Discriminating Sources and Flowpaths of

Anthropogenic Nitrogen Discharges to

Florida Springs, Streams and Lakes

S. T. BACCHUS

Applied Environmental Services, LLC, P.O. Box 174, Athens, GA 30603-0174

P. J. BARILE

Division of Marine Science, Harbor Branch Oceanographic Institution,

5600 U.S. 1 North, Ft. Pierce, FL 34946

Key Terms: Biogeochemical Tracers, CAFOs, Harmful Algal Blooms, Invasive Species, Sewage Effluent,

Stable Nitrogen Isotopes

ABSTRACT

Surface discharges of anthropogenic nutrients

historically have been the focus of Florida’s waterquality regulations. Groundwater contributions to

eutrophication of Florida’s surface waters are a more

recent focus. Florida’s naturally oligotrophic springs,

streams, and lakes are experiencing significant

anthropogenic nutrient contamination resulting from

groundwater discharges with elevated nitrate. Sources of nitrate contamination to these surface-water

ecosystems include sewage effluent, industrial animal

waste (concentrated animal feedlot operations) and

inorganic fertilizers. In this study, stable nitrogen

isotope (d15N) analysis of freshwater macrophytes

was combined with basic knowledge of watershed and

springshed land use and aquifer characteristics to

provide evidence of nitrogen contamination sources

and groundwater flowpaths. Selected naturally oligotrophic ecosystems included springs and a spring-run

stream within the Ocala National Forest (ONF) and

springs, a blackwater stream, and a sinkhole lake

on or adjacent to state lands. Elevated d15N values

(~þ 8 to 12&) in ONF macrophytes indicated nitrogen contamination from sewage effluent. Underground injections of effluent and other wastes at

ONF’s Alexander and Juniper Springs Recreation

Areas are the sole source of contaminants flowing

through the sandy, surficial aquifer at those study

areas. Samples from springs on state lands indicated

nitrogen contamination from various sources via

regional groundwater flowpaths. At Lake Placid’s

state lands, a dairy-waste lagoon was the groundwater source of nitrogen contamination via the sandy,

surficial aquifer. Bulow Creek d15N macrophyte

values (~þ 5 to 8&) suggested contamination from

both cattle and septic tank leachate. Results indicated

that uptake of anthropogenic nitrogen occurred in

invasive alien and nuisance native macrophytes in the

four freshwater ecosystem types evaluated.

INTRODUCTION

Sources and Discharges of Nutrient Contamination

Florida’s naturally oligotrophic (nutrient-limited) surface waters and ground waters, which are characteristic of

the southeastern Coastal Plain, have experienced severe

and escalating nitrogen (particularly nitrate) contamination during the past 50 years (Katz et al., 1999; Toth,

2001). Nitrate can be transported readily beneath the soil

zone because of its solubility and resistance to ion exchange (Fryar et al., 2000). Excessive nutrient (nitrogen

and phosphorus) contamination constitutes one of the

most significant water-quality problems facing the State

of Florida, resulting in regulations to mitigate this

problem and to protect waters of the state from further

nutrient discharges. Rules adopted by the state give

particular consideration to the antidegradation of waters

characterized by very low nutrient concentrations, including, specifically, the protection of those waters from

anthropogenic nutrient contamination (Florida Administrative Code 62-302.300 (13)). ‘‘Nutrient over-enrichment’’ has been used in a policy context to relate the

problem of nutrient contamination to ecosystem degradation (National Research Council, 2000). For example,

exceedingly low concentrations (10 ppb) of phosphorus

have led to the invasion of monotypic stands of cattails

and the decline of native sawgrass in the Florida

Everglades (Davis, 1994).

The primary sources of bioavailable anthropogenic

nitrogen entering Florida’s coastal and inland waters

include agricultural and urban fertilizers, human sewage

effluent, and industrial animal waste. The source of

industrial animal waste is referred to in state and federal

Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369 347
regulations as ‘‘concentrated animal feedlot operations’’

(CAFOs). In Florida, dairy cows, beef cattle, and

chickens are the primary producers of industrial animal

wastes.

Bioavailable anthropogenic nitrogen and other contaminants discharge to Florida’s coastal and inland waters

as surface and underground point sources and non-point

sources. Common point-source discharges from surface

sources that are regulated by the Florida Department of

Environmental Protection (FDEP) include discharges of

municipal effluent from sewage treatment plant (STP)

outfall pipes and other surface pipe discharges from industrial, commercial and municipal sources. An example

of common non-point sources regulated by FDEP is

stormwater runoff (overland flow), which can transport

urban, agricultural and industrial nutrients to surface

waters (Karr, 1990).

These surface discharges have been the primary focus

with respect to nutrient loading in Florida’s surface

waters. Comparable attention to nutrient-laden groundwater discharges to surface waters in Florida has not

occurred (Bacchus, 2001, 2002). Recently, FDEP has

begun investigating the impact of land use on local and

regional groundwater contributions to selected inland

surface waters in Florida where a primary component is

direct groundwater discharge via conduits in the regional

aquifer (Scott et al., 2002).

The physical characteristics of these local and regional

groundwater contributions have been described for the

submarine (drowned) carbonate platform within the

regional Floridan aquifer system. Both diffuse and direct

submarine groundwater discharges (SGD) are known to

occur along Florida’s coast (summarized by Bacchus,

2000, 2002). These discharges provide insight into the

flow characteristics of the regional aquifer system. Some

of the potential adverse impacts of SGD to coastal

ecosystems in Florida from nutrients and other contaminants injected into the aquifer system via shallow and

deep wells were addressed by Bacchus (2001, 2002).

Various groundwater flowpaths of local to regional scale

that may influence these SGD contributions, as well as

groundwater discharges to non-marine surface waters,

were described initially by Toth (1963).

Comparable diffuse and direct groundwater discharges

also occur throughout the exposed carbonate platform of

Florida and adjoining states comprising the southeastern

Coastal Plain. Recent investigations of selected, primarily

non-submerged springs in Florida have documented the

degradation of water quality as nutrients originating from

land-use changes are transported to the springs as

subsurface discharges (Jones et al., 1996, 1997; Toth,

1999, 2001, 2003; and Toth and Fortich, 2002). As part

of a multi-million dollar study of increasing groundwater

eutrophication in Florida, FDEP evaluated first-magnitude springs (Scott et al., 2002, p. 6), which were reported

as having an average flow of ‘‘100 cfs or more (64.6 mgd

or more).’’

The groundwater discharges described above are

enhanced by the karstic nature of the regional carbonate

Floridan aquifer system (Figure 1). The Floridan aquifer

is overlain by unconfined sandy surficial aquifers

throughout much of its region. The exchange of water

between these surficial aquifers and the underlying, semiconfined regional aquifer has been established throughout

the extent of the Floridan aquifer (Toth, 1999; Bacchus,

2000, 2002). Therefore, these surficial aquifers are an

integral part of the regional Floridan aquifer system. For

the purpose of our analysis, however, two groundwater

flow components are addressed: (1) local (unconfined,

surficial) and (2) regional (semi-confined Floridan). No

distinction is made between regional and subregional

(intermediate) aquifers, with both referenced as the semiconfined regional aquifer. The consequence of nutrient

contamination from these surficial aquifers and from

the regional aquifer is considered in our study described below.

Natural Ecosystem Characteristics

Many of the natural ecosystems and associated native

species of the southeastern Coastal Plain evolved under

nutrient-limited conditions and are not adapted to

abnormal anthropogenic nutrient loads (Sculthorpe,

1967; Bosserman, 1979; and Stinner, 1983). Consequently,

excessive soluble nutrients become available in the water

column and substrate for use by other organisms, such as

invasive alien and nuisance native species of microscopic

plants and macrophytes. Those conditions result in the

loss of competitive advantage for desirable native species

and the ultimate displacement of native species of plants

and animals. In the past, alien species have been referred

to as ‘‘exotic’’ species. Ten rare and endemic vascular

plants (macrophytes) have been inventoried and suggested to be threatened by alterations in the quality and

quantity of spring discharges in proximity to the springs

and spring-run streams in east-central Florida (Florida

Natural Areas Inventory, 2004).

Vegetative phase shifts were evident in studies

comparing anthropogenic and natural sources of nutrients

to aquatic ecosystems in Florida and related wetlands

(Valiela et al., 1975; Dierberg and Brezonik, 1978;

Stinner, 1983; and Bacchus, unpublished data). A largescale example of these vegetative phase shifts is the

replacement of the dominant native saw-grass (Cladium

jamaicense Crantz) by cattails (Typha spp.) in the northern

Everglades, following contamination with nutrient-laden

agricultural discharges (Davis, 1994; Vaithiyanathan and

Richardson, 1999). The natural nutrient limitations of

these ecosystems and the potential magnitude of anthropogenic sources of nutrients were demonstrated further in

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348 Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369
a study that found total phosphorus 24 times greater in

a depressional pond-cypress (Taxodium ascendensBrong.)

dome wetland in Florida receiving surface discharges of

treated sewage effluent than in comparable watershed

wetlands (Stinner, 1983). In that same study, total

phosphorus from inorganic fertilizers in an agroecosystem

and from a white ibis rookery were only 4.0 times greater

and 2.3 times greater, respectively, than total phosphorus in

the comparable natural ecosystem referenced above.

In recognizing the implications of anthropogenic

nitrate-nitrogen on natural ecosystems, FDEP (2000) noted

that concentrations ,1 mg/L cause a shift in the balance of

spring ecological communities, leading to intensified degradation of biological systems. The St. Johns River Water

Management District (SJRWMD) recognizes 0.2 mg/L

nitrate-nitrogen in ground water and discharge from

springs as a threshold for nitrogen contamination. This

threshold was established by their database (Toth, 1999).

By comparison, the physiological threshold of dissolved

inorganic nitrogen (nitrateþnitriteþammonium) reported

to support and saturate growth of Florida’s dominant red

tide organism and harmful marine macroalgae is ;1.0 lM

Figure 1. Areal extent of the regional Floridan aquifer system (submarine extent not shown); the six subregions designated for regional groundwater modeling (D, E, F, G, H, and unnamed subregions; from Krause and Randolph, 1989); and the general locations of the areas selected in

Florida for this study.

Subsurface Anthropogenic Nitrogen Discharges

Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369 349
(summarized by Barile, 2004), illustrating the similarities

between Florida’s naturally nutrient-limited freshwater

and marine ecosystems.

Species that replace desirable native species in

Florida’s freshwater ecosystems include invasive alien

species of aquatic macrophytes such as water hyacinth,

water lettuce, hydrilla, cyanobacteria (blue-green algae),

other filamentous algae, and nuisance native species.

Table 1 includes examples of invasive and nuisance

macrophytes in Florida, in addition to desirable native

macrophyte species, such as maidencane and pickerelweed. The invasive macrophytes become so dense in

Florida’s surface waters subjected to nutrient loading that

the quality of swimming, boating, fishing, and other

recreational opportunities is reduced or precluded. In an

effort to control these invasive species, the state spends

millions of dollars annually applying various herbicides.

As the plants die, their decomposition results in the

recycling of nutrients in the system, thus perpetuating the

problem. Recognizing the wide-spread and pervasive

nature of the nutrient pollution problem for surface waters

(National Research Council, 1992, 2000), Congress

passed the Aquatic Invasive Species Research Act (HR

1081) in 2003. Section 3 of the Act defined the term

‘‘aquatic ecosystem’’ as ‘‘a freshwater, marine, or

estuarine environment (including inland waters and

wetlands) located in the United States.’’

Harmful cyanobacteria blooms have become common

Table 1. Freshwater macrophyte species used for stable nitrogen isotope (d15N) analysis of selected springs, spring-run and blackwater streams,

and a sinkhole lake in Florida.

Common Name1 Scientific Name1 Growth Form Study Area/Site2

Cattail Typha spp. rooted emergent AS

BC

LP1,2,4,7,9

Common reed Phragmites australis (Cav.)

Trin. ex Steud.

rooted emergent LP8d

Duckmeat Spirodela polyrhiza (L.) Scheid. floating-leafed aquatic WS

Duckweed Lemna valdiviana Phil. floating-leafed aquatic AS

BC

LP13

PS

WS

Eelgrass Vallisneria americana Michx. submersed aquatic AS

JS

Elderberry Sambucus canadensis L. rooted emergent LP10–17

Eurasian water-milfoil Myriophyllum spicatum L. submersed aquatic LP6

Filamentous

algae/cyanobacteria

filamentous algae submersed aquatic AS

BC

JS

LP8a,d,9,15

OS

PS

WS

Frog’s bit Limnobium spongia (Bosc.) Stued. floating-leafed aquatic LP15

Hydrilla Hydrilla verticillata (L. f.) Caspary submersed aquatic PS

WS

Maidencane Panicum hemitomon Schult. rooted emergent LP4,5,6,8a,16

Musk-grass, stonewort Chara sp. submersed aquatic OS

Pickerelweed Pontederia cordata L. rooted emergent LP2,8b,9

Primrose-willow Ludwigia peruviana (L.) Hara. rooted emergent LP2,6,8a,8c,9–15,17

Torpedo grass Panicum repens L. rooted emergent LP1,2,7,8a

Water hemlock Cicuta mexicana Coult. & Rose rooted emergent AS

Water hyacinth Eichhornia crassipes (Mart.) Solms floating-leafed aquatic BC

WS

Water lettuce Pistia stratiodes L. floating-leafed aquatic AS

Yellow cow-lily Nuphar luteum (L.) Sibth. & Sm. floating-leafed aquatic LP3,6,8a,8b

OS

1

Nomenclature for vascular macrophytes is based on Godfrey and Wooten (1979, 1981).

2

Study areas: AS 1⁄4 Alexander Springs and Spring-Run Stream (upstream of 445 bridge, 10/13/02); BC 1⁄4 Bulow Creek (10/24/02); JS 1⁄4 Juniper

Springs (10/17/03); LP 1⁄4 Lake Placid, sites 1–17 as shown in Figure 4 (4/8/02); OS 1⁄4 Orange Grove Springs (10/10/02); PS 1⁄4 Peacock Springs

(10/10/02); WS 1⁄4 Wakulla Springs (2/25/05).

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350 Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369
in many east-central Florida and other freshwater systems

(Anderson et al., 2002; Livingston, 2005; and Phlips

et al., 2005). The toxins from these harmful algal blooms

have been identified as the causal factor in mass death of

manatee, a federally-listed endangered species with

critical habitat throughout Florida (U.S. Fish and Wildlife

Service, 2001), as well as other marine mammals, alligators, and numerous species of freshwater and coastal

fishes and invertebrates. In addition to wildlife mortality

resulting from cyanobacterial toxins, toxin-producing

blooms also have been associated with sublethal adverse

effects such as sexual reproduction abnormalities in

alligators (Ross, 2000). The production of microcystin

toxin in these systems also has threatened surface-water

sources used for potable water and been associated with

liver cancer in humans (Fleming et al., 2002).

Stable Nitrogen Isotopes in Aquatic Systems

Stable nitrogen isotopes (d15N) have been utilized in

ecosystem studies to trace food webs and discriminate

geochemical processes through biota, as well as to study

pollution source contamination in biological systems (see

reviews by Heaton, 1986; Peterson and Fry, 1987;

Handley and Raven, 1992; and Lajtha and Michener,

1994). Specifically, d15N has been utilized to discriminate

inorganic and organic nitrogen sources in aquatic

foodwebs. This technique particularly is valuable when

source values have been identified. The d15N of source

values can be discriminated as a result of fractionation,

whereby the lighter isotope, 14N, is taken up preferentially in metabolic processes compared to the heavier

isotope, 15N. For example, where atmospheric air is used

as a baseline of 0, inorganic nitrogen fertilizer has a d15N

of þ1 to 3&, organic nitrogen (which may include

natural, fixed nitrogen in soils and manure) generally

ranges fromþ3 to 9&, and human sewage effluent ranges

from ;þ8 to 20&, depending on treatment processes

(Heaton, 1986).

More recently, this method has been used in the

United States to evaluate the nitrogen composition or to

discriminate sources of nitrogen contamination in estuarine macrophytes (Cloern et al., 2002), marine macrophytes (Barile, 2004), and surface-water and groundwater

systems (Katz et al., 1999; Battaglin et al., 2001; Kendall

et al., 2001; Burns and Kendall, 2002; Chang et al., 2002;

deBruyn and Rasmussen, 2002; Ashkenas et al., 2004;

Pardo et al., 2004; and Wilcox et al., 2004). In spring

systems within our study region, Katz et al. (1999) and

Toth (1999, 2003) have used d15N to discriminate

sources of inorganic fertilizer nitrogen from organic

nitrogen, such as natural fixed nitrogen in soils and

manure and residential septic tank effluent in groundwater discharges. Where overlap occurs for reported

values, these values may originate from more than one

nitrogen source (Chang et al., 2002). We hypothesize that

d15N analysis of freshwater macrophytes, combined with

basic knowledge of watershed and springshed land use

and aquifer flow characteristics, can be used to

discriminate nitrogen contamination sources and groundwater flowpaths for sources such as industrial animal

feedlot operations, human sewage effluent, and inorganic

fertilizers used in agricultural operations.

STUDY AREAS AND METHODOLOGY

This study was initiated to identify probable sources of

subsurface nitrogen loading responsible for the conspicuous abundance of invasive alien and nuisance native

macrophytes in selected Florida freshwater ecosystems.

Stable nitrogen isotope (d15N) analysis of freshwater

macrophytes was combined with basic knowledge of

watershed and springshed land use and aquifer flow

characteristics to evaluate the sources and flowpaths for

groundwater discharges potentially containing anthropogenic nitrogen. Watershed land use included only

activities located within standard land surface topographic boundaries (commonly referenced as surface-water

divides) surrounding the target surface waters and

capable of resulting in groundwater discharge to those

target surface waters. Springshed land use included

activities located beyond watershed boundaries and

capable of contributing groundwater discharge to surface

waters in surrounding watersheds. Groundwater flow

components included sandy, surficial (local) aquifers and

the underlying (regional) karst Floridan aquifer. More

specific information regarding aquifer characteristics of

the Floridan aquifer system in the vicinity of our study

areas can be found in Rosenau and others (1977), Miller

(1986), Katz and others (1999), and Toth (1999; see

http://sjr.state.fl.us/programs/outreach/pubs/index.html).

Target alien species included Category I and II invasive

aquatic species identified by the Florida Exotic Pest

Plant Council (http://www.fleppc.org/). Both common

and scientific names for macrophytes evaluated in this

study are listed in Table 1; common names are used for all

further references to those plants. Target Category I

species included water hyacinth, hydrilla, and water lettuce. The primary target Category II species was Eurasian

water-milfoil. Target species were based on those identified as becoming a problem in the Flagler County area of

Bulow Creek because of increased anthropogenic nutrient

discharges associated with urbanization.

A formal description of Florida’s natural communities

was prepared by the Florida Natural Areas Inventory and

the Florida Department of Natural Resources (1990).

The following naturally oligotrophic community (aquatic

ecosystem) types were selected for this study: springs,

spring-run stream, blackwater stream, and sinkhole lake.

Criteria for selecting specific examples of these aquatic

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Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369 351
ecosystem types included a relatively protected, undeveloped watershed and springshed surrounding or

adjacent to the study areas; the presence of abundant

invasive macrophytes; and personal knowledge of

historic, pre-invasion conditions. The study areas and

sample collection sites selected for the study are

described below. Representative invasive macrophytes,

and benthic substrate and water column conditions at

selected study areas and comparative sites are shown in

Figure 2A–F.

Figure 2. Representative invasive macrophytes, and benthic substrate and water column conditions at: (A) swimming area in Wakulla Springs State

Park (note dense mat of floating duckweed, filamentous algae, and other invasive macrophytes); (B) Alexander Springs swimming area in ONF

Recreation Area (note continuous dark area of the benthic mat formed by the harmful algal bloom, except for area of exposed sand bottom cleared by

visitor use of the spring); (C) southern edge of the same swimming area (foreground), with wall located down-slope of the sewage effluent drainfield

(note faint ripples at lower right corner and dark flocculent areas of invasive algae); (D) northern perimeter of the same swimming area (note lack of

exposed sand substrate); (E) leaf blades of desirable native rooted submersed macrophyte (eelgrass, from ONF Salt Springs Marina) covered with

thick epiphytic invasive filamentous algae (note typical appearance of leaf tip in foreground, with algae removed, and penny for scale); and

(F) Wildcat Lake, ONF (note historically typical conditions of white sand bottom lacking macrophytes).

Bacchus and Barile

352 Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369
Springs and Spring-Run Stream Selected in the

Ocala National Forest and State Lands

The relative locations for the springs (Alexander and

Juniper Springs) and spring-run stream (Alexander

Spring-Run) selected as study areas in the Ocala National

Forest (ONF) are shown in Figure 1. Also included in

Figure 1 are the relative locations of springs selected as

study areas on state lands (Orange Grove, Peacock, and

Wakulla Springs).

Alexander and Juniper Springs and

Alexander Spring-Run

Surrounding Alexander Springs and immediately

downstream, the waters are overgrown with dense

macrophytes composed primarily of invasive nuisance

and alien species of algae and vascular plants, and much

of the natural stream bed is covered by a thick dark layer

of organic ooze. These symptoms are indicative of chronic

discharges of anthropogenic nutrients to the formerly

pristine, oligotrophic Alexander Springs and Spring-Run.

These aquatic systems, located in ONF (Lake County),

provided an ideal study area, based on the following

factors: (1) extensive undeveloped watershed within ONF;

(2) recent water-quality data from the Floridan aquifer

suggesting relatively limited anthropogenic nutrients contributed from the deep, regional aquifer source representing the springshed (Scott et al., 2002); and (3) previous

observations of the ecosystem condition since the 1960s,

prior to the shift to dominance by invasive macrophyte

species (Bacchus, unpublished data).

The FDEP report on first-magnitude springs of Florida

(Scott et al., 2002) included some erroneous and

misleading information in the description of Alexander

Springs. For example, the spring is not owned by ONF,

as stated in the report. The U.S. Forest Service only

manages the lands surrounding Alexander Springs, and

the U.S. Forest Service land immediately surrounding

Alexander Springs has been developed and is being

operated as a private concession. The report also indicates

that ‘‘native aquatic grasses are plentiful,’’ and the

‘‘bottom is mostly sandy’’ (Scott et al., 2002, p. 85).

Virtually no native aquatic grasses or other native species

occurred at Alexander Springs at the time of that study

through the present, and little of the originally sandy

bottom is visible because of the extensive noxious

filamentous algae (Figure 2B and C) and organic ooze.

No data were available from the FDEP study for

Juniper, Orange Grove, or Peacock Springs, because they

are not first-magnitude springs. Juniper Springs is located

within the same springshed as Alexander Springs, and is

;18 km (;11 mi) northwest of Alexander Springs, in

Marion County. Juniper Springs is exhibiting early signs

of nutrification, based on the recent abnormally dense

growth of native macrophytes and epiphytic filamentous

algae. Similar epiphytic algae has covered and killed

these desirable native plants at Alexander Springs.

Orange Grove, Peacock and Wakulla Springs

Orange Grove and Peacock Springs are located in

Peacock Springs State Recreation Area, Suwannee

County, ;3 km (;2 mi) north of the Suwannee River.

Peacock Springs is the headwaters of Peacock Slough and

is less than 2 km (1 mi) from Orange Grove Springs. That

recreation area has no facilities with underground

discharges. Although the area immediately surrounding

the springs is densely wooded, and primarily in a natural

state, the springshed includes both agricultural and

industrial animal land uses, combined with industrialscale irrigation. A portion of the extensive subterranean

karst conduits associated with Peacock Springs has been

mapped by cave divers. The general extent of those

conduits are shown in Figure 3, in comparison with karst

conduits in other spring systems mapped by cave divers

in Florida as examples of potential flowpaths and

springshed areas that may contribute to subsurface

nutrient discharge from the Floridan aquifer.

Wakulla Springs and the upper 5 km (3 mi) of the

Wakulla River are located in Wakulla Springs State

Park, Wakulla County, and designated as a ‘‘protected

wildlife sanctuary’’ (Scott et al., 2002, p. 134). The

watershed immediately surrounding Wakulla Springs

primarily is in a naturally-vegetated state. Neither the

watershed nor the springshed for Wakulla Springs

contains industrial feedlot operations. Two municipal

sewage treatment plants and associated effluent spray

fields are located within different watersheds, but the

same springshed, ;18 km (;11 mi) north-northwest and

north-northeast of Wakulla Springs, in Leon County.

The northwestern sewage treatment plant and effluent

spray field are located ;2 km (;1 mi) west of the

Munson Slough watershed. Discharge at the existing

wastewater treatment facility is reported as having ‘‘27.5-

MGD annual average daily flow,’’ with land application

of the reclaimed water ‘‘limited to 19.79 MGD’’ prior to

completion of additional areas for land application (FDEP

Permit Notice FLA 010139, FLA 010140, File FLA

183768-001-DW1R, 8/99). That watershed receives

considerable stormwater runoff from surrounding areas

in the watershed. Recent dye-tracer studies have documented rapid flow through karst conduits in the underlying regional Floridan aquifer (Davies et al., 2004;

Hazlett et al., 2004; and Kincaid et al., 2004), illustrating

the significance of springshed contributions.

Wakulla Springs was described in the FDEP report as

one of the largest and most dramatic of Florida’s springs.

Hydrilla was reported as previously covering much of the

spring pool and adjacent river bottom prior to removal by

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Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369 353
divers prior to release of the FDEP report. The FDEP

report describes the Wakulla River as ‘‘choked with this

exotic invasive plant species’’ (Scott et al., 2002, p. 132).

At the time of the 2005 sample collections for our

study, hydrilla had re-invaded the spring pool. Hydrilla

had not invaded Wakulla Springs or the upper reaches of

the Wakulla River in the 1960s, but was present by the

late 1970s or early 1980s (Bacchus, unpublished data).

The City of Tallahassee’s municipal sewage treatment

plant and spray field began operating prior to the invasion

and proliferation of hydrilla in Wakulla Springs and

immediately downstream.

Blackwater Stream in the Tomoka

Marsh Aquatic Preserve

Bulow Creek is a shallow meandering blackwater

stream in the northwestern portion of the Tomoka Marsh

Aquatic Preserve in Flagler County and is designated

‘‘Outstanding Florida Waters.’’ Bulow Creek and the

Aquatic Preserve are shown at http://www.dep.state.fl.us/

coastal/downloads/maps/default.htm with associated state

parks. Bulow Plantation Ruins State Historical Site is

shown as the northern boundary of Bulow Creek State

Park on that map. Bulow Creek, bounded by John

Anderson Highway on the east and Old Kings Road on the

west, is located within the watershed for the Tomoka

River, also ‘‘Outstanding Florida Waters,’’ and is a

manatee sanctuary. From its origin in the forested wetlands of Graham Swamp to the north, Bulow Creek flows

south for ;6.5 km (;4 mi), under Walter Boardman

Lane, meandering to its confluence with the Halifax and

Tomoka Rivers at Tomoka Basin and the Tomoka State

Park, as shown in the FDEP Aquatic Preserve map. The

banks of Bulow Creek are bordered by floodplain forests

with native species along the upper reaches. Increasing

expanses of freshwater marsh vegetation grade into tidal

marsh, dominated by black needle rush (Juncus roemerianus Scheele) in the lower reaches of Bulow Creek

(Florida Department of Natural Resources, 1990).

Relevant management issues and threats identified in

the FDEP Management Plan include protection of

manatee habitat and motor boat control, cumulative

impacts to water quality associated with point and nonpoint sources of pollution, restoration of disturbed areas,

control of alien plant species, and growth management

Figure 3. Submerged karst conduit networks in the Floridan aquifer associated with Peacock and Wakulla Springs, and similar Florida springs that

have been mapped as cave systems (Hazlett Kincaid, Inc., unpublished).

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354 Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369
(Florida Department of Natural Resources, 1990). Large

tracts of naturally vegetated (and densely forested) areas

still occur in Flagler County, but rapid urbanization of

that land is occurring. Sensitive aquatic systems in

Flagler County have been susceptible to the invasion and

spread of alien plants, partly because of lack of

information available to the public. These include areas

within the Tomoka Marsh Aquatic Preserve and Tomoka

Basin, the Bulow Creek system, and the Halifax River/

Intracoastal Waterway. Although the Tomoka Marsh

Aquatic Preserve and waters within Tomoka State Park

were designated as ‘‘Outstanding Florida Waters’’ in

1979 and 1991, respectively, alien species of macrophytes have invaded and proliferated to the extent that

they restrict recreational swimming and boating activities

in parts of these waters (Bacchus, unpublished data).

During an inspection of Bulow Creek by boat in 2002,

isolated dense patches of duckweed, water hyacinths, and

filamentous algae were observed at discrete locations in

the northern reach of Bulow Creek, in Flagler County.

One patch, primarily composed of water hyacinths,

occurred along the west side of the channel at the mouth

of a small tributary to Bulow Creek, 1.6 km (1 mi) north

(upstream) of the state historical site and ;2.5 km

(;1.5 mi) east of a cattle operation and open mine pit in

the Bulow Creek flood plain. A second patch, composed

of duckweed, water hyacinths, and filamentous algae,

occurred on the east side of the channel ;1.6 km (;1 mi)

south of the state historical site and ;120 m (;400 ft)

east of a single-family residence. Similar patches of

invasive macrophytes were not observed in the Flagler

County portion of Bulow Creek.

Sinkhole Lake in Central Florida

Lake Placid, formerly known as Lake Childs, is

a natural sinkhole lake located in west-central Highlands

County (Figure 1). It extends ;5 km (;3 mi) along the

north/south axis and 3.2 km (2 mi) along the east/west

axis. The limited residential development associated with

the lake (predominantly single-family residences) was

established primarily from the 1950s through the 1970s

and is clustered in the northeastern and northwestern

portions of the watershed. The northern and eastern

portions of the watershed include citrus groves and land

on which citrus previously was grown. The majority of

the southern portion of the Lake Placid watershed

remains naturally vegetated, containing both forested

seepage streams and wetlands, and sandhill uplands.

These areas include the state-owned Tobler Tract (from

west of Placid View Drive, east to Highway 17/railroad)

and Archbold Biological Station south of Lake Annie

(Figure 4).

Historically, Lake Placid was oligotrophic, as is

characteristic of natural sinkhole lakes in Florida (Florida

Natural Areas Inventory and Florida Department of

Natural Resources, 1990). Within the last decade, invasive macrophytes became established and proliferated

in various areas of Lake Placid’s littoral zone (Bacchus,

unpublished data).

A series of unpublished reports prepared by the

Highlands Soil and Water Conservation District describe

the water-quality problems that were reported as early as

April 26, 1999. A report by that date indicated that

nutrient levels in stormwater from the industrial dairy

operation upgradient of Lake Placid were ‘‘comparable

with that commonly observed in raw sewage’’ and ‘‘well

in excess of state water quality standards for any lake in

Florida’’ (Ford, 2001, p. 1).

Ford (2001) described nutrient levels that were in

excess of those possible from natural drainage. Ford’s

report also described bacterial results that were alarming,

with total and fecal coliform values at the central branch

under Highway 70 and at the mouth of the tributary to

Lake Placid that were too numerous to count (reported as

.80,000 cfu/l00 mL) and total coliform values of 38,000

cfu/100 mL at the west branch. The declining water

Figure 4. Map of macrophyte sample collection sites for Lake Placid.

Subsurface Anthropogenic Nitrogen Discharges

Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369 355
transparencies and increased algal blooms were attributed

to ‘‘loading of nutrients to the lake,’’ and was described

as a ‘‘large scale violation of the FDEP 62-302 surface

water-quality standards’’ (Ford, 2001, p. 2).

A subsequent report (Ford, 2002) revealed that the

water-quality problems were observed initially by

lakefront residents in July 2001. Numerous extensive

harmful algal blooms occurred following the waterquality problems observed that summer. Ammonia and

total coliform bacteria levels were described as being in

excess of state water-quality standards where flows were

entering Lake Placid from the tributary leading from the

industrial dairy operation to the southern end of the lake.

That report also indicated that during a meeting related to

the problem in December 2001, FDEP representatives

indicated that they did not feel that the dairy was

violating surface water-quality criteria (Ford, 2002, p. 2).

Water-quality monitoring conducted by the Highlands

County Lake Management Program in Lake Placid and

upstream in the tributary leading from the industrial dairy

operation revealed that ‘‘extremely high levels of

nutrients and bacteria, well in excess of FDEP 62-

302.530 limits were originating from drainage south of

State Road 70, in the vicinity of Posey Dairy’’ with

water-quality declines continuing for six months since the

initial decline of water quality in July 2001 (Ford, 2002,

p. 2). The natural tributary included in the water-quality

monitoring by the Highlands Soil and Water Conservation District is part of the state-owned Tobler Tract,

a densely forested natural area located between the

industrial dairy operation upstream and Lake Placid.

Correspondence from Keith Kleinmann, FDEP, dated

March 29, 2002, stated that the berms constructed on the

dairy in September 2001 and the extension constructed in

February 2002 were intact and functioning as intended,

and that there was no discharge from the dairy during the

inspection by FDEP inspector E. J. Jackson on March 27,

2002. On April 8, 2002, the macrophyte samples for our

study were collected from the Lake Placid study area. A

significant and constant discharge of ground water as

seepage flow was observed at the base of the sandy

‘‘berm’’ that had been constructed in the upper reach of

the natural tributary, in an effort to block stormwater

runoff from the dairy site.

On January 28, 2002, FDEP prepared an Administrative Agreement to allow the continued operation of the

dairy. That agreement stated that the dairy had 320 cows,

but was allowed to maintain a herd size as great as

850 cows.

Sample Collection and Stable Nitrogen Isotope

Analysis of Freshwater Macrophytes

The species and growth forms (e.g., rooted, floating)

of freshwater macrophytes varied among the study areas

described above, and determined which species could be

collected for d15N analysis. Table 1 includes the common

and scientific names, plant growth forms, and sample

dates for each study area and sample collection site. The

common name will be used for all further reference to the

plants sampled for analysis. At the springs, submersed

and floating aquatic vascular and non-vascular plants

were collected for analysis from the still, littoral zone

waters.

At Orange Grove Spring, one species of rooted,

aquatic macrophyte with floating leaves (yellow cow-lily)

also occurred in the littoral zone, and was collected for

analysis. For Alexander Spring-Run, Bulow Creek, and

Lake Placid, emergent, submersed, and floating aquatic

species were collected for analysis from the littoral zone.

Leaves from two woody emergent species in the littoral

zone of the Lake Placid tributary (common reed and

elderberry) and one woody emergent species in the

littoral zone of Alexander Spring-Run (water hemlock)

also were collected and analyzed. In the shallow channel

of Alexander Spring-Run’s upper reaches, dense stands

of filamentous algae were anchored on both mineral

substrate (e.g., carbonate rock) and on rooted, submersed

native vascular macrophytes (eelgrass). Macrophyte

samples from those shallow, flowing-water areas were

collected and evaluated with littoral zone samples from

this location.

Because the Lake Placid study area included three types

of potential sources for anthropogenic nitrogen contamination (feedlot manure, septic tank effluent, and

inorganic fertilizers) in surficial ground water at various

locations, numerous sample sites were required there

(1–17, as shown in Figure 4). Samples were collected from

various sites within the littoral zone of Lake Placid,

from small, natural tributaries near the southern end of

the lake, and from the dairy-waste lagoon in the upper

reach of one of those tributaries. Sample sites were selected

based on areas with uncharacteristically dense stands of

macrophytes and comparison areas. Sites were associated

with a lakeside conference center and homes (Sites 1 and

9, respectively); former/existing orange groves (Sites 4

and 5, respectively); naturally-vegetated shoreline without homes, groves or industry (Sites 2 and 3), including the

state-owned Tobler Tract natural area (Sites 6–8, 10–12, 16

and 17); and the dairy operation south of the lake (Sites 13–

15). The invasive macrophytes at Site 8 were so expansive

that four subsites (8a–d) were sampled at that location.

With the exception of Wakulla Springs and two

potential sources at Bulow Creek, the remaining study

areas included a single potential source of anthropogenic

nitrogen contamination via the surficial aquifer. Because

fewer sample sites were required at those study areas and

the sample sites were in closer proximity than those at

Lake Placid, data for the remaining study areas were not

Bacchus and Barile

356 Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369
reported by individual sample sites, but were compiled

for each study area.

Samples consisted of representative subsamples from

three plants for each species sampled. The initial subsamples (2002) were collected from the entire watercolumn portion of each submersed and floating aquatic

species (e.g., leaves, stems, and roots, except for roots

of macrophytes growing in the bottom substrate). For

extremely small plants (e.g., duckweed, Lemna minor),

;12 entire plants were required for each sample. For

emergent species with only leaves extending above the

water, and emergent species with woody stems, only

leaves were collected for analysis. For plants with

elongated, strap-like leaves (e.g., cattails), only the apical

portion (;15 cm) was collected. Entire leaves were

collected for all other emergent macrophytes.

After collection, the plant material was held in a sealed

cooler with ice during transport. After transport (,12

hours), samples were rinsed with deionized water, sorted,

and combined into a composite sample for each species.

The composite samples were dried for 48 hours at 608C.

Dried tissue was ground to a fine powder with a mortar

and pestle and stored in labeled vials until analysis for

stable nitrogen isotope ratios, using a Carlo-Erba N/A

1500 Elemental Analyzer and a VG Isomass mass

spectrometer with Dumas combustion. Dried samples

from subsequent samples, which were not analyzed

promptly after drying, were frozen until analysis. The

initial 100 samples were divided into sample replicates

prior to transfer to the laboratory, with samples analyzed

in duplicate to test for analytical precision (60.02).

Based on those results, subsequent samples were not

subdivided into sample replicates (n1⁄42–4). The standard

used for d15N analysis was N2 in air. The values for

d15N (&) were calculated as [(Rsample/Rstandard)  1] 3

103

, where R is equal to 15N/14N.

Data from initial samples from this study and from

subsequent data (Bacchus, unpublished) suggested that

d15N values may vary in different plant tissue (e.g., roots

vs. leaves) for the same location and sample event.

Therefore, leaf samples from species with dense watercolumn root systems (i.e., water lettuce and water

hyacinth) were not combined with root samples after

the 2002 sample period. Evaluations of Lake Placid sites

were made by combining the analytical results for all

species from each respective sample collection site.

RESULTS AND DISCUSSION

Ocala National Forest

Alexander Springs and Spring-Run

Results of FDEP’s single-point water-quality sample

events led to agency conclusions that all ground water

discharging at Alexander Springs was low in nutrients

and lacking bacteriological contaminants (Thorsen, 2002).

Samples in the FDEP study were collected by attaching

weights to tubing ‘‘which was then lowered into the

spring vent opening, ensuring the intake line was not

influenced by surrounding surface water’’ (Scott et al.,

2002, p. 13). A different FDEP sampling methodology

occurred at Wakulla and Homosassa Springs, where

samples were collected from pre-set pipes running into

the cave systems containing the spring vents (Scott et al.,

2002). The sampling methodology in the FDEP study

ensured that results were indicative only of water from

the spring’s primary discharge conduit in the deeper,

Floridan aquifer and did not reflect discharges from the

overlying sandy surficial (local) aquifer.

The FDEP report included results for a single sample

collected on September 12, 2001, with values for

unfiltered and filtered NO3 þ NO2 samples, reported as

0.04 and 0.044 mg/L (total nitrogen), and for unfiltered

and filtered NH3 þ NH4 samples, reported as 0.01 and

0.01 mg/L (total ammonia), respectively. The latter

values represented the method detection limits. Previous

water samples collected at Alexander Springs in 1946 and

1972 did not include values for those parameters, except

for a value of 0.03 mg/L for NO3þNO2. A similar, single

point sample was collected at the Alexander Springs vent

on July 18, 1995, in the study conducted by the

SJRWMD (Toth, 1999). The nitrate-nitrogen concentration for that sample was 0.07 mg/L. The samples from

both of those studies were below the 0.2 mg/L threshold

that was used to identify elevated nitrate-nitrogen in the

water samples (Toth, 1999).

Bacterial results from the FDEP study included

Escherichia coli, Enterococci, fecal coliform, and total

coliform, and have been used by the U.S. Forest Service

as a determination that the public swimming area

surrounding the spring and the down-stream waters

(Alexander Spring-Run) were free from bacterial contamination indicative of human sewage (Thorsen, 2002,

and unpublished records). All of the bacteriological

samples collected from Alexander Springs in the FDEP

study, however, exceeded the holding time limit.

Therefore, those results were not reliable.

Elevated d15N values (þ8 to 12&) in macrophytes

from Alexander Springs and Spring-Run (Figure 5) in our

study were similar to d15N values (þ8.7 to 9.9&) in

macroalgae from sewage-polluted coastal areas, such as

the east-central Florida coast and Boston Harbor (Barile,

2004). The lowest values of ;þ6 to 7& in the eastcentral Florida coastal study ‘‘were measured in October

and December, but are still within the ranges reported for

nearby sewage nitrogen sources’’ (Barile, 2004). The

total nitrogen load for the two counties included in the

coastal east-central Florida study by Barile (2004) was

1.94 tons/day, based on information from agency records.

Subsurface Anthropogenic Nitrogen Discharges

Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369 357
Therefore, the results for Alexander Springs suggest that

human sewage effluent was the dominant source of

nitrogen supporting the growth of the invasive macrophytes in the ecosystems at this study area surrounding

the spring.

The extensive harmful algal bloom throughout the

Alexander Springs swimming area was re-sampled on

October 21, 2004, as a comparison to the original

samples. The d15N value (þ10.2&) for filamentous

macroalgae was comparable to the original values for

Alexander Springs and Spring-Run. All samples collected

at this study area were collected during the season when

the swimming area is least used by swimmers and

waders. Those elevated d15N values are consistent with

d15N values reported by Toth (1999) for water samples

from nearby springs and watersheds contaminated with

nitrogen from septic tank effluent.

The extent of the harmful algal bloom in the

Alexander Springs swimming area is represented by the

dark area in Figure 2B, and comprises ;90 percent of

the swimming area. Attempts by visitors to avoid contact with the area of invasive macrophytes have been

observed during each inspection of Alexander Springs

for the past four years. Based on observations during

inspections and sampling, swimmers and waders avoided

the area where the flocculent algae and other invasive

macrophytes were. That limited area is the light-colored

area (bare sand) in Figure 2B. That area is located near

the steps entering the southwestern portion of the

swimming area where the visitor is seated. Figure 2D

illustrates the magnitude of the harmful algal bloom in

the northern perimeter of the swimming area, which is not

used by swimmers or waders. No exposed sand substrate

is visible in that area.

The watershed surrounding this study area is comprised of the ONF, with the only development being the

U.S. Forest Service’s Alexander Springs Recreation Area

and commercial concession buildings. Those conditions

suggest a single anthropogenic source of contaminant

discharge to those ecosystems from the surficial aquifer.

Those conditions also minimize the potential for

contaminant introduction via the deeper, regional aquifer

system that was sampled by the SJRWMD (Toth, 1999)

and FDEP (Scott et al., 2002).

The drainfield at the recreation area includes underground injection pipes that discharge sewage effluent

and other fluids (e.g., grey water) and solids from the

Alexander Springs Recreation Area camping locations

and commercial concession buildings. The land-surface

elevation of the drainfield is ;13 m (;40 ft) higher than

the land-surface elevation on the south side of the

swimming area. In Florida’s sandy, surficial aquifers, the

water table generally is near land surface at low

elevations, but is regarded to be a subdued reflection of

the topography at higher elevations (Toth, 1999).

The drainfield is located ;500 m (;1500 ft)

southwest of the swimming area, within the ‘‘buffer

area’’ identified by the Florida Natural Areas Inventory

(2004) for the protection of rare plant and animal species

and rare wetland communities associated with Alexander

Springs and Spring-Run. Several of the animal species

are listed by the state as threatened or species of special

concern, and four endangered vascular macrophytes,

including endemic species, are known to have been

associated with Alexander Spring and Spring-Run

(Florida Natural Areas Inventory, 2004). Walsh (2001)

provides more information for some of the rare and

endemic species that have been reported from Alexander

and other spring systems in Florida.

An enlarged view of the extensive invasive filamentous

algae in the swimming area is shown as dark flocculent

areas in Figure 2C, with faint ripples in the lower right

corner. The ripples suggest surficial groundwater discharge and are consistent with the thick clumps of invasive

algae along the shoreline wall downgradient of the

recreation area drainfield, based on land-surface elevations. The image in that figure was taken at the

southeastern end of the wall depicted in Figure 2B. The

algae is most concentrated adjacent to the wall at that

location. Historically the swimming area contained no

macrophytic algae, as was the case for similar Florida

springs evaluated in this study and elsewhere in Florida

(Florida Natural Areas Inventory and Florida Department

of Natural Resources, 1990; Bacchus, unpublished data).

Fogg et al. (1998, p. 422) documented that, in ‘‘contrast

to the other source types which are more areally extensive,

a septic tank is a point source that is itself heterogeneous,’’

Figure 5. Stable nitrogen isotope values (d15N mean 6 SD) for

macrophytes collected from Alexander, Juniper, Orange Grove,

Peacock, and Wakulla Springs; Alexander Spring-Run; and Bulow

Creek.

Bacchus and Barile

358 Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369
but the d15N values of the water samples ‘‘remain fairly

constant with depth after reaching the water table (;11

m).’’ That conclusion was based on their sample

collection borehole ‘‘drilled between two leach lines

spaced 7.5 m apart in the septic tank drainfield,’’ which

suggested that ‘‘solute plumes emanating from each drain

line did not spread far enough horizontally to intersect the

borehole location until a substantial depth was reached’’

(Fogg et al., 1998, p. 422). That depth was reported as

;6 m and is similar to the depth to the water table

reported at the Alexander Springs drainfield.

The d15N values of water samples from boreholes

associated with onsite sewage disposal sources in the

study by Fogg et al. (1998) approximated the lower

values for sewage effluent provided by Heaton (1986),

and were consistent with the values of our macrophyte

samples from the ONF study areas (Figure 5). The

combined d15N values for macrophytes from Alexander

Springs and Spring-Run shown in Figure 5 were

consistent with the þ8.6& and þ8.9& d15N values for

water samples collected from the spring vents at Wekiva

and Ponce de Leon Springs, respectively, which were

attributed to contamination by sewage, animal waste, or

a combination of both (Toth, 1999).

Wekiva and Ponce de Leon Springs are surrounded by

state lands, primarily composed of natural forested areas.

The springsheds for those two springs are urbanized, with

additional forested areas, and generally lack industrial

animal feed-lot operations. Those land-use characteristics,

combined with the rapidly increasing underground injection

of sewage effluent in Florida, suggests that the source of

nitrogen at Wekiva and Ponce de Leon Springs is sewage

effluent rather than animal waste. Our Alexander Springs/

Spring-Run study area is ;20 km (;12 mi) west of Ponce

de Leon Springs and ;43 km (;27 mi) north of Wekiva

Springs (see the Figure 2 location map from Toth, 1999).

Macrophyte taxa and growth forms at this Alexander

Springs/Spring-Run study area have shifted from the

historic sparse, rooted, native submersed aquatic species

(eelgrass), without epiphytic macroalgae, to opportunistic

invasive macrophytes. These invasive, primarily alien

species are not rooted in the substrate, and thus derive all

nutrients from the water column (e.g., duckweed,

filamentous algae, hydrilla, water hyacinths, and water

lettuce). This shift in taxa and growth forms, combined

with the abundance and location of opportunistic invasive

and alien macrophytes in this study area, are consistent

with anthropogenic nutrient-loading. The range in d15N

values at this study area may be attributed to differences in

uptake by different species (rooted vs. floating aquatics),

or to the U.S. Forest Service’s conversion from a package

STP to sewage treatment by a septic tank drainfield. The

extent of the adverse impact on rare and endemic species

from the combined nitrogen contamination and shift to

invasive macrophytes has not been determined.

The conditions described above, in conjunction with

comparably elevated d15N values from multiple sample

events at this study area, support the conclusion that

groundwater discharges from the sandy, surficial aquifer

on the southern side of the swimming area are the

primary source of nitrogen contamination in Alexander

Springs and Spring-Run. The ripples in the lower right

corner of Figure 2C also suggest that surficial groundwater discharge is occurring at discrete points, in addition

to diffuse surficial aquifer discharges throughout the

shallow swimming area.

Juniper Springs

At Juniper Springs, the other ONF spring study area

evaluated, no water-quality data were available from the

FDEP study. Duplicate point samples, however, were

collected from the vent at Juniper Springs on April 29,

1996, by the SJRWMD. Nitrate-nitrogen concentrations

for those duplicate samples were 0.07 and 0.08 mg/L,

below the 0.2 mg/L threshold established for that study

(Toth, 1999). The studies by FDEP and SJRWMD did not

include any of the additional areas evaluated in our study.

Macrophyte samples were collected from an abnormally dense stand of eelgrass, a native rooted submersed

macrophyte. The elongated leaf blades of the eelgrass were

covered with thick epiphytic invasive filamentous algae,

similar to those at the ONF Salt Spring Marina (Figure 2E).

The typical appearance of eelgrass is shown in the leaf tip

in the foreground of Figure 2E, with the algae removed.

Duckweed, the invasive floating-leaf aquatic macrophyte

near the base of the eelgrass leaf tip (foreground),

approximates the size of the date on the penny.

Ultimately those invasive epiphytes will prevent light

from reaching the leaf blades of the native macrophytes

and result in the demise of the eelgrass community, as has

occurred at Alexander Springs. The source of anthropogenic nitrogen at our Juniper Springs study area appears to

be consistent with the source at Alexander Springs, via the

localized, surficial groundwater flow system. The low

concentrations of nitrate-nitrogen from the spring vent

sampled by Toth (1999) and the endpoint values for septic

tank effluent from his studies conducted in the vicinity of

our study areas provide additional support for the conclusion about the sources of nitrogen contamination at the

ONF study areas and the assumption that fractionation has

not occurred in our macrophyte samples.

Springs on State Lands

Orange Grove and Peacock Springs

The d15N results from Orange Grove and Peacock

Springs in Figure 5 also represent the combined data from

the species present at each of those two relatively small

Subsurface Anthropogenic Nitrogen Discharges

Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369 359
study areas. The range in d15N values at Orange Grove

Springs, from þ5.2& for stonewort (or musk-grass, an

un-rooted, submersed aquatic) to þ8.8& for yellow cowlily (a rooted emergent), accounts for the largest standard

deviation (SD) observed at all of the springs study areas.

Those differences may be related to differences in growth

form of the two taxa or may represent two different

sources of nitrogen entering Orange Grove Spring.

A d15N value in the range exhibited by the un-rooted

macroalga stonewort is indicative of an organic nitrogen

source such as animal manure. Stonewort is recognized as

an indicator of deep-aquifer discharge (Rosenberry et al.,

2000; Bacchus, unpublished data for Florida), and

preferentially takes up minerals from the spring waters.

Those characteristics suggest potential uptake of nutrients

from those discharges. The abnormally dense mats of

stonewort and yellow cow-lily, combined with the

obviously unsuitable conditions for swimming (microalgal bloom covering the surface of the spring and lack of

flow), the lack of a septic tank and drainfield at this study

area, and the undeveloped watershed immediately

surrounding the spring, all suggest that the source of

nitrogen at this spring is animal waste transported to the

spring via the Floridan aquifer.

No vascular macrophytes were available for sampling

at Peacock Spring. The un-rooted filamentous macroalgae

collected from the Peacock Spring study area had the

lowest d15N signature of the springs that were evaluated in

our study (Figure 5). That d15N signature was comparable

to the signature (þ4.7&) of macroalgae from an inshore

site (Avenue J) at Big Pine Key, in the Lower Florida

Keys. That area has direct exposure to sewage discharges

and values were within the range reported for macroalgae

growing on sewage nitrogen (Lapointe et al., 2004).

A relatively controlled field study in California

reported a d15N signature for inorganic fertilizer in

groundwater samples similar to our d15N value for

Peacock Springs (Fogg et al., 1998). The d15N value of

nitrate in fertilizers, however, ‘‘is generally less than

þ3&’’ (Toth, 1999, p. 41), which is lower than the mean

and standard deviation for the Peacock Springs results.

Unlike the U.S. Forest Service recreation areas at

Alexander and Juniper Springs, the area immediately

surrounding Orange Grove and Peacock Springs has not

been sodded or otherwise developed with lawns, septic

tanks or drainfields. Therefore, the d15N results for the

macroalgal samples from Peacock Springs appear to be

indicative of either animal waste or sewage effluent

entering the spring from the springshed via regional flow,

rather than from the surficial aquifer.

Wakulla Springs

The FDEP springs report included results from a single

point-sample collected on September 27, 2001. Unfiltered

NO3 þ NO2 and NH3 þ NH4 concentrations from that

sample were 0.99 and 0.01 mg/L, respectively, whereas

filtered concentrations were 0.96 mg/L and 0.01 mg/L,

respectively. The unfiltered total-nitrogen value was

estimated and the values for total ammonia represented

the method detection limits. Bacterial analyses of water

samples collected from the Wakulla Springs vent during

that FDEP study were the same as those conducted for

Alexander Springs, again implying that sewage effluent

was not a factor in elevated concentrations of nitrogen.

Those samples, however, also exceeded the holding time

limit (Scott et al., 2002). Despite these shortcomings, the

FDEP results are indicative of anthropogenic nitrogen

discharging from the regional aquifer.

Dye-tracer studies have confirmed rapid groundwater

flow through the regional aquifer system from the vicinity

of the municipal effluent sprayfield in Leon County to

Wakulla Springs (Davies et al., 2004; Hazlett et al., 2004;

and Kincaid et al., 2004). Unlike the springshed for

Orange Grove and Peacock Springs, large-scale industrial

animal feedlot operations are not located in the Wakulla

Springs springshed or watershed.

The results of the dye-tracer studies combined with

elevated nitrogen levels in water samples from the

Wakulla Springs vents suggest that the springshed is

the source of the nitrogen taken up by the dense invasive

macrophytes at the Wakulla Springs study area. The

transport of nitrogen-contaminated ground water is

presumed to occur as conduit flow through the Floridan

aquifer (illustrated in Figure 3), with the probable source

being sewage effluent from the municipal sprayfield in

Leon County.

The d15N signature of the invasive macrophytes from

Wakulla Springs (provided in Figure 5) is comparable to

the lowest d15N values (;þ6 to 7&) measured in

October and December macroalgal samples from the

coastal east-central Florida study by Barile (2004). Those

values were attributed to nearby sewage nitrogen sources.

Lake Placid

Species Comparisons

A comparison of the same macrophyte species at each

of the Lake Placid sites and all study areas could have

provided information regarding potential species-specific

responses. Unfortunately, a comprehensive comparison

of species from all study areas, or even all sites within the

Lake Placid study area, was not possible because species

composition varied among study areas and among sites

within the same study area. Observations during site

inspections and sample collection suggested that vegetative phase shifts had occurred, as described in the section

below. Those observations and knowledge of surrounding land use supported the conclusion that nutrient

Bacchus and Barile

360 Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369
contamination from different sources and of different

magnitudes resulted in different species compositions.

Elderberry (a native rooted species) was one species

present at eight locations in the southern end of Lake

Placid where the industrial dairy operation is located. The

samples of this species, collected on the same date within

relatively close proximity, but exposed to different

conditions, facilitated a comparison of a single species.

Only leaf samples were collected and analyzed, for

reasons described above.

Samples from five of the elderberry collection

locations adjacent to Sites 11, 14, 15, 16 and 17 (Figure

4) were considered as ‘‘controls’’, because the plants were

rooted in an area at the sample site that was presumed not

to be exposed to surface water or shallow ground water

containing anthropogenic nitrogen from any of the

sources identified at the Lake Placid study area. Samples

from the remaining three sites were collected from plants

rooted in areas downgradient of the industrial dairy

operation (Figure 4, Sites 10, 12 and 13). No septic tanks

were located in proximity to Sites 12 and 13. Site 10 was

located upgradient (southwest) of homes with septic

tanks (Figure 4). Based on contour elevations from U.S.

Geological Survey 7.5 minute topographic maps of the

area, the approximate land-surface elevations for Sites

10, 11, 12, 13 and 15 (all downgradient from the dairy

operation) were ;37.5 m, ;38.7 m, ;40.5 m, ;41.8 m

and ;42.6 m (;123 ft, ;127 ft, ;133 ft, ;137 ft and

;140 ft), respectively.

A summary of the d15N analysis results for the

comparison of elderberry is provided in Table 2. The

mean for the samples identified as ‘‘controls’’ wasþ1.8&

(60.6&), whereas the mean for the samples presumed to

be exposed to surficial aquifer water containing nutrients

from the industrial dairy operation or possibly septic tank

leachate was þ9.7& (62.3&). The d15N range of þ1.0

to 2.5& for elderberry in the ‘‘control’’ group (Sites 11,

14, 15, 16 and 17) is presumed to represent natural fixed

nitrogen in the soil. Fogg et al. (1998, p. 425) found that

the ‘‘mean d15N for the soil organic matter sites was

about þ2.5&’’ which is similar to the ‘‘control sites’’ for

the rooted emergent macrophytes evaluated at the south

end of Lake Placid. The ‘‘fertilized’’ sites in their study

had both higher (;3.5&) and lower (~1.5&) values than

the soil organic matter values in their study. Therefore, it

is possible that results from some of the ‘‘control’’ sites

(e.g., Sites 16 and 17) may include input from an

inorganic nitrogen fertilizer source, such as orange

groves. In comparison, 21 samples of tropical macroalgal

samples from relatively unpolluted coastal waters of

southwestern Puerto Rico had a mean d15N of þ0.5&

(61.0&), indicative of nitrogen fixation as the source of

nitrogen supporting their growth (Lapointe et al., 2004).

The d15N range for the remaining elderberry speciescomparison sites wasþ6.4 to 12.3&. Bacteriological and

nutrient results from the Highlands Soil and Water

Conservation District’s water samples (referenced

above), which were collected from the same tributary

locations as sites 12 and 13 in our study, supported the

conclusion that those tributary locations contained

discharge from the dairy-waste lagoon south of Highway

70. The results of those water samples, combined with the

absence of septic tanks in that vicinity, support the

conclusion that dairy waste is the source of the elevated

d15N signature in elderberry leaves collected from Lake

Placid Sites 12 and 13 in our study.

The increase in mean d15N value from þ7.6& at Site

13 to þ9.6& downgradient at Site 12 approximates the

2.7& increase attributed to 10 percent denitrification of

NO3 by organic soils under controlled laboratory

conditions, described by Fogg et al. (1998). Our study

areas at springs that appear to have shallow groundwater

sources for nitrogen contamination (described above)

lack significant organic soils between the apparent source

of contaminant discharge to the surficial aquifer and the

downgradient surface-water sample sites. Organic soils

are associated with the extensive forested seepage

wetland (swamp) at the southern end of Lake Placid,

however, between the dairy and Lake Placid. Denitrification may be occurring in those organic soils as a plume

of nutrient-laden ground water originating from the dairywaste lagoon flows laterally through the surficial aquifer

toward Lake Placid. That scenario could account for the

elevated mean d15N values of þ9.6& and 12.0& at Sites

12 and 10, respectively, in elderberry that was growing in

the vicinity of the tributaries at those sites. A less likely

scenario is that septic tank leachate from homes along the

shore in the vicinity of Placid View Drive is flowing

Table 2. Mean 6 SD stable nitrogen isotope (d15N, N 1⁄4 2) values

for the rooted native macrophyte Sambucus canadensis L.

(elderberry) from control and nutrient-source sites associated with

Lake Placid, Florida.

Site1 Site Description1 d15N

Controls

11 control tributary channel, W branch þ2.52

14 swamp, SR 70 W of dairy þ1.6 6 0.4

15 roadside ditch, SR 70 E of dairy þ1.2 6 0.2

16 roadside ditch, SR 70, S Lake Annie þ2.2 6 0.2

17 roadside ditch, SR 17, N Lake Annie þ1.9 6 0.8

Mean þ1.8 6 0.6

Nutrient-Source

10 ditch, Placid View Drive þ12.0 6 0.3

12 dairy waste tributary, downstream/N þ9.6 6 2.5

13 dairy waste tributary, SR 70 þ7.6 6 1.2

Mean þ9.7 6 2.3

SR 1⁄4 State Road. 1

As shown in Figure 4.

2

Sole duplicate with negative value discarded as outlier/error.

Subsurface Anthropogenic Nitrogen Discharges

Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369 361
southwest, against the topographic gradient, to the

shallow root zone of elderberry sampled at Site 10.

Site Comparisons

Results of d15N analysis from the numerous species

collected from the 17 Lake Placid sample sites were

clustered into three groups characteristic of the three

categories of potential nitrogen discharge at this study

area (Figure 6). The group with the highest d15N

signature (;þ14&) was indicative of a mid-range value

for septic tank leachate (Peterson and Fry, 1987). That

group included macrophytes collected from a dense stand

in the littoral zone downgradient of a home on the

southwest shore of the lake (Figure 4, Site 9). No

comparable growth of macrophytes was observed in the

vicinity of any other homes on Lake Placid.

The group with the lowest d15N signature (;þ3& or

less) included the range of nitrogen from inorganic

fertilizer sources. As an example, Fogg et al. (1998,

p. 423) evaluated a site that had been under agricultural

production for more than 150 years and ‘‘augmented

annually with up to 250 lb N/acre from commercial

fertilizers.’’ The d15N results at that site for the ‘‘5

boreholes were consistent with each other, in addition to

being nearly uniform with depth.’’ The d15N values of

soil water at that location ranged from a low of 1.5&

to a high of þ4.3&. That range also includes natural

fixed nitrogen, however, as illustrated in the species

comparison discussion above. The mean d15N value for

natural fixed nitrogen at Lake Placid appears to be

þ1.8& 6 0.6 (Table 2). Those data suggest that some

of the sites included within the ‘‘fertilizer’’ group in

Figure 6 (e.g., Site 7) may reflect natural fixed nitrogen.

That group included plants collected from the Lake

Placid littoral zone (Figure 4, Sites 3, 5, 6 and 7), in

addition to tributaries/ditches (Figure 4, Sites 11, 14, 16,

and 17).

The remaining Lake Placid sites exhibited a d15N

signature attributed to organic nitrogen, presumably dairy

waste (e.g., manure) from the industrial dairy feedlot

operation upgradient of Lake Placid. That group included

plants collected from the Lake Placid littoral zone (Figure

4, Sites 1, 2, 4, and 8a–d) and tributaries/ditches (Figure

4, Sites 10, 12, 13, and 15).

The surrounding land at Sites 2, 6, 7, 8, 10, 11, 12, 16

and 17 primarily was naturally vegetated, without homes

or groves, or surface runoff from those land uses.

Samples from Sites 11, 14, 16 and 17 may have received

nitrogen from stormwater runoff. Samples from the

tributary flowing north from Lake Annie (Site 16) and

the associated downstream wetlands (Site 17) also may

be receiving nitrogen from groves located ;1 km (;0.5

mi) northeast, via lateral flow in the surficial aquifer.

Results of elderberry samples from sites listed in Table

2 are inconsistent with results provided for some of those

sites shown in Figure 6. This is because Site 11 was

the only species-comparison location where elderberry

plants (Table 2) were growing in the surface water (e.g.,

tributary channel) with the remaining macrophytes

evaluated and reported in Figure 6.

The isotopic signatures of the macrophytes from

various sample collection sites shown in Figure 6 suggest

that organic nitrogen from the dairy operation in the

southern portion of the watershed is entering Lake Placid

more than 3 km (;2 mi) downgradient (north) and

contributing to nitrogen contamination of the lake.

Nitrogen from this dairy feedlot operation appears to be

entering the lake via shallow groundwater discharge, as

well as via the natural tributary that flows from the dairy

into Lake Placid (Site 8a).

The conclusion described above is supported, in part,

by the pattern of macrophyte growth in the southern

portion of Lake Placid (Sites 8b–d), which is similar to

growth patterns associated with groundwater seepage

described by Rosenberry et al. (2000). This conclusion

also is supported by the significant shallow groundwater

seepage that was observed flowing out from under and

through the berm constructed at the dairy waste lagoon

and other areas along the northern perimeter of the

property containing the dairy operation.

Additional support for the magnitude of contaminated

discharge from the dairy operation was provided by

water-quality analysis conducted by the Highlands

County Lake Management Program. For example, on

August 23, 2001, NO3 þ NO2 was 0.75 mg/L and total

nitrogen was 3.48 mg/L in water samples collected at the

mouth of the primary tributary receiving discharges from

the dairy, in the vicinity of our Site 8a (Ford, 2002a). The

0.75 mg/L value for NO3þ NO2 from Lake Placid, in the

vicinity of the dairy, is comparable to the values for

Figure 6. Stable nitrogen isotope values (d15N mean 6 SD) for

macrophytes collected from sample sites associated with Lake Placid.

Bacchus and Barile

362 Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369
Wakulla Springs water samples collected from the spring

vent (Scott et al., 2002) and attributed to groundwater

input from the Leon County municipal sprayfield.

Nitrogen-contaminated discharges from the referenced

dairy also could be responsible for the isotopic signatures

of ;þ6& at Sites 1, 2, and 4, in the littoral zone of the

northern shore (Figure 4). The transport mechanism could

be wind-driven surface discharges from the tributary in

the vicinity of Site 8a or focused (discrete) subsurface

discharges. The d15N signatures for samples from the

dairy waste lagoon (Site 15) and the tributary mouth (Site

8a) are similar to the d15N means and standard deviations

for Sites 1 and 2 along the north shore of Lake Placid

(Figure 6). The greater standard deviation for samples

from Site 4 may be the result of groundwater discharge of

inorganic fertilizers applied before the orange grove

operation at that location was abandoned.

These results, suggesting extensive contamination of

Lake Placid from the industrial dairy operation, are

consistent with results of water-quality samples collected

by the Highlands Soil and Water Conservation District.

No sampling was conducted or required by FDEP to

determine the cause of the harmful algal blooms or

the increasing abundance of invasive macrophytes in

Lake Placid.

Bulow Creek

Results and Land-Use During Sample Collection

The more extensive data set for Lake Placid facilitates

interpretation of d15N for invasive macrophytes collected

in Bulow Creek. Results from Bulow Creek are included

with results from the spring-run stream and springs study

areas in Figure 5 and are within the range of both animal

waste (manure) and human waste (sewage effluent).

Those results represent the combined samples from the

mouth of a small tributary ;1.6 km (;1 mi) north of

the state historical site and from the opposite side of the

Bulow Creek channel ;1.6 km south of the state

historical site. The mouth of the tributary is shown as

a small protrusion from the western channel of Bulow

Creek, north of the green area labeled ‘‘Bulow Creek

State Park’’ on the Tomoka Marsh Aquatic Preserve map

(provided at http://www.dep.state.fl.us/coastal/downloads/

maps/default.htm).

The most probable source of nitrogen in the northern

sample subset is the cattle pasture in the flood plain west

of Old Kings Road (County Road 4011 on the Tomoka

Marsh Aquatic Preserve map). The d15N signature for

that subset was comparable to the d15N value for the

tributary discharging water to Lake Placid from the

industrial dairy operation (Figure 5, Site 8a).

No other large-scale or concentrated animal feedlot

operations are associated with the Bulow Creek portion

of the watershed. That operation appeared to include

fewer cows than the 320 cows reported by FDEP (FDEP

Case No. FLA284611, Administrative Agreement,

January 29, 2002) at the industrial dairy operation

adjacent to the Lake Placid study area. The Bulow Creek

cattle operation, however, was located closer to the water

body. The transport of animal wastes to Bulow Creek

from that source could be via shallow groundwater flow,

with contaminant access increased by mine pits excavated into the surficial aquifer in the cattle pasture.

Aquifer excavation has been shown to increase the threat

of groundwater contamination and lateral groundwater

flow (Zamora et al., 2003; Wilcox et al., 2004).

The southern sample subset at Bulow Creek was

collected from the opposite side of the stream channel at

an equal distance downstream of the state historical site

and adjacent to a single-family residence situated ;120 m

(;400 ft) east of the Bulow Creek stream channel. The

house was constructed in the early 1980s. A dense marsh

composed of native plant species occupies the littoral

zone from the natural channel of Bulow Creek to the

adjacent single-family residence. That sample subset was

within the lower range of sewage effluent.

One possible scenario is that leachate from the septic

tank drain field at that house flows through the littoral

zone marsh, and that nitrogen loads recently exceeded the

uptake capacity of the marsh plants and are discharging to

the surface waters of Bulow Creek. A second, more likely

scenario is that a discrete leachate plume is flowing

through the surficial aquifer, beneath the root zones of

those marsh plants, and recently began discharging

beneath the surface of Bulow Creek at that sample

collection site. The latter scenario is supported by the fact

that septic tank leachate augments groundwater flow in

a localized area of the surficial aquifer and could result in

focused subsurface point-source discharges to Bulow

Creek. Similar focused discharges appeared to be

occurring along the southern shoreline of Alexander

Springs, downgradient of the ONF drainfield. Comparable focused recharge was reported under natural depressional wetlands receiving wastewater in Texas, along

with other preferential pathways, such as through

desiccation cracks in the clay (Fryar et al., 2000).

Analogous natural spring discharges have been observed

along littoral zones in other ecosystems (Rosenberry et

al., 2000).

No additional houses or STPs were located within

;1 km (;0.5 mi) of the sample locations on either side

of Bulow Creek at the time of our study. The discrete

mass of invasive macrophytes at both locations where

these Bulow Creek subset samples were collected suggests that significant nitrogen sources were associated

with those two locations, similar to the discrete plumes

described by Fogg et al. (1998) and referenced above.

Other potential sources for the elevated d15N signaSubsurface Anthropogenic Nitrogen Discharges

Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369 363
tures at those locations in Bulow Creek occur in the

watershed between the two locations for the Bulow Creek

samples. Those potential sources include ;30 houses on

the east side of Bulow Creek in Flagler County, although

none are in proximity to the southern sample location. On

the west side of the creek, a recreational vehicle (RV)

park dating from the 1960s, with a mobile home park

added in the 1980s, is located along the northern

boundary of the state historic site. A mobile home park

adjacent to the southern boundary of the state historical

site was constructed in the late 1990s. The package STP

for that mobile home park is located ;1.2 km (;0.8 mi)

west of the Bulow Creek stream channel. The state

historical site includes public bathrooms and a ranger

residence that also may discharge sewage effluent to the

surficial aquifer, ultimately flowing into Bulow Creek.

The spatial separation of the potential sources and the

discrete clusters of invasive macrophytes at this study

area were similar to those at the Lake Placid industrial

dairy operation. Unlike the absence of other potential

nitrogen sources from the watershed in the vicinity of

the Lake Placid dairy operation, however, the watershed

in the vicinity of the Bulow Creek study area includes

numerous potentially large sources of human waste.

Therefore, additional research is required to confirm

that nutrients from the cattle operation are entering

Bulow Creek via surficial groundwater discharge. Additional information also is needed to confirm sources

and flowpaths for the sewage-effluent nutrient load to

Bulow Creek.

Permitted and Proposed Land-Use Changes

Among our study areas, the Bulow Creek watershed

has the most extensive permitted and proposed land-use

changes. Since this study was conducted, the cattle

operation is being replaced by a residential subdivision of

;750 homes proposed for the area surrounding the open

pit. Permits have been issued to expand the mobile home

park bordering the northern boundary of the state

historical site, as well as for the construction of a 108-

unit subdivision directly across from the mobile home

park that borders the state park on the north (SJRWMD

Permit Number 4-036-87306-1). A second subdivision

(reported as ‘‘200 acres’’) is proposed for 40 houses

adjacent to this proposed 108-unit subdivision (unpublished SJRWMD permit document). Additional land

on the west side of Bulow Creek is under the same

ownership and subject to similar development. If any of

these land-use changes occur, subsequent sample events

may provide significantly different results than those

reported in this study.

Unlike the lower reaches of Bulow Creek, where an

expansive littoral zone marsh occurs and can buffer

nutrient inputs to Bulow Creek, the area where highdensity developments have been permitted or proposed

lacks a similar buffering wetland. Individual septic tanks

and wells were proposed for all of the houses in the highdensity developments. Based on the apparent potential

for a single septic system on the east side of Bulow Creek

to support the growth of invasive macrophytes occurring

at the adjacent (downstream) sample collection site,

significant eutrophication is predicted to occur throughout Bulow Creek and in downstream coastal waters if

those developments proceed as permitted or proposed.

Fractionation, Denitrification and Lateral Flow

The assumption that the d15N values were not fractionated in plant material evaluated in this study is

supported by the fact that the Toth (1999) endpoint

values match the values in the macrophyte samples from

our springs study sites. This assumption also is supported

by the determination that fractionation in marine macrophyte samples is minimal (Barile, 2004). Isotopic fractionation can occur in water and soil samples collected

for d15N analysis that are not properly preserved during

the time between collection and analysis. Fogg and others

(1998) conducted a long-term storage study on frozen soil

cores to test the means of preserving samples recommended by the U.S. Environmental Protection Agency as

the best preservation technique available, refrigeration of

samples near freezing or below. Their study showed that

‘‘no significant changes occurred in either the NO3 concentration or d15N during the 56-week interval’’ (Fogg

et al., 1998, p. 420). The macrophyte samples in our

study were transported on ice, then dried and analyzed, or

dried and frozen until analysis if analysis was delayed.

Therefore, the assumption that fractionation in our macrophyte samples was insignificant should be valid.

Denitrification can result in an increase in d15N values.

Ground water from five irrigation wells sampled by Fogg

and others (1998, p. 424), however, revealed that

‘‘significant denitrification is occurring only in localized

pockets of the subsurface.’’ The results from those

irrigation wells were consistent with results from the

vadose zone for fertilizer signatures in their study.

Fogg and others (1998) also discussed scenarios other

than denitrification that could lead to increasing d15N

with depth. One scenario was ‘‘transient transport in

which elevated d15N deeper in the profile is due to an

older source that has since been replaced at the surface

by a source having lower d15N.’’ Another scenario they

described was ‘‘lateral flow contributing higher d15N

from off site.’’ That scenario was determined to be

plausible ‘‘if the off-site source is fairly close to the

profile or if perched conditions exist’’ (Fogg et al., 1998,

p. 421). Perched conditions could exist for the surficial

aquifers in our study. Denitrification is not considered to

be a factor in our sites characterized by lateral flow

Bacchus and Barile

364 Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369
through the sandy surficial aquifer prior to discharge into

surface waters. Exceptions include cases in which the

ground water may pass through extensive wetland areas

with organic soils, such as in the southern end of Lake

Placid.

Vegetative Phase Shifts

The sites evaluated in this study are typical of naturally

oligotrophic ecosystems in the southeastern Coastal

Plain. These ecosystems are characterized by the

occurrence of few macrophyte species that grow in

sparse stands when present, primarily as the result of low

nutrient concentrations in both the water column and

substrate. Examples of un-rooted macrophytes adapted to

grow under such naturally nutrient-limited conditions

include carnivorous species of Utricularia (bladderwort)

that capture and digest tiny organisms from the water

column as a source of nitrogen (Bosserman, 1979;

Godfrey and Wooten, 1981). Eelgrass is an example of

a rooted macrophyte adapted to these naturally nutrientlimited conditions. All are vascular macrophytes.

Vegetative phase shifts that are a consequence of

anthropogenic nutrients can occur as either an abnormal

abundance of macrophytes characteristic of these naturally nutrient-limited conditions, or as the occurrence of

invasive macrophytes. Conversely, the macrophytes

involved in these shifts can be used as indicator species

for anthropogenic nutrient contamination. In a study that

accounted for aerial deposition of nutrients, Stinner

(1983) found that duckweed did not occur naturally in

those oligotrophic aquatic ecosystems of the southeastern

Coastal Plain. The predominant species of duckweed that

occurred in the elevated nutrient plots in that study was

Lemna valdiviana, the same invasive species that was

predominant in our study areas. Biomass of submersed

macrophytes (Utricularia and Cabomba) has been shown

to increase significantly when exposed to fertilization,

compared to reference aquatic ecosystems. Invasive

submersed macrophytes such as Cabomba have been

shown to exhibit directional responses to anthropogenic

sources of nutrients, establishing sufficient biomass

within two years of nutrient additions to result in

an ‘‘overwhelming proportion’’ compared to naturallyoccurring Utricularia. As a rooted submersed macrophyte, Cabomba is able to acquire nutrients from both the

water column and the sediments (Sculthorpe, 1967).

Both chronic and acute anthropogenic nutrient discharges to naturally nutrient-limited ecosystems, including advanced wastewater treatment discharges, have

resulted in vegetative phase shifts. Point-source surface

discharges of sewage effluent (Dierberg and Brezonik,

1978) and sewage sludge (Valiela et al., 1975) to

wetlands (also considered aquatic sites) resulted in such

shifts, including species replacement compared to control

sites. Similar vegetative phase shifts should be expected

to occur from subsurface point-source and non-pointsource discharges of sewage effluent to aquatic sites in

our study areas, particularly Alexander, Juniper, and

Wakulla Springs.

The stages of vegetative phase shifts appear to be, from

initial to final stage: (1) abnormal abundance of native

vascular macrophytes adapted to naturally nutrient-limited

conditions; (2) occurrence of invasive non-vascular

macrophytes suspended in the water column, or as epiphytes covering desirable native macrophytes (Figure 2E)

and typically bare submerged substrate (Figure 2B and

2C), particularly when nutrient discharges occur as

subsurface discharges; (3) occurrence of invasive macrophytes on the water surface or in the water column (Figure

2A and 2D), primarily as floating or submersed growth

forms, and rooted emergents; and (4) coverage of ;50

to 100 percent of the water surface, the water column,

the native macrophytes, and/or the benthic substrate by

invasive vascular and non-vascular macrophytes.

This series of vegetative phase shifts can be used to

describe the stage of the nutrient contamination continuum each study area represented at the time of the

evaluation. Juniper and Orange Grove Springs were in

transition between Stages 1 and 2. Peacock Springs was

in transition between Stages 2 and 3. Bulow Creek would

be classified as Stage 3. The main Alexander Springs

swimming area (south shore, Figure 2C) would be

classified as Stage 2, but the invasive macrophytes

(primarily suspended on the bottom and in the water

column) probably are being displaced by swimmers and

waders. Figure 2B illustrates the less popular southern

portion of the swimming area, whereas Figure 2D is

characteristic of conditions in the unused, northern

portion of the swimming area and the upper reaches of

Alexander Spring-Run, which would be classified as

Stage 4. Wakulla Springs (Figure 2A) also meets the

criteria of Stage 4, despite attempts to control the invasive

macrophytes by herbicides and by mechanical and

manual removal. Conditions of the Lake Placid sites

downslope of the industrial dairy operation also would be

classified as Stage 4. Conditions at the ONF Wildcat

Lake Recreation Area public access (Figure 2F), adjacent

to the self contained waterless public toilets that

contribute no discharge to the lake, are representative of

pre-contamination conditions at the study areas.

SUMMARY AND CONCLUSIONS

Stable nitrogen isotope (d15N) analysis of macrophytes

represents time-integrated data capable of providing

inferences about adverse environmental impacts and the

source(s) of nitrogen contaminants, in addition to

complementing time-specific water-quality data. Using

d15N analysis, we have demonstrated that uptake of

Subsurface Anthropogenic Nitrogen Discharges

Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369 365
anthropogenic nitrogen has occurred in invasive alien and

nuisance native macrophytes exhibiting harmful growth

in Florida’s freshwater ecosystems. We suggest that d15N

analysis of freshwater macrophytes, combined with other

information (e.g., watershed and springshed land-use

information, groundwater flow characteristics, and, to

a lesser extent, hydraulic-head maps and water-quality

analyses based on discrete data) can provide evidence of

nitrogen contamination sources and groundwater flowpaths in other karst aquifer systems.

Study areas were selected in locations of Florida where

special protection of surface waters is required by law.

These included aquatic ecosystems within and adjacent to

the ONF, and state lands, including Aquatic Preserves

and Outstanding Florida Waters, where pre-invasion

conditions of those ecosystems were known. The natural community types evaluated in this study included

springs (Alexander, Juniper, Orange Grove, Peacock and

Wakulla), a spring-run stream (Alexander Spring-Run),

a blackwater stream (Bulow Creek), and a sinkhole lake

(Lake Placid). Invasive macrophytes that were evaluated

in this study included those designated as Category I

and II species by the Florida Exotic Pest Plant Council,

and examples of both invasive alien and nuisance

native species.

Stable nitrogen isotope analysis of macrophytes from

naturally-oligotrophic freshwater aquatic ecosystems in

subregions of the Floridan aquifer system confirmed

contamination from uptake of anthropogenic nitrogen

(manure, sewage effluent and/or inorganic fertilizer).

Those results were augmented with knowledge of historic

(pre-invasion) conditions of ecosystem integrity; characteristics of the selected natural communities and associated aquifer system; potential sources of anthropogenic

bioavailable nitrogen in the watershed and springshed

surrounding each study area; and other available data.

Based on the methods used in our study and values from

anthropogenic nitrogen sources in water samples that

matched macrophyte values in our study, fractionation

above source values was presumed to be negligible. The

results of this study indicate that the federal and state

designations have failed to protect the natural ecological

integrity of the aquatic systems evaluated in this project.

Macrophyte samples from Alexander Springs and

Spring-Run had d15N signatures suggesting contamination with human sewage effluent. A previous study by

FDEP concluded that Floridan-aquifer discharge did not

contribute significant amounts of nutrients to Alexander

Springs. Because of the National Forest designation, the

watershed immediately surrounding the spring is undeveloped, except for the commercial concessionaire

facilities associated with the U.S. Forest Service’s

Alexander Springs Recreation Area. The other ONF

spring study area, Juniper Springs, exhibited early

vegetative phase shift conditions similar to those at the

ONF Salt Spring Marina, where the rooted native eelgrass

is abnormally dense, but the elongated leaf blades are

covered with epiphytic macrophytes. The source of

anthropogenic nitrogen at the Juniper Springs study area

also appears to be lateral flow through the sandy surficial aquifer.

Among springs on state lands evaluated in this study,

the watershed surrounding Orange Grove and Peacock

Springs exhibited the least disturbance, with no on-site

sewage effluent disposal or developed recreation facilities

comparable to those at the Alexander and Juniper Springs

study areas. Macrophytes from Orange Grove Spring had

d15N signatures indicative of organic nitrogen from

animal waste (manure) and possibly from human sources

transported to the spring via the Floridan aquifer. The

invasive, filamentous macrophytic algae analyzed from

Peacock Spring had a d15N signature comparable to that

of macroalgae from an inshore site in the Lower Florida

Keys with direct exposure to sewage discharges, and

within the range reported for macroalgae growing on

sewage nitrogen. Although the source of the nitrogen at

Peacock Springs is not known, the nitrogen appears to be

discharging from the Floridan aquifer.

The invasive macrophytes sampled at Wakulla Springs

had a d15N signature indicative of treated sewage

effluent. This is consistent with dye-tracer studies

showing rapid groundwater flow through the regional

aquifer system from the vicinity of the municipal effluent

sprayfield in Leon County and with elevated nitrogen

levels in water samples from the Wakulla Springs vents.

Results from discrete clusters of invasive macrophytes

in Bulow Creek suggested nitrogen discharges within the

range of both animal waste and sewage effluent. A

potential source for nitrogen from animal waste was the

cattle operation and open mine pit located in the flood

plain. The cattle operation was located a comparable

distance from the west channel of Bulow Creek to the

industrial dairy operation’s distance south of Lake Placid.

The other discrete cluster of macrophytes evaluated from

Bulow Creek was associated with a single-family

residence. No additional houses or STPs were located

near those discrete clusters of invasive macrophytes.

Other potential sources of sewage effluent contamination

to adjacent portions of Bulow Creek include the state

historical site and the associated mobile home and RV

parks. Additional single-family residences also may

contribute to elevated nitrogen concentrations in Bulow

Creek. Shallow groundwater flow, with discrete subsurface discharge, is the most probable transport

mechanism for nitrogen in invasive Bulow Creek

macrophytes.

Land use in the watershed immediately surrounding

Lake Placid, a natural sinkhole lake, was the most varied

of the study areas. That land use included an industrial

dairy-waste lagoon and associated feedlot operation

Bacchus and Barile

366 Environmental & Engineering Geoscience, Vol. XI, No. 4, November 2005, pp. 347–369
(CAFO), single-family residences with septic tanks, and

active and abandoned orange groves. These varied uses

required a more extensive network of sample collection

sites. The primary source of anthropogenic nitrogen at the

Lake Placid site appeared to be localized flow of animal

waste from the existing dairy operation waste lagoon.

In conclusion, results from this study indicated that

uptake of anthropogenic nitrogen has occurred in

invasive alien and nuisance native macrophytes in the

four freshwater ecosystem types evaluated and was

sufficient to support dense growth of invasive macrophytes at all of the ‘‘protected’’ areas that were evaluated.

The source of the anthropogenic nitrogen in invasive

macrophytes from Alexander and Juniper Springs, and

Alexander Spring-Run in the Ocala National Forest’s

Recreation Areas appears to include underground discharge of sewage effluent and other wastes via the

shallow surficial aquifer. The primary source of nitrogen

contamination at Wakulla Springs appears to be sewage

effluent from the Leon County municipal sprayfield,

transported by karst conduits through the Florida aquifer

system. For the remaining springs evaluated in our study,

nitrogen from unidentified sources appears to be

discharging from the Floridan aquifer. At Lake Placid,

a dairy-waste lagoon contributed nitrogen via the sandy,

surficial aquifer as well as via a tributary stream flowing

through state lands. The Bulow Creek study area appears

to be receiving contamination from both cattle and septic

tank leachate, at least in part as localized surficial

groundwater discharge. Additional research is needed to

identify specific sources at study areas where multiple

potential sources occur (e.g., Orange Grove and Peacock

Springs). We suggest that d15N analysis of freshwater

macrophytes, combined with basic knowledge of watershed and springshed land use and aquifer characteristics,

can provide evidence of nitrogen contamination sources

and groundwater flowpaths.

ACKNOWLEDGMENTS

The quality of the manuscript was improved significantly by the comments of an anonymous reviewer,

J. Humphrey, and the editors. Collection of macrophyte

samples from Bulow Creek and Lake Placid was

facilitated by boats provided by N. Giambolvo and G.

Gramling, respectively, with additional field assistance

provided by G. Duggins, E. Gissendanner, T. Warnke,

and D. White. Funding for the laboratory analysis of

samples was provided, in part, by E. Gissendanner, the

Flagler Regional Chapter of Floridians for Environmental

Accountability and Reform, and Wetlands Alert. Permission to use images of the mapped submerged cave

systems in the Floridan aquifer was provided by HazlettKincaid, Inc. The images in Figure 2A and Figure 2E

were taken by T. Warnke.

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