Laguna Cartagena (approximately 160 ha or about 400 acres) is a shallow (maximum depth 2 m) freshwater lagoon and associated wetland situated in the Lajas Valley, Municipality of Lajas, in southwestern Puerto Rico and can be described as a eutrophic ecosystem.

The Laguna Cartagena ecosystem receives water (including pollutants) from multiple creeks, canals, and ditches. Margara Creek (or “Quebrada”) is a natural stream that delivers water to Laguna Cartagena. Runoff is received from the east by Lago Loco near Yauco (several kilometers to the east) and flows from this reservoir through the Principal Irrigation Canal (irrigation source water) along the base of the adjacent northern hills and then to agricultural fields in the Lajas Valley. From there drainage water flows through the Principal Drainage Canal to Laguna Cartagena (Figure 1). In 1961, Margara Canal (the continuance of Margara Creek) was constructed to conduct unused irrigation water, some drainwater, and runoff from north of the lagoon in order to maintain lagoon water levels [17] . Margara Canal, and the Principal Drainage Canal adjacent to Road 305 at the UPR Lajas Agricultural Experiment Substation are the major channelized sources that bring water (both drainwater and runoff) to a single inlet canal, the Principal Drainage Canal in Laguna Cartagena, just to the west of the town of Magüayo, and then to the main body of the lagoon. The Principal Drainage Canal was constructed in 1950s and thus made Laguna Cartagena a permanent body of water [18] [19] [20] [21] [22] . This historically oligotrophic wetland system then continued to be directly affected by phosphorus loading from inorganic agricultural fertilizer until the end of subsidized fertilizer use and sugar cane cultivation in the late 1990s, although a legacy of these practices remains [21] .

Laguna Cartagena has acted as both a sink and source of TP and SRP with internal phosphorus loading (internal eutrophication) from the lagoon’s own sediments, causing higher phosphorus concentrations in lagoon water, and downstream to other ecosystems. This behavior has been influenced by external factors including water level fluctuations and rainfall and by intensified

management interventions performed by US Fish and Wildlife Service (USFWS) including excavation and removal of sediment and vegetation, draining, and burning [21] [22] . The overgrowth of Typha domingensis, Pistia stratiotes (water lettuce) and Eichhornia crassipies (water hyacinth) and the presence of extensive floating peat mats (Figure 1) continue to occupy large portions of the lagoon’s surface area and cause losses of the open water habitat [21] [22] .

Water samples were collected in triplicate at mid-column depths from the channelized external sources of P and N that enter Laguna Cartagena. These external sources as shown in Figure 1 are: 1) distant rural domestic discharge from the hills to the north (Cerro Alto) of Laguna Cartagena (>1.5 km, point (a)); 2) local runoff from agricultural, domestic or industrial discharge to Margara Creek immediately south of where this creek passes under Road 306 (<1.5 km, point (b)); 3) Margara Canal—the continuance of Margara Creek (drainwater and runoff, point (c)); 4) Principal Drainage Canal adjacent to Road 305 at the UPR Lajas Agricultural Experiment Substation (near point (d)); and 5) Principal Drainage Canal in the Refuge to the west of the town of Magüayo, i.e., the Combined Inlet (drainwater and runoff) (near point (e)). All nutrients that enter the lagoon from this drainage basin come together and enter through the combined inlet canal, just west of point (e) that eventually flows west to Boquerón Bay.

Sampling occurred at two-week intervals from October 2013 through November 2014. All samples for P (SRP and TP) analyses were collected in glass bottles, preserved with H₂SO₄, and transported in ice to the Universidad del Turabo (now Ana G. Méndez University, Gurabo). These samples were stored at a low temperature and analyzed within 10 days collection using the persulfate digestion and ascorbic acid methods [23] . A Thermo Fisher Scientific, Genesys 20, with a wavelength range between 352 to 1100 nm was used to measure sample absorption at 880 nm.

Rainfall data were obtained weekly from a rain gauge located approximately 6 km north of the lagoon in the upper portion of the watershed, a location representative of regional rainfall. Weekly rainfall data were recorded from September 2013 to November 2014. Dissolved oxygen (DO, mg/L), pH, Total dissolved solids (TDS, mg/L), temperature (˚C), and turbidity (cm) were measured in the field for each sampling event. Instruments used for measurement of these parameters were a Hanna Instruments (HI 98186) for DO, AZ Instruments Corp, model 8685 for pH and temperature, an EC/TDS/˚C Martini (EC 59) meter for TDS and a turbidity tube for turbidity. Nitrate ( NO 3 − ), nitrite ( NO 2 − ), and ammonia (NH₃) were determined using a HACH DR900 colorimeter. Nitrate ( NO 3 − ) was measured using the cadmium reduction method 353.2. Nitrite ( NO 2 − ) was measured with the USEPA diazotization method, and ammonia using the salicylate method. All samples for N (nitrate, nitrite, and ammonia) were collected in glass bottles and stored at a low temperature and analyzed within 24 hours.

Mean SRP, TP, nitrate, nitrite, and ammonia values were calculated for each sample (triplicate sampling, n = 3 for each sample). ANOVA (Analyses of Variance) was used to determine significant differences between SRP, TP, nitrate, nitrite, and ammonia for the major canals that bring water to the lagoon. Correlation analyses were used to evaluate the possible correlations of SRP, TP, nitrate, nitrite, and ammonia with rainfall one week and two weeks prior sample collection. Results were considered significant when a p-value was less than 0.05.

This study was conducted under the authorization of Biosafety Committee (IBC) Protocol B03-017-13 of the Ana G Mendez University System Office of Compliance.

Monthly rainfall (mm) for the Laguna Cartagena drainage basin recorded from September 2013 to November 2014 (Figure 2) indicated heavy rainfall during the months of August to November (except for Oct 2013), and allowed identification of specific rainfall events that occurred one and two weeks prior to water sample collection.

There were significant differences between SRP and TP for the major canals that bring water to the lagoon (One Way ANOVA: p = 0.000 and p = 0.000). The University of Puerto Rico (UPR) Agricultural Experimental Substation Drainage Canal (SRP mean 443.5 µg/L and TP mean 2469.3 µg/L) and Cerro Alto northern hill sides (SRP mean 310.2 µg/L and TP mean 1626.2 µg/L) provided the highest concentrations of SRP and TP (Figure 3) with Cerro Alto exhibiting the

highest concentrations of phosphorus (P) in periods of high rainfall. The lowest SRP and TP concentrations were found in Margara Creek (SRP mean 151.1 µg/L and TP mean 641.2 µg/L). On 23 Feb, 15 Mar and 31 May SRP was less than 100 µg/L at all canals that bring water to Laguna Cartagena. However, on 23 Feb the SRP for the Principal Drainage Canal in the Refuge to the west of Magüayo (the Combined Inlet) was 130.6 µg/L. On 31 Aug, this canal presented an event of high TP (4665.2 µg/L) concentrations.

For nitrite there were no significant differences between the channelized external sources of P that enter the lagoon (One Way ANOVA: p = 0.504). Nitrate ( NO 3 − ) and ammonia (NH₃) concentrations between the sampling sites were different (One Way ANOVA: p = 0.000 and p = 0.002, respectively). On 13 Jul, nitrate ( NO 3 − ) was undetectable in all canals. The highest NO 3 − concentrations were found in Margara Creek ( NO 3 − mean = 15.3 µg/L) and the UPR Agricultural Experimental Substation Drainage Canal ( NO 3 − mean 12.1 µg/L). Nitrate ( NO 3 − ) concentrations were zero (undetectable) in the Combined Inlet, except for 23 Feb (6.7 µg/L) and 15 Mar 2014 (6.7 µg/L) and 31 May 2014 (3.3 µg/L). The UPR Agricultural Experimental Substation Drainage Canal and Cerro Alto provided the highest concentrations of ammonia, with means of 496.7 µg/L and 82.2 µg/L, respectively (Figure 4).

Physicochemical parameters (Table 1) at each sampling point showed that the UPR Agricultural Experimental Substation and Cerro Alto had the highest measurements for turbidity (12.1 cm) and TDS (434 mg/L), respectively. Mean temperatures for all nonpoint source locations ranged from 25.5˚C to 27.6˚C, (within acceptable regulatory limits (<32.2˚C) [24] ). Values for pH were 7.2 - 8.1, which are considered optimal.

The Principal Drainage Canal (Combined Inlet) had the lowest DO, with mean of 1.79 mg/L) and range of 0.20 mg/L to 4.23 mg/L.

SRP and TP concentrations at Cerro Alto were significantly correlated (p < 0.05)

(^a) DO: Dissolved oxygen was determined with a Hanna Instruments (HI 98186); (^b) pH: pH was determined with an AZ Instruments Corp, model 8685; (^c) TDS: Total Dissolved Solid was determined with the EC/TDS/˚C (EC 59); (^d) Temp: Temperature was measured with an AZ Instruments Corp; (^e) Turbidity: The turbidity was determined by observation tube method; Higher numbers denote greater clarity, less turbidity.

with rainfall at one week (r = 0.567, p = 0.018; r = 0.581, p = 0.015, respectively), and two weeks (r = 0.536, p = 0.026; r = 0.502, p = 0.040, respectively) prior to sample collection (Table 2). Neither SRP nor TP were significantly correlated with rainfall event at Margara Creek, Margara Canal, or UPR Agricultural Experimental Substation Drainage Canal (Table 2). SRP at the Combined Inlet west of Magüayo was significantly correlated with both rainfall at one week before sampling, and two weeks before sampling (r = 0.784, p = 0.000 for one week and r = 0.751, p = 0.001 for two weeks, respectively) (Table 2). However, TP was not significantly correlated with either rainfall at one week, or two weeks before sampling (Table 2).

Nitrate ( NO 3 − ), nitrite ( NO 2 − ), and ammonia (NH₃) concentrations at the channelized external sources of nutrients that enter Laguna Cartagena were not significantly (p > 0.05) correlated with rainfall at one or two weeks prior to sample collection. However, ammonia (NH₃) at Margara Canal was significantly correlated with rainfall event at both one week (r = 0.848, p = 0.000) and two weeks (r = 0.789, p = 0.002) prior to sampling (Table 3).

^a: Soluble Reactive Phosphorus; ^b: Total Phosphorus; ^c: Pearson correlation coefficient; ^d: p-value for the correlation.

^a: Pearson correlation coefficient; ^b: p-value for the correlation.

The channelized waterways that carry water to the lagoon can be classified as hypereutrophic (>100 µg/L) [11] for TP concentrations but oligotrophic (<200 µg/L) [11] for nitrogen (nitrogen depleted). Water Quality Standards set by the Puerto Rico Department of Natural and Environmental Resources for TP and nitrogen (nitrate, nitrite, and ammonia) establish limits for estuarine waters of 1000 µg/L and 5000 µg/L, respectively [24] . By these standards, the Principal Drainage Canal at the UPR Agricultural Experimental Substation exceeded the limits for total phosphorus (TP) on 15 of 17 occasions, with concentrations between 1239.6 µg/L and 5133.8 µg/L. Alto exceeded these limits on 9 of 17 occasions, with TP concentrations between 1020.3 µg/L and 6350.4 µg/L. However, the main channelized external influent water to the lagoon did not exceed regulatory limits for nitrogen.

There was a significant difference between SRP and TP for the major canals that bring water to the lagoon. Agriculture (rice and cattle) is the predominant land use at the UPR Lajas Agricultural Experiment Substation, the predominant nonpoint source of nutrient pollution (Soluble Reactive Phosphorus (SRP), Total Phosphorus (TP), and ammonia (NH₃)) in the principal channelized water sources to the lagoon. Nutrients are likely derived from fertilizers applied to the Substation’s rice fields and also as a result of high a density livestock (dairy cattle), and the nitrogenous wastes from the cattle oxidation pond. Intensive agricultural practices use fertilizers containing high concentrations of nutrients (P and N), which show up in the runoff [25] .

The upper Lajas Valley area (between Road 101 and the Principal Irrigation Canal) is a large productive drainage basin with beef cattle and pineapple currently as the major crops, but previously (until the late 1990s) dominated by sugar cane and pineapple.

Flux rates of nutrients to water from fertilizer and manure depend on various factors including specific farming practices, the nature and amount of fertilizer applied, soil type, leaving tilled soil exposed to rainfall and overland flow, soil drainage, vegetative cover, season, and timing of rainfall after application [26] . Farmers may overfertilize because they are unaware of the specific nutrient content of their soils or the needs of their crops [27] .

The second important cause of external surface water degradation (Soluble Reactive Phosphorus (SRP), Total Phosphorus (TP) and ammonia (NH₃) is the discharge from rural households, as indicated from the results of nearby Cerro Alto. Most homes in the drainage basin discharge greywater directly to the environment—to the hillsides behind the homes. Lakes and streams near urban suburban and rural areas are often adversely affected by storm water runoff [28] [29] . These storm water runoff are important diffuse sources of phosphorus and nitrogen [28] [29] .

Rural domestic discharges include household cleaning products, the septic leachate and runoff from failed septic systems (wastewater), and especially the direct discharge of rural domestic greywater to the hillside pastures that drain through the small tributaries that converge at the Cerro Alto sampling point. Other discharges may include urban runoff from construction, and runoff from lawns and streets. Most of the domestic waste collecting tanks (septic tanks) in Lajas are constructed without any lining materials and can leak liquid waste to the ground, causing nutrients to overflow to open canals during the rainy season. However, a lack of corresponding high N concentrations suggests that the elevated TP levels seen at the Cerro Alto location and lower sampling sites result from soaps and phosphate detergents used by the residences that directly discharge their domestic greywater effluents to the environment.

The water quality results in this study showed that dissolved oxygen (DO) levels measured in all canals were more than 2 mg/L, (13 of 17 occasions). Such low DO concentrations will not support fish from in the Principal Drainage Canal at the UPR Agricultural Experimental Substation. DO levels with value from 8 to 11 mg/L are usually required in a nutritionally balanced stream, while 4 mg/L is a minimum value needed to support most species of fish [27] . On few occasions the DO levels measured in the canals were greater than 11 mg/L. This is not surprising, given that DO depends on the movement of aerated water from turbulence and the currents caused by wind, water flow, and thermal upwelling [1] .

The high turbidity levels (1.20 to 52.87 cm) from the Principal Drainage Canal at the UPR Agricultural Experimental Substation are due to the high amounts of suspended inorganic and organic matter, including nutrients and pollutants. The overall pH values for all samples were basic (alkaline).

In general, these results demonstrated the impact of humans on the lagoon ecosystem by the introduction of chemicals and biological constituents to the water that flows to the lagoon.

SRP concentrations at Cerro Alto and the Principal Drainage Canal in the Refuge of Magüayo (the Combined Inlet) were significantly correlated with rainfall events at one week (r = 0.567, p = 0.018; r = 0.784, p = 0.000, respectively), and two weeks (r = 0.536, p = 0.026; r = 0.751, p = 0.001, respectively) prior to sample collection. Also, TP concentrations at Cerro Alto were significantly correlated with rainfall at one week (r = 0.581, p = 0.015), and two weeks (r = 0.502, p = 0.040) prior sample collection.

Nitrate ( NO 3 − ), nitrite ( NO 2 − ), and ammonia (NH₃) concentrations at the channelized external sources of nutrients that enter Laguna Cartagena were not significantly (p > 0.05) correlated with rainfall events at one and two weeks prior to sampling collection, except for ammonia at Margara Canal was significantly correlated with rainfall events at one week (r = 0.848, p = 0.000) and two weeks (r = 0.789, p = 0.002) prior to sampling. Although increased precipitation is related to N loading in surface water [30] , study of the N cycle is complicated due to natural factors such as vegetation or soil cover, soil properties, geology, and morphology [15] [31] that can affect N valence states (−3 to +5).

From Cerro Alto to Margara Canal, phosphorus (P) concentrations are likely to be less because nutrients may be diluted and flushed away rapidly downstream. However, concentrations in the Combined Inlet at Magüayo increase again due to high phosphorus concentrations from the UPR Agricultural Experimental Substation. This study also found no evidence of any discharge of “sewage”, per se, or concentrated nitrogenous wastes from the adjacent Magüayo neighborhood. The channelized external sources of P and N that enter Laguna Cartagena through the Principal Drainage Canal (Combined Inlet) in the Refuge collect nonpoint sources that have had no treatment or control. In general, precipitation is strongly associated with high levels of P loading that enters Laguna Cartagena. Relationships of precipitation with P loading also have been noted in studies elsewhere [29] .