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Chapter 3: Methodology for Deriving U.S. EPA's Proposed Criteria 

for Springs and Clear Streams 

Springs and their associated spring runs are a unique class of aquatic ecosystem, highly treasured 

for their biological, economic, aesthetic, and recreational value. Because their principal water 

source is ground water, most springs have water that is extremely transparent, and rich with 

dissolved ions due to prolonged contact with subterranean limestone (Brown et al. 2008). 

Globally, the largest number of springs (approximately 600–700) occurs in Florida. Springs are 

often classified based on their flow rate, which ranges from more than 2,800 L/sec (first 

magnitude) to less than 0.47 L/sec (eighth magnitude). Many of the larger spring ecosystems in 

Florida have likely been in existence since the end of the last major ice age, approximately 

15,000 to 30,000 years ago (Martin 1966; Munch et al. 2006). During this period, plant and 

animal communities have evolved to become highly adapted to the unique water quality and 

conditions found in the springs. The productivity of the diverse assemblage of aquatic flora and 

fauna is primarily determined by light availability and secondarily affected by the availability of 

macro and micro nutrients and by the ambient ground water temperature. 

Springs also represent an important resource for human utilization, both by indigenous peoples 

(as supported by archaeological evidence) and by present-day Floridians and tourists, who use 

them for a variety of recreational purposes (Scott et al. 2002). People are interested in and 

fascinated by the intrinsic aesthetics of clear, cool water vigorously emanating from 

underground. A number of the spring boil areas (i.e., the limestone vent where the majority of 

aquifer water is discharged to the surface, sometimes in a turbulent manner) have been modified 

to facilitate swimming, recreation, and even “health spas.” Currently, all of the largest springs in 

Florida, whether privately or publicly owned, are managed as recreational parks, which, in turn, 

attract a large number of visitors and generate many millions of dollars in revenue on an annual 

basis. Correspondingly, many springs have suffered declines (generally) in their condition 

(e.g., up-rooting of vegetation, bank erosion, litter) due to visitation by ever-increasing numbers 

of people. 

Other more serious factors with the potential to permanently alter Florida’s spring ecosystems 

have been increasingly recognized over the last two decades. The two most significant 

anthropogenic factors that have been linked with adverse changes in spring ecosystems are: 

1. Pollution of ground water, principally with nitrate-nitrogen (NO3-nitrogen),1

 resulting from 

human land use changes, cultural practices, and general population growth. 

2. The simultaneous reduction in ground water supply through consumptive human 

withdrawals. 

Human influences, in the form of nonpoint source pollution, are one of the most critical issues 

affecting Florida’s springs. Nutrients (predominantly nitrogen) associated with urban and 

agricultural activities (including fertilization and waste disposal), seep through soils and are 

transported to springs by way of underground pathways. However, in excessive amounts, 

 

1

 Nitrate nitrogen (NO3-nitrogen) is a closely associated inorganic nitrogen compound to nitrite nitrogen (NO2-nitrogen). Because 

of the analytical methods used to quantify them, they are expressed together as NO3-NO2. Nitrite concentrations, however, tend 

to be a small proportion of the combined NO3- NO2 concentration. 
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nutrients can be harmful to the environment, leading to eutrophication and potentially allowing 

periphyton (algae) and invasive plant species to displace native plants, which results in an 

ecological imbalance. Problematic growths of nuisance algae and noxious plants result in 

reduced habitat and food sources for native wildlife, excess organic carbon production, 

accelerated decomposition, and lowered substrate quality, all of which affect the overall health 

and aesthetics of Florida’s springs (Brown et al. 2008). 

Prior to wide-scale development of Florida springs and their watersheds, native submerged 

aquatic vegetation (SAV), primarily Sagittaria kurziana and Vallisneria americana, dominated 

the underwater regions near most spring boils. Evidence indicates that macroalgae likely 

occurred naturally in Florida springs, but not in the excessive abundance commonly observed 

today. Stevenson et al. (2004) found that the majority of the Florida springs studied had nuisance 

growths of algae, primarily Vaucheria and Lyngbya wollei. Vaucheria has been reported in 

spring seeps in areas around the world with very limited human activity, but typically not in 

great abundance. Additionally, historic records of Lyngbya wollei exist from Silver Spring 

(Pinowska et al. 2007a), but prior to the last 20–30 years there were no records of nuisance 

Lyngbya growth. 

Within the past 20–30 years, however, anecdotal observations at several springs suggest that 

nuisance algae species have proliferated and are now out-competing and replacing SAV. As 

benthic algal mats accumulate, they kill beneficial SAV through direct smothering or indirectly 

through shading (Dennison and Abal 1999; Doyle and Smart 1998). Once the native SAV is 

displaced, other nonnative taxa such as Hydrilla verticillata (hydrilla) can re-colonize bare 

substrates, leading to other biological and ecological changes. For example, springs have become 

increasingly inhospitable to certain fish, snails, crayfish, turtles, and other animals that depend 

on the spring habitat. The loss of the native SAV, as well as the increased dominance of nuisance 

filamentous algae, results in community metabolism changes and disappearance of higher-order 

animals. This chain of events reflects the significant adverse structural and functional changes 

that have occurred in spring ecosystems. 

Numerous biological studies have documented excessive algal growth at many major springs. In 

some of the more extreme examples, such as Silver Springs and Weeki Wachee Springs, algal 

mat accumulations have become several feet thick. The thick benthic algal mats are detrimental 

to aquatic life and cause significant problems for recreational use. The profuse growth of 

macroalgae has been linked to increased nutrient levels in the springs (Florida Springs Task 

Force 2000). In a recent survey of 60 first- and second-magnitude springs in Florida, the most 

commonly observed algal taxa were filamentous mat-forming cyanobacteria of the genus 

Lyngbya, and the xanthophyte Vaucheria (Stevenson et al. 2004). When algal cells senesce and 

die, they cause localized depletion of oxygen and the release of ammonia and hydrogen sulfide, 

which can further degrade water quality (Joint Nature Conservation Committee 2004). In 

general, the spread of macroalgae in Florida has resulted in reductions in fish and crab harvests, 

lost tourism, and removal costs (O’Neil and Dennison 2005; Bonn and Bell 2003). Lyngbya 

wollei proliferation is especially problematic because more than 70 biologically active 

compounds have been isolated from this species, many of which are toxic and/or carcinogenic to 

humans and can therefore inhibit the recreational use of the resource (Osborne et al. 2001). 
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Lyngbya are a source of lyngbyatoxin and aplysiatoxin, which produce a condition known as 

“swimmer’s itch” (Cardellina et al. 1979; Mynderse et al. 1977). Lyngbya wollei can produce a 

variety of paralytic shellfish poisons (e.g., saxitoxin) and other toxins capable of producing 

dermatitis in humans (Carmichael et al. 1997; Onodera et al. 1997; Stewart et al. 2006; Teneva et 

al. 2003) and deaths in domestic and wild animals that consume algal mats (Edwards et al. 1992; 

Falconer 1999; Gugger et al. 2005; Hamill 2001; Saker et al. 1999). 

It is hypothesized that the shift from SAV to blue-green algae in Florida springs is being driven 

by land-use change in springsheds, specifically increased loading of dissolved nutrients, 

especially NO3, and organic matter into the Floridian aquifer system from agriculture and 

urbanization. For example, in Ichetucknee Springs, mean annual NO3 concentration increased 

from 0.35 mg/L in 1975 to 0.70 mg/L in 2001 (Scott et al. 2004). During that period, anecdotal 

observations indicate that some areas in the river, including reaches below Blue Hole and 

Mission Springs, became dominated by Lyngbya. 

Detailed study of two spring ecosystems, Silver Springs and the Rock Creek/Wekiva Spring 

complex, suggests that there is evidence for ecosystem scale effects of NO3 enrichment. Using a 

subsidy stress theory to explain the decline in gross primary production (GPP), it is suggested 

that increases in NO3 act to increase productivity for a time, but then act as a stress to depress 

productivity above a certain threshold (Knight and Notestein 2008). One study presents 

compelling evidence for a decrease in overall productivity in Silver Springs from values 

measured in a 1950 study as compared to today (Knight and Notestein 2008). This declining 

ecosystem productivity documented at Silver Springs was highly correlated with increasing NO3-

nitrogen concentrations during the 50-year period of available data. Declining spring flows, 

increased shading by riparian trees, and altered fish populations were also observed to be 

correlated with declining ecosystem production at Silver Springs and could offer alternate or 

cumulative explanations of the observed ecosystem changes. 

The second study conducted in the Wekiva River and Rock Springs Run also found an inverse 

correlation between nutrient (total nitrogen (TN) and total phosphorus (TP)) concentrations and 

ecosystem metabolism. These spring run ecosystems exhibited other significant environmental 

stresses caused by humans, including decreases in discharge, intensive exotic plant management 

efforts, and disturbance due to recreational activities. Studies of whole ecosystem responses to 

nutrients that would result in direct evidence that increased nutrient levels alone could result in 

decreased ecosystem productivity and/or photosynthetic efficiency were not available (Knight 

and Notestein 2008). 

In addition to increased NO3 concentrations, there are numerous other biotic and abiotic factors 

that have also changed in some, but not all, springs. These changes include increased recreational 

use, decreased water output, decreased dissolved oxygen (DO) concentrations in spring 

discharge, increased need for aquatic weed control, greater abundance of invasive species, and 

increased salinities. By themselves or in combination, these factors might either accentuate or 

mask the effects of nutrients in specific spring systems, and they might help explain the observed 

variations in response to increasing NO3 levels observed in Florida’s springs over the last several 

decades. For example, it is believed that aquatic plant control techniques (e.g., herbicide 

applications or mechanical harvesters) that are used to suppress excessive growth of nonnative 
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plants have the potential to serve as severe disturbances that can further promote succession 

towards algae-dominated spring ecosystems. 

The specific mechanisms and interactions between NO3-NO2 enrichment and these other 

confounding factors that cause the observed changes in Florida springs are not fully understood. 

However, due to the obvious degradation of other aquatic habitats worldwide as a result of 

increased nutrient loading, there is justifiable concern for potential negative consequences in 

Florida spring systems. The potential consequences of nutrient enrichment in springs include an 

increase in opportunistic primary producers, increased organic matter deposition, greater number 

of nuisance algae species and amount of algal biomass, decreased plant and animal productivity 

and diversity, reduced water quality, and not insignificantly, a reduction of the aesthetics these 

ecosystems have long provided. 

3a. Basis for Nitrate-Nitrite Criteria 

Nitrogen is one of the essential elements for plant life, and NO3 is a form of nitrogen that is 

readily utilized by aquatic plants and algae. However, NO3 in excess can lead to the development 

of nuisance aquatic plant problems (Rabalais 2002). As a result of human land use changes, 

cultural practices, and general population growth, there has been an increase in the level of 

pollutants, especially NO3, in ground water over the last several decades. 

Because there is no geologic store of NO3, all the NO3 emerging in spring vents originates from 

that deposited on the land surface. Because there are generally limited biogeochemical 

mechanisms or processes to retain or remove NO3 once it has been introduced into the ground 

water below the root zone, it is transported through the ground water largely as a conservative 

solute. Consequently, a significant portion of the NO3 introduced at the land surface, especially 

when in excess of biological demand, finds its way into ground water and ultimately into the 

spring system. 

Historically, natural background NO3 concentrations in spring discharges are thought to have 

been 0.05 mg/L or less (Maddox et al. 1992), which is sufficiently low to restrict the growth of 

native algae and vegetation under natural conditions. Increasing human populations have altered 

the global nitrogen cycle and other biogeochemical cycles through land use changes, fertilizer 

use, fossil fuel combustion, and other pathways. Population increases and land use changes have 

resulted in nutrient enrichment. Florida’s karst region has experienced unprecedented population 

growth and changes in land use over the past several decades, with a consequential transfer of 

nutrients to the relatively unprotected ground water. Katz et al. (1999) used isotopic analyses to 

show that substantial portions of the NO3-NO2 found in the Upper Floridan Aquifer and in spring 

discharges are derived from anthropogenic activities such as fertilizer application for agriculture 

and residential uses, livestock waste, and human waste. 

Figure 3-1 shows the changes in NO3 concentration in Weeki Wachee Spring discharge as 

related to the population increase in Hernando County, Florida. The spring NO3 concentrations 

follow a pattern very similar to the population curve with a 10 to 15 year lag. The lag period 

between changes on the land surface and the subsequent effect on spring discharges is expected 

since aging measurements of water emerging from springs suggest that on average, it has spent 

between 10 and 30 years in the subsurface. However, these studies have also shown that a 
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significant portion of water (30–70%) has residence times less than 4 years and that the relative 

age contributions vary significantly between springs, depending on the characteristics of the 

springshed. 

Of 125 spring vents sampled by the Florida Geological Survey in 2001–2002, 52 (42%) had NO3

concentrations exceeding 0.50 mg/L and 30 (24%) had concentrations greater than 1.0 mg/L 

(Scott et al. 2004). Therefore, over 40% of the springs sampled had at least a tenfold increase in 

NO3 concentrations above background and approximately one-quarter of them demonstrated at 

least a 20-fold increase. Similarly, a recent evaluation of water quality in 13 first-magnitude 

springs shows that mean NO3-NO2 levels increased from 0.05 mg/L to 0.9 mg/L between 1970 

and 2002 (Scott et al. 2004; Figure 3-2). Overall, data suggest that NO3-NO2 concentrations in 

many spring discharges have increased from 10- to 350-fold over the past 50 years, with the 

level of increase closely correlated with the anthropogenic activity and land-use changes within 

the springshed. 

As a result of the increased NO3-NO2 levels in ground water and spring discharges, downstream 

NO3-NO2 loads are also increasing rapidly in many watersheds. For example, the NO3-NO2

concentrations of several springs in the Suwannee River Basin have increased in the last 40 years 

from less than 0.1 mg/L to more than 5 mg/L (Hornsby and Ceryak 2000, cited in Katz et al. 

1999) with the NO3-enriched spring discharge resulting in a two- to three-fold increase in the 

level of NO3 exported to the Gulf by the Suwannee River. Consequently, the Suwannee Sound 

has experienced phytoplankton blooms in excess of 11 μg/L of chl. a, resulting in its placement 

on the list of Impaired Waters. 

As NO3-NO2 concentrations have increased during the past 20 to 50 years, many Florida springs 

have concurrently undergone a number of adverse environmental and biological changes, as 

described previously. There is a general consensus in the scientific community that NO3 is an 

important factor leading to the observed changes in spring ecosystems and their associated 

biological communities. Nitrogen, particularly NO3-NO2, appears to be the most problematic 

nutrient problem in Florida’s karst region. 

There are four primary reasons for greater concern about NO3-NO2 compared to phosphorus. 

First, increases in NO3-NO2 concentrations are nearly omnipresent in areas where anthropogenic 

loading to the land’s surface has occurred. Second, once in the ground water, denitrification is 

negligible and NO3-NO2 appears to be transported as a conservative solute. Third, although 

Florida’s geology is naturally rich in phosphorus, there does not appear to be a trend of 

increasing phosphorus concentrations in spring discharges. While NO3-NO2 concentrations have 

increased significantly in most spring discharges, phosphorus concentrations have remained 

relatively constant over the past 50 years. Fourth, since springs are naturally rich in phosphorus, 

the majority of Florida springs are likely to have been historically nitrogen-limited (Inglett et al. 

2008; Knight and Notestein 2008). In their historical natural state, most Florida springs contain 

high levels of bioavailable soluble reactive phosphorus levels of between 30 and 60 ppb, which 

have been shown to be sufficient to support the observed growth of algae and aquatic plants. 

Because NO3-NO2 has been linked to many of the observed detrimental impacts in spring 

systems, there is an immediate need to reduce NO3-NO2 concentrations in spring vents and in upgradient ground water. To restore and preserve springs, activities in springsheds that contribute 
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to the transfer of nitrogen to the ground water must be properly managed to achieve acceptable 

levels. Due to the relatively long lag times between activities on the land’s surface and the 

resulting effect on the spring discharge, it will likely be several years before the effects of NO3

control measures will be seen. The first step in the process is to develop a numeric NO3-NO2

criterion for spring systems that will be protective of this unique resource. In addition, a NO3-

NO2 criterion will help identify systems at risk of degradation from excessive NO3 pollution, as 

well as those where detrimental changes have already occurred and restoration is needed. EPA’s 

analysis of a NO3 criterion, which was based largely on and agrees with FDEP’s results, is 

discussed below. 

i. Criteria Development Methods 

FDEP has worked to derive response-based thresholds that will definitively link nutrient 

thresholds to biological and environmental risk. FDEP has used multiple lines of evidence taken 

from the results of different types of research, as well as empirical data available from various 

monitoring programs, to develop NO3 criteria for springs, which ultimately were applied to all 

clear streams (< 40 platinum-cobalt units (PCU)). The information evaluated includes: 

 Results from laboratory dosing studies conducted at various scales 

 In situ algal monitoring 

 Real-world surveys of biological communities and nutrient levels in Florida springs 

 Data regarding NO3 concentrations found in minimally disturbed reference streams 

Similar to the methods being used to establish numeric nutrient criteria for lakes and streams, 

FDEP has used multiple lines of evidence taken from the results of different types of research, as 

well as empirical data available from various monitoring programs, to develop NO3 criteria for 

clear streams, including springs. This information and its use in deriving the recommended NO3

criteria for springs are discussed in greater detail below. 

ii. Laboratory Studies 

The results of laboratory experiments have provided much valuable information about the 

response of algae (particularly algal growth response) to increasing nutrient concentrations under 

specific highly controlled conditions. Experimental systems, however, usually do not include all 

the complexities and ecological processes that affect the response to nutrients in natural 

waterbodies. The limitations of the small-scale experimental platform must be taken into account 

when applying the results to natural full-scale systems. 

Nutrient amendment bioassay work was conducted by Cowell and Dawes (2004) to determine 

the NO3 concentration required to achieve a reduction in biomass of Lyngbya wollei, a nuisance 

blue-green benthic algal species that dominates many spring systems due to elevated NO3

concentrations. Using Lyngbya cultures incubated in a series of NO3 amendments, they found 

that both the biomass and growth rates were low in treatment groups with NO3 concentration at 

or below 300 μg/L, while the growth rates and biomass were significantly higher in treatments 

with NO3 concentrations at or greater than 600 μg/L (Cowell and Dawes 2004). In addition, the 

experiment also showed that the biomass and growth rate in treatment groups with NO3

concentrations from 70 to 300 μg/L were similar, suggesting that further reduction of NO3
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concentration below the 300 μg/L level would probably not achieve significant additional 

reductions of L. wollei abundance and growth. They concluded that a NO3 concentration of 300 

μg/L should be sufficient to control L. wollei growth. 

Similarly, Stevenson et al. (2007) found that growth of small L. wollei mats in NO3-dosed 

raceways approached maximum levels at NO3 concentrations above 518 to 546 μg/L. In similar 

studies using Vaucheria, growth rates were low at NO3 concentrations below 69 μg/L and 

increased substantially from 69 to 644 μg/L. Further growth rate increases at NO3 concentrations 

above 644 μg/L were minimal. 

In smaller-scale microcentrifuge tube microcosm experiments conducted to evaluate the growth 

response of individual macroalgal filaments to precise levels of NO3 dosing at high phosphate 

levels, Stevenson et al. (2007) found that the growth rate of Lyngbya wollei was minimized at 

NO3 concentrations below 34 μg/L. Growth rates increased substantially at NO3 concentrations 

from 34 to 230 μg/L and approached maximum levels at concentrations above 230 μg/L. For 

unexplained reasons, the growth rate of Vaucheria did not respond to NO3 additions in the 

microcentrifuge tube microcosm experiments. 

As discussed by Stevenson et al. (2007), the differences in results between the raceway and 

microcentrifuge tube experiments were likely related to the differences in scale of the 

experiments. In the microcentrifuge tube microcosms using individual macroalgal filaments, 

very accurate control of nutrient levels was possible. In the larger-scale raceways using small 

algal mats, substantial nutrient depletion was possible and could not be accounted for, which 

resulted in a higher estimate of regulating NO3 concentrations. Recognizing the limitations of the 

laboratory experiments, Stevenson et al. (2007) recommended using the ED90 (NO3-NO2

concentration that produces 90% of the maximum growth) determined from the highly controlled 

microcentrifuge tube experiments as a preliminary NO3 criterion that could be refined using 

additional information. The best estimates for the NO3 ED90 s determined from the laboratory 

experiments were 230 μg/L for Lyngbya wollei and 261 μg/L for Vaucheria species. 

iii. Field Surveys 

Numerous surveys of macroalgae and nutrients in springs have been conducted to demonstrate 

the cause-effect relationships between elevated nutrient concentrations and macroalgal growth, 

and to evaluate the nitrogen or phosphorus concentrations associated with proliferations of 

macroalgae. The benefit of using results of field surveys for nutrient criteria development is the 

direct applicability of observed nutrient concentrations and biological responses. 

In a survey of Florida springs, macroalgae were found at 59 of the 60 sampled sites, and an 

average of 50% of the spring bottoms were covered by macroalgae; the thickness of the 

macroalgal mats was commonly 0.5 meter or more and as thick as two meters in one spring boil 

(Stevenson et al. 2004). Lyngbya wollei and Vaucheria spp. were the two most common taxa of 

macroalgae that occurred in extensive growths in the studied springs, however 23 different 

macroalgal taxa were observed in the spring survey. 

During the surveys, the abundance of Vaucheria species within the springs was found to be 

positively related to nitrogen and phosphorus concentrations. Nonlinear models of Vaucheria 
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percent cover and thickness along the TN and NO3 gradients explained substantially more 

variation than a linear model, with a clear threshold in Vaucheria response at 0.454 mg/L as NO3

(i.e., 0.591 mg/L as TN). Excessive growth and cover of Vaucheria were found at sites with NO3

concentrations at or above the 0.454 mg/L threshold, with Vaucheria abundance being 

significantly less at sites with lower NO3 levels (Stevenson et al. 2007). This excessive growth of 

macroalgae is considered to constitute an imbalance of the natural biological communities and 

not in compliance with Florida’s narrative nutrient criteria. Therefore, to provide for a margin of 

safety, a protective numeric NO3 criterion would need to be below the observed 0.454 mg/L NO3

Vaucheria threshold. 

iv. TMDL Development Activities 

The Wekiva River and Rock Springs Run are spring-influenced systems that are on the state’s 

impaired waters list due to evidence of an imbalance in aquatic flora characterized by excessive 

algal growth and lower ecosystem metabolic activities. There was also evidence that the 

impairment of the Wekiva River and Rock Springs Run was caused by elevated NO3 and 

phosphorus levels. The mean NO3 concentration in the Wekiva River and Rock Springs Run 

ranged between 60 and 70 μg/L, which is significantly higher than levels found at nearby 

minimally disturbed reference sites with similar characteristics (Juniper and Alexander Springs). 

Additionally, the Wekiva and Rock Springs NO3-NO2 levels were above the threshold NO3

concentration identified by Stevenson et al. (2004) to be associated with nuisance Vaucheria

growth (Gao 2008). 

During the development of the TMDL for these waterbodies, protective nutrient concentration 

targets were derived using periphyton and water quality data collected from the Suwannee River 

and two tributaries, the Withlacoochee River and Santa Fe River (Hornsby et al. 2000). These 

data were considered applicable to the Wekiva River and Rock Springs Run because the 

Suwannee River is heavily influenced by spring inflow and, in the absence of anthropogenic 

inputs, the algal communities would be expected to be generally similar in composition to those 

in the Wekiva River and Rock Springs Run. 

An evaluation of periphytometer data collected during the period from 1990 through 1998 from 

13 sites along the Suwannee River showed positive correlations for both periphyton biomass 

versus NO3 concentration and cell density versus NO3 concentration. The functional 

relationships of cell density versus NO3 concentration and periphyton biomass (represented as 

ash-free dry weight, or AFDW) versus NO3 concentration are shown in Figures 3-3 and 3-4, 

respectively. Data presented in these figures represent long-term average biomass, cell densities, 

and NO3 concentrations at the stations across the Suwannee River system (Niu 2007). 

As can be seen for both cell density and biomass, periphyton abundance significantly increased 

when NO3 concentration increased above approximately 350 μg/L. The data were further 

evaluated using a change point analysis to better define the NO3 concentration that might 

significantly impact the periphyton biomass and cell density. The change point analysis fits a 

step function through observed data by examining the probability of each data point as the 

change point. For both periphyton cell density and periphyton biomass, change point step 

functions were shown to be the best model among the models tested, which supports the use of 

change point analysis. 
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For the relationship between cell density and NO3 concentration, the change point step function 

identified two populations of sites. The first set of sites had cell densities near 163,000 cells/cm2

(P = 0.009), which was considered the baseline condition under which no significant NO3 impact 

was detected. The second group of sites had cell densities near 616,000 cells/cm2

 (P = 0.0001), 

which was significantly elevated above the baseline condition. The change point analyses also 

indicated that the critical increase in mean algal cell density occurred as the mean NO3

concentration increased from 286 to 401 μg/L (Niu 2007). This suggests that to prevent the 

periphyton cell density from increasing to the higher level, the NO3 target concentration should 

be established below 401 μg/L. 

Similarly, the change point analysis of the relationship between periphyton biomass and NO3

concentration identified two populations of sites. The first set of sites had a periphyton biomass 

near 1.73 g/m2

 (p< 0.0001), which was considered the baseline condition under which no 

significant NO3 impact was detected. The second group of sites had an increased algal biomass 

near 4.15 g/m2

 (p = 0.0001), which was significantly elevated above the baseline condition. The 

change point analyses also indicated that the critical increase in mean periphyton biomass 

occurred as the mean NO3 concentration increased from 401 to 420 μg/L (Niu 2007). This 

suggests that to prevent the periphyton biomass from increasing to the higher level, the NO3

target concentration should be established below 420 μg/L. 

Periphyton cell density exhibited a sensitive response to increasing NO3 concentrations and was 

used as the basis for the NO3 target concentration. A periphyton cell density increase was 

observed for NO3 concentrations between 0.286 mg/L and 0.401 mg/L. The value of 0.286 mg/L 

was chosen to be the TMDL NO3 target concentration for the Wekiva River and Rock Springs 

Run systems because it provided a conservative criterion with an adequate margin of safety that 

is reasonably protective of the biological communities within these systems (Gao 2008). 

Following adoption of the TMDL, the change point analysis was repeated using additional data 

collected from 1990 through 2007 for the same 13 sites located along the Suwannee River. To 

account for any long-term temporal changes at a site, the period of record was divided into four 

periods. The average periphyton abundance and NO3-NO2 data for each period for each site were 

used to repeat the change point analysis. The results were very similar to those obtained from the 

original analyses as described above. A NO3 concentration change point of 440 μg/L was 

determined for both periphyton cell density and biomass (Figures 3-5 and 3-6). Since these 

change points represent the lower concentration range for the group of sites with significantly 

higher periphyton abundance, as compared to the baseline group, a protective NO3 criterion 

should include an appropriate safety margin to ensure that sites do not reach this level. 

Figures 3-7 and 3-8 illustrate the same observed relationships between periphyton cell density 

versus NO3 and algal biomass versus NO3 concentrations, respectively, with field and laboratory 

results illustrated using red lines. The 0.44 mg/L change point represents the upper NO3-NO2

concentration where the observed biological changes occur. Additionally, the 0.23 mg/L NO3-

NO2 threshold based on the laboratory studies, which represents the lower bound of the range in 

which biological changes occur, is also shown on the graphs for reference. Both graphs clearly 

indicate that algal abundance is restricted at NO3 concentrations below approximately 0.35 mg/L, 

with the potential for increased algal cell density and biomass increasing substantially near the 

0.44 mg/L change point. 
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In addition to the observed increases in periphyton cell density and biomass, shifts in the 

structure and taxonomic composition of the periphyton community were also observed across 

this NO3-NO2 gradient. The abundance of taxa indicative of eutrophic conditions (Van Dam et 

al. 1994) increased significantly with increasing NO3-NO2 concentrations above approximately 

0.35 mg/L (Figure 3-9). Similar to the changes in the algal abundance and biomass, the 

abundance of the eutrophic taxa are similar at sites with NO3-NO2 concentrations below 0.35 

mg/L, while the abundance of these taxa increases with increasing NO3-NO2 concentrations 

above 0.35 mg/L. The significant alterations in community composition, in combination with the 

increases in cell density and biomass, clearly demonstrate that increased NO3-NO2 levels in the 

range between the 0.23 mg/L laboratory threshold and the 0.44 mg/L change point based on field 

observations were associated with an imbalance of aquatic flora (Rule 62-302, FAC). Because 

the 0.23 mg/L laboratory limit is likely unnecessarily protective and the 0.44 mg/L change point 

is not adequately protective, this range must be refined to develop an appropriate criterion. 

Based on the analyses performed, multiple lines of evidence indicate that the biological changes 

appear to occur at sites with NO3-NO2 concentrations above approximately 0.40 mg/L. Since the 

0.44 mg/L change point represents the upper bound of the range in which the biological changes 

occur, an appropriate safety factor to ensure that sites do not reach this level should be applied to 

the change point to derive a protective NO3-NO2 criterion. 

To define this safety factor, two complementary analyses were conducted and the results 

averaged (Figure 3-10). The full confidence interval procedure resulted in a protective threshold 

of 0.33 mg/L, and the upper half of the confidence interval method yielded a protective threshold 

of 0.38 mg/L. The average of these complementary methods resulted in the final protective 

threshold of 0.35 mg/L. 

v. Analysis of Nutrient Gradient Study Rapid Periphyton Data 

In Appendix B-11, an analysis of algal responses (Rapid Periphyton Survey) to nutrients and 

other variables is presented. The analysis demonstrated that periphyton thickness was 

significantly correlated with NO3-NO2, canopy cover, and water color, but not with TP. It should 

be noted that color is a strong confounding factor in these analyses because it is correlated to 

some extent with canopy cover, NO3-NO2 concentration, and TP concentration. The analyses of 

the rapid periphyton assessment data are provided in greater detail in Appendix B-1. Note that in 

streams where color was less than 40 PCU and canopy was relatively open, there was a 

significant correlation between NO3-NO2 and excess algal growth (defined as >50% of the 

measurements having algal thickness exceeding 2 centimeters). Although there was uncertainty 

associated with this response, the range included the protective 0.35 mg/L threshold found for 

clear spring systems. Based on this finding, and to ensure protection against excess algal growth 

in Florida streams, it is reasonable to apply the 0.35 mg/L NO3-NO2 limit to all streams with 

color measurements of less than 40 PCU. 

vi. Summary and Conclusions 

Multiple lines of evidence indicate that reducing NO3-NO2 concentrations in clear stream 

systems should reduce macroalgal growth rates. Although response-based NO3-NO2 criteria were 

originally investigated for application in springs, additional analyses indicated that such criteria 
TSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

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would be appropriate in streams with color less than 40 PCU. Control of NO3-NO2 is expected to 

result in a reduced frequency, intensity, and duration of nuisance macroalgal growth in clear 

streams and to prevent biological imbalances (i.e., to restrict growth and accumulations of 

nuisance macroalgae and to preserve the native periphyton community structure). 

The most accurate and conservative experimental results––those from microcentrifuge tube 

experiments––suggest that NO3 concentrations less than 0.230 mg NO3-N/L are needed to slow 

the growth of Lyngbya wollei. Similarly, to reduce the growth of Vaucheria under laboratory 

conditions, NO3 concentrations below approximately 0.261 mg NO3-N/L would be required. 

Results of periphyton field surveys conducted at a large number of spring systems indicated that 

NO3 concentrations would need to be reduced below the observed 0.454 mg/L threshold to 

reduce the nuisance abundance and cover of Vaucheria species in Florida springs (Pinowska et 

al. 2007a). Since the 0.454 mg/L threshold represents the lower range of NO3 concentrations for 

sites with excessive algal growth and cover, an appropriate safety factor is needed to turn the 

threshold into a protective criterion. 

In addition, nearly two decades of scientific results from periphytometers deployed in the 

relatively low color (generally <40 PCU) Suwannee, Santa Fe, and Withlacoochee (north) rivers 

clearly indicated that significant increases in cell density and biomass, along with alterations in 

taxonomic community structure (which are indicative of an imbalance), occur as NO3

concentrations approach the 0.441 mg/L change point. 

A margin of safety, derived by averaging the upper half-range and full-range 95% confidence 

intervals, was applied to the 0.44 mg/L change point to derive the final 0.35 mg/L NO3-NO2

criterion. At monthly concentrations below 0.35 mg/L, there is a high confidence (95% 

confidence interval) that adverse responses will not be observed. 

Rapid Periphyton Survey analyses indicated that NO3-NO2 was significantly correlated with 

excess algal growth in clear streams (< 40 PCU). As a result, EPA will apply the same protective 

0.35 mg/L NO3-NO2 criterion developed by FDEP to all streams with color below 40 PCU. 

3b. Duration and Frequency for Proposed Springs Criteria 

The duration and frequency expression of an annual geometric mean not to be surpassed more 

than once in a three-year period is consistent with the expressions of duration and frequency for 

other water body types (e.g., lakes, streams, canals) for TN and TP for the same reasons EPA 

selected a three-year period for those waters (please see sections 1.d and 2.d above). EPA 

determined that this frequency of excursions should not result in unacceptable effects on aquatic 

life as it will allow the springs and clear streams aquatic systems enough time to recover from 

the occasionally elevated year of nutrient loadings. Likewise, the long-term arithmetic average of 

annual geometric means not exceed the criterion-magnitude concentration is also consistent with 

the expression used for other waters. 

EPA also considered as an alternative, expressing the criterion as a monthly median not to be 

surpassed more than 10% of the time. Stated another way, the median value over any given 

calendar month shall not be higher than the criterion-magnitude value in more than one out of 

every ten months. This alternative is consistent with the expression that FDEP proposed in June 
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2009 for its State rule and the expression in the TSD for Florida’s Inland Waters that EPA sent 

out for external scientific peer review in July 2009. The rationale for this alternative is that field 

data indicate that the response in springs is correlated to monthly exposure at the criterionmagnitude concentration value and a 10% frequency of excursions is a reasonable and fully 

protective allowance given small sample sizes in any given month (i.e., the anticipated amount of 

data that will be available for assessment purposes in the future). The clear streams nitrate-nitrite 

criterion was derived by FDEP based on multiple lines of evidence, with the primary lines of 

evidence being mesocosm dosing experiments and field studies. These two main studies were 

conducted by FDEP over very different time frames. 

One set of mesocosm studies was conducted by FDEP for periods just under one month (i.e., 21 

to 28 days), while the algal biomass field survey was conducted over an 18-year period and was 

analyzed using four to five year averaging periods. While lab studies indicate that algal 

communities can respond to excess nitrate-nitrite over a short period of time, the mesocosm and 

other dosing studies indicate that this response occurs on the order of a month, which would 

support a monthly expression of the criterion. Further, there is no evidence to suggest that the 

short-term responses observed under controlled lab settings equate to impairment of the 

designated use in conditions experienced in State waters. In fact, the more realistic field surveys 

demonstrated that excess biomass does not become persistent in clear streams until average 

nitrate-nitrite exceeds 0.441 mg/L. The 0.441 mg/L threshold was adjusted by FDEP to 0.35 

mg/L to provide a fully protective criterion with a low likelihood of excess algal growth. This 

threshold adjustment is consistent with the restricted growth of diatom taxa indicative of 

eutrophic conditions. 

The 10% excursion frequency would recognize that in most cases the monthly “median” would 

actually be based on a single sample, given that most springs are only sampled monthly at the 

most. A 10% excursion frequency may be considered a reasonable and fully protective allowance 

given small sample sizes in any given month, essentially requiring that the monthly median 

nitrate-nitrate concentrations thought to be fully supportive of relevant designated uses be met 

90% of the time. 

3c. Alternative Approach Considered by U.S. EPA 

i. Nitrate-Nitrite Criteria for all Waters as an Independent Criterion 

Stressor-response analyses from FDEP’s rapid periphyton survey in streams demonstrated that 

periphyton thickness is significantly correlated with NO3-NO2 (Appendix B-1). This relationship 

is similar to that described in spring systems, in which periphyton mean cell density and 

biomass, parameters determining thickness, increased as a function of increasing NO3-NO2 

concentration. Based on the rapid periphyton survey results and the consideration that NO3-NO2 

loads in springs and streams can flow to other surface waters, U.S. EPA is seeking comment on 

an alternative approach where the 0.35 mg/L NO3-NO2 criterion for springs and clears streams 

would apply to all surface fresh waters, in addition to the proposed criteria for those waters. The 

application of the NO3-NO2 criterion to all surface fresh waters would also provide a way to 

gauge anthropogenic contributions to TN across all waters of the state, not just to springs and 

clear streams. 
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Figure 3-1. Changes in NO3 concentration in Weeki Wachee Spring discharge and population of 

Hernando County, Florida. Note that NO3 concentrations follow a pattern very similar to the 

population curve with a 10- to 15-year lag. 

Figure 3-2. Increased NO3 concentrations in discharges from 13 selected first-magnitude springs 

(Alexander, Chassahowitzka Main, Fanning, Ichetucknee Main, Jackson Blue, Madison Blue, 

Manatee, Rainbow Group composite, Silver Main, Silver Glen, Volusia Blue, Wakulla, and 

Wacissa #2 Springs) between the 1970s and the early 2000s. Source: Scott et al. 2004. 
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Mean NOx vs. Mean Cell Density

0

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0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Mean NOx (mg/L)

Cells / cm2

Figure 3-3. Relationship between mean nitrate/nitrite (NOx) concentration and mean periphyton 

cell density from sampling sites on the Suwannee, Santa Fe, and Withlacoochee rivers. Source: 

Mattson et al. 2006. 

Figure 3-4. Relationship between mean nitrate/nitrite (NOx) concentration and mean periphyton 

biomass from sampling sites on the Suwannee, Santa Fe, and Withlacoochee Rivers. Source: 

Mattson et al. 2006. 

Mean NOx vs. Mean Biomass

1

2

3

4

5

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Mean NOx (mg/L)

2 AFDW g / m
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Mean NOx (Mg/L)

Mean Cell Density (/Square-CM)

0.0 0.2 0.4 0.6 0.8

0 2*10^5 6*10^5 10^6

Figure 3-5. Change point analysis for data from the 13 stations at the Suwannee River System 

(mean cell density vs. mean NOx). Change point = 0.44 mg/L. The 95% confidence interval for 

the change point based on 1000 bootstrapping samples is 0.378 to 0.629. 

Mean NOx (Mg/L)

AFDW (g/Square-Meter)

0.0 0.2 0.4 0 .6 0.8

0246

Figure 3-6. Change point analysis for data from the 13 stations At the Suwannee River System 

(mean biomass vs. mean NOx). Change point = 0.44. The 95% confidence interval for the change 

point based on 1000 bootstrapping samples is 0.441 to 0.584 μg/L. 
TSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

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y = 127582e2.351x

R2 = 0.7215

0

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0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

Nitrate + Nitrite (mg N/L)

Mean Cell Density (#/cm2) 

Figure 3-7. Relationship between mean nitrate plus nitrite concentration and mean periphyton 

abundance for sampling sites on the Suwannee, Santa Fe, and Withlacoochee rivers. Values with 

NO3 concentrations above and below 0.35 mg/L are shown in different colors. Red dotted lines 

are at 0.23 mg/L and 0.44 mg/L, representing results from lab and field. 

Figure 3-8. Relationship between mean nitrate plus nitrite and mean periphyton biomass for 

sampling sites on the Suwannee, Santa Fe, and Withlacoochee Rivers. Values with nitrate 

concentrations above and below 0.35 mg N/L are shown in different colors. Red dotted lines are 

at 0.23 mg N/L and 0.44 mg N/L, representing results from lab and field studies, respectively. 
TSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

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y = 2345.1e3.6176x

R2 = 0.6838

0

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0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Nitrate + Nitrite (mg N/L) 

Mean Cell Density van Dam 4-7 (#/cm2) 

Figure 3-9. The mean cell density of Van Dam eutrophic indicator diatoms increases with 

increasing nitrate plus nitrite concentrations. The red dotted lines are at 0.23 mg/L and 

0.44 mg/L, representing results from lab and field studies, respectively. 

y = 127582e2.351x

R2 = 0.7215

0

200000

400000

600000

800000

1000000

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1600000

0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800 0.900 1.000

Nitrate+Nitrite (mg/L)

Mean Cell Density (Cells/cm2

)

Figure 3-10. An example of the full-range confidence interval procedure to develop a nitrate plus 

nitrite criterion. The full confidence interval procedure resulted in a protective threshold of 0.33 

mg/L. The upper half of confidence interval method yielded a protective threshold of 0.38 mg/L. 

The average of these complementary methods resulted in the final protective threshold of 

0.35 mg/L.