 g dry wt -m-2 between 

February and April and began increasing in May, reaching maximum values of - 200 g dry 

wt am-2 in September (Fig. 18). Between February and May, the rhodophyte Laurencia 

poiteaui and the chlorophyte Cladophorafiliginosa were abundant and blooms of the 

chlorophyte Cladophora crispata developed as an epiphyte on sponges, seagrasses, and soft 

corals between late March and July. Blooms of the blue green epiphyte Lyngbya gracilis 

covered all benthic biota in June, which was accompanied by blooms of Lyngbya 

meneghinina and Lyngbya semiplena in August. Biomass of calcareous macroalgae showed a 

bi-modal pattern with maximum values in March and August (Fig. 19). 

C:N: P, 6 ~JN, and APA of Benthic Macroalgae 

The rhodophyte Laurencia poiteaui displayed significant variation in tissue C:N:P 

contents among the three stations over the study period. At AJ, PR, and LK, respectively, the 

% C of dry weight averaged 20.48 + 1.20,22.84 + 2.8, 16.49 f 2.32; % N averaged 1.32 f 

0.30, 1.49 f 0.22, and 0.97 + 0.36; and % P averaged 0.028 f0.011,0.024 f0.008, and 0.033 

rt 0.01 1. The mean % N of dry weight in L poiteaui was significantly higher at the nearshore 

AJ (t = 5.9, P< 0.001) and PR (t = 3.75, P = 0.007) compared to the more offshore LK. 

However, there was no significant (P > 0.05) difference in mean % P of L poiteaui among 

the three stations. 

There were also significant differences in the mean C:P and N:P ratio, but not the C:N 

ratio, of Luurencia poiteaui among the three stations. At AJ, PR, and LK, respectively, the 

C:N ratio averaged 19.1 + 4.9, 18.1 k- 2.9, and 21.1 f 4.4; the C:P ratio averaged 21 8 1 k- 920, 

2917 f 1573, and 1408 f 407; and the N:P ratio averaged 1 10 f 21,156 f 59, and 69 f 26. 

The C:P ratios of L. poiteaui were significantly higher at the nearshore AJ ( t = 2.79, P = 

0.031) and PR (t = 2.53, P = 0.039) compared to the more offshore LK. Similarly, the N:P 

ratios of L. poiteaui were significantly higher at the nearshore AJ ( t = 7.62, P = 0.0002) and 

-PR (t = 4.05, P = 0.0048) compared to the more offshore LK. However, there was no 

"significant (P > 0.05) difference in the C:N ratio of L. poiteaui among the three stations. , 

At the offshore LK station, there was a trend of increasing % N (Fig. 20) and % P (Fig. 

21) in Cladophorafuliginosa tissue from winter to summer. During the entire period of study 
( n = 9 ), % C of C. fuliginosa averaged 22.37 f 2.6, % N averaged 1.07 + 0.15, and % P 

averaged 0.034 f 0.012 % dry weight. The tissue % P increased from 0.037 % to 0.062 % 

between April and May with the onset of rain in late May (Fig. 18). Coincidental with the 

increase in % P of C. fuliginosa tissue at LK was a significant (F = 1 1.54, P = 0.0004) 

seasonal increase in APA from values < 20 pM ~043-s~ dry wtah-1 between February and 

April to values > 50 pM ~043-e~ dry wt-h-1 in May following the onset of rain (Fig. 22). 

The APA of Laurencia poiteaui was significantly affected by location (F = 18.8 1, P = 

0.00003) and season (F = 15.42, P = 0.0009). The mean APA of L poiteaui was highest at 

AJ (51.6 pM SRP- g dry wt.-1-h-1) , intermediate at PR (36.8 pM SRP- g dry wt.-1-h-1) and 

lowest at LK (22.0 pM SRP g dry wt.-1-h-1). APA of L. poiteaui increased from low 

values in winter to high values in summer at AJ (Fig. 23), PR (Fig. 24), and LK (Fig. 25), 

respectively. 

The 615~ values of Laurencia poiteaui varied among the three stations and over time 

during the study (Figs. 26,27, and 28). The mean 615~ of Laurencia poiteaui was highest 

at AJ (4.69 +- 1.14 oloo), with lower values at PR (3.03 +- 0.46 0100) and LK (3.00 +- 1.04 

0100). The mean 615~ value of L. poiteaui at AJ was significantly greater (t = 3.51, P = 

0.009) than those from PR and LK, and values from the latter two stations were not 

statistically different from each other (t = 0.057, P = 0.955). 

The 815~ values of the chlorophyte Cladophora fuliginosa at LK increased following 

rainfall in May and the wind event in July (Fig. 29). Between April 24 and May 23, the 615~ 

values increased from < 3.0 0100 to > 5.00 0100 following the onset of heavy rain and 

increases in DIN at LK; the 615~ values again increased on July 14 from values < 1 .OO 0100 

to > 5.00 0100 following high northeast winds and DIN enrichment. 

Epiphytes, C:N:P, and 615~ of Thalassia testudinum 

The epiphyte loads on Thulassia testudinum were generally high at all three stations, 

especially LK. The epiphyte:seagrass blade ratio averaged 0.92 + 0.43 at AJ, 0.94 + 0.28 at 

<PR, and 1.85 + 0.81 at LK. The highest epiphyte loads at AJ were observed in April (Fig. 30) 

,while those at PR and LK occurred in June (Figs. 31 and 32). Over the entire study, the 9 

epiphyte loads at LK were significantly greater than those at PR (t = -4.309, P = 0.001) and 

AJ (t = -3.937, P = 0.003). The epiphytic algal community at LK between April and June 

included the phaeoph ytes Cladosip hon occidentalis and Lophosiphonia saccorhiza, the 
rhodophytes Chondria sp., Ceramium fastigatum, and Grzfithsia sp., and the blue green alga 

Lyngbya gracilis. Following a dense bloom of the chlorophyte Cladophora montagneaena in 

September and October at AJ, we observed an extensive die-off of manatee grass, 

Syringodium jiliforme. 

The C:N:P contents in blade tissue of Thulassia testudinum varied differently for N 

versus P. Over the entire study, the % N at AJ, PR, and LK averaged 2.40 f 0.20, 1.82 f 

0.17, and 1.70 f 0.1 1; these three average values were significantly (t > 2.87, P < 0.018) 

different from each other. The % P at AJ, PR, and LK averaged 0.13 f 0.02,0.10 f 0.02, and 

0.16 f 0.02; these three values were also significantly (t > -3.29, P < 0.009) different from 

each other. At AJ, PR, and LK, respectively, the C:N ratio averaged 16.29 f 1.05,20.92 f 

1.12, and 22.54 f 1.19; the C:P ratio averaged 658 f 83,827 f 134, and 549 f 90; and the 

N:P ratio averaged 40.6 f 5.7,39.5 f 5.7, and 24.3 f 3.3. 

The average 615~ value of Thulassia testudinum was highest at the offshore LK 

station, in contrast to the blade epiphytes which had the highest average value at the inshore 

AJ station. At AJ, PR, and LK, the 615~ value of T. testudinum averaged 2.80,2.77, and 

3.36, or generally increasing with distance from shore. At the inshore AJ station, no clear 

temporal pattern in the 615~ values of T. testudinum was evident (Fig. 33) whereas at the 

PR and LK stations the 615~ values increased from winter to summer (Figs. 34 and 35). 

The 615~ values of the blade epiphytes of T. testudinum were higher than the host plant 

(Figs. 36,37, and 38) and increased during February and March when high winds and low 

tides resulted in elevated water column DIN. 

Discussion 

Episodic Water Column Enrichment 

This study demonstrated significant linkages among episodic physical events, landbased wastewater nutrient discharges, and enrichment of dissolved nutrient concentrations in 

coastal waters of the FKNMS as far offshore as LKNMS. The observation of maximum DIN 

concentrations at AJ during the lowest tide of the study on March 19 illustrates the 

importance of tidal pumping via ebbing tides to enhanced rates of submarine groundwater 3 discharge (SGD) of wastewater nutrients. Lapointe et al. (1990), using a heat-pulsing 

groundwater flowmeter, found that lateral rates of shallow groundwater flow increased by - 

three-fold during ebbing tides as compared to flooding tides. This effect would be maximum 
during extreme low tides, such as that on March 19 when the highest DIN concentration (7.75 

pM) was observed. In addition to increased rates of SGD, the extreme low tides also result in 

minimal dilution of the increased wastewater nutrient load with lower nutrient offshore water, 

further increasing nutrient concentrations in the coastal receiving waters. 

High winds were another important physical mechanism causing nutrient enrichment. 

Elevated DIN and SRP concentrations were measured at all stations during the high 

northeasterly winds in February and July. These increased nutrient concentrations potentially 

resulted from several contributing factors. First, wind-driven advection associated with these 

events could increase cross-island hydrostatic potentials by piling up water on the Florida 

Bay side of the Florida Keys, thereby increasing rates of SGD and enrichment of coastal 

waters towards Hawk Channel and the offshore reefs (e.g. see Shinn et al. 1994). High winds 

cause sediment resuspension and enhanced advection and diffusion of pore water nutrients 

derived from SGD into the water column. High northeasterly winds also cause offshore 

transport of water from inshore to offshore in the lower Florida Keys; we observed this effect 

on March 12 and during the following days when high northeasterly winds transported highly 

turbid waters several miles offshore (south) of LK. 

Rainfall was also an important mechanism causing episodic nutrient enrichment. 

Rainfall results in increased infiltration of septic tank and cesspit wastewater plumes into 

shallow groundwaters and also increases lateral groundwater flow rates, thereby increasing 

wastewater nutrient loads and DIN and SRP concentrations in adjacent surface waters of the 

Florida Keys (Lapointe et al. 1990). Although rainfall in the Florida Keys can have 

significant DIN concentrations (- 15.0 pM, Lapointe and Matzie 1996), an extremely heavy 

rainfall event of 6" over several hours would enrich a shallow 2 m water column by only - 1 

pM. Following the first rain events between May 15 and 23 when a total of - 4 inches of 

rain fell, DIN concentrations increased from < 1 pM to > 3 pM at AJ, from < 0.5 pM to > 2.5 

pM at PR, and from < 0.5 pM to > 1.5 pM at LK. Obviously, the relatively low amount of 

rainfall over this period combined with the magnitude of this increase in DIN over several 

days implicates DIN sources other than rainfall alone. The increase in DIN, together with 

increased 615~ values of Laurecia poiteai at AJ and Cladophorafuliginosa at LK following 

this "first flush", provide clear evidence that wastewater derived DIN was contributing 

significantly to the DIN pool throughout the entire study area. , 

These findings underscore the earlier conclusions of Lapointe and Matzie (1996) that 

high frequency sampling is neccessary to resolve on the episodic "pulses" of wastewater 
nutrient discharges and their ecological effects in the FKNMS. This is important not only to 

resolve on linkages between land-based nutrient enrichment and the initiation of algal 

blooms, but also for longer-term monitoring programs designed to assess "status and trends" 

of the FKNMS. The current water quality monitoring program in the FKNMS is spatially 

intensive but conducted at only quarterly intervals (4 days out of the 365 days in a year) and 

therefore not representative of the highly variable temporal domain as demonstrated in the 

present study. The ongoing monitoring program is further confounded by the fact that 

sampling occurs over varying tidal cycles and other physical forcing events, all of which are 

important regulators of nutrient concentrations, algal blooms, and the status of water quality 

in the FKNMS. The need for medium (monthly) and high frequency (daily) sampling has 

been recognized the Great Barrier Reef Marine Park Authority (GBRMPA) and is currently 

in use in their long-term monitoring programs for eutrophication on the Great Barrier Reef 

(Brodie and Furnas 1992). 

Land-based Wastewater Nutrients and Algal Blooms 

Several lines of evidence from this study support the hypothesis that land-based 

wastewater nutrient discharges from the Florida Keys enhance blooms of phytoplankton, 

macroalgae, and seagrass epiphytes as far offshore as LKNMS. First, the concentrations of 

DIN, SRP, and chl a were all maximum at the inshore AJ station and the gradients 

consistently decreased with increasing distance from shore, pointing to a land based-source of 

nutrients and phytoplankton biomass in the study area. Second, the spatial pattern of 

decreasing 615~ values of Laurencia poiteaui and epiphytes on Thalassia testudinum with 

increasing distance from AJ to LK, combined with episodic increases in DIN and 615~ 

values of Cladophora fuliginosa at LK following rain and wind events, provides unequivicol 

evidence that wastewater DIN contributes to macroalgal blooms throughout the study area. 

Third, the increase in biomass of phytoplankton, macroalgae and seagrass epiphytes at all 

stations following the increase in DIN after the wet season began in May demonstrates the 

widespread ecological effects of even low levels of nutrient enrichment from wastewater. 

These results corroborate earlier studies that linked land-based wastewater nutrient discharges 

to algal blooms in nearshore waters of the Florida Keys (Lapointe and Clark 1992) and 

*offshore to LKNMS (Lapointe and Matzie 1996). The wastewater enrichment throughout our 

study area is consistent with current meter studies that showed long-term net cumulative 3 

transport from inshore waters between Big Pine Key and Marathon in a southwesterly 

direction towards the offshore bank reefs (Lapointe et al. 1992, Smith 1994, Pitts, 1994) and 
also previous observations of an "ammonium wake" in downstream waters of LKNMS during 

the wet season (Lapointe et al. 1992). 

Our results also refute recent speculations that offshore upwelling of deep, cold water 

is the primary source of nutrients fueling the algal blooms in nearshore waters of the Florida 

Keys (e.g. see Szmant and Forrester 1996). There are no supporting data for that hypothesis 

and considerable conflicting evidence. For example, in addition to the nutrient and chl a 

gradients decreasing from nearshore to offshore, the predominant DIN species at AJ during 

this study was NH4+, the DIN form in septic tank-contaminated groundwaters (Lapointe et 

al. 1990) and throughout surface waters of the Florida Keys (Lapointe and Clark 1992). The 

maximum concentrations of NH4+ at PR and LK were - 3-fold higher than the maximum 

concentrations of NO3- during this study and showed distinct pulses associated with physical 

forcing (i.e. high winds, low tides, and rain events) that we observed to enhance offshore 

transport of the NH4+ and chl-a rich inshore waters. Because NOj- is the predominant DIN 

species associated with upwelling, the episodically pulsed NH4+ enrichment that we 

observed throughout the study area was obviously not of upwelling origin. This conclusion is 

further evidenced by the lack of persistent cold water intrusions in the study area and the 

well-known long-term transport pattern in an offshore -- not onshore -- direction in the 

middle and lower Florida Keys (Lapointe et al. 1992, Pitts, 1994, Smith, 1994). Thus, 

although upwelling events have been documented on short temporal scales at LKNMS 

(Lapointe and Smith 1986), these events are either anomalous (i.e., upwelling at LKNMS in 

July 1986 was associated with 2-3 days of 20-25 knot westerly winds) or of relatively shortterm duration (i.e., time scale of hours to days) and do not reflect the dominant, long-term 

offshore nutrient transport process. 

The time course data on DIN, APA, and C:N:P ratios of Cladophora fuliginosa at LK 

illustrates the dynamics as to how episodic NH@-rich wastewater discharges initiate and 

sustain macroalgal blooms. The pulse of DIN (mostly NH4+) at LK following the "first 

flush" rain event in May coincided with the maximum tissue % N in C. fuliginosa. 

Controlled experimental studies in Bermuda with a related alga, Cladop hora prolifera, 

showed that DIN enrichment also resulted in increased tissue % N, which in turn caused 

increased APA due to increased N:P ratios that induced SRP-limitation (Lapointe and 

vO'Connell 1989). An increase in APA also occurred in C. fuliginosa following the DIN pulse * 

in May, which enhanced sequestering of SRP from dissolved organic phosphorus (DOP) 

pools. The end result was a parallel increase in tissue % P of C. fuliginosa even though no 

increased SRP concentrations were observed during this period at LK. Tliese findings 
demonstrate the importance of rapid assimilation of NH4+ during episodic enrichment 

events and subsequent reliance on APA to sequester SRP from the dissolved organic 

phosphorus (DOP) pool to sustain balanced growth. 

Our results also suggest that tissue C:N:P analysis alone may not be a reliable index of 

wastewater nutrient enrichment in macroalgae. The mean % N of the rhodophyte Laurencia 

poiteaui was higher at PR (1.49 % dry wt.) compared to AJ (1.32 % dry wt.) despite the fact 

that mean DIN concentrations were significantly higher at AJ, the station in closest proximity 

to land-based wastewater nitrogen loads. However, the wastewater impact at AJ was clearly 

indicated by the significantly higher 615~ values of L. poiteaui at AJ compared to this alga 

at PR and LK. The lack of an enrichment signal in tissue N of L. poiteaui is most likely due 

to the confounding effects of variable light on the growth rate and tissue N of macroalgae 

(Lapointe and Duke 1984). For example, the L. poiteaui at AJ may have experienced higher 

light levels compared to plants at the more exposed and often turbid PR; this effect would not 

only increase the productivity of the L. poiteaui at AJ but also reduce its tissue %N. Future 

wastewater monitoring studies using macroalgae should emphasize the use of 615~ values, 

rather than simple tissue C:N:P contents, due to the confounding effects of variable light 

(and other factors) on algal growth rates and tissue nutrient pools. 

1 In comparison, Thalassia testudinum showed considerable tissue N enrichment at AJ 

(2.40 % dry wt.) compared to both PR (1 32% dry wt.) and LK (1.71 % dry wt.). However, 

1 the 615~ values of T. testudinum, unlike the macroalgae and blade epiphytes, showed no 

I decreasing trend in the 615~ values offshore and actually had the highest values at LK. We 

interpret this as evidence that T. testudinum relies largely on nitrogen fixation for its 

nitrogenous supply (Capone and Taylor 1977) and as such may be more influenced by pore 

water P enrichment associated with wastewater discharges. In oligotrophic carbonate-rich 

waters of San Salvador, Bahamas, porewater P enrichment (but not N) increased growth of 

the seagrass Syringodiumfilifomze (Short et al. 1990). The die-off of both 7'. testudinum 

and Syringodiumfiliforme at AJ in September may have been related to light limitation and 

carbon imbalance from a recurrent bloom of Cladophora montagneaena that formed dense 

mats over the seagrasses. Alternatively, tissue N was high in T. testudinum at AJ and may 

.reflect nitrogen toxicity, particularly during this period of seasonally maximum temperatures 

,(Burkholder et al. 1991). 3 

Nutrient Enrichment and Biotic Phase Shifts 
The relative importance of different types of primary producers in coastal ecosystems 

(i.e. phytoplankton, macroalgae, seagrasses, corals) is a key characteristic of ecosystem 

structure and has important consequences for ecosystem function. Numerous studies have 

shown large-scale phase-shifts in primary producers result from changes in limiting 

resources, most notably nutrient availability. Coastal eutrophication in tropical and 

subtropical regions during the past decades has resulted in trends away from seagrasses and 

corals and towards epiphytes, macroalgae, and phytoplankton. The high biomass of 

phytoplankton (all stations were above the eutrophic threshold for coral reefs of 0.5 ug/l, Bell 

1992), fleshy macroalgae (100 - 300 g dry wt m-21, and seagrass epiphytes (epiphyte:blade 

ratios up to 3: 1 at Looe Key), and the proliferation of macroalgal "indicator species", i.e. 

Chdophora spp., Lyngbya spp) clearly show that the entire study area is now in an advanced 

stage of eutrophication. 

Comparison of the chl a data at LK in this study with previous annual averages for this 

station dating back to 1987 illustrate the disturbing trend of eutrophication at LK during the 

past decade. The lowest annual average chl a measured, c 0.1 ugll, was in the drought year 

of 1990 when the FKNMS was established; since then, annual chl a concentrations increased 

to 0.25 ug/l in 1992 and 0.68 ug/l in the present study (1996), a value well above threshold 

level considered eutrophic for coral reefs (see Bell 1992; Fig. 39). Although we found 

significant wastewater DIN impacts throughout our study area, including LK, the dramatic 

escalation of chl a at LK are also likely related to Florida Bay outflows that have been 

influenced by increasing phytoplankton blooms and turbidity in central and western Florida 

Bay since 199 1 (Lapointe et al. 1996). The chl a and turbidity in Florida Bay is advected by 

prevailing currents southward through tidal channels of the middle and lower Keys and 

offshore to the outer bank reefs (Lapointe et al. 1992, Smith 1994, Pitts 1994). These blooms 

have expanded with the increased freshwater runoff and associated nitrogen loads from the 

Everglades since 199 1 (Lapointe et al. 1996) that enhance DIN-limited phytoplankton 

populations in the central and western bay (Tornas 1996). This trends towards increased 

phytoplankton blooms and turbidity in Florida Bay has resulted from policies aimed at 

increasing freshwater flows to lower the salinity in Florida Bay (USEPA 1996). Thus, it is 

.unlikely that corrective actions for controlling wastewater nutrient loads in the Florida Keys 

.will, by itself, be adequate to fully restore water quality over the large regional scale of the 3 

FKNMS. Larger-scale control of nutrient loads from the south Florida watershed will be 

needed if the trend of eutrophication in Florida Bay and the FKNMS is to be reversed. 
Literature Cited 

Banner, A. H. 1974. Kaneohe Bay , Hawaii: urban pollution and a coral reef ecosystem. 

Proc. 2nd Inter. Coral Reef Symp., Brisbane 2:685-702. 

Bell, P. R. F. 1992. ~uko~hication ad coral reefs: Some examples in the Great Barrier Reef 

lagoon. Water Research 26553-568. 

Bell, P. R. F. and I. Elmetri. 1995. Ecological indicators of large-scale eutrophication in the 

Great Barrier Reef lagoon. Ambio 24:208-215. 

Brodie, J. and M. Furnas. 1993. Long term monitoring programs for eutrophication and the 

design of a monitoring program for the Great Barrier Reef. Proc. 7th. Int. Coral Reef 

Symp. 1: 77-83. 

Burkholder, J. M., K. M. Mason, and H. B. Glasgow, Jr. 1992. Water-column nitrate 

enrichment promotes decline of eelgrass Zostera marina: evidence from seasonal 

mesocosm experiments. Mar. Ecol. Prog. Ser. 8 1 : 163-178. 

Burnison, B. K. 1980. Modified dimethyl sulfoxide (DMSO) extraction for chlorophyll 

analysis of phytoplankton. Can. J. Fish. Aq. Sci. 37:729-733. 

Caperon, J., S. A. Cattell, and G. Krasnick. 1971. Phytoplankton kinetics in a subtropical 

estuary:eutrophication. Limnol. Oceanogr. 16: 599-607. 

Capone, D. G. and B. F. Taylor. 1977. Nitrogen fixation (acetylene reduction) in the 

phyllosphere of Thalassia testudinum. Mar. Biol. 40: 19-28. 

Duarte, C. M. 1995. Submerged aquatic vegetation in relation to different nutrient regimes. 

Ophelia 41 97- 1 12. 

GESAMP. 1990. The State of the Marine Environment. Joint Group of Experts on the 

Scientific Aspects of Marine Pollution. Rep. and Stud. 39. United Nations 

Environmental Programme. 

Ginsberg, R. N. Compiler. 1994. Proceedings of the colloquium on global hazards to coral 

reefs:health, hazards, and history. Rosenstiel School of Atmospheric Science, 

University of Miami. 

Lapointe, B. E. 1997. Nutrient thresholds for bottom-up control of macroalgal blooms on 

* coral reefs in Jamaica and southeast Florida. Limnol. Oceanogr. in press. 
Lapointe, B. E., and C. S. Duke. 1984. Biochemical strategies for growth of Gracilaria 

tikvahiae (Rhodophyta) in relation to light intensity and nitrogen availability. J. 

Phycol. 20:488-95. 

Lapointe, B.E: and J. D. O'Connell. 1989. Nutrient-enhanced growth of Cladophora 

prolifera in Harrington Sound, Bermuda: eutrophication of a confined, phosphoruslimited marine ecosystem. Est. and Coast. Shelf Sci. 28:347-360. . 

Lapointe, B. E., J. D. O'Connell, and G. S. Garrett. 1990. Nutrient couplings between on-site 

sewage disposal systems, groundwaters, and nearshore surface waters of the Florida 

Keys. Biogeochemistry 10: 289-308. 

Lapointe, B. E. and M. Clark. 1992. Nutrient inputs from the watershed and coastal 

eutrophication in the Florida Keys. Estuaries 15465-476. 

Lapointe, B. E., N. P. Smith, P. A. Pitts, and M. Clark. 1992. Baseline characterization of 

chemical and hydrographic processes in the water column of Looe Key National 

Marine Sanctuary.. Final Report to U. S. Dept. of Commerce, NOAA Office of Ocean 

and Coastal Resource Management, contract # NA86AA-H-CZ07 1, Washington, D. C. 

103 pp. 

Lapointe, B. E., M. M. Littler, and D. S. Littler. 1993. Modification of benthic community 

structure by natural eutrophication: the Belize Barrier Reef. Proc. 7th Inter. Coral Reef 

Symp., Guam 1 :323-334. 

Lapointe, B. E., D. A. Tomasko, and W. R. Matzie. 1994. Eutrophication and trophic state 

classification of seagrass communities in the Florida Keys. Bull. Mar. Sci. 54: 696- 

717. 

Lapointe, B. E. and W. R. Matzie. 1996. Effects of stormwater nutrient discharges on 

eutrophication processes in nearshore waters of the Florida Keys. Estuaries 19:422- 

435. 

Lapointe, B. E., W. R. Matzie, and D. Tomasko. 1996. Modeling relationships between 

external nutrient loading, water quality, and biotic phase-shifts: examples from Tampa 

Bay, Sarasota Bay, and Florida Bay. Program and Abstracts. Florida Bay Science 

Conference, December 10 - 12,1996. Florida Sea Grant, University of Florida, 1 1 3 pp. 

Lapointe, B. E., M. M. Littler, and D. S. Littler. 1997. Macroalgal overgrowth of fringing 

* coral reefs at Discovery Bay, Jamaica: bottom-up versus top-down control. Proc. 8th 

Inter. Coral Reef Symp., Panama, in press. 
Lindau, C. W., R. D. Delaune, W. H. Patrick, Jr. and E. N. Lambremont. 1989. Assessment 

of stable isotopes in fingerprinting surface water inorganic nitrogen sources. Water, 

Air, and Soil Pollution 48:489-496. 

McClelland, J. W., I. Valiela, and R. H. Michener. 1996. Nitrogen stable isotope signatures in 

estuarine food webs: a record of increasing urbanization in coastal watersheds. Lirnnol. 

Oceanogr., in press. 

National Academy of Sciences (NAS). 1994. Priorities for Coastal Science, National 

Academy Press. 

Parsons, T. R., Y. Maita, and C. M. Lalli. 1984. A manual of chemical and biological 

methods for seawater analysis. Pergarnon Press. 173 pp. 

Paul, J. H., J. B. Rose, S. Jiang, C. Kellogg, and E. A. Shinn. 1995 a. Occurrence of fecal 

indicator bacteria in surface waters and the subsurface aquifer in Key Largo, Florida. 

Applied and Environmental Microbiology 6 1 : 2235-2241. 

Paul, J. H., J. B. Rose, J. Brown, E. A. Shinn, S. Miller, and S. R. Farrah. 1995 b. Viral tracer 

studies indicate contamination of marine surface waters by sewage disposal practices 

in Key Largo, FL. Applied and Environmental Microbiology 6 1 : 2230-2234. 

Pitts, P. A. 1994. An investigation of near-bottom flow patterns along and across Hawk 

Channel, Florida Keys. Bull. Mar. Sci. 54:610-620. 

Schmitt, R. J. and C. W. Osenberg. 1995. Detecting ecological impacts; concepts and 

applications in coastal habitats. Academic Press, Inc., San Diego, CA, USA. 

Shinn, E. A., R. S. Reese, and C. D. Reich. 1994. Fate and pathways of injection-well 

effluent in the Florida Keys. U. S. Geological Survey, Open-File Report 94-276. 116 

PP. 

Short, F. T., W. C. Dennison, and D. G. Capone. 1990. Phosphorus-limited growth of the 

tropical seagrass Syringodiumfiliforme in carbonate sediments. Mar. Ecol. Prog. Ser. 

62: 169- 1 74. 

Smith, N. P. 1994. Long-term Gulf-to-Atlantic transport through tidal channels in the Florida 

Keys. Bull. Mar. Sci. 54:602-609. 

, 'Smith, S. V., W. J. Kimmerer, E. A. Laws, R. E. Brock, and T. W. Walsh. 1981. Kaneohe 

Bay sewage diversion experiment: perspectives on ecosystem response to nutritional 

perturbation. Pacific Science 35: 279-397. 
Szmant, A. M. and A. Forrester. 1996. Water column and sediment nitrogen and phosphorus 

distribution patterns in the Florida Keys, USA Coral Reefs 15:21-41. 

Tomas, C. R. 1996. The role of nutrients in initiating and supporting Florida Bay microalgal 

blooms and primary production. Abstract. Florida Bay Science Conference, December 

10 - 12, 1996. Florida Sea Grant, University of Florida, 113 pp. 

Tornasko, D. A. and B. E. Lapointe. 1991. Productivity and biomass of Thalassia testudinum 

as related to water column nutrient availability and epiphyte levels: field observations 

and experimental studies. Mar. Ecol. Prog. Ser. 75:9-17. 

USEPA, 1996. Water quality protection program for the Florida Keys National Marine 

Sanctuary. First Biennial Report to Congress. December, 1996, 32 pp. 

Windom, H. L. 1992. contamination of the marine environment from land-based sources. 

Marine Pollution Bulletin 35:32-36. 
Figure Legend 

Fig. 1. Map of the Florida Keys showing locations of three monitoring stations (AJ, PR, LK) 

used in this study. 

Fig. 2. Map of Big Pine Key showing locations of the inshore station AJ in Big Spanish 

Channel and the PR station just south of Munson Island. 

Fig. 3. Map of Looe Key National Marine Sanctuary showing locations of LK 1 on the reef 

flat (shallow seagrass meadow) just behind the reef crest and LK 2 in the adjacent back 

reef zone that is macroalgal dominated. 

Fig. 4. Rainfall during the period of study. 

Fig. 5. DIN concentrations at Avenue J. Values represent means (n=3). 

Fig. 6. DIN concentrations at the Patch Reef. Values represent means (n=3). 

Fig. 7. DIN concentrations at Looe Key. Values represent means (n=3). 

Fig. 8. SRP concentrations at Avenue J. Values represent means (n=3). 

Fig. 9. SRP concentrations at the Patch Reef. Values represent means f 1 S.D. (n=3). 

Fig. 10. SRP concentrations at Looe Key. Values represent means + 1 S.D. (n=3). 

Fig. 11. Chlorophyll a concentrations at Avenue J. Values represent means + 1 S.D. (n=3). 

Fig. 12. Chlorophyll a concentrations at the Patch Reef. Values represent means f 1 S.D. 

(n=3). 

Fig. 13. Chlorophyll a concentrations at Looe Key. Values represent means + 1 S.D. (n=3). 

9 

Fig. 14. Biomass of fleshy macroalgae at Avenue J. Values represent means k 1 S. D. (n = 

10). 
Fig. 15. Biomass of calcareous macroalgae at Avenue J. Values represent means + 1 S. D. (n 

= 10). 

Fig. 16. Biomass of fleshy macroalgae at the Patch Reef. Values represent means + 1 S. D. 

(n = 10). 

. Fig. 17. Biomass of calcareous macroalgae at the Patch Reef. Values represent means + 1 S. 

D. (n = 10). 

Fig. 18. Biomass of fleshy macroalgae at Looe Key. Values represent means + 1 S. D. (n = 

10). 

Fig. 19. Biomass of calcareous macroalgae at Looe Key. Values represent means + 1 S. D. (n 

= 10). 

Fig. 20. Tissue nitrogen in the chlorophyte Cladophorafuliginosa at Looe Key. 

Fig. 21. Tissue phosphorus in the chlorophyte Cladophora fuliginosa at Looe Key. 

Fig. 22. Alkaline phosphatase activity of the chlorophyte Cladophora fuliginosa at Looe Key. 

Values represent means + 1 S.D. (n. = 4). 

Fig. 23. Alkaline phosphatase activity of the rhodophyte Laurencia poiteaui at Avenue J. 

Values represent means + 1 S.D. (n = 4). 

Fig. 24. Alkaline phosphatase activity of the rhodophyte Laurencia poiteaui at the Patch 

Reef. Values represent means + 1 S.D. (n = 4). 

Fig. 25. Alkaline phosphatase activity of the rhodophyte Laurencia poiteaui at Looe Key. 

Values represent means + 1 S.D. (n = 4). 

Fig. 26. 615~ of Laurecia poiteaui at Avenue J. Values represent means f 1 S.D. (n=2). 

.Fig. 27. 615~ of Laurecia poiteaui at the Patch Reef. Values represent means f 1 S.D. 3 

(n=2). 

Fig. 28. 615~ of Laurecia poiteaui at Looe Key. Values represent means.+ 1 S.D. (n=2). 
Fig. 29. Correlation of dissolved inorganic nitrogen (DIN) concentrations and 615~ of 

Cladophora fuliginosa at Looe Key. 

Fig. 30. Epiphyte:seagrass blade ratio for Thalassia testudinum at Avenue J. 

Fig. 3 1. Epiphyte:seagrass blade ratio for Thalassia testudinum at the Patch Reef. 

Fig. 32. Epiphyte:seagrass blade ratio for Thalassia testudinum at Looe Key. 

Fig. 33. 615~ of Thalassia testudinum at Avenue J. Values represent means + 1 S.D. (n=2). 

Fig. 34. 615~ of Thalassia testudinurn at Match Reef. Values represent means + 1 S.D. 

(n=2). 

Fig. 35. 615~ of Thalassia testudinum at Looe Key. Values represent means + 1 S.D. (n=2). 

Fig. 36. 615~ of Thalassia testudinum epiphytes at Avenue J. Values represent means + 1 

S.D. (n=2). 

Fig. 37. 615~ of Thalassia testudinum epiphytes at the Patch Reef. Values represent means 

k 1 S.D. (n=2). 

Fig. 38. 615~ of Thalassia testudinum epiphytes at Looe Key. Values represent means k 1 

S.D. (n=2). 

Fig. 39. Annual mean values of chlorophyll-a at Looe Key (LK 1) between 1987 and 1996. 

Values represent means + 1 S. D. (16< n < 83). 

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Figure 32 



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HARBOR BRANCH 

ENVIRONMENTAL LABORATORY $@; 

I 

5600 U.S. 1 NORTH *~UWR~PH\' 

FORT PIERCE. FLORIDA 34946 fmnrlrrMI 

APPENDIX I 

HIGH FREQUENCY MONITORING OF WASTEWATER NUTRIENT 

DISCHARGES AND THEIR ECOLOGICAL EFFECTS IN THE FLORIDA KEYS 

MARINE SANCTUARY 

QUALITY ASSURANCE SUMMARY 

NUTRIENT ANALYSIS 

PRESENTED TO: 

BRIAN LAPOINTE, Ph.D. 

HARBOR BRANCH OCEANOGRAPHIC INSTITUTION, INC. 

ANALYSIS BY: 

HARBOR BRANCH ENVIRONMENTAL LABORATORY 

LABORATORY DIRECTOR: 

N. MYRON GUNSALUS, JR. 

A DIVlSlON OF HARBOR BRANCH OCEANOGRAPHIC INSTITUTION, INC. 

August 12,1997 
HARBOR BRANCH ENVIRONMENTAL LABORATORY q?@! 

5 

0c~4~~c~&?*\G ~USTITUTIOH 

QA Summary 

Page 1 of 5 

0 VER VIEW 

Harbor Branch Environmental Laboratory (HBEL) is committed to producing high 

quality data that is legally defensible, scientifically sound and fulfills the usability 

requirements for our clients' application in terms of analytical accuracy, precision, and 

completeness. HBEL has implemented the QNQC program to ensure that the 

generation of monitoring and analytical measurement data are of known adequate quality 

to meet the requirements of each project's statement of work. 

The Harbor Branch Environmental Laboratory pursues excellence in environmental 

testing and conducts applied research to understand and protect the natural environment. 

PROJECT GOALS 

Samples were collected during and following meteorological events that exceed normal 

weather conditions and held on ice in the dark until amval in the laboratory where they 

were fiozen. The samples were then thawed and analyzed for NH,, NO,, NO,, and SRP 

at Harbor Branch Environmental Laboratory by methods derived fiom widely accepted 

chemistries used in previously published studies in the determination of low level 

nutrients in surface waters. The results obtained from these analyses will be evaluated to 

determine the development of phytoplankton and macroalgal blooms as a result of 

nutrient enrichment following episodic events. 

For the duration of the project, a previously developed Quality Assurance Project Plan 

(QAPP) consisting of quality control goals was in place to ensure accurate reproducible 

data and to meet quality expectations for data usability. The data quality goals included 

target method detection limits, recommended holding times, and five quality assurance 

parameters: precision, accuracy, representativeness, comparability, and completeness. 

When quality objectives were not met for all samples, they were noted with corrective 

actions and narrated in the report. 

METHOD DETECTION LIMIT (MDL) 

Method Detection Limits were performed on "bluewater," saltwater matrix in accordance 

to CFR. 40 Part 136 Appendix B. These studies demonstrated achievable detection limits 

below the QAPP specified MDLs. The project detection limits were utilized'instead of 

the statistically derived MDLs. 
F 

HARBOR BRANCH ENVIRONMENTAL LABORATORY 

QA Summary 

Page 2 of 5 

HOLDING TIMES 

The QAPP recommended holding time of twenty-eight days was exceeded for several 

sample sets in this project. These exceedances and the performance of several analytical 

series for the various sample sets provided an opportunity for observation of the stability 

of these nutrients upon freezing and thawing as noted in the following discussion. 

Exceedances were noted in the final analytical report. 

DISCUSSION OF ANALYTE STABILITY 

In the course of work performed, the following informal observations on analyte stability 

were made: Nitrate, nitrite and soluble reactive phosphorous(SRP) are fairly stable 

throughout the freezing and thawing process when frozen for longer periods of time than 

the recommended holding time specified in the QAPP. Ammonium appears to be far less 

stable, with potential for loss or gain in the freezing and thawing process. Also, although 

ambient lab conditions can adversely affect any data collected, Ammonium and SRP 

were noticeably more sensitive to lab environmental conditions. 

Soluble Reactive Phos~hate (SRP) 

For SRP care must be taken to avoid phosphate contamination of the analytical aliquot 

through reagent impurities or ambient laboratory conditions. This was accomplished 

through the monitoring of the reagent supply, locating the nutrient auto analyzer in the 

most phosphate fiee section of the laboratory, and accomplishing the analytical run as 

quickly as possible once the analytical aliquot was poured for analyses. 

Ammonium 

For ammonium, control of the loss or gain of ammonium from the analytical aliquot and 

the sample was of concern and more difficult than SRP to control. Great care was taken 

to minimize loss by immediate analyses upon opening of the sample bottle after the 

sample had been thawed and gently mixed. Air space in the bottle upon collection could 

have an affect on final results. Also, atmospheric lab ammonium can be absorbed into 

the analytical aliquot and the sample itself. Minimizing exposure to air during handling 

and analyses reduced ammonium absorption. Without these kind of procedures, NH, can 

be lost or gained. 

From our observations, it appears the most reliable way to produce ammonium results 

which are representative of the sample upon collection is to avoid the complications of 

fieezing and ambient conditions. It is recommended that fresh, unfrozen samples 

collected with no air space be analyzed as soon as possible on the day of collection. 
HARBOR BRANCH ENVIRONMENTAL LABORATORY 

i+&! 

I 

%NOCRAP*" 

~~STITUTIO* 

QA Summary 

Page 3 of 5 

ACCURACY 

The following table was used to define the data quality objectives for accuracy and 

precision. These are different than the goals outlined in the QAP. These are derived 

from data points obtained from the lab. 

LCL = Lower Control Limit MS = Matrix Spike 

UCL = Upper Control Limit MSD = Matrix Spike Duplicate 

A nalyte Achieved recovery limits Target Recovery Lirnits 

LCL UCL 

NH, MS 69.18 127.10 90-1 10 

LCS 74.44 121.77 

NO, MS 77.70 1 14.90 90-1 10 

LCS 79.1 1 1 16.36 

NO2 MS 73.43 121.94 90-1 10 

LCS 82.72 1 16.06 

SRP MS 71.37 1 17.20 90-1 10 

LCS 83.30 108.37 

Continuing Calibration - Continuing Calibration 

+I- 10% and run after every 10 samples +I- 10% and after every 10 samples 

Linearity R2 2 0.995 Linearity R2 2 0.995 

PRECISION 

Achieved Target 

1 20 % Relative Percent Difference - < 10 % Relative Percent Difference 

REPRESENTA TIVENESS * 

Samples were collected in triplicate from the test sites and analyzed separately. Also, 

please refer to the section on accuracy and precision of the analytical results. 
HARBOR BRANCH ENVIRONMENTAL LABORATORY 

QA Summary 

Page 4 of 5 

COMPARABILITY 

Harbor Branch Environmental Laboratory participated in a number of split studies and 

round robin analyses monitoring the comparability of data generated by the nutrient auto 

analyzer department. 

INTERLABORATORY SPLIT SAMPLE STUDIES 

Low Level Marine Nutrient Split - Attachment A 

Samples were split with Ron Jones' Laboratory at Florida International University. 

Please see attachment A for comparability spread sheet. 

The calculations used to determine comparability are: 

%RSD - Percent Relative Standard Deviation 

Where: A = concentration in sample A 

B = concentration in sample B 

RPD - Relative Percent Dzference 

RPD = /A-BI X 200 

A+B 

Where: A = concentration in sample A 

B = concentration in sample B 

I - Industrial Statistic 

Where: A = concentration in sample A 

B = concentration in sample B 
HARBOR BRANCH ENVIRONMENTAL LABORATORY 

QA Summary 

Page 5 of 5 

This data indicates that overall interlaboratory comparability is acceptable for the QC 

goals and objectives of this project. The split study demonstrated that dissolved 

inorganic nutrients were determined to be at the same relative levels of concentration by 

both laboratories. 

For individual samples, Nitrite and Nitrate exhibited a good level of reproducibility 

between the two laboratories, Ammonium exhibited an acceptable level of 

reproducibility, and SRP exhibited a weaker level of reproducibility. Possibilities for this 

pattern of reproducibility are numerous; including the consistency of distribution in the 

environment, stability in the fi-eezing process, stability of the analyte in ambient 

laboratory conditions, low level reagent impurities, and the robustness of the chemistries 

employed in the analytical processes at these very low levels of analyte concentration. 

The comparability of values for each sampling site per event is even greater when 

averages of the triplicate samples sets collected for each site are considered. 

FDEP Surface Water Study for Total Phosphate - Attachment B 

During the time frame of this project's duration, HBEL participated in two Florida 

Department of Environmental Protection (FDEP) round robin surface water studies for 

the evaluation of Total Phosphate. Compared accuracy and data reproducibility for 

HBEL was of a consistently high quality. 

COMPLETENESS 

Documentation of the extent to which the database fulfills QAPP quality control 

objectives has been provided for each criterion in the final analytical report and this 

quality assurance summary. 
HARBOR BRANCH ENVIRONMENTAL LABORATORY 

ATTACHMENT A 

INTERLABORATORY SPLIT SAMPLE STUDIES 
iFM-EPA 

DATE 

9/23/96 

9/23/96 

9/23/96 

9/23/96 

9/23/96 

9/23/96 

9/23/96 

9/23/96 

9/23/96 

9/24/96 

9/24/96 

9/24/96 

9/24/96 

9/24/96 

9/24/96 

9/24/96 

9/24/96 

9/24/96 

9/25/96 

9/25/96 

9/25/96 

9/25/96 

9/25/96 

9/25/96 

9/25/96 

9/25/96 

9/25/96 

- 

A JA 

A JB 

A JC 

PRA 

PRB 

PRC 

LKA 

LKB 

LKC 

A JA 

A JB 

A JC 

PRA 

PRB 

PRC 

LKA 

LKB 

LKC 

A JA 

A JB 

A JC 

PRA 

PRB 

PRC 

LKA 

LKB 

LKC 

HBOl 

NO2 

0.208 

0.154 

0.222 

0.152 

0.143 

0.1 64 

0.066 

0.037 

0.052 

0.146 

0.124 

0.067 

0.089 

0.086 

0.108 

0.121 

0.106 

0.089 

0.184 

0.121 

0.106 

0.195 

0.065 

0.083 

0.150 

0.118 

0.105 

FIU 

NO2 

0.270 

0.230 

0.220 

0.210 

0.160 

0.170 

0. I00 

0.060 

0.040 

0.100 

0.060 

0.080 

0.080 

0.080 

0.080 

0.110 

0.080 

0.070 

0.210 

0.150 

0.090 

0.050 

0.050 

0.050 

0.120 

0.110 

0. I I0 

HBOl 

NO3 

0.674 

0.464 

0.326 

1.057 

0.994 

1.287 

0.328 

0.156 

0.100 

0.244 

0.097 

0.087 

0.540 

0.601 

0.381 

0.722 

0.523 

0.424 

0.420 

0.168 

0.147 

0.323 

0.153 

0.157 

1.370 

1.145 

1.204 

averaaes 0.121 0.116 19.378 . 27.405 0.137 0.522 0.645 24.429 3
HFM-EPA 

DATE 

9/23\96 

9/23/96 

9/23/96 

9/23/96 

9/23/96 

9/23/96 

9/23/96 

9/23/96 

9/23/96 

9/24/96 

9/24/96 

9/24/96 

9/24/96 

9/24/96 

9/24/96 

9/24/96 

9/24/96 

9/24/96 

9/25/96 

9/25/96 

9/25/96 

9/25/96 

9/25/96 

9/25/96 

9/25/96 

9/25/96 

9/25/96 

smamlLAJA 

A JB 

A JC 

PRA 

PRB 

PRC 

LKA 

LKB 

LKC 

A JA 

A JB 

AJC 

PRA 

PRB 

PRC 

LKA 

LKB 

LKC 

A JA 

A JB 

A JC 

PRA 

PRB 

PRC 

LKA 

LKB 

LKC 

HBOl 

NH4 

2.380 

2.540 

2.490 

0.392 

0.469 

0.254 

0.204 

0.218 

0.107 

3.668 

3.500 

4.680 

0.454 

0.107 

0.292 

0.422 

0.266 

0.281 

1.350 

1.420 

2.440 

0.324 

0.020 

0.029 

0.355 

0.089 

0.207 

HBOl 

SRP 

0.026 

0.025 

0.000 

0.000 

0.017 

0.040 

0.000 

0.034 

0.080 

0.068 

0.095 

0.078 

0.049 

0.000 

0.000 

0.015 

0.034 

0.01 1 

0.01 7 

0.01 7 

0.022 

0.000 

0.027 

0.000 

0.117 

0.01 9 

0.016 

NU 

SRP 

0.140 

0.070 

0.060 

0.060 

0.020 

0.030 

0.040 

0.010 

0.070 

0.070 

0.040 

0.080 

0.000 

0.000 

0.010 

0.030 

0.010 

0.000 

0.280 

0.190 

0.170 

0.020 

0.000 

0.010 

0.050 

0.020 

0.020 

averaaes 1.073 1.369 35.71 8 50.51 2 0.253 0.030 0.053 87.352 1
HFM-EPA 

DATE 

9/23/96 

HBOl 

DIN 

AJA 3.26 

AJB 3.16 

AJC 3.04 

PRA 1.60 

PRB 1.61 

PRC 1.70 

LKA 0.60 

LKB 0.41 

LKC 0.26 

A JA - 4.06 

AJB 3.72 

AJC 4.83 

PRA 1.08 

PRB 0.79 

PRC 0.78 

LKA 1.27 

LKB 0.89 

LKC 0.79 

AJA 1.95 

AJB 1.71 

A JC 2.69 

PRA 0.84 

PRB 0.24 

PRC 0.27 

LKA 1.88 

LKB 1.35 

LKC 1.52 

averaaes 1.72 2.13 20.103 28.430 0.142