### FINAL 

### REPORT
"HighFrequency 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 

### 33

### 043
Date: August 28, 1997

### Acknowledgements
We thank our volunteer staff, including Colleen Murphey, Margaret Vogelsang, Nicole Logan, Andy Cannon, and Damon Marunyak for assistancewith field sampling, processing 

### of samples, data analysis, and graphics. Dr. Clinton Dawes (Universityof 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 aregrateful to G. P. Schmahl (Looe Key National Marine Sanctuary)and Bill Kruczynski (USEPA)for their assistancein various aspects of the study. Samplingin 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 enrichmentas a criticalmanagementissue. This study assessedhow physical forcing mechanisms (rainfall,wind, tides) linked land-basedwastewater discharges with the initiation of algal blooms in shallow (< 10m) inner-shelf waters between Big Pine Key and hoeKey National Marine Sanctuary (LKNMS) fromJanuary to October 1996. Three stations were selected along the offshore gradientthat 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 concentrationsof dissolved inorganicnitrogen (DIN =NH4++ N03- 

## +N02-; SRP = soluble reactivephosphorus);phytoplankton biomass (chlorophyll a, chl a); biomass, species composition, alkalinephosphatase activity, tissue carbon:nitrogen:phosphorus (C:N:P) contents, and natural abundanceof 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 selectedphysical events to resolve on temporal relationships between the events, nutrient enrichment of the water column, and initiation of algalblooms.
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 concentrationsof 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 concentrationsat all three stations during the mid-summerperiod. Chl a concentrationswere high at all stations throughoutthe study and averaged 1.86ug/l at 

### AJ (n 

### = 87), 0.55 ugh at 

### PR (n = 86), and 0.60 ugll at LK (n = 83). Fleshy macroalgal biomass averaged 

### < 100g dry wt



### .m-2 at all three stations in winter and early springbut 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 

### hurenciapoiteaui was abundant at all three stations and 

### 6 15~ values of this plant were highest at AJ (+ 5.0 oloo), intermediate .atPR (+ 3.5 oloo), and lowest at LK (

## + 3.0 oloo) 

### -- indicating increasing wastewater 

### N xontributions to this alga with increasing proximity to shore. Large-scaleblooms of ' 

### Cladophorafuliginosa were first observed at LK in February and this alga became the dominantbenthic cover by late spring. Transient, short-termincreases in 615~ of 

### C. fuliginosa from 

### -1 to 5 oloo were associated with increased DIN concen~ations and tissue 

### Nfollowing the first major rain event in late May. Increased APA of C.fuliginosa at 

### LK was also associated with increased DIN in lateMay and resulted in significantincreasesin tissue 

### P of this alga. Thehighest algal epiphyteloads on Thlassia testudinumoccurred at 

### LK rather 

### than the inshore stations, a result of high 

### DIN concentrations combined with high submarine irradiance at this station; these high epiphyteloads 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 scientificliterature.
In summary,these results corroborateprevious findings that episodic stormwater discharges of land-based wastewater nutrients initiateharmful algal blooms in nearshore waters of the 

### FKNMS, including the offshorebank reef at 

### LKNMS. Our results also underscore the need for high frequency water quality sampling to obtain statisticallyrobust annual estimates of contaminantconcentrationsneeded 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 managementissue. A major
concern is the effect of increased nutrient inputs from land-basedwastewater discharges on the oligotrophiccoral reef and seagrass communities. Eutrophication caused by land-based human activitiesis a major mechanism altering coastal ecosystemsworldwide (GESAMP 1990,NAS 1994)and can cause dramatic ecosystem changein tropical and subtropical ecosystems. Bell (1992) criticallyreviewed case studies of eutrophication in coral reef regions and noted that enrichmentabove concentrationsof

# - 1.0

### pI14dissolved inorganic nitrogen 

### @IN) and 0.1 pA4 solublereactive phosphorus (SRP) caused the demise of coral reefs in Barbados, KaneoheBay, 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 causedblooms of phytoplankton (Caperon et al. 1971) and the green "bubblealga" Dictyosphaeriacavemosa (Banner 1974). Macroalgalblooms have correlatedtightly with nutrient enrichment on reefs in Jamaica (Lapointe 1997,Lapointe et al. 1997),southeastFlorida (Lapointe 1997),Belize (Lapointe et al. 1993),and the inner Great Barrier Reef lagoon (Bell and Elmetri 1996).Water column nutrient enrichment of seagrassmeadows 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;Lapointeet al. 1994). Based on empirical evidencefrom the many case studies around the world, eutrophication is now considered a leading cause of both seagrass aapointeet al. 1994,Duarte 1995)and coral reef (Bell 1992,Ginsberg 1993) decline worldwide.

Because restoration of ecosystems alteredby eutrophicationis difficult, early diagnosis and management of the problem is desirable. Taxonomic or biotic shiftshave been used as indicators of eutrophication (Schmitt and Osenberg 1995)but because the appearance of "indicator species" signals an advanced stage of the problem, considerableenvironmental damageis 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~) as a tracer and found that septic tank 

### DIN loads contributed significantlyto 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). Wealso measured 615~ in both 

### T. testudinum and macroalgal tissue as a tracer of wastewater DIN enrichment.

Materials and Methods

The study was conductedbetween January and October, 1996, at three shallow (1-2m) 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 (AvenueJ, "AJ", 240 40' 351 N, 810 20' 320 W) downgradient of

# - 1,000septic 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' 800N, 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' 564N, 810 24' 245 W, a shallow seagrass meadow used for sampling all parameters exceptfor 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 subsurfacebuoys 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 abovebottom) water samples were collected in triplicate at the three stations into clean 250 ml Nalgene bottles, filteredthrough 0.45 pm 

### GFIF filters, and held on icein the darkuntil return to the lab where they were frozen. Subsequently,they were analyzedfor NH4+, N03- plus NO2-, and SRP on a Bran and Luebbe TRACS Analytical Console at the HBOI EnvironmentalLaboratory in Ft. Pierce, FL (seeAppendix 

### I for Quality Assurance Summary). For chl a, three 100ml replicate water samples were filtered (after adding 1mg 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 10rnl of dimethyl sulfoxide and then with an added 15ml of 90% acetone at 5 OC overnight and measured fluorometricallybefore and after acidification for the measurement of chlorophyll and phaeopigment concentrations (Burnison 1980;Parsons et al. 1984). Fluorescence .measurementswere made using a Turner Designs 10-000Rfluorometer equippedwith 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 14and 19February as daily averagewind speed increased from 5 to 15knots during a winter northeaster and on July 14when winds blew from the northeast at 15knots. Sampleswere collected between 15and 20 March as a function of tidal stageranging from ahigh tide on 15March (+1.0 ft) to low tides on 18March (-0.33 ft), 19 March (- 0.43 ft), and 20 March (-0.47 ft). Sampleswere collectedthrough four differentrain events on 11March (- 1 .O inch), 15to 25 May (- 4.0 inches), 1to 

### 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 fromthe Key West National Weather ServiceStation as recorded in theNational ClimaticDatabase. Monthly sampling of macroalgae included species composition,biomass, tissue C:N:P ratios and alkalinephosphatase 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. Subsamplesof two macroalgae fromeach 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 ServicesLaboratory in Solomons,MD. Blade epiphytelevels were also measured on T. testudinum at the three stationsby the methods describedby Lapointe et al. (1994). APA of macroalgae was measured (n 

### =4) by the spectrophotometricmethods detailedin Lapointe et al. (1994).

We analyzed dried tissue of abundantmacroalgae and 

### T. testudinum sampled monthly fiom each station for natural abundances of stablenitrogen isotoperatios (615

### N =15~/14~). Analyses for total N and 15N atom % were performed by Isotope Analytical Services,Inc. (Los Alarnos, New Mexico). The sampleswere processed through a Carlo-Erba NIA 1500 elementalanalyzerusing Dumas combustion. Thepurified nitrogen gas was then measured by a VG Isomass mass spectrometer.
The data were analyzedusing 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, 615N), 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

DissolvedInorganic Nutrients and Chla in theWater Column

There was a significantinteraction (F

### = 3.124, P =0.00008) between location and time
on the mean DIN concentrations, which decreased with increasing distancefrom 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 

### f0.68 pM (n =99) at LK. Maximum DIN concentrationswere 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 concentrationsof NH4+ were

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

Considerablevariability in DIN concentrationsresulted from physical forcing. Elevated DIN concentrations at the inshore AJ station were associated with high winds, rain, and low tides. For example, DIN (primarilyNH@) increased from

# - 1.0pM to 4.0 pM as strong northeasterly winds developed between 15and 19February. The highest DIN concentrationsmeasured during the entire study, 7.75 pM, occurred during the low tide sampling on March 19 (Fig. 5). SignificantDIN pulses also followedrain events on 11 March, 15to 24 May, 1July, and 23 September. DIN also increased at both PR and LK with high northeasterly winds between 15and 19February and on July 14when the maximum DIN concentrationsoccurred at these stations (Figs. 2 

### and 3). DIN spikes occurred at PR and 

### LK following rain events on 24 May, 

### 1July, 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 significantinteraction (F= 1.82, 

### P =0.029) between location and time on the mean SRP concentrations,which decreased with increasing distance from shore. Concentrationsof 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 

### f0.03 pM (n 

### = 99) at LK. The highest SRP concentrations at all three stationswere associated with the high northeast wind event *between15and 19February (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 significantinteraction (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 averagechl 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 

### f2.11 ugh (n = 87) at AJ, 0.55 

### f0.77 ugh (n = 86) atPR, 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 intenserainfall (> 7 " rain between 15and 25 May, Fig. 11). A similarpattern was observed at 

### PR where the highest chl a concentrations occurred on July 1 (Fig. 12)following heavy rain (Fig. 4). The seasonalpattern 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 CompositionofBenthic Macroalgae

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

# - 25 

### g dry wt-m-2during February to peak values of

# - 90 g dry wt-m-2in late May following the onset of the rainy season (Fig. 14). During February and March, the rhodophytes Laurenciapoiteaui andHeterosiphonia wurdmanni dominated the fleshy macroalgal biomass 

### and blooms of the chlorophytes Cladophoracrispula and Chaetomorphagracilis appearedin late April and May. The rhizophytic chlorophyte Caulerpasertulariodes became abundant in June following heavy rainfall and blooms of the chlorophyte Cladophoramontagneaena and the blue green Lyngbya majescula developedin August and September,respectively. Biomass of the calcareous macroalgae (primarilyHalimeda opuntia)increased from low values of

# - 150g dry wt -m-2in 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 increasingto maximum values of

# - 300 g dry wt in September following the onset of rain in late May (Fig. 16).Between February and May, the rhodophytesLaurenciapoiteaui and Heterosiphonia wurdmanni dominatedthe fleshy macroalgal biomass and blooms of Ceramiumnitens appeared as 

### an epiphyteon Gorgonians 

### "in late April. Extensive blooms of the blue-green Lyngbya gracilis (withLyngbya 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 PRshowed a decreasingtrend from February to October with maximum values 

### < 140g dry wt m-2 (Fig. 17).

Biomass of fleshy macroalgae at LK averaged

# - 40 to 50 g dry wt -m-2between 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 rhodophyteLaurencia poiteaui and the chlorophyte Cladophorafiliginosa were abundant and blooms of the chlorophyte Cladophoracrispata developed

### as an epiphyte on sponges, seagrasses, and soft corals between late March and July. Blooms of the blue green epiphyteLyngbya gracilis covered all benthicbiota 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, andAPA ofBenthic Macroalgae

Therhodophyte Laurenciapoiteaui 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 

### f2.32; % N averaged 1.32 f 0.30, 1.49 

### f0.22, and 0.97

# +0.36; and % P averaged 0.028 f0.011,0.024 f0.008, and 0.033 rt0.01 1.The mean %N of dry weight in 

### Lpoiteaui was significantly higher at the nearshore AJ (t =5.9, P< 0.001) and PR (t = 3.75, P 

### =0.007) comparedto the more offshore LK. However, there was no significant (P 

### >0.05) differencein mean % P of

### Lpoiteaui 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 Luurenciapoiteaui 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 2181

### k- 920, 2917 f 1573,and 1408f 407; and the N:P ratio averaged 110f 21,156 

### f59, 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) comparedto the more offshoreLK. Similarly, the N:P ratios 

### of L.poiteaui were significantlyhigher 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) differencein 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 entireperiod 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 f0.012 % dry weight. The tissue % P increased from0.037 % to 0.062 % between April and May with the onset of rain in late May (Fig. 18). Coincidentalwith the increase in % P of C.fuliginosa tissue at LK was a significant (F = 11.54, P =0.0004) seasonal increasein APA from values 

### <20 

### pM ~043-s~ dry wtah-1 between February and April to values 

### > 50 

### pM ~043-e~ dry wt-h-1in May following the onset of rain (Fig. 22).

The APA of Laurenciapoiteaui was significantly affectedby location 

### (F = 18.81,P = 0.00003) and season (F = 15.42, P 

### =0.0009). The mean APA of

### Lpoiteaui 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 Laurenciapoiteaui varied among the three stations and over time during the study (Figs. 26,27, and 28). The mean 615~ of Laurenciapoiteaui was highest at AJ (4.69 

### +- 1.14oloo), 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 statisticallydifferentfrom each other (t =0.057, P =0.955).

The 815~ values of the chlorophyteCladophora fuliginosa at LK increased following 

### rainfall in May and the wind eventinJuly (Fig. 29). Between April 24 

### and May 23, the 615~ values increased from

### < 3.0 0100 to 

### >5.00 

### 0100following the onset of heavy rain and increases in 

### DIN at LK; the 615~ values again increased on July 14from 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 epiphyteloads on Thulassia testudinum were generallyhigh 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 epiphyteloads at 

### AJ were observedin April (Fig. 30) ,while those at PR and LK occurredin June (Figs. 31 and 32). Over the entire study, the

9 epiphyteloads at LK were significantlygreater 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 phaeophytes Cladosip hon occidentalis and Lophosiphonia saccorhiza,therhodophytes Chondria sp., Ceramium fastigatum, and Grzfithsia sp., and the blue green alga Lyngbya gracilis. Following a densebloom of the chlorophyte Cladophora montagneaena in Septemberand October at AJ, we observed an extensivedie-off of manatee grass, Syringodium jiliforme.

The C:N:P contents in blade tissue of Thulassiatestudinum varied differently for N
versus P. Over the entire study,the % N at AJ, PR, and LK averaged 2.40 

### f 0.20, 1.82f 0.17, and 1.70

### f 0.1 1;these three averagevalues were significantly (t 

### >2.87, P 

### <0.018) differentfromeach other. The % P at

### AJ,PR, and LK averaged 0.13 

### f 0.02,0.10 

### f0.02, and 0.16 

### f 0.02; these three values were also significantly (t 

### >-3.29, P 

### <0.009) differentfrom each other. At AJ, PR, and LK, respectively, the C:N ratio averaged 16.29 

### f 1.05,20.92f 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 f5.7, and 24.3 f 3.3.

The average 615~ value of Thulassiatestudinum 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. testudinumwere 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 ColumnEnrichment

This study demonstrated significantlinkages among episodicphysical 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 concentrationsat

### AJ during the lowest tide of the study on March 

### 19illustrates the importance of tidal pumping via ebbing tides to enhancedrates of submarinegroundwater

3 discharge 

### (SGD) of wastewater nutrients. Lapointe et al. (1990), using a heat-pulsing groundwaterflowmeter, found that lateral rates of shallow groundwaterflow increasedby 

# - three-fold during ebbing tides as compared to flooding tides. This effect would be maximumduring extreme low tides, such as that on March 19when the highest DIN concentration (7.75 pM) was observed. In addition to increased rates of SGD, the extremelow tides also result in minimal dilution of the increased wastewater nutrient load with lower nutrient offshore water, furtherincreasingnutrient concentrationsin the coastalreceiving waters. High winds were another importantphysical mechanism causing nutrient enrichment. Elevated DIN and SRP concentrationswere measured at all stations during the high northeasterly winds in February and July. Theseincreased nutrient concentrationspotentially resulted from several contributing factors. First, wind-driven advection associatedwith these events could increase cross-islandhydrostatic 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 sedimentresuspension 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 frominshore to offshore in the lower Florida Keys; we observed this effect
on March 12and during the following days when high northeasterly winds transported highly turbid waters several miles offshore (south) of LK.

Rainfall was also an importantmechanism causing episodic nutrient enrichment. Rainfall results in increased infiltration of septictank and cesspit wastewater plumes into shallow groundwaters and also increases lateral groundwater flow rates, thereby increasing wastewaternutrient loads and 

### DIN and 

### SRP concentrations in adjacent surfacewaters of the Florida Keys (Lapointeet al. 1990). Although rainfall in the Florida Keys can have significantDIN concentrations (- 15.0 pM, Lapointe and Matzie 1996),an extremelyheavy rainfall event of 6" over severalhours would enrich a shallow 2 m water column by only 

# - 1 

### pM. Following the firstrain events between May 15and 23 when a total of

# -4 inches of rain fell, DIN concentrationsincreased from 

### < 1pM to 

### > 3 

### pM at AJ, from 

### <0.5 pM to 

### > 2.5 pM at PR, and from 

### <0.5 pMto 

### > 1.5 pM at LK. Obviously, the relatively low amount of rainfall over this period combined with the magnitude of this increasein DIN over several days implicates 

### DIN sources other than rainfall alone. The increasein DIN, together with increased 615~ values of Laureciapoiteai at AJ and Cladophorafuliginosa at LK following this "first flush",provide clear evidencethat wastewater derived DIN was contributing significantly to the DIN pool throughout the entire study area.

, These findingsunderscore the earlier conclusions of Lapointe and Matzie (1996) that high frequency sampling is neccessary to resolve on the episodic "pulses"of wastewaternutrient discharges and their ecological effects in the FKNMS. This is importantnot only to resolve on linkagesbetween land-based nutrient enrichment and the initiation of algal blooms, but also for longer-term monitoring programs designed to assess "statusand trends" of the FKNMS. The currentwater 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 demonstratedin the present study. The ongoing monitoringprogram is further confoundedby the fact that sampling occurs over varying tidal cycles and other physical forcing events, all of which are importantregulators of nutrient concentrations, algal blooms, and the status of water quality in the FKNMS. The need for medium (monthly) and high frequency (daily) samplinghas been recognized the Great Barrier Reef Marine Park Authority 

### (GBRMPA) and is currently in use in their long-termmonitoring programs for eutrophication on the Great Barrier Reef (Brodie and Furnas 1992).
Land-based WastewaterNutrients andAlgal Blooms

Several lines of evidencefrom this study supportthe hypothesis that land-based wastewater nutrient dischargesfrom the Florida Keys enhance blooms of phytoplankton, macroalgae, and seagrass epiphytesas far offshore 

### as LKNMS. First, the concentrationsof DIN, SRP, and chl a were all maximum at the inshore

### AJ station and the gradients consistently decreasedwith increasing distancefrom shore, pointing to a landbased-source of nutrients and phytoplankton biomass in the study area. Second, the spatialpattern of decreasing 615~ values of Laurenciapoiteaui and epiphytes on Thalassiatestudinum with increasing distance from

### AJ to LK, combined with episodicincreases in 

### DIN and 615~ values of Cladophora fuliginosa at LK followingrain and wind events,provides unequivicol evidence that wastewater 

### DIN contributes to macroalgal blooms throughout the study area. Third, the increasein biomass ofphytoplankton,macroalgae and seagrass epiphytes at all stations followingthe increasein 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-basedwastewater nutrient discharges to algal blooms in nearshore waters of the Florida Keys (Lapointeand Clark 1992)and *offshore to LKNMS (Lapointe and Matzie 1996). The wastewater enrichment throughout our study area is consistentwith currentmeter studies that showed long-term net cumulative

3 transportfrominshore waters between Big Pine Key and Marathon in a southwesterly direction towards the offshorebank reefs (Lapointeet al. 1992,Smith 1994,Pitts, 1994)andalso previous observations of an "ammoniumwake" in downstream waters of LKNMS during the wet season (Lapointe et al. 1992).

Our results also refuterecent speculations that offshoreupwelling of deep, cold water is the primary sourceof nutrients fueling the algalblooms 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 formin septictank-contaminated groundwaters (Lapointeet 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 distinctpulses 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+ enrichmentthat we observed throughoutthe study area was obviouslynot of upwelling origin. This conclusion is further evidencedby the lack of persistent cold water intrusions in the study area and the well-known long-term transportpattern in an offshore 

### -- not onshore 

### -- direction in the middle and lower Florida Keys (Lapointeet al. 1992,Pitts, 1994,Smith, 1994). Thus, although upwelling eventshave been documentedon short temporal scales at LKNMS (Lapointe and Smith 1986),these events areeither anomalous (i.e., upwelling at LKNMS in July 1986was 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 offshorenutrient transport process.
The time course data on DIN, APA, and C:N:P ratios of Cladophorafuliginosa at LK illustrates the dynamics as to how episodicNH@-rich wastewater discharges initiateand sustain macroalgalblooms. 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 increasedAPA due to increased N:P ratios that induced SRP-limitation (Lapointe and vO'Connell1989). An increasein APA also occurredin 

### C. fuliginosa following the DIN pulse * in May, which enhanced sequestering of SRP from dissolved organicphosphorus (DOP) pools. The end result was a parallel increasein tissue % 

### P of C. fuliginosa even though no increased SRP concentrations were observed during this period at LK. Tliese findingsdemonstratethe importanceofrapid assimilation of NH4+ during episodic enrichment events and subsequentreliance on APA to sequester SRP fromthe dissolved organic phosphorus (DOP) pool to sustain balanced growth.

Our results also suggestthat tissue C:N:P analysis alone may not be a reliable index of wastewater nutrient enrichmentin 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 significantlyhigher at AJ, the station in closest proximity to land-based wastewater nitrogen loads. However, the wastewater impact at AJ was clearly indicatedby the significantlyhigher 615~ values of L. 

### poiteaui at AJ compared to this alga at PR and LK. Thelack 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 experiencedhigher 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 studiesusing macroalgae should emphasize the use of 615~ values, rather than simpletissue C:N:P contents, due to the confoundingeffects of variable light (and other factors)on algal growth rates and tissue nutrient pools.

1 In comparison, 

### Thalassia testudinum showed considerabletissueN enrichment at AJ 

### (2.40 % dry wt.) compared to both PR (132% 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 interpretthis as evidencethat 

### T. testudinumrelies largely on nitrogen fixation for its nitrogenous supply (Capone and Taylor 1977)and as such may be more influencedby pore water P enrichment associated with wastewater discharges. In oligotrophiccarbonate-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 Septembermay have been related to light limitation and carbon imbalancefrom a recurrent bloom of 

### Cladophoramontagneaena that formed dense mats over the seagrasses. Alternatively, tissue N was high in 

### T. testudinum at AJ and may .reflectnitrogen toxicity, particularly during this period of seasonallymaximum temperatures ,(Burkholder et al. 1991).

3



### Nutrient Enrichment and Biotic Phase ShiftsThe relative importance of different types of primary producers in coastal ecosystems (i.e. phytoplankton,macroalgae, seagrasses, corals) is a key characteristicof ecosystem structure and has important consequences for ecosystem function. Numerous studies have shown large-scalephase-shifts in primary producers result from changes in limiting resources, most notably nutrient availability. Coastal eutrophication in tropical and subtropicalregions 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 eutrophicthreshold for coral reefs of 0.5 ug/l, Bell 1992),fleshy macroalgae (100 

### - 300 g dry wt m-21, and seagrassepiphytes (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 1987illustrate 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 1990when the FKNMS was established; since then, annual chl a concentrationsincreased to 0.25 ug/l in 1992 and 0.68 ug/l in the present study (1996), a value well above threshold level consideredeutrophicfor coral reefs (see Bell 1992;Fig. 39). Although we found significantwastewater DIN impacts throughout our study area, including LK, the dramatic escalation of chl a at LK are also likelyrelated to Florida Bay outflows that have been influenced by increasing phytoplankton blooms and turbidity in central and western Florida Bay since 1991 (Lapointeet al. 1996). The chl a and turbidity in Florida Bay is advectedby prevailing currents southward through tidal channels of the middle and lower Keys and offshoreto the outer bank reefs (Lapointe et al. 1992,Smith 1994,Pitts 1994).These blooms have expanded with the increased freshwaterrunoff and associated nitrogen loads from the Everglades since 199

### 1(Lapointeet 

### al. 1996)that enhance DIN-limitedphytoplankton 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 freshwaterflows to lower the salinity in Florida Bay (USEPA 1996). Thus, it is .unlikelythat 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-scalecontrol 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: Someexamples 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. 81:163-178. 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 differentnutrient regimes. Ophelia4197-112.
GESAMP. 1990. The State of the Marine Environment. Joint Group of Experts on the ScientificAspects of Marine Pollution. Rep. and Stud. 39. United Nations EnvironmentalProgramme. 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 strategiesfor growth of Gracilaria tikvahiae (Rhodophyta) in relation to lightintensity 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 couplingsbetween 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 eutrophicationin the Florida Keys. Estuaries 15465-476. Lapointe, B. E., N. P. Smith, P. A. Pitts, and M. Clark. 1992. Baseline characterization of chemical and hydrographicprocesses in the water column of Looe Key National Marine Sanctuary.. Final Report to 

### U. S. Dept. of Commerce, NOAA Office of Ocean and Coastal ResourceManagement, contract # NA86AA-H-CZ07 1,Washington, D. C. 103pp. Lapointe, B. E., M. M. Littler, and D. S. Littler. 1993. Modification of benthic community structureby 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. Eutrophicationand 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 eutrophicationprocesses 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, SarasotaBay, and Florida Bay. Program and Abstracts. Florida Bay Science Conference,December 10

### - 12,1996.Florida Sea Grant, University of Florida, 

### 11 

### 3

### pp. Lapointe, 

### B. E., M. M. Littler, and D. S. Littler. 1997. Macroalgalovergrowth 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 stableisotopesin fingerprinting surface water inorganic nitrogen sources. Water, Air, and SoilPollution 48:489-496. McClelland, 

### J. W., 

### I. Valiela, and R. H. Michener. 1996.Nitrogen stableisotope signatures in estuarine food webs: a record of increasing urbanization in coastal watersheds. Lirnnol. Oceanogr., inpress. 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. 173pp. Paul, 

### J. H., J. B. Rose, S. Jiang, C. Kellogg, and 

### E. A. Shinn. 1995 a. Occurrence of fecal indicatorbacteria in surfacewaters and the subsurfaceaquifer in Key Largo, Florida. Applied and EnvironmentalMicrobiology 6 1:2235-2241. Paul, J. H., J. B. Rose, J. Brown, E. A. Shinn, S. Miller, and S. R. Farrah. 1995b. Viral tracer studies indicate contamination of marine surface waters by sewagedisposalpractices in Key Largo, FL. Applied and EnvironmentalMicrobiology 61:2230-2234. Pitts, 

### P. A. 1994. An investigation of near-bottomflow patterns along and across Hawk Channel, Florida Keys. Bull. Mar. Sci. 54:610-620. Schmitt, R. J. and C. W. Osenberg. 1995. Detecting ecologicalimpacts; concepts and applicationsin 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-FileReport 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 channelsin 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 ecosystemresponse to nutritional perturbation. Pacific Science 35: 279-397.Szmant, A. M. and A. Forrester. 1996. Water column and sediment nitrogen and phosphorus distribution patternsin 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, 113pp. Tornasko, 

### D. A. and B. 

### E. Lapointe. 1991. Productivity and biomass of Thalassia testudinum asrelated to water column nutrient availability and epiphyte levels: field observations and experimental studies. Mar. Ecol. Prog. Ser. 75:9-17. USEPA, 1996.Water qualityprotection program for the Florida Keys National Marine Sanctuary. First Biennial Report to Congress. December, 1996, 32pp. 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 stationjust south of Munson Island. Fig. 3. Map of Looe Key National Marine Sanctuary showing locations of LK 1on the reef flat (shallow seagrass meadow)just behind the reef crest and LK 2 in the adjacentback 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 concentrationsat the Patch Reef. Values representmeans (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 concentrationsat the Patch Reef. Values representmeans 

### f 1S.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 

### Laurenciapoiteaui at Avenue J. Values represent means

# + 1 

### S.D. (n =4). Fig. 24. Alkaline phosphatase activity of the rhodophyte 

### Laurenciapoiteaui at the Patch 

### Reef. Values represent means

# + 1 

### S.D.(n =

### 4). Fig. 25. Alkaline phosphatase activity of the rhodophyte 

### Laurenciapoiteaui at Looe Key. Values represent means

# + 1 

### S.D. (n =4). Fig. 26. 615~ of 

### Laureciapoiteaui at Avenue 

### J. Values represent means f 1

### S.D.(n=2). .Fig. 27. 615~ of 

### Laureciapoiteaui at the Patch Reef. Values represent means f 1 

### S.D.

3 (n=2). Fig. 28. 615~ of 

### Laureciapoiteaui at Looe Key. Values represent means.+ 1 S.D.(n=2).Fig. 29. Correlationof 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:seagrassblade 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 testudinumat AvenueJ. Values represent means

# + 1 S.D. (n=2). Fig. 34. 615~ of Thalassia testudinurnat MatchReef. Values represent means

# + 1 S.D. (n=2). Fig. 35. 615~ of Thalassia testudinumat 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 Thalassiatestudinum 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-aat Looe Key (LK 1)between 1987 and 1996. Values represent means

# + 1 S. D. (16< n 

### < 83).

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APPENDIX I

HIGH FREQUENCY MONITORING OF WASTEWATER NUTRIENT DISCHARGES AND THEIR ECOLOGICAL EFFECTS IN THE FLORIDA KEYS MARINE SANCTUARY

QUALITY ASSURANCE SUMMARY NUTRIENTANALYSIS

PRESENTED TO:
BRIAN LAPOINTE, Ph.D. HARBOR BRANCH OCEANOGRAPHIC INSTITUTION, INC.

ANALYSIS BY:
HARBOR BRANCH ENVIRONMENTALLABORATORY

LABORATORY DIRECTOR: N. MYRON GUNSALUS,JR.

A DIVlSlON OF HARBOR BRANCH OCEANOGRAPHIC INSTITUTION, INC. August 12,1997HARBOR BRANCH ENVIRONMENTAL LABORATORY 

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5 0c~4~~c~&?*\G ~USTITUTIOH QA Summary

Page 1 of 5 0VERVIEW

Harbor Branch EnvironmentalLaboratory (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 implementedthe QNQCprogram to ensure that the generation ofmonitoring and analytical measurement data are ofknown adequatequality to meet the requirements of each project's statement ofwork.
The Harbor Branch EnvironmentalLaboratory pursues excellencein environmental testing and conducts applied research to understand and protect the natural environment.

PROJECT GOALS Sampleswere 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 EnvironmentalLaboratory by methods derived fiom widely accepted chemistriesused in previously published studies in the determinationof low level nutrients in surfacewaters. The results obtained from these analyseswill be evaluated to determinethe development ofphytoplankton and macroalgalblooms as a result of nutrient enrichmentfollowing episodicevents.
For the duration ofthe project, a previously developed Quality Assurance Project Plan (QAPP) consisting of quality control goals was in place to ensure accuratereproducible data and to meet quality expectations for data usability. The data quality goals included target method detection limits, recommendedholding times, and five quality assurance parameters: precision, accuracy,representativeness, comparability, and completeness. When quality objectiveswere not met for all samples, they were noted with corrective actions and narrated in the report.
METHODDETECTIONLIMIT (MDL)
Method Detection Limits were performed on "bluewater," saltwatermatrix in accordance to CFR. 40 Part 136Appendix B. These studies demonstrated achievabledetection limits below the QAPP specifiedMDLs. The project detection limits were utilized'insteadof the statistically derived MDLs.F

HARBOR BRANCH ENVIRONMENTAL LABORATORY

QA Summary Page 2 of 5
HOLDING TIMES
The QAPP recommended holding time oftwenty-eight days was exceeded for several sample sets in this project. These exceedancesand the performance of several analytical series for the various sample setsprovided an opportunity for observationof the stability ofthese 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 ofwork performed, the following informalobservationson 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 conditionscan adversely affect any data collected, Ammonium and SRP
were noticeably more sensitiveto lab environmental conditions.
Soluble Reactive Phos~hate (SRP)
For SRP care must be taken to avoid phosphate contamination ofthe analytical aliquot through reagent impurities or ambient laboratory conditions. This was accomplished through the monitoring ofthe reagent supply, locatingthe nutrient auto analyzer in the most phosphate fiee sectionofthe laboratory, and accomplishing the analytical run as quickly as possible oncethe analytical aliquot was poured for analyses.
Ammonium
For ammonium, control ofthe loss or gain of ammonium from the analytical aliquot and the sample was ofconcern and more difficult than SRP to control. Great care was taken to minimize lossby immediate analyses upon opening ofthe samplebottle after the samplehad been thawed and gently mixed. Air space in the bottle upon collectioncould have an affect on finalresults. Also, atmospheric lab ammonium can be absorbed into the analytical aliquot and the sample itself. Minimizing exposureto air during handling and analyses reduced ammonium absorption. Without these kind ofprocedures, NH, can be lost or gained.
From our observations, it appears the most reliable way to produce ammoniumresults which are representative ofthe sample upon collection is to avoid the complications of fieezing and ambient conditions. It is recommendedthat fresh, unfrozen samples collected with no air spacebe 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 followingtable was used to define the data quality objectives for accuracy and precision. These are differentthan 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 SpikeDuplicate
Analyte Achieved recovery limits TargetRecovery Lirnits
LCL UCL NH, MS 69.18 127.10 90-110 LCS 74.44 121.77 NO, MS 77.70 114.90 90-110 LCS 79.1 1 116.36
NO2 MS 73.43 121.94 90-110 LCS 82.72 116.06
SRP MS 71.37 117.20 90-1 10 LCS 83.30 108.37 ContinuingCalibration - Continuing Calibration +I- 10%and run after every 10 samples +I- 10% and after every 10 samples Linearity R22 0.995 Linearity R22 0.995

PRECISION
Achieved Target

120 % Relative Percent Difference < 10 % Relative Percent Difference -

REPRESENTATIVENESS * Samples were collected in triplicate from the test sites and analyzed separately. Also, please refer to the sectionon accuracy and precision of the analytical results.HARBOR BRANCH ENVIRONMENTAL LABORATORY

QA Summary Page 4 of 5

COMPARABILITY
Harbor Branch EnvironmentalLaboratory participated in a number of split studies and round robin analysesmonitoringthe comparability ofdata generatedby the nutrient auto analyzer department.

INTERLABORATORY SPLIT SAMPLE STUDIES
Low Level Marine Nutrient Split 

### - Attachment A Sampleswere split with Ron Jones' Laboratory at Florida International University. Please see attachment Afor comparabilityspread sheet.
The calculationsused to determine comparabilityare:
%RSD -Percent Relative StandardDeviation

Where: A=concentration in sampleA B =concentration in sampleB

RPD -Relative Percent Dzference
RPD = /A-BI X 200 A+B
Where: A=concentrationin sampleA B =concentration in sampleB

I -Industrial Statistic

Where: A=concentration in sampleA B =concentration in sampleBHARBOR BRANCH ENVIRONMENTAL LABORATORY

QA Summary Page 5 of 5

This data indicatesthat overall interlaboratory comparability is acceptable for the QC goals and objectives ofthis project. The split study demonstratedthat dissolved inorganic nutrients were determined to be at the same relative levels of concentrationby both laboratories.
For individual samples,Nitrite and Nitrate exhibited a good level ofreproducibility between the two laboratories,Ammonium exhibited an acceptable level of reproducibility,and SRP exhibited a weaker level ofreproducibility. Possibilitiesfor this pattern ofreproducibility are numerous; includingthe consistency ofdistributionin the environment, stability in the fi-eezingprocess, stabilityofthe analyte in ambient laboratoryconditions, low level reagent impurities, and the robustness of the chemistries employed in the analyticalprocesses at these very low levels of analyte concentration.
The comparabilityof values for each sampling site per event is even greater when averages of the triplicate samples setscollected for each site are considered.

FDEP Surface Water Study for Total Phosphate

## - Attachment B During the time frame ofthis project's duration,HBEL participated in two Florida Department ofEnvironmentalProtection (FDEP) round robin surfacewater studies for the evaluation ofTotal 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 objectiveshas been provided for each criterionin the final analytical report and this quality assurance summary.HARBOR BRANCH ENVIRONMENTAL LABORATORY



# ATTACHMENT A

INTERLABORATORY SPLIT SAMPLE STUDIESiFM-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 

# -

AJA AJB AJC PRA PRB PRC LKA LKB LKC AJA AJB AJC PRA PRB PRC LKA LKB LKC AJA AJB AJC 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 34.548 '0.173

AnACHMENT A Interlaboratory SplR Sample Studies Page 1 of 3HFM-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 AJB AJC PRA PRB PRC LKA LKB LKC AJA AJB AJC PRA PRB PRC LKA LKB LKC AJA AJB AJC 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 0.011 0.017 0.01 7 0.022 0.000 0.027 0.000 0.117 0.019 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 50.512 0.253 0.030 0.053 87.352 123.534 0.618

A'TTACHMENTA Interlaboratory Split Sample Studies Page 2 of 3HFM-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 AJA
- 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 AJC 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

ATTACHMENTA lnterlaboralorySpli Sample Studies Page 3 of 3