TSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

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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 un ique class of aquatic eco system, highly treasured for their biological, economic, aesthetic, and recr eational 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 numb er of springs (approximately 600–700) occurs in Florida. Springs are often classified based on their flow rate, wh ich ranges from more than 2,800 L/sec (first magnitude) to less than 0.47 L/sec (eighth magnit ude). Many of the larger spring ecosystems in Florida have likely been in exis tence since the end of the last major ice age, approximately 15,000 to 30,000 years ago (Martin 1966; Munch etal. 2006). During th is period, plant and animal communities have evolved to become highly adapted to the unique water quality and conditions found in the springs. Th e 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 fo r human utilization, both by indigenous peoples (as supported by archaeological evid ence) and by present-day Flor idians and tourists, who use them for a variety of recreational purposes (Sco tt 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, some times in a turbulent manner) have been modified to facilitate swimming, r ecreation, and even “health spas.” Curre ntly, 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 suffere d declines (generally ) in their condition (e.g., up-rooting of vegetation, ba nk erosion, litter) due to visita tion 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 change s in spring ecosystems are: 1. Pollution of ground water, princi pally with nitrate-nitrogen (NO 3 -nitrogen), 1 resulting from human land use changes, cultural prac tices, and general population growth. 2. The simultaneous reduction in ground wa ter 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 (predom inantly nitrogen) associated with urban and agricultural activ ities (including fertiliza tion and waste disposal), seep through soils and are transported to springs by wa y of underground pathways. Howe ver, in excessive amounts,

1 Nitrate nitrogen (NO 3 -nitrogen) is a closely associated inorgani c nitrogen compound to nitrite nitrogen (NO 2 -nitrogen). Because of the analytical methods used to quantif y them, they are expressed together as NO 3 -NO 2 . Nitrite concentrations, however, tend to be a small proportion of the combined NO 3 - NO 2 concentration.TSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-2 nutrients can be harmful to the environment, le ading 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 qua lity, 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 notin the excessive abunda nce commonly observed today. Stevenson et al. (2004) found that the major ity of the Florida spri ngs 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. Additionall y, historic records of Lyngbya wollei exist from Silver Spring (Pinowska et al. 2007a), but prio r to the last 20–30 years there were no records of nuisance Lyngbya growth.

Within the past 20–30 years, however, anecdotal ob servations at several springs suggest that nuisance algae species have proliferated and are now out-competing an d replacing SAV. As benthic algal mats accumulate, they kill benefici al 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 biol ogical and ecological changes. For example, springs have become increasingly inhospitable to cert ain fish, snails, crayfish, turtle s, 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 metabol ism changes and disappearance of higher-order animals. This chain of events reflects the sign ificant adverse structural and functional changes that have occurred in spring ecosystems.

Numerous biological studies have documented ex cessive algal growth at many major springs. In some of the more extreme examples, such as S ilver Springs and Weeki Wachee Springs, algal mat accumulations have become several feet thic k. The thick benthic algal mats are detrimental to aquatic life and cause signi ficant 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 depl etion of oxygen and the release of ammonia and hydrogen sulfide, which can further degrade water quality (Joi nt Nature Conservation Committee 2004). In general, the spread of m acroalgae in Florida has resulted in re ductions in fish and crab harvests, lost tourism, and removal costs (O’N eil and Dennison 2005; Bonn and Bell 2003). Lyngbya wollei p roliferation 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 inhib it the recreational use of the resource (Osb orne et al. 2001).TSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-3 Lyngbya are a source of lyngbyatoxin and aplysi atoxin, which produce a condition known as “swimmer’s itch” (Cardellina et al. 1979; Mynderse et al. 1977). Lyngbya wolle i can produce a variety of paralytic shellfish poisons (e.g., saxi toxin) and other toxins capable of producing dermatitis in humans (Carmichael et al. 1997; Onode ra et al. 1997; Stewart et al. 2006; Teneva et al. 2003) and deaths in domestic and wild animal s that consume algal mats (Edwards et al. 1992; Falconer 1999; Gugger et al. 2005; Hamill 2001; Saker et al. 1999).

It is hypothesized that th e shift from SAV to blue-green algae in Florida springs is being driven by land-use change in springsheds, specificallyincreased loading of dissolved nutrients, especially NO 3 , and organic matter into the Floridian aquifer system from agriculture and urbanization. For example, in Ichetucknee Springs, mean annual NO 3 concentration increased from 0.35 mg/L in 1975 to 0.70 mg/L in 2001 (Scott et al. 2004). During th at 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 eviden ce for ecosystem scale effects of NO 3 enrichment. Using a subsidy stress theory to explai n the decline in gross primary production (GPP), it is suggested that increases in NO 3 act to increase productivity for a time , but then act as a stress to depress productivity above a certain threshold (Knight and Notest ein 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 toda y (Knight and Notestein 2008). This declining ecosystem productivity documented at Silver Sp rings was highly correlated with increasing NO 3 - nitrogen concentrations during the 50-year peri od of available data. Declining spring flows, increased shading by riparian trees, and altere d fish populations were also observed to be correlated with declining ecosystem production at Silver Springs and could offer alternate or cumulative explanations of th e 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 ecosyst ems exhibited other significant environmental stresses caused by humans, including decreases in discharge, intensive ex otic plant management efforts, and disturbance due to recreational activ ities. 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 photosynth etic efficiency were not available (Knight and Notestein 2008).

In addition to increased NO 3 concentrations, there are numerous other biotic and abiotic factors that have also changed in some, but not all, sp rings. These changes incl ude increased recreational use, decreased water output, decreased dissolved oxygen (DO) concentrations in spring discharge, increased need for a quatic weed control, greater a bundance 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 sy stems, and they might help explain the observed variations in response to increasing NO 3 levels observed in Florida’ s springs over the last several decades. For example, it is believed that a quatic plant control t echniques (e.g., herbicide applications or mechanical harv esters) that are used to suppr ess excessive growth of nonnativeTSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-4 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 NO 3 -NO 2 enrichment and these other confounding factors that cau se the observed change s 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, in creased organic matter deposition, greater number of nuisance algae species and amount of algal bi omass, decreased plant and animal productivity and diversity, reduced water qua lity, and not insignificantly, a re duction 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 NO 3 is a form of nitrogen that is readily utilized by aquatic plan ts and algae. However, NO 3 in excess can lead to the development of nuisance aquatic plant probl ems (Rabalais 2002). As a result of human land use changes, cultural practices, and general popu lation growth, there has been anincrease in the level of pollutants, especially NO 3 , in ground water over the last several decades.

Because there is no geologic store of NO 3 , all the NO 3 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 NO 3 once it has been introduced into the ground water below the root zone, it is transported thr ough the ground water largely as a conservative solute. Consequently, a si gnificant portion of the NO 3 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 NO 3 concentrations in spring di scharges are thought to have been 0.05 mg/L or less (Maddox et al. 1992), which is sufficiently low to re strict the growth of native algae and vegetation under na tural 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 seve ral decades, with a consequential transfer of nutrients to the relatively unprotec ted ground water. Katz et al. ( 1999) used isotopic analyses to show that substantia l portions of the NO 3 -NO 2 found in the Upper Floridan Aquifer and in spring discharges are derived from anthropogenic activities such as fertilizer application for agriculture and residential uses, livestoc k waste, and human waste.

Figure 3-1 shows the changes in NO 3 concentration in Weeki Wachee Spring discharge as related to the population increase in Hernando County, Florida. The spring NO 3 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 subseque nt effect on spring discharges is expected since aging measurements of wate r emerging from springs suggest that on average, it has spent between 10 and 30 years in the subsurface. Howe ver, these studies have also shown that aTSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-5 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 NO 3 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 NO 3 concentrations above background and approxima tely one-quarter of them demonstrated at least a 20-fold increase. Similarly, a recent eval uation of water quality in 13 first-magnitude springs shows that mean NO 3 -NO 2 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 NO 3 -NO 2 concentrations in many spring discharges have increased from 10- to 350-fold over the past 50 years, with the level of increase closely correlate d with the anthropogeni c activity and land-use changes within the springshed.

As a result of the increased NO 3 -NO 2 levels in ground water and sp ring discharges, downstream NO 3 -NO 2 loads are also increasing rapidly inmany watersheds. For example, the NO 3 -NO 2 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 NO 3 -enriched spring discharge resulting in a two- to three-fold increase in the level of NO 3 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 NO 3 -NO 2 concentrations have increased during the past 20 to 50 years, many Florida springs have concurrently undergone a number of advers e environmental and biological changes, as described previously. There is a general cons ensus in the scientific community that NO 3 is an important factor leading to th e observed changes in spring ecos ystems and their associated biological communities. Nitrogen, particularly NO 3 -NO 2 , appears to be the most problematic nutrient problem in Florida’s karst region.

There are four primary reasons for greater concern about NO 3 -NO 2 compared to phosphorus. First, increases in NO 3 -NO 2 concentrations are nearly omnipr esent in areas where anthropogenic loading to the land’s surface has occurred. Second, once in the gr ound water, denitrification is negligible and NO 3 -NO 2 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 NO 3 -NO 2 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 nitroge n-limited (Inglett et al. 2008; Knight and Notestein 2008). In their historical natural state, most Florida springs contain high levels of bioavailable soluble reactive pho sphorus 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 NO 3 -NO 2 has been linked to many of the obs erved detrimental impacts in spring systems, there is an immediate need to reduce NO 3 -NO 2 concentrations in spring vents and in upgradient ground water. To restore and preserve springs, activities in spri ngsheds that contributeTSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-6 to the transfer of nitrogen to the ground water must be properl y 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 li kely be several years before the effects of NO 3 control measures will be seen. The first step in the process is to develop a numeric NO 3 -NO 2 criterion for spring systems that will be protective of this unique resource. In addition, a NO 3 - NO 2 criterion will help identify systems at risk of degradation from excessive NO 3 pollution, as well as those where detrimental ch anges have already occurred and restoration is needed. EPA’s analysis of a NO 3 criterion, which was based largely on a nd agrees with FDEP’s results, is discussed below.



### i. Criteria Development Methods FDEP has worked to derive response-based thre sholds that will defi nitively 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 NO 3 criteria for springs, which ultimately were applied to all clear streams (< 40 platinum-cobalt units (P CU)). The information evaluated includes:

 Results from laboratory dosing stud ies conducted at various scales

 In situ algal monitoring

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

 Data regarding NO 3 concentrations found in minima lly 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 NO 3 criteria for clear streams, including springs . This information and its use in deriving the recommended NO 3 criteria for springs are discu ssed in greater detail below.



### ii. Laboratory Studies The results of laboratory experiments have pr ovided much valuable information about the response of algae (particul arly algal growth response) to increasing nutrientconcentrations under specific highly controlled conditions. Experiment al systems, however, us ually do not include all the complexities and ecological processes that affect the response to nutrients in natural waterbodies. The limitations of the small-scale expe rimental platform must be taken into account when applying the results to natural full-scale systems.

Nutrient amendment bioassay work was conduc ted by Cowell and Dawes (2004) to determine the NO 3 concentration required to achi eve a reduction in biomass of Lyngbya wollei , a nuisance blue-green benthic algal speci es that dominates many spring systems due to elevated NO 3 concentrations. Using Lyngbya cultures incubated in a series of NO 3 amendments, they found that both the biomass and growth rate s were low in treatment groups with NO 3 concentration at or below 300 μg/L, while the gr owth rates and biomass were si gnificantly higher in treatments with NO 3 concentrations at or greater than 600 μg /L (Cowell and Dawes 2004). In addition, the experiment also showed that the biomass a nd growth rate in treatment groups with NO 3 concentrations from 70 to 300 μg/L were sim ilar, suggesting that further reduction of NO 3TSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-7 concentration below the 300 μg/L level woul d probably not achieve significant additional reductions of L. wollei abundance and growth. They concluded that a NO 3 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 NO 3 -dosed raceways approached maximum levels at NO 3 concentrations above 518 to 546 μ g/L. In similar studies using Vaucheria , growth rates were low at NO 3 concentrations below 69 μg/L and increased substantially from 69 to 644 μg/L . Further growth rate increases at NO 3 concentrations above 644 μg/L were minimal.

In smaller-scale microcentrifuge tube microcosm experiments conducted to evaluate the growth response of individual macroalgal fi laments to precise levels of NO 3 dosing at high phosphate levels, Stevenson et al. (2007) found that the growth rate of Lyngbya wollei was minimized at NO 3 concentrations below 34 μg/L. Growthrates increased substantially at NO 3 concentrations from 34 to 230 μg/L and approached maximum le vels at concentrations above 230 μg/L. For unexplained reasons, th e growth rate of Vaucheria did not respond to NO 3 additions in the microcentrifuge tube microcosm experiments.

As discussed by Stevenson et al. (2007), the di fferences in results be tween the raceway and microcentrifuge tube experiments were likely related to the differen ces in scale of the experiments. In the microcentrifuge tube micr ocosms using individual macroalgal filaments, very accurate control of nutrient levels was possi ble. In the larger-sca le raceways using small algal mats, substantial nutrient depletion was possible and could not be accounted for, which resulted in a higher es timate of regulating NO 3 concentrations. Recognizing the limitations of the laboratory experiments, Stevenson et al . (2007) recommended using the ED90 (NO 3 -NO 2 concentration that produces 90% of the maximum growth) determined from the highly controlled microcentrifuge tube experiments as a preliminary NO 3 criterion that could be refined using additional information. The best estimates for the NO 3 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 concen trations associated wi th 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 growth s in the studied springs, however 23 different macroalgal taxa were obse rved 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 VaucheriaTSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-8 percent cover and thickn ess along the TN and NO 3 gradients explained substantially more variation than a linear model, with a clear threshold in Vaucheria response at 0.454 mg/L as NO 3 (i.e., 0.591 mg/L as TN). Exce ssive growth and cover of Vaucheria were found at sites with NO 3 concentrations at or above the 0.454 mg/L threshold, with Vaucheria abundance being significantly less at sites with lower NO 3 levels (Stevenson et al. 2007). This excessive growth of macroalgae is considered to constitute an imba lance of the natural biol ogical communities and not in compliance with Florida’s narrative nutrient criteria. Therefore, to provide for a margin of safety, a protective numeric NO 3 criterion would need to be below the observed 0.454 mg/L NO 3 Vaucheria threshold.



### iv. TMDL Development Activities The Wekiva River and Rock Springs Run are spri ng-influenced systems that are on the state’s impaired waters list due to evidence of an imba lance in aquatic flora characterized by excessive algal growth and lower ecosystem metabolic ac tivities. There was also evidence that the impairment of the Wekiva River and Ro ck Springs Run was caused by elevated NO 3 and phosphorus levels. The mean NO 3 concentration in the Wekiva River and Rock Springs Run ranged between 60 and 70 μg/L, which is sign ificantly higher than levels found at nearby minimally disturbed reference sites with similar characteristics (Juniper and Alexander Springs). Additionally, the Wekiva and Rock Springs NO 3 -NO 2 levels were above the threshold NO 3 concentration identified by Stevenson et al . (2004) to be asso ciated with nuisance Vaucheria growth (Gao 2008).

During the development of the TMDL for these waterbodies, protective nutrient concentration targets were derived using peri phyton and water quality data co llected from the Suwannee River and two tributaries, th e Withlacoochee River and Santa Fe River (Hornsby et al. 2000). These data were considered applicable to the We kiva 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 positi ve correlations for both periphyton biomass versus NO 3 concentration and cel l density versus NO 3 concentration. The functional relationships of cell density versus NO 3 concentration and periphyton biomass (represented as ash-free dry weight, or AFDW) versus NO 3 concentration are shown in Figures 3-3 and 3-4, respectively. Data presented in these figures repr esent long-term average biomass, cell densities, and NO 3 concentrations at the stations across the Suwann ee River system (Niu 2007).

As can be seen for both cell density and bioma ss, periphyton abundance significantly increased when NO 3 concentration increased above approximately 350 μg/L. The data were further evaluated using a change point an alysis to better define the NO 3 concentration that might significantly impact the periphyton biomass and ce ll density. The change point analysis fits a step function through observed data by examini ng 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 amon g the models tested, which supports the use of change point analysis.TSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-9 For the relationship between cell density and NO 3 concentration, the change point step function identified two populations of sites. The first se t of sites had cell de nsities near 163,000 cells/cm 2 (P = 0.009), which was considered the base line condition under which no significant NO 3 impact was detected. The second group of site s had cell densities near 616,000 cells/cm 2 (P = 0.0001), which was significantly elevated above the baseli ne condition. The change point analyses also indicated that the critical increase in mean algal cell density occurred as the mean NO 3 concentration increased from 286 to 401 μg/L (N iu 2007). This suggests that to prevent the periphyton cell density from increasing to the higher level, the NO 3 target concentration should be established below 401 μg/L.

Similarly, the change point analysis of the relationship between periphyton biomass and NO 3 concentration identified two populations of sites. The first set of sites had a periphyton biomass near 1.73 g/m 2 (p< 0.0001), which was considered the baseline condition under which no significant NO 3 impact was detected. The second group of sites had an increased algal biomass near 4.15 g/m 2 (p = 0.0001), which was significantly el evated above the baseline condition. The change point analyses also i ndicated that the critical incr ease in mean periphyton biomass occurred as the mean NO 3 concentration increased from 401 to 420 μg/L (Niu 2007). This suggests that to prevent the pe riphyton biomass from increasing to the higher level, the NO 3 target concentration should be established below 420 μg/L.

Periphyton cell density exhibited a se nsitive response to increasing NO 3 concentrations and was used as the basis for the NO 3 target concentration. A periphy ton cell density increase was observed for NO 3 concentrations between 0.286 mg/L and 0.401 mg/L. The value of 0.286 mg/L was chosen to be the TMDL NO 3 target concentration for the Wekiva River and Rock Springs Run systems because it provided a conservative criter ion with an adequate margin of safety that is reasonably protective of th e biological communities with in 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 NO 3 -NO 2 data for each period for each site were used to repeat the change point analysis. The resu lts were very similar to those obtained from the original analyses as described above. A NO 3 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 s ites with significantly higher periphyton abundance, as compared to the baseline group, a protective NO 3 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 NO 3 and algal biomass versus NO 3 concentrations, respectivel y, with field and laboratory results illustrated using red lines. The 0.44 mg/L change point represents the upper NO 3 -NO 2 concentration where the observed biological changes occur. Additionally, the 0.23 mg/L NO 3 - NO 2 threshold based on the laboratory studies, whic h 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 abund ance is restricted at NO 3 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.TSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-10 In addition to the observed increases in periphyt on cell density and biomass, shifts in the structure and taxonomic composition of the pe riphyton community were also observed across this NO 3 -NO 2 gradient. The abundance of taxa indicative of eutrophic conditions (Van Dam et al. 1994) increased significantly with increasing NO 3 -NO 2 concentrations above approximately 0.35 mg/L (Figure 3-9). Similar to the change s in the algal abundance and biomass, the abundance of the eutrophic taxa are similar at sites with NO 3 -NO 2 concentrations below 0.35 mg/L, while the abundance of these taxa increases with increasing NO 3 -NO 2 concentrations above 0.35 mg/L. The significant alterations in co mmunity composition, in combination with the increases in cell density and biomass, clearly demonstrate that increased NO 3 -NO 2 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 imbalan ce of aquatic flora (Rule 62-302, FAC). Because the 0.23 mg/L laboratory limit is likely unnecessarilyprotective 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 NO 3 -NO 2 concentrations above approximately 0.40 mg/L. Since the 0.44 mg/L change point represents the upper bound of the range inwhich 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 NO 3 -NO 2 criterion.

To define this safety factor, two compleme ntary 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 complementar y methods resulted in the final protective threshold of 0.35 mg/L.



### v. Analysis of Nutrient Gradien t Study Rapid Periphyton Data In Appendix B-11, an analysis of algal response s (Rapid Periphyton Survey) to nutrients and other variables is presented. The analysis demonstrated that periphyton thickness was significantly correlated with NO 3 -NO 2 , 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, NO 3 -NO 2 concentration, and TP concentration. The analyses of the rapid periphyton assessment data are provided ingreater detail in Appe ndix B-1. Note that in streams where color was less than 40 PCU and canopy was relatively open, there was a significant correlation between NO 3 -NO 2 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 incl uded 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 reas onable to apply the 0.35 mg/L NO 3 -NO 2 limit to all streams with color measurements of less than 40 PCU.



### vi. Summary and Conclusions Multiple lines of evidence indicate that reducing NO 3 -NO 2 concentrations in clear stream systems should reduce macroalgal grow th rates. Although response-based NO 3 -NO 2 criteria were originally investigated for application in springs, additional analyses indicated that such criteriaTSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-11 would be appropriate in streams with color less th an 40 PCU. Control of NO 3 -NO 2 is expected to result in a reduced frequency, intensity, and dur ation of nuisance macroa lgal 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 experiment al results––those from microcentrifuge tube experiments––suggest that NO 3 concentrations le ss than 0.230 mg NO 3 -N/L are needed to slow the growth of Lyngbya wollei . Similarly, to reduce the growth of Vaucheria under laboratory conditions, NO 3 concentrations below approximately 0.261 mg NO 3 -N/L would be required. Results of periphyton field survey s conducted at a large number of spring systems indicated that NO 3 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 threshol d represents the lower range of NO 3 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) Suwann ee, Santa Fe, and Withlacoochee (north) rivers clearly indicated that significan t increases in cell density and bi omass, along with alterations in taxonomic community structure (which are i ndicative of an imbalance), occur as NO 3 concentrations approach the 0.441 mg/L change point.

A margin of safety, derived by averaging the upp er half-range and full-range 95% confidence intervals, was applied to the 0.44 mg/L cha nge point to derive the final 0.35 mg/L NO 3 -NO 2 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 an alyses indicated that NO 3 -NO 2 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 NO 3 -NO 2 criterion developed by FDEP to all streams with color below 40 PCU.



### 3b. Duration and Frequency fo r Proposed Springs Criteria The duration and frequency expression of an annua l geometric mean not to be surpassed more than once in a three-year period is consistent with the expressi ons of duration and frequency for other water body types (e.g., lakes, streams, canal s) for TN and TP for the same reasons EPA selected a three-year period for those waters (please s ee sections 1.d and 2.d above). EPA determined that this frequency of excursions s hould not result in unacceptable effects on aquatic life as it will allow the springs and clear str eams aquatic systems enough time to recover from the occasionally elevated year of nutrient loadin gs. Likewise, the long-ter m arithmetic average of annual geometric means not exceed the criterion-magnitude concentrat ion is also consistent with the expression used for other waters.

EPA also considered as an alternative, expressi ng 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 va lue in more than one out of every ten months. This alternativ e is consistent with the expre ssion that FDEP proposed in JuneTSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-12 2009 for its State rule and the e xpression in the TSD for Florida’ s Inland Waters that EPA sent out for external scientific peer review in July 2009. The rationa le 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% freque ncy of excursions is a reasonable and fully protective allowance given small sample sizes in any given month (i.e., th e 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 FD EP for periods just under one month (i.e., 21 to 28 days), while the algal biomass field surv ey was conducted over an 18-year period and was analyzed using four to five year averaging pe riods. 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 thatthis 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 experi enced 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 protec tive criterion with a lo w likelihood of excess algal growth. This threshold adjustment is consistent with the re stricted growth of diatom taxa indicative of eutrophic conditions.

The 10% excursion frequency woul d 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 consider ed a reasonable and fully protective allowance given small sample sizes in any given month, e ssentially 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 Co nsidered by U.S. EPA

i. Nitrate-Nitrite Criteria for all W a ters as an Independent Criterion Stressor-response analyses from FDEP’s rapid peri phyton survey in streams demonstrated that periphyton thickness is signi ficantly correlated with NO 3 -NO 2 (Appendix B-1). This relationship is similar to that described in spring system s, in which periphyton mean cell density and biomass, parameters determining thickness, increased as a function of increasing NO 3 -NO 2 concentration. Based on the rapid periphyton surv ey results and the c onsideration that NO 3 -NO 2 loads in springs and streams can flow to othe r surface waters, U.S. EPA is seeking comment on an alternative approach where the 0.35 mg/L NO 3 -NO 2 criterion for springs and clears streams would apply to all surface fresh waters, in additionto the proposed criteria for those waters. The application of the NO 3 -NO 2 criterion to all surface fresh waters would also provide a way to gauge anthropogenic contributions to TN across all waters of th e state, not just to springs and clear streams.TSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-13 Figure 3-1. Changes in NO 3 concentration in Weeki Wachee Spring discharge and population of Hernando County, Florida. Note that NO 3 concentrations follow a pattern very similar to the population curve with a 10- to 15-year lag.

Figure 3-2. Increased NO 3 concentrations in discharges from 13 selected first-magnitude springs (Alexander, Chassahowitzka Main, Fanning, Iche tucknee Main, Jackson Blue, Madison Blue, Manatee, Rainbow Group composite, Silver Main , Silver Glen, Volusia Blue, Wakulla, and Wacissa #2 Springs) between th e 1970s and the early 2000s. Source: Scott et al. 2004.TSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-14 Mean NOx vs. Mean Cell Density

0 100000 200000 300000 400000 500000 600000 700000 800000 900000 1000000

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 (NO x ) 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 (NO x ) concentration and mean periphyton biomass from sampling sites on the Suwannee, Santa Fe, and Withlaco ochee 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) 2AFDW g / mTSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-15 Mean NOx (Mg/L)Mean Cell Density (/Square-CM)

0.0 0.2 0.4 0.6 0.80 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 NO x ). 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 0.80246

Figure 3-6. Change point analysis for data from the 13 stations At the Suwannee River System (mean biomass vs. mean NO x ). Change point = 0.44. The 95% conf idence interval for the change point based on 1000 bootstrapping sa mples is 0.441 to 0.584 μg/L.TSD for Proposed Numeric Nutrient Criteria for FL Inland Surface Fresh Waters

3-16 y = 127582e 2.351x

R

2 = 0.7215

0 200,000 400,000 600,000 800,000 1,000,000 1,200,000 1,400,000

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 co ncentration and mean periphyton abundance for sampling sites on the Suwannee, Sant a Fe, and Withlacoochee rivers. Values with NO 3 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, represen ting results from lab and field.

Figure 3-8. Relationship between mean nitrate plus nitrite and meanperiphyton 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 s hown 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

3-17 y = 2345.1e 3.6176x

R 2 = 0.6838

0 5,000 10,000 15,000 20,000 25,000 30,000 35,000 40,000 45,000 50,000

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 = 127582e 2.351x

R 2 = 0.7215

0 200000 400000 600000 800000 1000000 1200000 1400000 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 confiden ce interval procedure to develop a nitrate plus nitrite criterion. The full confidence interval pr ocedure resulted in a protective threshold of 0.33 mg/L. The upper half of confidence interval me thod yielded a protective threshold of 0.38 mg/L. The average of these complementary methods re sulted in the final pr otective threshold of 0.35 mg/L.