Prepared in cooperation with the Orange County Utilities Department

Potential for Denitrification near Reclaimed Water 

Application Sites in Orange County, Florida, 2009

By Michael J. Byrne Sr., Richard L. Smith, and Deborah A. Repert

Open File Report 2012–1123

U.S. Department of the Interior

U.S. Geological Survey
ii

U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia 2012

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Suggested citation:

Byrne, M.J. Sr., Smith, R.L., and Repert, D.A., 2012, Potential For Denitrification near Reclaimed Water 

Application Sites in Orange County, Florida, 2009; U.S. Geological Survey Open-File Report 2012-1123, 54 p.

Any use of trade, product, or firm names is for descriptive purposes only and does not imply 

endorsement by the U.S. Government.

Although this report is in the public domain, permission must be secured from the individual 

copyright owners to reproduce any copyrighted material contained within this report.
iii

Acknowledgments 

The authors are grateful to Orange County employee Kim Kunihiro for her logistical support 

and advice. Coinvestigator Sharon E. Kroening began this project and provided the foundation for this 

research. Several U.S. Geological Survey researchers, including David Sumner, Leel Knowles, Andy 

O’Reilly, Rick Spechler, Nick Sepulveda, and Louis Murray, helped design the final test and 

provided guidance. 
iv
v

Contents

Abstract ......................................................................................................................................................................... 1

Introduction.................................................................................................................................................................... 1

Purpose and Scope.................................................................................................................................................... 2

Previous Investigations .............................................................................................................................................. 2

Water Quality and Flow to Springs............................................................................................................................. 4

Methods of Investigation................................................................................................................................................ 5

Water Sampling.......................................................................................................................................................... 5

Sample Preparation and Incubation........................................................................................................................... 5

Analytical Procedures ................................................................................................................................................ 6

Potential Rates of Denitrification.................................................................................................................................... 6

Limitations................................................................................................................................................................ 14

Summary ..................................................................................................................................................................... 14

References Cited......................................................................................................................................................... 15

Appendix 1. Sources of Nitrate and Evaluating the Potential for Denitrification in the Surficial 

and Upper Floridan Aquifers, Central Florida............................................................................................................... 18

Appendix 2. Laboratory Report of Tritium/He Measurements for USGS Project......................................................... 32

Figures

Figure 1. Map showing location of study area.............................................................................................................. 3

Figure 2. Graphs showing time course of nitrous oxide production by well water and spring-water 

samples during laboratory incubations to measure denitrification potential. ................................................... 10

Figure 3. Graphs showing time course of nitrate and nitrite concentration change in well water 

and spring-water samples during laboratory incubations................................................................................ 12

Tables

Table 1. Site identification and ambient water-quality data Site identification and ambient water-quality data............. 8

Table 2. Denitrification rates of water samples based on laboratory analysis. ............................................................. 9
vi

Conversion Factors

Multiply By To obtain

Length

foot (ft) 0.3048

mile (mi) 1.609 kilometer (km)

Area

acre 4,047 square meter (m2)

acre 0.4047 hectare (ha)

Volume

gallon (gal) 3.785 liter (L) 

Flow rate

million gallons per day (Mgal/d) 0.04381 cubic meter per second (m3/s)

Abbreviations

DOC dissolved organic carbon

RIB rapid infiltration basin

TN total nitrogen

USGS U.S. Geological Survey

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F=(1.8×°C)+32

Nitrate mg/L as N may be converted to Nitrate mg/L as follows: Nitrate mg/L = Nitrate mg/L as N x 4.43
1

Potential for Denitrification near Reclaimed Water 

Application Sites in Orange County, Florida, 2009

By Michael J. Byrne Sr., Richard L. Smith, and Deborah A. Repert

Abstract

The potential for denitrification was tested in water samples from four Upper Floridan aquifer 

wells near a reclaimed water application site, in west Orange County Florida, and two adjacent springs. 

Results of the study indicate that denitrifying bacteria are present in the groundwater and spring water 

samples, and that these bacteria can readily denitrify the waters when suitable geochemical conditions

exist. The acetylene block technique was used to assess nitrous oxide in the samples that was produced 

by denitrification. The laboratory incubation experiment consisted of four different treatments to each of 

the six samples: (1) ambient water (no added nitrate or glucose), (2) ambient water amended with 

1.4 milligrams per liter (mg/L) nitrate as nitrogen (N), (3) ambient water amended with 5.0 mg/L nitrate 

as N, and (4) ambient water amended with 5.0 mg/L nitrate as N and 10 mg/L glucose as C6H12O6. A

companion set of incubations using treatment 2 tracked changes in nitrate and nitrite concentration with 

time. The rate of denitrification in treatment 2 ranged from 0.059 to 0.124 milligram per liter per day 

nitrogen [(mg/L)/d N] and in treatment 3 ranged from 0.071 to 0.226 (mg/L)/d N. At all of the sampling 

sites, treatment 4 yielded denitrification rates at least an order of magnitude greater than those measured 

for the other treatments; rates ranged from 2.3 to 4.4 (mg/L)/d N. The electron donor supply, dissolved 

organic carbon, in the groundwater and springwater is sufficient to remove at least 1.1-1.4 mg/L nitrate

as N in 20 to 30 days, as indicated by nitrous oxide production rates under ambient conditions 

(treatment 1). The even higher nitrate removal observed with addition of supplemental carbon in 

treatment 4 suggests that carbon is a limiting nutrient in this reaction. Denitrifying activity might 

explain the low ambient nitrate concentrations in the Upper Floridan aquifer in this area. 

Introduction

Springs in central Florida are threatened by elevated concentrations of nitrate, which lead to 

algal blooms, fish kills, and oxygen depletion (Walsh and others, 2009). The sources of the nitrate 

include fertilizer, manure, and septic waste, all of which are carried by rainfall into the Upper Floridan

aquifer through natural diffuse downward leakage as well as numerous drainage wells, and karstic 

features such as sinkholes (Katz and others, 1999). The nitrate-rich groundwater then enters the springs 

through artesian groundwater discharge. Rapid infiltration basins (RIBs) are shallow basins established 

to dispose of applied, nitrate-containing reclaimed water and additionally to recharge the Upper Floridan

aquifer (Adamski and German, 2003). RIBs initially recharge the surficial aquifer that overlies the 

Upper Floridan aquifer, then most of the water in the surficial aquifer eventually flows downward to the 

Upper Floridan aquifer in this area (Adamski and German, 2003).
2

Springs have also been affected by groundwater withdrawals. Groundwater consumption in 

Orange County, Florida, which is predominantly from the Upper Floridan aquifer, has increased from 

87 million gallons per day (Mgal/d) in 1965 to 267 Mgal/d in 2005 (Marella, 2009). Increased 

groundwater consumption has contributed to decreased water levels in the Upper Floridan aquifer and 

decreased discharge to the springs in Orange County (Adamski and German, 2003). 

Nitrate concentrations in the springs are elevated above background concentrations of 

0.2 milligram per liter (mg/L) nitrate as nitrogen (N) (Walsh and others, 2009). Nitrate can be naturally 

reduced to an inert nitrogen gas by a bacterial process called denitrification, which can serve as a 

remediation mechanism if the appropriate conditions (a suitable electron supply, such as organic carbon,

and anoxia) are present:

NO3

− → NO2− → NO + N2O → N2 (g)

The process begins with nitrate and the end product of denitrification is non-reactive nitrogen gas 

(Smith and Duff, 1988). An understanding of the degree to which denitrification occurs, or the “rate of 

denitrification,” in the Upper Floridan aquifer is needed to predict nitrate transport to the springs and the 

potential amount of nitrate removed within the transport interval.

In 2004, the U.S. Geological Survey (USGS), in cooperation with the Orange County Utilities

Department, initiated a study to determine: (1) the source of the nitrate in the Upper Floridan aquifer 

beneath a RIB site located in west Orange County, Florida (fig. 1); (2) whether denitrification occurs 

within the aquifer; (3) the age (time since recharge) of the water in the aquifer in this area; and (4) an 

estimate of the rate of denitrification in the Upper Floridan aquifer. The first two objectives were 

addressed by Kroening (2007; app. 1). The third objective was addressed by Schlosser and others

(2008; app. 2). The final objective of the study is addressed in this report. Estimates of denitrification 

rates of water from selected springs, in addition to the Upper Floridan aquifer, also are included in 

this publication.

Purpose and Scope

The purpose of this report is to describe potential rates of denitrification in water samples from 

the Upper Floridan aquifer, Wekiwa Springs, and Rock Springs in west Orange County, Florida (fig. 1). 

In addition, this report incorporates findings from the previous reports completed as part of the study to 

provide the reader with a single report that addresses all of the study objectives. The “potential” rate of 

denitrification is defined as the capacity of denitrifying bacteria present in the water to reduce nitrate 

concentrations when certain optimal conditions exist, (presence of denitrifying bacteria, ample supply of 

carbon to act as an electron donor source, and reducing conditions related to a low concentration of 

dissolved oxygen) and does not necessarily reflect what is occurring in the aquifer under ambient 

conditions. The potential rate of denitrification was calculated as the rate of nitrate reduction to nitrous 

oxide within water samples using the acetylene block technique in short-term laboratory incubations 

(Balderston and others, 1976; Yoshinari and others, 1977). For the current effort, a total of six water 

quality samples were collected at six sites (four groundwater wells, two springs) in late 2009. 

Previous Investigations

Sumner and Bradner (1996) described the fate and transport of nutrients within the surficial 

aquifer beneath a rapid infiltration basin along the Lake Wales Ridge in Orange County. Removal of 

nitrogen by denitrification from the percolating reclaimed water was minimal within the surficial aquifer 

in the immediate vicinity of the basin, probably because of the lack of reducing conditions and a relative 
3

Figure 1. Map showing location of study area.
4

paucity of organic carbon substrates. They also noted that phosphorus concentrations were decreased by 

about 90 percent from concentrations in applied reclaimed water after moving through the upper 15 feet 

(ft) of the soil profile. This most likely was a result of adsorption onto abundant iron and aluminum 

hydrous oxyhydroxide coatings on sand grains. A number of models have been developed that describe 

the northeasterly groundwater flow in the Upper Floridan aquifer in Orange County and predict the 

areas of aquifer recharge (Murray and Halford, 1996; O’Reilly, 1998; Sepulveda, 2002). Katz and others 

(1999) used nutrients, isotopes, and major ions to study the spring water in the Suwannee River Basin 

and found that (1) the age of the spring water was greater than 20 years, (2) nitrate contamination was 

positively correlated with volume of fertilizer use, and (3) nitrate and dissolved oxygen concentrations 

in groundwater were positively correlated. Toth and Fortich (2002) also described how nitrate 

contamination in Rock Springs and Wekiwa Springs was related to fertilizer use in those areas. Walsh 

and others (2009) found that nitrate contamination had decreased in Rock Springs and Wekiwa Springs 

by 2007, but was increasing in other springs.

Denitrification in groundwater can be quantified or inferred using several different techniques. 

Smith and Duff (1988) used the acetylene block technique to determine rates of denitrification in an

unconfined sand and gravel aquifer, and discovered that available carbon limited the reaction. Katz and 

Böhlke (2000) used isotopic signatures of nitrogen to identify denitrification in a carbonate aquifer in

the Suwannee River Basin. Kroening (2007; app. 1) found excess nitrogen gas, the end product of 

denitrification, in the Upper Floridan aquifer beneath the RIBs site that is the focus of this report, and 

used nitrogen isotopes to identify fertilizer as the source of the nitrate. Nitrogen-15-enriched nitrate also 

has been injected into groundwater during natural gradient tracer tests to quantify denitrification in situ

(Smith and others, 2004). 

Water Quality and Flow to Springs

Historical and recent groundwater quality, as well as estimates of how fast groundwater moves 

from the RIBs to nearby springs, are presented in this section. The Orange County Northwest Water 

Reclamation Facility RIBs are located near the west border of Orange County, Florida, more than 

7 miles (mi) from Wekiwa Springs and Rock Springs (fig. 1). These RIBs were constructed in 

1987, and have a treatment capacity of 7.5 Mgal/d (Morrell and others, 2002). Tertiary wastewater

treatment before application consists of grit removal, activated sludge aeration, sand filtration, and 

disinfection using chlorine.

Although nitrate and dissolved oxygen concentrations are relatively high in the surficial aquifer, 

indicating potential water-quality degradation beneath the RIBs, results from Kroening (2007) indicate 

that these effects do not extend to the Upper Floridan aquifer. Nitrate and dissolved-oxygen levels in 

the surficial aquifer beneath the RIBs were 1.20 to 6.30 mg/L nitrate as N and 1.5 to 8.4 mg/L as 

O2, respectively (Kroening, 2007; app. 1). Water in the Upper Floridan aquifer beneath the RIBs is 

suboxic (0.1-0.2 mg/L as O2), and the nitrate concentration is considered to be at background levels 

(less than 0.2 mg/L nitrate as N; Kroening, 2007, app I). Dissolved oxygen concentrations are low in 

Wekiwa Springs (0.40 mg/L as O2) and in Rock Springs (1.38 mg/L as O2) (Walsh and others, 2009).

In 1961, the nitrate concentration in Wekiwa Springs was 0.61 mg/L as N (Toth and Fortich, 

2002); however nitrate concentrations in 2009 in Wekiwa Springs (1.12 mg/L as N) and Rock Springs 

(1.38 mg/L as N) were elevated above the background concentration of 0.2 mg/L as N (Walsh and 

others, 2009). A peak nitrate concentration (2.0 mg/L as N) was measured in Wekiwa Springs in 

1995; values have since declined (Toth and Fortich, 2002; Walsh and others, 2009). The declining trend 

appears to be related to a decrease in citrus acreage, which peaked at 65,000 acres in 1968, dropped 

substantially to 45,000 acres in 1984 following a hard freeze, and declined further to 10,000 acres by 
5

2001 (Toth and Fortich, 2002). Toth and Fortich (2002) used isotope data to determine that (1) the 

source of nitrate was fertilizer and animal waste at Wekiwa Springs, and fertilizer at Rock Springs, 

and (2) the mean age of the water since recharge to the Upper Floridan aquifer at both springs was

approximately 17 years.

Groundwater age and time of travel from the RIBs to the springs were estimated from a 

groundwater flow model and groundwater age-dating and geochemical methods. Model projections, 

based on particle tracking (MODPATH 4.2) simulations using a calibrated groundwater flow model of 

the area (O’Reilly, 1998), indicate that it takes more than 100 years for the water in the Upper Floridan

aquifer beneath the RIBs to reach Wekiwa Springs (Nicasio Sepulveda, U.S. Geological Survey, oral 

commun., 2011). Based on isotopic tritium/helium-3 analysis of replicate samples from one well

(MW-2), the water currently in the Upper Floridan aquifer beneath the RIBs is estimated to have entered 

the aquifer 19 years before present (Schlosser and others, 2008; app. 2). Boron, an element used in 

detergent, was elevated in the RIBs water, but there were only trace levels in the Upper Floridan aquifer.

Kroening (2007; app. 1) suggested the Upper Floridan aquifer beneath the RIBs does not contain water 

from the RIBs based on the low concentration of boron present. Based on analysis of nitrogen and 

argon gases in groundwater, Kroening (2007; app. 1) inferred that excess nitrogen gas exists in the 

Upper Floridan aquifer. The excess dissolved nitrogen gas (1-5 mg/L as N), above the naturally 

occurring concentration in water at the time of infiltration, was attributed to denitrification. Based on 

the age of the water in the aquifer and the amount of excess nitrogen gas, a rate of denitrification of

5×10-4 mg/L as N per day was estimated for this process. Nitrogen isotopic analyses of the excess 

nitrogen (N2) indicated that the source of the nitrogen in the Upper Floridan aquifer was fertilizer, 

probably from the orange groves that occupied the site prior to the establishment of the RIBs

(Kroening, 2007; app. 1).

Methods of Investigation

The potential rate of denitrification of water in the Upper Floridan aquifer and Wekiwa and 

Rock Springs was assessed using laboratory incubations. Sample collection and analysis were made in

accordance with standard USGS protocols. Further details on the method are provided in Smith and 

others (2004).

Water Sampling

Water samples were collected on November 9 and 30, 2009, from four Upper Floridan aquifer 

wells (MW-8B, MW-5R, MW-2, and Phipps) located on the RIBs property (fig.1), and two nearby 

springs, Wekiwa Springs and Rock Springs (fig. 1). Groundwater samples were collected by lowering a 

submersible pump into the open-hole interval of each well and near the main boil of both springs, and 

pumping at 2.5 liters per minute (L/min.) until the field readings of specific conductance, pH, dissolved 

oxygen, and temperature stabilized. A total of 4 liters (L) of water was collected from each site using 

sterile pump tubing, and was stored in 1-L amber glass bottles that had previously been baked at 

500°C. The bottles were flushed with 3 to 5 L of sampled water, filled completely, and capped to avoid 

an air space. The four bottles from the six sites were then sealed, placed on ice, and shipped overnight to 

the USGS laboratory in Boulder, Colorado for testing the next day.

Sample Preparation and Incubation

For each of the six samples, one 1-L bottle was filtered and preserved for nitrate, nitrite, 

ammonium, dissolved organic carbon (DOC) and total dissolved nitrogen (TN) analyses (Savoie and 
6

others, 1998). A second 1-L bottle of unfiltered water for each sample was placed immediately into an 

anaerobic glovebox. In the glovebox, the water was dispensed into triplicate 60-milliliter (mL) serum 

bottles for each of four treatments to assess denitrification potential. The bottles were stoppered and 

flushed with helium to replace the glovebox gas (a mixture of nitrogen, hydrogen, and carbon dioxide 

gases). Anoxic, stock solutions of 250 mg/L NaNO3 as N and 500 mg/L glucose as C6H12O6, were then

added, as appropriate, to prepare the following treatments: (1) ambient (no added nitrate or glucose), 

(2) amended with 1.4 mg/L nitrate as N, (3) amended with 5 mg/L nitrate as N, and (4) amended with

5 mg/L nitrate as N and 10 mg/L glucose as C6H12O6. The glucose plus nitrate addition was included to 

serve as a positive control to confirm that proper conditions existed for denitrification to occur. Oxygenfree acetylene gas was then added to the headspace of each bottle. Acetylene blocks the reduction of 

nitrous oxide (an intermediate in the denitrification pathway) to nitrogen gas. Accordingly, in the 

presence of acetylene, nitrous oxide production over time is used to quantify rates of denitrification.

Rates are designated as “potential” denitrification when in situ conditions are altered, such as by adding 

nitrate and (or) glucose. After the addition of acetylene, the sample bottles were placed on a rotator, set 

to 4 revolutions per minute, and incubated at room temperature (22°C). The bottles were analyzed for 

nitrous oxide concentration in the headspace after 1 hour, at daily intervals for 10 days, and (thereafter) 

once every few days for 1 to 2 months. At the end of the experiment, water samples were removed by 

syringe from the serum bottles, filtered, and preserved for nitrate, nitrite, ammonium, and 

DOC/TN analysis.

Nitrate/nitrite concentrations also were monitored over time in a separate, companion set of 

incubation bottles as a control for the experiment to measure denitrification without the acetylene block.

These were triplicate, 100-mL unfiltered water samples in serum bottles to which nitrate was added to

obtain a concentration of 1.4 mg/L as N. The bottles were flushed with helium to replace the glovebox 

gas, but no acetylene was added. This second set of bottles was rotated at room temperature, and water 

samples were removed by syringe and filtered and preserved for nitrate and nitrite analysis each time a 

nitrous oxide measurement was made in the headspace of the acetylene-amended bottles. At the end of 

the experiment, water samples were removed from the companion bottle set and filtered and preserved

for nitrate, nitrite, ammonium, and DOC/TN analyses.

Analytical Procedures

Nitrous oxide production was measured using an HNU gas chromatograph equipped with an 

electron capture detector. Concentrations were calibrated using authentic standard gas mixtures. Nitrate 

and nitrite were analyzed using a Dionex Model DX500 ion chromatograph with an AS4A-SC 

4-millimeter (mm) analytical column, sodium carbonate/bicarbonate mobile phase, and conductivity 

detection. Ammonium was measured using a Dionex Model DX300 ion chromatograph with a CS12A 

4-mm analytical column, a gradient mobile phase of 5- to 25-mN (millinormal) sulfuric acid, and 

conductivity detection. Dissolved organic carbon and total dissolved nitrogen were analyzed 

simultaneously on a Shimadzu combined TOC-VCSN and TNM-1 analyzer with high temperature 

combustion and chemiluminiscent detection. Alkalinity was measured using a Radiometer Model 

ABU93 auto-titrator.

Potential Rates of Denitrification

The potential rate of denitrification was calculated as the rate of nitrate reduction to nitrous 

oxide within water samples, quantified by nitrous oxide production. All samples tested exhibited 

potential denitrifying activity, as evidenced by the accumulation of nitrous oxide in the presence of 
7

acetylene (fig. 2). All groundwater samples except the Phipps Well sample required an addition of 

nitrate or nitrate plus glucose for denitrification, whereas both springwater samples demonstrated 

denitrifying activity under all conditions tested. Most of the incubations showed an initial 1 to 2 day lag 

period, followed by a period of linear (steady) nitrous oxide production, and a subsequent tapering off in 

production with the exhaustion of the nitrate pool. Estimates of rates of denitrification were determined 

using linear regression over the initial portion of the nitrous oxide production curve (fig. 2). In three of 

the four wells, MW-2, MW-5R, and MW-8B, the ambient nitrate concentrations were too low (below 

0.04 mg/L as N) to establish a denitrification rate using treatment 1. In the Phipps Well sample, the 

ambient nitrate concentration was 0.13 mg/L as N (table 1 and the denitrification rate for treatment 1 

was 0.050 milligram per liter nitrogen per day [(mg/L)/d] N (table 1). When nitrate was added for 

treatments 2 and 3, the denitrification rate doubled (table 2). Wekiwa Springs water had an ambient 

nitrate concentration of 1.12 mg/L as N and the denitrification rate for treatment 1 was 

0.072 (mg/L)/d N; treatments 2 and 3 yielded higher denitrification rates. Rock Springs water had an 

ambient nitrate concentration of 1.38 mg/L as N and a denitrification rate of 0.071 (mg/L)/d N for 

treatment 1; treatments 2 and 3 yielded higher denitrification rates. Water from well MW-2 had a 

denitrification rate of 0.059 (mg/L)/d N for treatment 2 with a higher rate of 0.087 (mg/L)/d N for

treatment 3 (table 2). At all of the sampling sites, treatment 4 yielded denitrification rates at least an 

order of magnitude greater than those measured for the other treatments; rates ranged from 2.3 to 

4.4 (mg/L)/d N as shown in figure 2 and table 2.

At four of the six sites, the calculated denitrification rate was similar in treatments 2 and 

3 (fig. 2). This finding may indicate that when nitrate exceeds about 1.4 mg/L as N, the denitrification 

rate is not limited by available nitrate. Rock Springs deviated the most; the denitrification rate in this 

sample for treatment 3 was 2.4 times as high as treatment 2 (table 2, fig. 2). For all the samples, the 

order of magnitude increase in the denitrification rate produced by the addition of carbon in the form of 

glucose (treatment 4) indicates that carbon availability may be the primary control on nitrous oxide 

production. Ambient dissolved organic carbon (1-3 mg/L), which is the electron donor supply in the 

groundwater and spring water, is sufficiently abundant to remove at least 1.1-1.4 mg/L nitrate as N in 

20 to 30 days, even in situations where the background nitrate concentration in groundwater is low. The 

rate of nitrate consumption was determined at all sites for the companion set of sample bottles that were 

amended with 1.4 mg/L nitrate as N using the linear section of the nitrate consumption curves in 

figure 3 (table 2). At most sites, the rate of nitrate consumption exceeded the rate of nitrous oxide 

production; this difference is primarily attributable to nitrite production. One exception, however, was 

the sample from Rock Springs, for which the rate of nitrate consumption for treatment 3 and nitrous 

oxide production were comparable [0.226 (mg/L)/d N and -0.218 (mg/L)/d N, respectively, table 2]. The 

slowest rate of nitrate consumption, 0.09 (mg/L)/d N, occurred in the groundwater sample from well

MW-5R, and the fastest, 0.27 (mg/L)/d N, occurred in the Wekiwa Springs sample. Consumption rates 

for all other sites ranged between 0.17 and 0.23 (mg/L)/d N.

Using the ambient nitrous oxide production rates, the number of days required to consume 

available nitrate was calculated for the three sites that had above-background nitrate concentrations. The 

available nitrate would be consumed in 15, 2.6, and 20 days at Wekiwa Springs, Phipps Well, and Rock 

Springs, respectively, assuming that the rates remain constant as nitrate concentrations decrease.

In a separate laboratory control sample experiment, bottles, without an acetylene block and 

amended with 1.4 mg/L nitrate as N were also measured over several weeks. In these samples all 

available nitrate was removed through denitrification in 4 to 6 days, with an initial lag, leaving only 

background concentrations of 0.04 mg/L nitrate as N.
8

Table 1. Site identification and ambient water-quality data Site identification and ambient water-quality data.

[mg/L, milligrams per liter; mg/L as N, milligrams per liter as nitrogen]

Site name Well number Date of 

sample

Well 

depth, in 

feet 

below 

land 

surface

Water 

temperature, 

in oC

pH, in 

standard 

units

Specific 

conductance, in 

μS/cm

Dissolved 

oxygen, 

in mg/L

Nitrate, in 

mg/L 

as N

Totalb

Nitrogen, in 

mg/L as N

Dissolved 

Organic 

Carbon, in 

mg/L as C

MW-2 283803081314901 11/09/09 160 25.7 7.46 504 0.23 0.042 0.436 2.229

MW-5R 283754081312101 11/09/09 165 25.7 7.84 333 0.16 0.020 0.076 1.064

MW-8B 283809081321001 11/09/09 170 26.0 7.95 278 0.32 0.032 0.116 1.263

Phipps Well 283738081305801 11/30/09 165 24.1 7.53 308 NA 0.129 0.197 1.088

Rock Springs 2234610 11/30/09 --- 23.4 7.61 269 1.38a 1.385 1.521 0.673

Wekiwa 

Springs 2234600 11/30/09 --- 24.0 7.30 357 0.40a 1.117 1.254 1.129

a Median concentration from Walsh and others (2009).

b Difference between Total Nitrogen and Nitrate is predominately ammonia
9

Table 2. Denitrification rates of water samples based on laboratory analysis.

[(mg/L)/d N, milligrams nitrogen per liter per day; mg/L nitrate, milligrams nitrate-nitrogen per liter; NA, not applicable]

Site name Date

1st treatment 

ambient 

denitrification rate, 

in (mg/L)/d N

2nd treatment 

ambient sample 

amended with 

1.4 mg/L nitrate 

denitrification rate, 

in (mg/L)/d N

3rd treatment 

ambient sample 

amended with 

5.0 mg/L nitrate 

denitrification rate, 

in (mg/L)/d N

4th treatment 

ambient sample 

amended with 

5.0 mg/L nitrate and 

10 mg/L glucose 

denitrification rate, 

in (mg/L)/d N

Nitrate consumption 

rate sample 

amended with 

1.4 mg/L nitrate, in

(mg/L)/d N

MW-2 11/10/09 NA 0.059 0.087 2.3 -0.227

MW-5R 11/10/09 NA 0.083 0.071 4.0 -0.090

MW-8B 12/15/08 NA 0.120 NA NA NA

MW-8B 11/10/09 NA 0.118 0.127 2.3 -0.205

Phipps Well 12/01/09 0.050 0.099 0.105 4.4 -0.173

Wekiwa Spring 12/01/09 0.072 0.124 0.142 2.4 -0.269

Rock Spring 12/01/09 0.071 0.095 0.226 3.4 -0.218
10

Figure 2. Graphs showing time course of nitrous oxide production by well water and spring-water samples during 

laboratory incubations to measure denitrification potential.
11

Figure 2. Graphs showing time course of nitrous oxide production by well water and spring-water samples 

during laboratory incubations to measure denitrification potential.—Continued
12

Figure 3. Graphs showing time course of nitrate and nitrite concentration change in well water and spring-water 

samples during laboratory incubations.
13

Figure 3. Graphs showing time course of nitrate and nitrite concentration change in well water and spring-water 

samples during laboratory incubations.—Continued

 
14

Limitations

Smith and others (1996) in a prior study of a surficial aquifer, suggest that potential rates of 

denitrification determined with the anoxic laboratory samples are likely greater than rates that occur in

the aquifer due to elevated dissolved oxygen concentration in the shallow aquifer of that study. 

However, because the Upper Floridan aquifer has low dissolved oxygen concentrations, it is doubtful 

that a disparity in laboratory and aquifer dissolved oxygen would bias applicability of the laboratoryderived denitrification rates to the Upper Floridan aquifer. The addition of nitrate and glucose would 

tend to stimulate denitrification during laboratory incubations since the relevant bacteria require nitrate 

and glucose to colonize and, therefore, would bias laboratory-derived denitrification rates higher than in 

situ aquifer rates. The laboratory results also are only relevant to denitrifying bacteria and carbon 

suspended in the groundwater; the experiments did not measure rates of denitrification for bacteria 

attached to possible carbon-rich biofilms on aquifer solids. If attached bacteria actively consume nitrate 

by denitrification in in situ aquifer conditions, neglect of biofilm denitrification would bias laboratoryderived denitrification rates lower than in situ aquifer rates. The data presented in this study suggest that 

carbon is the limiting nutrient for this reaction in the Upper Floridan aquifer. 

Low nitrate concentrations in the Upper Floridan aquifer in this area make tracer studies 

impractical under ambient aquifer conditions and retrieval of injected enhanced-nitrate tracers is 

challenging in the fast-moving, conduit flow within this aquifer. 

Summary

Water samples from four Upper Floridan aquifer wells and two springs were tested to determine 

the potential for denitrification near reclaimed water application sites in west Orange County, Florida. 

Tests were conducted to quantify the potential rates of denitrification using the acetylene block 

technique; the potential rate of denitrification was calculated as the rate of nitrate reduction to nitrous 

oxide within water samples, quantified by nitrous oxide production. The experiment consisted of four 

different treatments: (1) ambient water (no added nitrate or glucose); (2) ambient water amended with 

1.4 mg/L nitrate as N, (3) ambient water amended with 5.0 mg/L nitrate as N, and (4) ambient water 

amended with 5.0 mg/L nitrate as N and 10 mg/L glucose as C6H12O6. Nitrate/nitrite concentrations also 

were monitored over time in a separate, companion set of incubation bottles subjected to treatment 2 but 

without addition of an acetylene block (laboratory control). These control samples were denitrified in 

4-6 days with an initial lag and final concentrations of 0.04 mg/L nitrate as N. 

Results of the study indicate that denitrifying bacteria were present in the groundwater and 

spring water samples, and these bacteria readily facilitated denitrification when placed in suitable 

conditions. In three of the four well sites, the ambient nitrate concentrations were too low (less than 

0.04 mg/L as N) to establish a denitrification rate using treatment 1. But in the fourth well, where the 

ambient nitrate concentration was 0.13 mg/L as N, the nitrous oxide production rate for treatment 1 was 

0.050 milligram per liter per day [(mg/L)/d] N. When nitrate was added for treatments 2 and 3, the 

denitrification rate increased. In four of the six sites, the denitrification rate was similar in treatments 

2 and 3. This finding may indicate that when nitrate exceeds about 1.4 mg/L as N, the denitrification

rate is not limited by available nitrate. The denitrification rate, in all the samples ranged from 0.059 to 

0.124 (mg/L)/d N (treatment 2) and 0.071 to 0.226 (mg/L)/d N (treatment 3). Treatment 4 yielded 

nitrous oxide production rates at least an order of magnitude greater than those measured for the other 

treatments, at all of the sampling sites; rates ranged from 2.3 to 4.4 (mg/L)/d N. The electron donor 

supply, dissolved organic carbon, in the groundwater and springwater is sufficient to remove at least 

1.1-1.4 mg/L nitrate as N in 20 to 30 days, as indicated by nitrous oxide production rates under ambient 
15

conditions (treatment 1). The even higher nitrate removal observed with addition of supplemental 

carbon in treatment 4 suggests that carbon is a limiting nutrient in this reaction. Denitrifying activity 

might explain the low ambient nitrate concentrations in the Upper Floridan aquifer. The in situ rates of 

denitrification were calculated as 5×10-4 mg/L as N per day based on groundwater age and excess 

nitrogen gas concentrations data collected by Kroening(2007). The potential rates of denitrification 

determined in the low dissolved oxygen laboratory incubations during this study may represent in situ

rates of denitrification because, in contrast to previous studies of shallow aquifers with oxygenated 

conditions that retard denitrification, low dissolved oxygen concentrations prevail in the Upper 

Floridan aquifer.

The potential for denitrification exists in the Upper Floridan aquifer near a reclaimed water 

application site in west Orange County, Florida. The necessary conditions for denitrification, including 

the presence of denitrifying bacteria, relatively low dissolved oxygen concentrations, and ample

dissolved organic carbon are met. The laboratory-derived rates of denitrification in this study represent a 

reasonable approximation for actual rates in the Upper Floridan aquifer in the study area. Fertilizer

appears to be the primary source of nitrate in the Upper Floridan aquifer beneath the rapid infiltration 

basin sites. 

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Florida: U.S. Geological Survey Water-Resources Investigations Report 03-4257, 113 p.

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Murray, L.C., and Halford, K.J., 1996, Hydrogeologic conditions and simulation of ground-water flow 

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He dating of shallow 

groundwater: Earth and Planetary Science Letters, v. 89, p. 353-362.

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no. 08-1.15.0.

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systems in peninsular Florida: U.S. Geological Survey Water-Resources Investigations 

Report 02-4009, 130 p. 

Smith, R.L., Böhlke, J.K., Garabedian, S.P., Revesz, K.M., and Yoshinari, T., 2004, Assessing 

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Smith, R.L., and Duff, J.H., 1988, Denitrification in a sand and gravel aquifer: Applied and 

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transformation beneath a rapid infiltration basin, Reedy Creek Improvement District, Orange County, 

Florida: U.S. Geological Survey Water-Resources Investigations Report 95-4281, 51 p. 

Toth, D.J., and Fortich, C., 2002, Nitrate concentrations in the Wekiva groundwater basin with 

emphasis on Wekiwa Springs: Palatka, Fla., St. Johns River Water Management District Technical 

Publication SJ2002-2.
17

Turrin, B., [n.d.], Introduction to T/3He dating of ground water sampling and measurement methods (no 

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the 1978 National Bureau of Standards tritiated-water standards: International Journal of Applied

Radiation and Isotopes, v. 31, p. 611- 614.

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H/3He dating 

background: Accessed on December 29, 2011, at http://water.usgs.gov/lab/3h3he/background/.

Walsh, S.J., Knowles, L., Jr., Katz, B.G., and Strom, D.G., 2009, Hydrology, water quality, and aquatic 

communities of selected springs in the St. Johns River Water Management District, Florida: U.S. 

Geological Survey Scientific Investigations Report 2009-5046, 115 p.

Walsh, V. and Price, R. 2010, 3

H and 3HeTritium and Helium Apparent Age dating of Groundwaters: 

Accessed on December 29, 2011, at http://www2.fiu.edu/~pricer/Radioisotopes.pdf.

Yoshinari, T., Hynes, R., and Knowles, R., 1977, Acetylene inhibition of nitrous oxide reduction and 

measurement of denitrification and nitrogen fixation in soil: Soil Biology and Biochemistry, v. 9, 

p. 177-183.
18

Appendix 1. Sources of Nitrate and Evaluating the Potential for Denitrification in 

the Surficial and Upper Floridan Aquifers, Central Florida

The following article by Sharon E. Kroening is reprinted from Proceedings of the Fourth 

National Outdoor Action Conference on Aquifer Restoration, Ground Water Monitoring and 

Geophysical Methods with permission of the National Ground Water Association. Copyright 1990. 

Refer to Kroening (2007) in the references cited section for the complete report citation.
Sources of Nitrate and Evaluating the Potential for Denitrification in the Surficial and 

Upper Floridan Aquifers, Central Florida

Sharon E. Kroening 

U.S. Geological Survey 

Orlando, Florida 

Abstract

The sources of nitrate and potential for denitrification were evaluated in the karstic 

Upper Floridan aquifer (UFA) and overlying surficial aquifer beneath and upgradient from a 

reclaimed water facility in Central Florida. Nitrate-N concentration (<0.02 to 6.3 mg/L) in 

ground water in this area likely originated from past fertilizer use in citrus agriculture or 

treated wastewater applied to rapid-sand infiltration beds. Chemical, isotopic, and dissolved 

gas data were evaluated from water samples collected from nine wells to determine nitrate 

sources and if conditions were conducive for denitrification. Surficial aquifer wells ranged 

from 3.7 to 23.8 m deep, and UFA wells ranged from 46 to 102 m deep. δ15N and δ18O 

values of nitrate in well water samples indicated that treated wastewater likely is the source 

of nitrate in the surficial aquifer wells, whereas the sources of nitrate in the UFA were likely 

treated wastewater and fertilizer. Denitrification likely occurs in the UFA, but may only 

occur to a limited extent in the surficial aquifer. Dissolved oxygen concentrations generally 

were less than 1 mg/L in the UFA and greater than 2 mg/L in the surficial aquifer. Dissolved 

gas and isotopic data indicated that denitrification had occurred in water from 4 of 5 UFA 

wells. Excess nitrogen gas (N2), the end product of denitrification, was estimated for four 

UFA wells based on the concentrations of dissolved N2 and argon in ground water. Excess N2

concentrations ranged from 1 to 5 mg/L. δ15N values associated with N2 in these same wells 

also were substantially greater (+0.97 to +2.14 ‰) than for N2 in equilibrium with airsaturated water. Denitrification may naturally attenuate some nitrate in the UFA before it is 

transported to natural springs in the area. 

Introduction

Karstic aquifer systems supply water to approximately 25 percent of the world’s 

population (Williams, 1993) and are extremely susceptible to nitrate contamination due to the 

rapid recharge of these systems through sinkholes and other subsidence features, sinking 

streams, and preferential flow paths created by conduits. High nitrate concentrations in these 

systems pose a threat to the quality of potable water supplies and may affect the ecosystems 

surrounding karstic springs, which evolved under nutrient-limited conditions in parts of the 

U.S. (Stevenson et. al., 2004). Specifically, the nuisance growth of filamentous algae may be 

associated with increased nitrate concentrations and were identified as a problem for 

recreational use and aquatic life support in springs (Stevenson et. al., 2004). 

 

The karstic Upper Floridan aquifer (UFA) is the primary source of water in Central 

Florida (St. Johns River Water Management District, 2006a) and discharges to numerous 

19
natural springs. Approximately 3.3x106

 m3/day were withdrawn from the UFA in Central 

Florida in 2000 (Marella, 2004). Ground water use is projected to increase about 60 percent 

by the year 2025 due to projected population increases for the area (St. Johns River Water 

Management District, 2006b). Ground water from the UFA is being withdrawn near the 

maximum sustainable rate (St. Johns River Water Management District, 2006a), and 

measures are being implemented to replenish this aquifer system to help ensure that adequate 

water supplies are available to meet projected water-supply demands and sustain the ground 

water inflow to the many natural springs. Artificial recharge with treated wastewater, or 

reclaimed water, is one management technique which is becoming widely utilized to 

recharge the UFA in Florida and aquifer systems in other parts of the U.S. (York et. al. 2002; 

U.S. Environmental Protection Agency 2004; St. Johns River Water Management District 

2006a). In many of these applications, reclaimed water is discharged to rapid-sand infiltration 

basins (RIBs) and allowed to percolate into the ground (Brooke and Godlewski 1995; 

O’Reilly 1998; U.S. Environmental Protection Agency 2004). 

Artifically-recharged water from RIBS, while providing beneficial recharge to the 

ground water system, may potentially contaminate the UFA with nitrate. Wastewater 

disposal to RIBS in other parts of the U.S. resulted in nitrate contamination of the ground 

water (Aulenbach and Tofflemire, 1975; LeBlanc, 1984). Disposal of treated wastewater for 

over 60 years in the Cape Cod, Massachusetts resulted in a 3.5 km-long plume containing 

nitrate concentrations greater than 3 mg/L (LeBlanc, 1984). Knowledge of the source and 

fate of nitrate in the ground water system is necessary in order to develop effective 

management strategies to prevent or remediate nitrate contamination. Denitrification, nitrate 

reduction to N2 gas, generally is considered the primary nitrate attenuation mechanism in 

contaminated aquifers (Korom 1992). Numerous studies have documented the potential for 

denitrification in surficial sand and gravel aquifers (Wassenaar 1995; Starr and Gillham 

1993; Smith and Duff 1988). Denitrification in karstic aquifers, such as the UFA, often is 

believed to be insignificant due to the rapid transport velocities of oxygenated water through 

these systems (Coxon 1999), however, the potential for denitrification has been reported in

several karstic aquifers based on dissolved gas or stable isotope data (Panno et. al. 2001; 

Wilson et. al. 1990; Mariotti et. al. 1988). 

This paper presents the results of a study conducted from 2004-2006 by the U.S. 

Geological Survey in cooperation with Orange County, Florida to determine the nitrate 

sources in the karstic UFA and surficial aquifer system (SAS) beneath and upgradient from a 

wastewater treatment and reuse facility (WWTRF). Chemical data were used to determine 

whether conditions in these aquifers were conducive for denitrification to occur. Nitrogen 

and oxygen isotope ratios on the nitrate in water samples and N isotope ratios on the N2 gas 

dissolved in the water samples were examined for evidence of denitrification and were used 

to determine nitrate sources. Dissolved gas (N2 and Ar) concentrations were used to estimate 

recharge temperatures and the amount of NO3

- consumed by denitrification. N2O 

concentration data were used to identify whether active areas of denitrification were present.

20
Study Area 

The study was conducted in the SAS and UFA in a nine km2

area in Central Florida

(Fig. 1). The mean annual air temperature in this area is approximately 22.0°C (Adamski and 

German, 2005). Regional ground-water flow in the UFA generally is from the southwest to 

the northeast (Kinnaman, 2006), and ground water discharge occurs through several springs 

to the north and northeast of the study area, most notably Wekiwa and Rock Springs. The 

UFA is characterized by primary and secondary porosity (Spechler and Halford 2001), and

generally is separated from the SAS by a confining unit consisting of low permeability 

Miocene or early Pliocene-age carbonate and siliceous deposits, except where the confining 

unit is breached by sinkholes. Within the study area, the UFA is mantled by about 140 to 150 

feet of post-Miocene and Miocene-age deposits, and the uppermost deposits generally are 

described as consisting of fine-grained sand which grades into silty and clayey fine sands 

(Morrell et. al. 2002). 

A WWTRF occupies approximately one-third of the study area and has been operated 

since 1987 to treat domestic waste. Other land uses in the study area include agriculture and 

low- and medium-density residential developments, which are primarily located to the north 

and east of the WWTRF. Current agricultural land use consists of ornamental nurseries, 

abandoned tree crops, and pasture. Prior to the construction of this facility, citrus agriculture 

likely was the predominant land use (Florida Department of Environmental Protection, 

2004). 

Rainfall and treated wastewater effluent are the predominant sources of recharge 

water to SAS and UFA in the study area. Central Florida typically receives about 130 cm of 

rainfall per year, with the majority occurring from June through September (Adamski and 

German 2004). The WWTRF discharged approximately 61.8 x 106

 m3 of treated wastewater 

to 13 RIBs from 1990-2003. Most of the wastewater was discharged to RIB 1 (34 percent), 5 

(16 percent), and 12 (12 percent), which have the greatest infiltration rates (1.6-1.9 m/day) 

(Post Buckley Schuh & Jernigan, Inc., 1993). The other RIBs each received less than 6 

percent of the treated wastewater. 

Methods

Site Selection

Nine SAS and UFA wells were sampled for this study at five sampling sites (Fig. 1). 

Eight of these wells were located in the vicinity of the WWTRF and one UFA well was 

located upgradient of the facility. Existing UFA wells, ranging from about 46 to 102 m deep 

were sampled at sites 2 (45.7 m deep), 4 (51.8 m deep), and 5 (102 m deep). A 64.0-m deep 

UFA well was installed at site 3 in December 2004 to replace an existing monitoring well. At 

site 1, a vertical sequence of SAS wells (wells 1-A, 1-B, 1-C, and 1-D), ranging from 3.7 to 

23.8 m deep, and one UFA well (well 1-E, 62.5 m deep) were installed in December 2004

(Fig. 2) to replace an existing UFA monitoring well and characterize any vertical gradient in 

21
Figure 1. Location of the study area, rapid-infiltration basins, and sampling sites.

22
the denitrification potential. SAS wells were screened to 1.5-3.0 m intervals. The UFA wells 

were not screened and the open-hole interval for these wells ranged from 3.0-31.7 m. UFA 

Well 5 had the longest open-hole interval. The open-hole intervals for the other UFA wells 

were 15.2 m or less. 

Lithologic and ground-water level data collected at site 1 in 2004 showed the 

direction of the vertical head gradient was downward and there was substantial confinement 

between most of the SAS wells. Ground-water level measurements showed there was a 

downward vertical head gradient among the SAS and UFA wells at site 1 during all sampling 

events. Lithologic data collected prior to drilling at this site showed clay layers were 

interbedded among the sands comprising the SAS, which resulted in substantial water 

confinement between most of the SAS layers. The most substantial confinement occurred 

between wells 1-B through 1-D. The median difference in ground water levels between wells 

1-B and 1-C was 9.4 m, and the median difference in ground water levels between wells 1-C 

and 1-D was 3.4 m. Of all the SAS wells, ground water levels in wells 1-A and 1-B were the 

most similar, with a median difference in ground water levels of 0.2 m. 

Figure 2. Placement of well cluster at site 1.

Water-quality sampling 

Water samples were collected from October 2004 to April 2006. All wells except 

number 5 were sampled using a stainless steel submersible pump with PVC tubing. Well 5 

supplied potable water to a toll plaza. Water samples were withdrawn from this well at a 

point upgradient of the pressure tank using the dedicated submersible pump. Samples 

collected for analysis of N2O dissolved in the ground water were collected only in 2006, and 

samples were not collected from well 1-A because the well was dry. Most samples were 

collected using standard techniques (Wilde et. al., 1999) after a minimum of three well-bore 

volumes of water were purged and field readings of specific conductance, pH, dissolved 

oxygen, and temperature stabilized. A closed flow-through chamber was used to measure 

water temperature, specific conductance, and pH. Dissolved oxygen concentrations were 

measured using a polargraphic probe during October 2004-2005 and using a colorimetric 

reagent in ampoules and a battery-operated spectrophotometer (Chemetrics Inc. Calverton 

23
VA, USA) (use of trade names is for identification purposes only and does not imply 

endorsement by the U.S. Government) in 2006. Water samples for N and O isotope analysis 

were filtered using an encapsulated 0.45 μm filter into 125-mL plastic bottles and kept frozen 

until analysis. Water samples for N2, Ar, CO2, and CH4 determinations were collected in 150-

mL serum bottles without headspace. For N2O determinations, 20-mL water samples were 

collected in a syringe and injected into stoppered 30-mL serum vials containing 0.2 mL of 

12.5 N KOH in a He headspace. 

Analytical procedures 

NO3

-

, NO2

-

, and NH4

+ were analyzed in water samples using established methods 

(EPA 300.0; Fishman, 1993) at the USGS laboratory in Denver, Colorado or the Orange 

County laboratory in Orlando, Florida. Organic carbon and boron were analyzed using

established methods (EPA 200.7, SM5310C) at the Orange County laboratory in Orlando, 

Florida. Dissolved N2, Ar, CO2, and CH4 gases in the samples were analyzed by gas 

chromatography after extraction in headspaces of glass samplers (Busenberg et. al. 1998). 

The isotopic composition of N2 in the water samples, δ15N of N2, was measured in the 

leftover headspace after the N2, Ar, CO2, and CH4 analysis. The headspace was pressurized 

with He, expanded into a 0.1 mL loop, then flushed with He through a 5A mole sieve gas 

chromatograph to separate O2+Ar from N2 (modified from Revesz et al., 1999; Smith et al., 

2006). These peaks were passed through an open split to the ion source of a Finnigan Delta 

Plus XL mass spectrometer (use of trade names is for identification purposes only and does 

not imply endorsement by the U.S. Government) and monitored in continuous-flow mode at 

m/z 28, 29, 32, 34, 36, and 40. Relative peak areas at m/z 28 and 40 (from N2 and Ar) were 

used to confirm sample integrity between the concentration measurements and the isotopic 

analyses. Values of δ15N are reported with respect to atmospheric N2 (0 ‰) and were 

calibrated by analyses of air-saturated water samples (+0.65 ‰) that were collected in serum 

bottles and treated the same way as the samples, with estimated uncertainties of 

approximately ± 0.1 ‰ (1-sigma). N and O isotopes in aqueous NO3

-

, δ15N of NO3

-

 and δ18O 

of NO3

-

, were analyzed by bacterial conversion of nitrate to nitrous oxide and subsequent 

measurement on a continuous flow isotope ratio mass spectrometer as described by Sigman 

et. al. (2001), Casciotti et. al. (2002), Coplen et. al. (2004), and Revesz and Casciotti (2005). 

Nitrogen isotope ratios are reported relative to N2 in air (Mariotti, 1983). δ15N of NO3

- values 

were normalized to values of +4.72 ‰ for International Atomic Energy isotopic standard 

NO-3 and +180.0 ‰ for USGS-32. δ

18O of NO3

- values were reported on a scale such that

the Vienna Standard Mean Ocean Water (VSMOW) was 0 ‰ and the Standard Light 

Antarctic Precipitation was -55.5 ‰ relative to the VSMOW (Coplen, 1988, 1994). The 

reported δ18O data were calibrated by assuming the following values for internationally 

distributed nitrate isotopic reference materials (Böhlke and others, 2003): IAEA-NO-3 

(+25.6 ‰), USGS32 (+25.7 ‰), USGS34 (-27.9 ‰), and USGS35 (+57.5 ‰). The analytical 

uncertainties for δ15N of NO3

-

 and δ18O of NO3

- results were ±0.5 and ±1.0 ‰, respectively

(2-sigma). N2O concentrations were determined by gas chromatography with an electron 

capture detector as described by Smith and Duff (1988) at the USGS laboratory in Boulder, 

Colorado. 

24
Reconstruction of Initial Nitrate Concentration and Isotopic Composition 

The initial concentration and isotopic composition of NO3

-

 was estimated for wells 

where excess N2 gas resulting from denitrification was estimated to be present. The initial 

NO3

- concentration present in the ground water prior to the onset of denitification was 

estimated as the sum of the measured NO3

- and excess N2. The initial isotopic composition of 

the NO3

-

 concentration, δ15N of NO3

-o, present in the ground water prior to the onset of 

denitification, was estimated by an isotopic mass balance as described by Böhlke et. al. 

(2002). 

Results and Discussion

Chemical and Isotopic Composition of the Treated Effluent

The treated effluent discharged to the RIBS contained high concentrations of NO3

-

-N, 

organic carbon, and boron. Measured NO3

- concentrations from 2004-2006 ranged from 

1.37-6.93 mg/L as N. Organic carbon and boron concentrations measured in an effluent 

sample in November 2004 were 6.1 mg/L as C and 0.24 mg/L, respectively. δ15N of NO3

-

values from effluent samples had a wide variation (+9.46 to +29.07 ‰) which resulted from 

periodic denitrification of the effluent at the WWTRF. 

Evidence for Denitrification

Chemical conditions were conducive for denitrification to occur in the UFA but 

generally not in the SAS. The UFA wells contained organic carbon, varying amounts of NO3

-

, and little O2. Dissolved O2 concentrations in water from all UFA wells were less than 1 

mg/L. Ground waters exhibiting evidence of denitrification typically have concentrations less 

than approximately 2 mg/L (Böhlke et. al., 2002). Organic carbon was present in all UFA 

wells at concentrations ranging from 0.4-1.7 mg/L, and was highest (0.9-1.7 mg/L) in water 

samples from well 5. NO3

- was present in water from UFA wells 1-E, 2, and 4, but was not 

detectable in samples from wells 3 and 5. NO3

- concentrations were highest in well 1-E 

(1.78-3.17 mg/L as N) and 4 (0.44-0.77 mg/L as N). NO3

- was detected at low concentrations 

(0.02-0.05 mg/L as N) in water from well 2 from 2004-2005 but was not detected in water 

samples collected in 2006. Water from the SAS wells typically contained oxic water with 

higher concentrations of organic carbon and NO3

- compared to the UFA. Dissolved O2

concentrations in the SAS wells ranged from 1.5-8.4 mg/L, and organic carbon 

concentrations ranged from 0.6-3.9 mg/L. NO3

-

 concentrations generally were elevated in the 

SAS wells compared to the UFA and ranged from 1.20-6.30 mg/L as N. Aerobic degradation 

of organic carbon likely was occurring in the part of the SAS represented by well 1-B, which 

resulted in increased CO2 concentrations and lower pH values in water from this well. Water 

from well 1-B had PCO2 values ranging from 5.1-7.3 kPa, which were approximately 150-

200 times higher than values in ambient air. Water pH values also were lower in well 1-B 

(5.1-6.2 units) due to the high PCO2 values and were about 1 unit lower than the pH of water 

from well 1-A which represents an overlying part of the SAS. 

25
Anomalously high apparent recharge temperatures calculated based on the N2 and Ar 

data in 4 of the 5 UFA wells indicated there was an additional source of N2 in the ground

water, which was attributed to denitrification. Concentrations of dissolved N2 and Ar in water 

from the SAS wells and UFA well 5 were consistent with atmospheric equilibrium of the air 

during recharge with the addition of small amounts of excess air (<4.8 cm3 STP/L). Apparent 

ground water recharge temperatures ranged from 14.0°C to 22.7°C in the SAS wells and 

from 20.3 to 22.2°C in UFA well 5. The wide variation in apparent recharge temperatures 

from the SAS wells likely resulted from the seasonal temperature fluctuations of the treated 

effluent discharged to the RIBS combined with the high infiltration rate. Apparent recharge 

temperatures were anomalously high in UFA wells 2 (30.1-36.9°C), 3 (36.8-37.8°C), and 4 

(30.2-33.9°C), and were near or slightly higher than the mean annual air temperature in well 

1-E (21.5-25.4°C). It was interpreted from these high recharge temperatures there was 

another source of N2 in the ground water besides recharge water in equilibrium with 

atmospheric air and the entrainment of excess air. This additional source of N2 most likely 

resulted from the denitrification of NO3

-

. The amount of N2 resulting from denitrification was 

estimated to range from near zero to 4.8 mg/L, assuming the water in these wells was 

recharged at the median recharge temperature calculated for well 5 (21.4°C), which is close 

to the mean annual air temperature for the area. Concentrations were lowest in well 1-E (near 

zero to 1.5 mg/L) and highest in wells 2 (2.8-4.6 mg/L) and 3 (4.7-4.8 mg/L). The amount of 

excess N2 may be underestimated for well 1-E. Water may be recharged to this well at a

temperature similar to those determined for the SAS wells (14.0°C to 22.7°C). A lower 

recharge temperature would result in a higher estimate of the amount of N2 resulting from 

denitrification, and would range from 2.7-4.1 mg/L at 15°C and 1.4-2.8 mg/L at 18°C. 

Elevated δ15N of N2 values measured in wells 1-E and 2-4 confirmed denitrification 

had occurred in these UFA wells. δ15N of N2 values in wells 1-E and 2-4 (+0.94 to +2.14 ‰) 

were notably higher than values associated with air-saturated water, approximately +0.75 ‰. 

δ15N of N2 values in the SAS wells (+0.61 to +0.75 ‰) and UFA well 5 (+0.70 to +0.84 ‰) 

were similar to air-saturated water values. The isotopic enrichment associated with 

denitrification was estimated to range from -2 to -8 ‰, which generally is smaller compared 

to the ranges reported for denitrification (Kendall and Aravena, 2000). Smaller enrichment 

factors reported in this study likely resulted from the well water samples containing a mixture 

of denitrified and undenitrified waters. 

Detections of N2O and NO2

-

 indicated active denitrification occurred in selected parts 

of the UFA. N2O and NO2

-

-N were detected in water samples from two UFA wells, 1-E and 

4. N2O concentrations in well 1-E ranged from 20.4-25.7 μg/L, and NO2

-

 concentrations in 

this well ranged from below method reporting limits (0.008 mg/L as N) to 0.04 mg/L as N. 

N2O concentrations were lower in well 4 compared to well 1-E and ranged from 3.1-11.9 

μg/L, and NO2

- concentrations in this well ranged from below method reporting limits (0.016 

mg/L as N) to 0.04 mg/L as N. N2O also was detected in SAS wells 1-B, 1-C, and 1-D at 

concentrations ranging from 3.1-20.2 μg/L, although the highest concentrations were 

measured in well 1-D (16.6-20.0 μg/L). These N2O detections may have resulted from: 1) the 

initial onset of denitrification before any appreciable build-up of N2 in the ground water

because some O2 concentrations measured were near the upper limit for ground waters 

exhibiting evidence for denitrification, and δ15N of N2 values were similar to those in airsaturated water, 2) the water in the well contains a mixture of denitrified and nondenitrified 

waters, or 3) nitrification of ammonium in the ground water. However, ammonium only was 

26
detected at concentrations near method detection limits in the water from these wells (0.01 

mg/L), which is considerably lower than concentrations previously reported in ground water 

in which nitrification was occurring (Smith et. al., 2006). This suggests the potential for 

nitrification in the SAS may be limited.

Sources of Nitrate and Initial Nitrate Concentrations 

Measured δ15N of NO3

-

 and δ18O of NO3

- values indicated that the source of nitrate 

was treated effluent in the SAS. δ15N of NO3

-

 and δ18O of NO3

- values in water from these

wells were consistent with values measured in the effluent and were within the typical range 

of values in wastewater (Fig. 3). Elevated boron concentrations also indicated the source of 

the high nitrate concentrations in the SAS wells was treated wastewater. Boron is a common 

component of detergents and often is used as a tracer of wastewater in ground water systems 

(Repert et. al., 2006; Verstraeten et. al., 2005). Boron concentrations measured in the SAS 

wells ranged from 0.17-0.23 mg/L and were slightly lower than those measured in the treated

effluent. 

Reconstructed initial N isotope values indicated that treated wastewater and fertilizer

were the NO3

- sources in the UFA wells. Estimated initial nitrate concentrations in the UFA 

wells ranged from 2.58-6.77 mg/L, depending upon the recharge temperature assumed for 

well 1-E. Initial nitrate concentrations in wells 2-4 ranged from 3.10-4.82 mg/L. In well 1-E, 

the initial nitrate concentrations ranged from 2.58-3.67 mg/L as N if the assumed recharge 

temperature was 21.4°C and was higher for lower assumed recharge temperatures (for 

example 5.77-6.77 mg/L as N at a recharge temperature of 15°C). δ15N of NO3

-o values were 

estimated for three UFA wells: 1-E, 2, and 4. δ15N of NO3

-o values could not be estimated for 

wells 3 and 5 because there was insufficient NO3

- in water samples to determine its current 

isotopic composition. δ15N of NO3

-o values in wells 1-E and 2 (Fig. 3), which are located 

near two of the most heavily-loaded RIBS, indicated the nitrate source likely was treated 

wastewater. Lower δ15N of NO3

-o values in well 4 indicated the nitrate source was past 

27
fertilizer use, which may have originated from citrus agriculture. The distribution of boron 

concentrations in the UFA wells supports this interpretation of the isotope data. The highest 

boron concentrations in the UFA were measured in wells 1-E (0.06-0.10 mg/L) and 2 (0.06-

0.08 mg/L), and concentrations in the other UFA wells ranged from 0.01-0.03 mg/L. 

Summary and Conclusions

The potential for denitrification and sources of nitrate were evaluated in the karstic 

Upper Floridan aquifer (UFA) and overlying surficial aquifer beneath and upgradient from a 

wastewater treatment and reuse facility in Central Florida. Prior to the construction of this 

facility, the land use likely was citrus agriculture. Nitrate-N concentration (<0.02 to 6.3 

mg/L) in ground water in this area likely originated from past fertilizer use in citrus 

agriculture or treated wastewater applied to rapid-sand infiltration beds. Chemical, isotopic, 

and dissolved gas data were evaluated from water samples collected from 9 wells to 

determine nitrate sources, the potential for denitrification, and identify areas of active 

denitrification. Sampled surficial aquifer wells ranged from 3.7 to 23.8 m deep, and UFA 

wells ranged from 46 to 102 m deep. δ15N of NO3

-

 and δ18O of NO3

- values in well water 

samples indicated that treated wastewater likely is the source of nitrate in the surficial aquifer 

wells, whereas reconstructed initial δ15N of NO3

- values indicated the sources of nitrate in the 

UFA were treated wastewater in wells closest to the RIBS and fertilizer in the well located 

further away from the facility. Denitrification likely occurs in the UFA, but may only occur 

to a limited extent in the surficial aquifer. Dissolved oxygen concentrations generally were 

less than 1 mg/L in the UFA and greater than 2 mg/L in the surficial aquifer. Dissolved gas 

and isotopic data indicated that denitrification had occurred in water from 4 of 5 UFA wells. 

Excess nitrogen gas (N2), the end product of denitrification, was estimated to be present in 

water from four UFA wells based on the concentrations of dissolved N2 and Ar in the ground 

water. Excess N2 concentrations which likely resulted from denitrification typically ranged 

from 1 to 5 mg/L. δ15N values associated with N2 in these same wells also were substantially 

greater (+0.97 to +2.14 ‰) than for N2 in equilibrium with air-saturated water. 

Denitrification may naturally attenuate some nitrate in low dissolved oxygen zones of the 

UFA before it is transported to natural springs in the area. The chemical character of 

groundwater in karst can change rapidly during recharge events (Doctor et al., 2006). More 

frequent sampling of well water during conditions of water table fluctuation, especially 

during periods of RIB loading, may provide additional information on controls on conditions 

leading to denitrification within the study area.

Acknowledgments

This study was funded jointly by the US Geological Survey (USGS) and Orange County, 

Florida. The author greatly appreciates the permission given by the Orlando-Orange County 

Expressway Authority to sample water from their private well. The author thanks J.K. 

Böhlke and Dick Smith of the USGS for analysis of stable isotopes of aqueous N2, allowing 

the author to use laboratory equipment to analyze N2O in water samples, and insightful 

discussions. Field assistance from Orange County and Trudy Phelps of the USGS is 

28
gratefully acknowledged. The manuscript was significantly improved from reviews from Dan 

Doctor and Lan Tornes of the USGS. 

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Biographical Sketch

Sharon Kroening is a hydrologist with the U.S. Geological Survey in Orlando, 

Florida; U.S. Geological Survey, 12986 Research Parkway, Orlando, Florida 32826; 

kroening@usgs.gov. 

32
Appendix 2. Laboratory Report of Tritium/He Measurements for USGS Project

Prepared by the Noble Gas Laboratory Lamont-Doherty Earth Observatory of Columbia 

University, New York. Explanatory information provided for the reader precedes the reprinted 

document. Refer to Schlosser and others (2008) in the references cited section for the complete 

report citation.

 

33
Tritium/helium measurements may be used to determine the elapsed time since water in an

aquifer lost contact with the atmosphere, commonly referred to as the “age” of the water. Tritium is a 

radioactive form of hydrogen containing 1 proton and 2 neutrons, often written as 3

H. Tritium is a rare, 

naturally occurring isotope with typical concentrations near 1-3 tritium units (TU) in current rain 

samples. From 1952 to 1963, tritium was also produced as a byproduct of above ground testing of 

nuclear bombs. Tritium is incorporated into the water molecule, making it an effective environmental 

tracer that is useful in age dating relatively young groundwater. Tritium concentrations peaked in 1963 

at over 1,000 TU in precipitation at some locations in the U.S. In Miami from the mid-1960s to 1990, 

the tritium concentration in precipitation ranged from 35 TU to the background concentration of 1-3 TU 

(Walsh and Price, 2010). 

The half-life of tritium is 12.43 years (Unterwegar and others, 1980) and the end product is 

radioactive helium, 3

Hetrit (Turrin, 2012). As a result of radioactive decay since the peak production of 

tritium, an accurate measurement of the concentration of 3

Hetrit is also required to estimate groundwater 

age. Helium is predominately in the form of 4He, but 3He is naturally occurring in the earth’s mantle and 

the atmosphere. In order to determine the precise concentration of 3Hetrit in groundwater, the following 

variables need to be accounted for: concentrations of 4He and neon dissolved in groundwater and the 

temperature and elevation at which recharging water was no longer in equilibrium with the atmosphere 

(Turrin, 2012). For further detail on 3H/3He dating methods, see Schlosser and others (1988). 

The nine water samples presented in this lab report were collected to determine the age of water 

in the surficial and Upper Floridan aquifer from wells located at or near the Northwest Water 

Reclamation Facility in Orange County, Florida (fig. 1). Not all of these results are included in the main 

report because some analyses were inconclusive or not used for the study. Description of well depths 

and location reported by Kroening (2007) are included in appendix I; however, the well names were 

different in that report. Cluster wells 1-B, 1-C, 1-D in Kroening (2007) are reported in this appendix as 

Cluster Well 2, Cluster Well 3, and Cluster Well 4, respectively. Cluster Well 1-E in Kroening (2007) 

is Monitoring Well 8R, and sites 2, 3, 4, and 5 are reported here as Monitoring Well 2, Monitoring Well 

5R, Phipps Well and Toll Plaza Well, respectively. Keene Road Well 14, which was not used in this 

study, is an Upper Floridan Well located in the landfill on the northeast boundary of the RIBs site. This 

well is 172 ft deep with a 10 ft open interval. Three wells are in the surficial aquifer and the remaining 

6 wells are in the Upper Floridan aquifer. Information on well depths, casing, and hydrogeology are in 

Kroening (2007) under Site Selection. 

The wells were sampled January 24, 25, 31 of 2006, using the method described by Schlosser 

(undated). Nine sets of duplicate samples were submitted to the laboratory. Analysis methodology is 

described by Schlosser and others (1988). Laboratory results and measured concentrations are listed in 

this appendix. For further explanation of the meaning of uncorrected age and corrected age data listed in 

the laboratory report see U.S. Geological Survey (undated). Tritium was measured in samples from all

nine wells, and ranged in concentration from 0.08-5.46 TU. The 3

He, 4He, and neon concentrations were 

measured in the samples from five wells. Neon concentration was also measured in the sample from one 

additional well. Various reasons are listed in the laboratory report for the possible problems associated 

with the samples. Two sites where problems affected laboratory analysis were Cluster Well 4 (surficial 

aquifer) and replicate samples from Monitoring Well 2 (Upper Floridan aquifer). For the purpose of this 

study, 19 years was used as an estimate for the age of the water from the Upper Floridan aquifer. 

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