On 09 June 2017 at 09:20, surface water samples were collected from the Guist Creek Marina (38.20810˚, 85.15720˚) in ten 1 L glass containers for treatment in the laboratory. Guist Creek Lake is a human-made reservoir that was created from impounding Guist Creek in 1961. Guist Creek Lake is 1.3 km² in area with 43 km of shoreline and is located in Shelby County, Kentucky, USA [21]. The lake is owned by the Kentucky Department of Fish and Wildlife Resources and the mean and maximum depths are 5 m and 14 m, respectively [21]. Guist Creek Lake is part of the Salt River and Ohio River Basins and has been the subject of toxic algae advisories in recent years. On 01 August 2014, the Kentucky Division of Water declared an advisory for Guist Creek Lake, in addition to three other Kentucky lakes. The Shelbyville Water and Sewer Commission was drawing drinking water from Guist Creek Lake at the time of this study (partnerships with the Louisville Water Company have since been established). Guist Creek Lake is just one of several Kentucky reservoirs that has had water drawn while having had toxic cyanobacteria advisories in recent history. Guist Creek Lake also has fish consumption guidelines in place for mercury. The lake and main tributary (Guist Creek) are on the Commonwealth of Kentucky’s impaired waters list as reported to Congress via Section 303(d) of the Clean Water Act. The 303(d) report indicates that nutrients and eutrophication are the cause for impairment in the lake and creek.

Guist Creek Lake drains a substantial portion of Shelby County, which is mostly pastureland (50.6%) with a mixture of deciduous forest (26.7%), cultivated crops (11.6%), developed open space (4.6%), and several other less frequent land uses (6.5%). Land use data were obtained from the National Land Cover Database [22] [23]. The lake and catchment are in the Bluegrass region of Kentucky which has naturally occurring high phosphorus levels in both the water and soil as part of the high limestone content associated with the unique geology [24].

Ten 1 L samples were collected at Guist Creek Lake and were transported immediately to Eastern Kentucky University in coolers, where they were further equally distributed into eight glass jars of 900 ml of water in the environmental health laboratory within 90 minutes of collection. In total, seven water samples were spiked with one or more of the following treatment substances: 3.0 mg of triple phosphate, Ca(H₂PO₄), (0-45-0); 3.0 mg of ammonium nitrate, NH₄NO₃ (34-0-0); and/or 3.0 mg of urea, CO(NH₂)₂, (46-0-0). The control sample received no treatment. In total, all possible combinations were created using the same additions of nutrients. The treatment receiving the greatest amount of input was the triple phosphate (Ca(H₂PO₄) + ammonium nitrate + urea treatment, which received 3.0 mg of each. The treatment fertilizers were acquired from an area farm supply store. The decision to add fertilizer used (and obtained) from within the region for spiking into sample water was informed by a 2017 study in Kentucky [25].

Upon adding nutrients, magnetic stirrers were used on a slow setting for 150 minutes to ensure adequate mixing and dissolving of fertilizer into the lake sample treatments. In addition, two 24-hour electrical timers were set to turn on four (4) 250 v 660 w glow lamps at 06:30 am and to turn these lamps off at 21:10, daily, to simulate natural sunrise and sunset. Each lamp was positioned approximately 63 cm above the open containers. Light intensity was not measured. Container lids were not used, and glass containers were used to hold the lake water to maximize light entry thereby better mimicking the natural lake water environment. The simulated daytime temperature of approximately 30˚C in the sample water was maintained by the generally warm summer building temperatures at the university and the glow lamps. Before all analyses, samples were gently stirred for 30 min on Day 3, Day 6, and Day 7.

An initial assessment of water quality before nutrient addition was conducted on 09 June 2017. An assessment of phycocyanin pigment and in vivo chlorophyll a was obtained on 12 June 2017. A final assessment of all water quality parameters was performed on 15 June 2017. Total phosphorus ( PO 4 3 − ), total nitrate (NO₃-N), and nitrogen-ammonia + ammonium (NH₃-N) concentrations were determined with a Hach DR 2800 spectrophotometer using Hach Method TNT 843 (acid persulfate digestion), Hach Method 8171 (Cd reduction method), and Hach Method 8038 (Nessler reagent method), respectively. Turbidity was determined using a Lutron TU-2016 turbidimeter. Phycocyanin and in vivo chlorophyll a were determined from 0.2 aliquots of sample water placed into individual PCR tubes. The relative fluorescence units were then read by a handheld Beagle Biosciences dual-channel fluorometer (manufactured by AmiScience, Fremont, California) and compared against a standard curve. No extracted chlorophyll a concentrations were determined. Phycocyanin fluorometry has been used in studies of freshwater lakes as a specific indicator of cyanobacteria biovolume as well as harmful cyanobacteria bloom risk [26] [27] [28].

The initial mean total phosphorus level (0.27 mg·L⁻¹) for Guist Creek Lake was hypereutrophic according to Carlson’s Trophic State Index [29]. Table 1 demonstrates that phosphorus levels on the final day (Day 7) fell below quantification for the total phosphorus method used in this study (<0.15 mg·L⁻¹) in treatments not receiving the phosphorus fertilizer treatment. In treatments with

^aNO₃-N: PO 4 3 − Molar Ratio; ^b(NO₃-N + NH₃-N): PO 4 3 − Molar Ratio.

phosphorus fertilizer, phosphorus levels remained 4.0 – 6.7x greater than the pre-existing hypereutrophic levels. T-testing demonstrated that nitrate levels in all groups, including ammonium nitrate-treated and urea-treated bioassays, were significantly lower (p < 0.05) at Day 7 than the initial nitrate level of 4.20 mg·L⁻¹ (Table 1). Ammonia + ammonium (NH₃-N) levels remained at or above initial levels (0.30 mg·L⁻¹) in all treatments at Day 7, with the highest level (0.53 mg·L⁻¹) being observed in the ammonium nitrate + urea treatment.

The initial water (Day 0) was as turbid or more turbid than all treatment groups (Table 2). The most turbid treatment groups were the groups with the highest phycocyanin and chlorophyll a results (Figure 1, Figure 2). The phycocyanin (PC) results suggest cyanobacteria growth was at first nitrogen-limited as the ammonium-nitrate treatment resulted in the highest PC value on day 3 among the single nutrient treatments (Figure 1). Similarly, the other nitrogen treatment with urea produced a greater PC value on day 3 than the phosphorus fertilizer treatment. These results were not however reflected in the chlorophyll a results (Figure 2); whereby individual nitrogen and phosphorus additions had only a slight impact on chlorophyll a compared to the no treatment control, which declined. In tandem, P + N, resulted in noticeable increases in chlorophyll a relative to single nutrient treatments and the no treatment bioassay (Figure 2).

Nitrogen and phosphorous enter lakes through several routes including runoff, air deposition, and rainfall. Nitrogen sources can enter water systems at relatively high concentrations directly or indirectly from fertilizers, industrial waste, and

sewage. In areas of Kentucky where phosphorus levels are naturally high [24], nitrogen fertilizers are more often used by agriculture and can enter surface water and groundwater. In the southern US, a secondary N-limitation has been described [30] for primary productivity making runoff from these N-sources noteworthy for investigation. By comparing Carlson’s Trophic State Index [29] for total phosphorus with Kratzer and Brezonik’s Index for nitrate [30] we see higher expected primary productivity with total phosphorus (TSI = 84.9; very hypereutrophic) compared to total nitrate (TSI = 75.2; hypereutrophic). For this study, we used in vivo chlorophyll a for calculating Carlson’s TSI for chlorophyll a.

Here, we observed lower chlorophyll a values than expected by the TSI values for either total phosphorus and nitrate. Carlson’s TSI [29] for chlorophyll a using the non-extracted value recorded here produced a TSI of 51 (mildly eutrophic) in the initial untreated water. If chlorophyll a levels were doubled by extraction, the TSI would increase to 58 (eutrophic), but still be lower than expected by both the TSI values for P and N. This pattern is not unique in Kentucky, as Reeder [25] observed a similar trend in Eastern Kentucky Lakes (TSI_TP > TSI_Nitrate > TSI_chla). One possible reason for this discrepancy in summer TSI values is that turbidity is presumed to cause light limitations in many Kentucky lakes [25].

In an effort to identify a nutrient pattern associated with cyanobacteria bloom production at Guist Creek Lake, a secondary N-limitation was artificially created in this study, following a massive infusion of total phosphorus (>3 mg·L⁻¹) in several of the treatments. Furthermore, artificially low N:P ratios were then simultaneously created, which presumably favored the growth of N-fixing cyanobacteria and low N:P tolerant Microcystis [31] [32]. Such a scenario (or a milder version) could potentially play out following heavy rainfall in many of Kentucky’s agricultural watersheds. The chemical composition of nitrogen fertilizers used throughout the United States is gradually shifting to urea in lieu of ammonium nitrate. In fact, almost 90% of nitrogen fertilizers are now composed of urea [20]. In this study, urea, like ammonium nitrate, was also capable of stimulating cyanobacterial growth (and potentially bloom formation). This study provides evidence for future studies on nutrient dynamics related to blooms of unknown origin to consider monitoring urea, which is often not included (or measured) in most water quality analyses [20], including this study.

Figure 1 demonstrated that both, urea and ammonium nitrate, increased cyanobacterial growth more than phosphorus fertilizer alone at Guist Creek Lake. Ultimately, either urea or ammonium nitrate were able to facilitate cyanobacterial growth when phosphorus was already abundant; thereby making nitrogen instrumental in facilitating cyanobacterial blooms in this lake as observed in other hypereutrophic lakes [14] [33]. Given the relationship between TSI values reported here and elsewhere in Kentucky [25], the impact of nitrogen on cyanobacteria bloom potential and formation throughout Kentucky and beyond warrants greater investigation and potential changes in nutrient management policies.

Since the 1970s, freshwater ecologists have frequently recognized the importance of available phosphorous as a limiting factor for inland freshwater productivity [34]. There has been an assumption in Kentucky that a reduction in phosphorous inputs would reduce harmful cyanobacterial blooms, essentially treating cyanobacteria similar to chlorophytes. For shallow, smaller hypereutrophic lakes with P loaded sediments, greater attention may be needed towards nitrogen management. Guist Creek Lake is 1.3 km² with a 5 m mean depth and would be described as a small shallow lake. Recent research examining 573 lakes observed nitrogen limitations (and nitrogen-phosphorus co-limitations) frequently in shallow lakes, with the nitrogen limitation being even more likely as the trophic status increases [35]. The initial sample from Guist Creek Lake had a very high trophic status index value of 84.9 based upon total phosphorus, suggesting Guist Creek Lake could experience nitrogen limitations that favor cyanobacteria dominance like other small shallow lakes globally [35].

For evaluation of the plausibility of the scenario presented in this study, recent 2020 study results from several mesocosm studies done on Wascana Lake can be explored. Specifically, the small lake located in Saskatchewan, Canada is also shallow (7.5 m maximum depth), small (0.5 km²), and hypereutrophic with similar phosphorus concentrations as observed in Guist Creek Lake. As nitrogen loading occurred in Wascana Lake, particularly urea, cyanobacteria dominance increased along with cyanotoxin concentrations until reaching and exceeding a potentially toxic nitrogen level [36].

Beyond small lakes, even larger lakes, such as Lake Erie, notably the western basin, have been recently linked to having cyanobacteria blooms attributable to nitrogen limitations and/or nitrogen-phosphorus co-limitations [37]. The bioassay study on Guist Creek Lake has a number of research limitations but does provide evidence of the potential need for nitrogen (or dual nutrient) management strategies being implemented in the watershed of Guist Creek if bloom conditions are to be prevented. Cyanobacteria research experts recommend mitigating the adverse impact of harmful bloom events and suggest the single nutrient reduction paradigm for freshwater be replaced by a dual nutrient (N and P) reduction paradigm better suited for the current rapidly changing global climate and existing terrestrial and aquatic nutrient loads [38]. Extending beyond benefits to freshwater lakes, nitrogen reductions have been long-established as being essential in the United States for mitigating hypoxia and harmful algal bloom events in the Gulf of Mexico [39] [40]. As rainfall continues to increase across the US, modifications to existing fertilizer application approaches are recommended to diminish the adverse impacts of nitrogen on the Gulf of Mexico [41] and many productive lakes (small and large) as part of a dual nutrient management strategy [20] [38] [42].