The River Influence Controls Water Quality and Spatio-Temporal Microalgal Distribution in Pacific Estuaries (Padre Ramos and Salinas Grandes) of Nicaragua

Estuaries are coastal wetlands that deserve special attention because they are vulnerable, biodiversity- and service-rich environments. However, estuaries of tropical areas have been scarcely studied regarding water quality and biodiversity in spite of strong developments of their agricultural and livestock sectors. Two estuaries on the Pacific Nicaragua in which snapper cages had been set up were studied regarding water physicochemical properties, microalgae and cyanobacteria along with their dynamics over a hydrological year. Both environments showed excellent water quality (oligotrophy and absence of harmful organisms) arising from their fluvial features. During the rainy season, there was a decline in chemical compounds and microorganisms. In the dry period nutrients and salinity promoted different assemblages of microscopic primary producers that were never too dense. Nutrient inputs from shrimp farms, agricultural crops and snapper cages did not appear to increase eutrophication and cyanobacterial blooms did not occur. Furthermore, the strong fluvial character of these estuaries seems to prevent the entry of harmful dinoflagellates from marine areas.


Introduction
The ecological and service value of wetlands, especially coastal wetlands, is now recognized. We also know that they are highly vulnerable to global changes [1], such as changes in sea level, desiccation, deforestation or the abusive use of pesticides [2][3][4].
However, the amount of available information related to coastal wetlands in different parts of the planet is not comparable; temperate zones have been studied in greater depth compared to tropical ones. A paradigmatic example is the scarcity of information concerning the estuaries of Central America. These wetland areas have a high level of biodiversity; they help to control the coastlines in the face of rising sea levels, and their fish and bivalves, etc. are the source of livelihood for an impoverished and vulnerable population. Moreover, before we get the chance to learn more about them, they are being profoundly altered by farms upstream, or in their catchment area [5], due to extensive shrimp aquaculture, whose exploitation is not always controlled, or deforestation and extensive agriculture which produces inputs of allochthonous material (i.e., pollutants, loads of organic and inorganic material, etc.) as well as extreme climatic processes [6,7] among other factors. On the other hand, coastal processes also affect estuary waters, hence toxic tidal events can end up damaging the biota of the estuaries [8] and, more recently, the over-exploitation of coastal fish with a market value, such as the spotted rose snapper (Lutjanus guttatus), endangers the symbiosis of fishing communities linked to the estuaries. These issues should be viewed as a One Health concern [9].

Obtaining Variables In Situ, Sampling and Analysis
Salinity, dissolved oxygen, pH and temperature were measured a few centimeters below the surface, at the middle depth of the water column and 20 cm from the bottom; the data did not vary significantly and were averaged. When the sampling location was near a floating cage for snappers, measurements were taken after having gone past the cage in the direction of the current. Dissolved oxygen (DO, mg/L) was measured with a Handheld Multiparameter Instrument oximeter (model 556, YSI), and salinity (ppm) was measured with a handheld salinity refractometer (J&G SC). The pH was measured using a pH meter (Eco Testr pH2), and Transparency (cm) was measured using a Secchi disk.
Using a hydrographic bottle, 2 L of water were extracted at the middle depth of the water column. Then, 1 L of this water was used to carry out the chemical analyses and it was kept refrigerated; the other liter of water was fixed with lugol for the subsequent study of the MIC. Both samples arrived for processing at the UNAN-León laboratory on the same day the sampling took place.
To obtain the nitrate, ammonium and orthophosphate concentration, the reagents from the MQuantTM test strips kit (Merck [35,36]) were used.
The liter of sampled water settled for three days in 250 mL test tubes in order to be able to concentrate the seston in 20 mL, the volume that was used in the analysis. The taxonomic classification and measurement of the body-size of the most abundant The EPR includes mangroves, beaches, shrimp farms, islets, etc. The ESG also houses beaches where salt is currently produced [33]. Both have a dry, warm climate, a tropical savanna climate according to the Köppen classification, with a dry season from November to April and a wet season the rest of the year. Rainfall is 1500-1800 mm per year [35]. Temperatures fluctuate around 36 degrees Celsius. The winds are strong in the dry season, and they are intensified between February and April, reaching speeds of up to 30 km/h [33]. Sampling took place in April, September, October, November and December 2018, and January and February 2019; the exact days were chosen to ensure that the high tide occurred between 9-12 am to allow transport by boat along the course of the estuaries, whose depth was approximately 5 m. In EPR, six sites were sampled ( Figure 1b); the extremes being in Poza de la Vaca (12 • 50 20.9 N, 87 • 32 33.2 W) and El Sistema (12 • 45 20.9 N, 87 • 27 21.0 W). All the sites had a floating cage nearby for the rearing of snapper, except for Quilaca (site P4). In the EPR, the distance between the furthest sites is 13.5 km, extending to 19 km if we include the derivations (Figure 1b). In ESG, 9 sites were sampled between La Garita (12 •  these sites are found along the Chiquito river, a distance of 7.5 km (Figure 1c). The only site with a snapper cage was Garita (site P1). All the sites were geo-referenced (Table S1). The snapper cages are 5 × 5 × 5 m cubic bags and their maximum production is 1800 kg of snapper in 8 months.

Obtaining Variables In Situ, Sampling and Analysis
Salinity, dissolved oxygen, pH and temperature were measured a few centimeters below the surface, at the middle depth of the water column and 20 cm from the bottom; the data did not vary significantly and were averaged. When the sampling location was near a floating cage for snappers, measurements were taken after having gone past the cage in the direction of the current. Dissolved oxygen (DO, mg/L) was measured with a Handheld Multiparameter Instrument oximeter (model 556, YSI), and salinity (ppm) was measured with a handheld salinity refractometer (J&G SC). The pH was measured using a pH meter (Eco Testr pH2), and Transparency (cm) was measured using a Secchi disk.
Using a hydrographic bottle, 2 L of water were extracted at the middle depth of the water column. Then, 1 L of this water was used to carry out the chemical analyses and it was kept refrigerated; the other liter of water was fixed with lugol for the subsequent study of the MIC. Both samples arrived for processing at the UNAN-León laboratory on the same day the sampling took place.
To obtain the nitrate, ammonium and orthophosphate concentration, the reagents from the MQuantTM test strips kit (Merck [35,36]) were used.
The liter of sampled water settled for three days in 250 mL test tubes in order to be able to concentrate the seston in 20 mL, the volume that was used in the analysis. The taxonomic classification and measurement of the body-size of the most abundant populations were done with a conventional LW microscope at 400× magnification. The taxonomic classification of the MIC was carried out, differentiating between the large groups of algae: Chlorophytes, Diatoms, Dinophytes, Euglenophytes and Cyanobacteria [37]. The organisms were identified at the finest possible taxonomic resolution following standard references (cited in [38]) as well as some local recommendations for organisms of possible marine origin and from tropical areas [39][40][41]. For the counting and subsequent calculation of the population densities, Neubauer and Sedgwick-Rafter chambers [42] were used to analyze the 20 mL concentrate at 100× magnification; both chambers were filled and counted six times to get average values. Algal biomass (expressed as biovolume, mm 3 /L) was calculated with density and cell volume, measuring sizes from at least 20 individuals and following [43]. The total chlorophyll a concentration was estimated taking into account the chlorophyll a concentration per unit of biovolume established for each MIC group by [37].

Statistical Analysis
Means comparisons were carried out with repeated measure ANOVA (post-hoc Tukey's pairwise test). Compliance with the requirements was verified (normality with the Shapiro-Wilks test; homogeneity with the Levene test). When these did not comply, a non-parametric comparison was made, obtaining the Kruskal-Wallis H (post-hoc Mann-Whitney's test). Principal components analysis (based on correlation coefficient; [44]) was carried out to order samples and their limnological variables (temperature, pH, dissolved oxygen, salinity, concentration of nitrate, ammonium and orthophosphate plus MIC biovolume). Canonical Correspondence Analysis (CCA; [45]) was also used. CCA of the sites and taxa abundance, where each site has given values for the seven physico-chemical environmental variables. Thus the taxa abundances are considered to be a response to the environmental variables gradient. The calculations were done using the free statistical program package PAST [46].

Limnology of the Estero del Padre Ramos
The temperature range throughout the year studied, including all the sites, was 27.7-31.2 • C. A decreasing trend can be observed from the first to the last month of sampling ( Figure 2). The ANOVA analysis highlighted two thermal seasons (Table S2): April to September with 30.4 ± 0.6 • C, and October to February with 28.5 ± 0.6 • C (mean ± standard deviation). The pH range varied between 7.5-8.7, and showed three periods (Table S2): from April to October, from November to January, and February, with averages of 7.8 ± 0.3, 8.4 ± 0.3 and 8.3 ± 0.2, respectively ( Figure 2). The DO concentration range was 3.1-7.2 mg/L ( Figure 2). Three significantly different mean periods were observed (Table S2): April (4.3 ± 0.4 mg/L), from September to November (5.1 ± 0.7 mg/L) and from January to February (6.5 ± 0.5 mg/L). Salinity ( Figure 2) fluctuated between 20 ppm and 36 ppm, and the annual means were significantly different (Table S2). From lowest to highest, the salinity ranges were: September-October (26.4 ± 3.7 ppm), April and November (32.5 ± 2.1 ppm and 32.7 ± 1.8 ppm) and January-February (35.2 ± 1.8 ppm).
ter 2021, 13, x FOR PEER REVIEW variation was 102 in April and 34 in February, Figure 4). The average statistically different. There were no differences in the mean biovolume calculated for each sampling site ( Figure 4). The estimated chlorophyll a co covaried with the biovolume (R 2 = 0.85, p < 0.001). Estimated chlorophyll a did 2.3 µg/L, nor did it show significant differences between months. It ranged b ± 0.70 µg/L (April) and 0.45 ± 0.10 µg/L (January).
The ordering into main components (Figure 5a) chronologically se samples from the EPR. The April, September and October samples are lo negative section of component 1, together with a higher biovolume concentra and soluble phosphorus as well as a higher temperature. At the positive extr January and February samples, along with nitrogen compounds and salinity. 2 separates April-September (biovolume and phosphorus vector, respectively separating November (salinity vector) from January-February (nitrate co vector; Figure 5a).  (Table S2; Su material). The lower-case letters indicate significant differences in pairwise comparison values when p < 0.001.  (Table S2; Supplementary material). The lower-case letters indicate significant differences in pairwise comparisons. Significant values when p < 0.001.
Nitrate concentration varied between undetectable values and 12 mg/L, and two periods were observed ( Figure 3; Table S2). The minimum concentrations were from April to November, while the period from January to February had an average concentration of 9.2 ± 1.6 mg/L. The ammonium concentration was minimal from April to January at all the sites, and increased in January and February; as was the case with nitrate. However, in these last two months a great variation between sites was observed, from undetectable concentrations to 2.5 mg/L in Poza de la Vaca ( Figure 3); an ANOVA indicated that there were no significant differences between months. The orthophosphate concentration (mg/L) varied considerably between the sites on each sampling date ( Figure 3; Table S2), and it was higher in September and October (0.025 ± 0.024 mg/L), with peaks at two sites near the estuary mouth (P1-P3; Figure 1b).   Figure 4). The averages were not statistically different. There were no differences in the mean biovolume over time calculated for each sampling site ( Figure 4). The estimated chlorophyll a concentration covaried with the biovolume (R 2 = 0.85, p < 0.001). Estimated chlorophyll a did not exceed 2.3 µg/L, nor did it show significant differences between months. It ranged between 1.04 ± 0.70 µg/L (April) and 0.45 ± 0.10 µg/L (January).    Table S3). Populations belonging to seven different genera were found (Table S4), the most abundant, and also the most conspicuous, were Coenocystis, Crucigeniella, Monoraphidium and Dictyosphaerium. Diatoms showed their highest density in October and November (372 ± 173 ind/mL and 328 ± 172 ind/mL, respectively; Table S3). This is the group of algae with the greatest diversity; populations of 23 genera were found (Table S4). We found a pattern of population substitution over time which is related to the hydrological cycle. From September to November (rainy season) the most abundant populations belonged to the genera and species Biddulphia, Nitzschia (i.e., N. sigma and N. sinuata), Pleurosigma, Surirella and Skeletonema costatum. However, in January and February (dry season), the genera Aulacoseira and Navicula appeared as dominant. Only a few cyanobacteria individuals (Tables S3 and S4) were detected, and they were always filamentous, belonging to the genera Oscillatoria (in P1 in October) and Anabaena (in P3-P4 in January and February). Dinophyte peaks occurred in October and November (2.6 ± 1.3 ind/mL and 1.0 ± 0.7 ind/mL) and appeared along the estuary. For the rest of the months the average abundance of Dinophytes did not exceed 0.3 ind/mL.  Table S3). Populations belonging to seven different genera were found (Table S4), the most abundant, and also the most conspicuous, were Coenocystis, Crucigeniella, Monoraphidium and Dictyosphaerium. Diatoms showed their highest density in October and November (372 ± 173 ind/mL and 328 ± 172 ind/mL, respectively; Table S3). This is the group of algae with the greatest diversity; populations of 23 genera were found (Table S4). We found a pattern of population substitution over time which is related to the hydrological cycle. From September to November (rainy season) the most abundant populations belonged to the genera and species Biddulphia, Nitzschia (i.e., N. sigma and N. sinuata), Pleurosigma, Surirella and Skeletonema costatum. However, in January and February (dry season), the genera Aulacoseira and Navicula appeared as dominant. Only a few cyanobacteria individuals (Tables S3 and S4) were detected, and they were always filamentous, belonging to the genera Oscillatoria (in P1 in October) and Anabaena (in P3-P4 in January and February). Dinophyte peaks occurred in October and November (2.6 ± 1.3 ind/mL and 1.0 ± 0.7 ind/mL) and appeared along the estuary. For the rest of the months the average abundance of Dinophytes did not exceed 0.3 ind/mL.

Limnology of the Estero de Salinas Grandes
The temperature range throughout the year studied, includin °C, the coldest months being January and February (Figure 7) highlighted the temperature gradient (Table S2) from September The pH range varied between 6.7-9.0, showing different peri maximum value was measured in November-December (averag 0.46). The DO concentration range was 0.5-6.6 mg/L (Figure between months were not significant. The oxygen concentration, higher in the areas close to the mouth of the estuary, decreasing the interior (Figure 7; Figure 1c). Salinity fluctuated between 10 pp annual means were significantly different (Figure 7; Table S2); from salinity ranges were: September 22.8 ± 8.0 ppm, October and Nove ppm (30.1 ± 2.8 ppm, 29.9 ± 3.3 ppm, respectively), and Januar combined mean of 34.8 ± 0.7 ppm.

Limnology of the Estero de Salinas Grandes
The temperature range throughout the year studied, including all sites, was 26.1-30.4 • C, the coldest months being January and February (Figure 7). An ANOVA analysis highlighted the temperature gradient (Table S2) from September to February (Figure 7). The pH range varied between 6.7-9.0, showing different periods (Table S2) and its maximum value was measured in November-December (average of both months 8.01 ± 0.46). The DO concentration range was 0.5-6.6 mg/L (Figure 7) and the differences between months were not significant. The oxygen concentration, as well as the pH, was higher in the areas close to the mouth of the estuary, decreasing when moving towards the interior (Figure 7; Figure 1c). Salinity fluctuated between 10 ppm and 35 ppm, and the annual means were significantly different (Figure 7; Table S2); from lowest to highest, the salinity ranges were: September 22.8 ± 8.0 ppm, October and November values close to 30 ppm (30.1 ± 2.8 ppm, 29.9 ± 3.3 ppm, respectively), and January and February with a combined mean of 34.8 ± 0.7 ppm. Water 2021, 13, x FOR PEER REVIEW 10 of 19  The nitrate concentration was practically undetectable in the first sampling months (values lower than 0.1 mg/L and 11.8 mg/L (Figure 7). Moreover, large differences were observed between months (Table S2), with the months from December to January being similar (combined average of 6.6 ± 3.0 mg/L). The ammonium concentration was practically undetectable in all the samples, and the average of all of them was 0.10 ± 0.27 mg/L. Only in Nacascolo (P7) in January did it exceed 1 mg/L (Figure 8). The mean orthophosphate concentration ranged from 0.11 ± 0.19 mg/L in September to 0.15 ± 0.15 mg/L in February; however, these variations were not significantly different. It is noteworthy that in November in the first two sites the concentration of orthophosphate was undetectable, in December it was undetected in the first three sites, and in January and February it was in the first four sites.  (Table S2; Supplementary material). The lower-case letters indicate significant differences in pairwise comparisons. Significant values when p < 0.001.
The nitrate concentration was practically undetectable in the first sampling months (values lower than 0.1 mg/L and 11.8 mg/L (Figure 7). Moreover, large differences were observed between months (Table S2), with the months from December to January being similar (combined average of 6.6 ± 3.0 mg/L). The ammonium concentration was practically undetectable in all the samples, and the average of all of them was 0.10 ± 0.27 mg/L. Only in Nacascolo (P7) in January did it exceed 1 mg/L (Figure 8). The mean orthophosphate concentration ranged from 0.11 ± 0.19 mg/L in September to 0.15 ± 0.15 mg/L in February; however, these variations were not significantly different. It is noteworthy that in November in the first two sites the concentration of orthophosphate was undetectable, in December it was undetected in the first three sites, and in January and February it was in the first four sites. Water 2021, 13, x FOR PEER REVIEW 11 of 19 When the average concentration observed at all the sites was taken into account (Figure 9), the total MIC biovolume was significantly different between months. We can distinguish two sets (F = 6.5; p < 0.001): September, October and November with a combined mean of 0.39 ± 0.20 mm 3 /L, and December, January and February which reached 0.25 ± 0.17 mm 3 /L (Figure 9). Comparison of the average biovolume of all the months studied in each site shows us an ordering from lower to higher concentration as we move away from the estuary mouth ( Figure 9). Thus, between P1 and P3 the average was 0.14 ± 0.10 mm 3 /L, and between P8 and P9 it was 0.54 ± 0.10 mm 3 /L. The estimated chlorophyll a covaried significantly with the biovolume (R 2 0.98, p < 0.001), and ranged from 1.52 ± 0.06 µg/L from September to November to 2.10 ± 0.11 µg/L in the period from December to February. The ordering in principal components of the samples from the ESG (Figure 5b) was spatial, and it ordered the samples based on their distance from the sea. In the positive part of component 1 were the sites closest to the outlet of the water from the estuary, together with the salinity vector and the oxygen concentration vector. At the opposite end were the samples from the sites farthest from the sea, together with a higher MIC biovolume. On the other hand, component 2 separated the months of December, January and February together with the nutrients of the months of September, October and November with higher temperatures (Figure 5b). When the average concentration observed at all the sites was taken into account (Figure 9), the total MIC biovolume was significantly different between months. We can distinguish two sets (F = 6.5; p < 0.001): September, October and November with a combined mean of 0.39 ± 0.20 mm 3 /L, and December, January and February which reached 0.25 ± 0.17 mm 3 /L (Figure 9). Comparison of the average biovolume of all the months studied in each site shows us an ordering from lower to higher concentration as we move away from the estuary mouth ( Figure 9). Thus, between P1 and P3 the average was 0.14 ± 0.10 mm 3 /L, and between P8 and P9 it was 0.54 ± 0.10 mm 3 /L. The estimated chlorophyll a covaried significantly with the biovolume (R 2 0.98, p < 0.001), and ranged from 1.52 ± 0.06 µg/L from September to November to 2.10 ± 0.11 µg/L in the period from December to February. The ordering in principal components of the samples from the ESG (Figure 5b) was spatial, and it ordered the samples based on their distance from the sea. In the positive part of component 1 were the sites closest to the outlet of the water from the estuary, together with the salinity vector and the oxygen concentration vector. At the opposite end were the samples from the sites farthest from the sea, together with a higher MIC biovolume. On the other hand, component 2 separated the months of December, January and February together with the nutrients of the months of September, October and November with higher temperatures (Figure 5b). The main algal groups that made up the MIC biovolume in ESG were Chlor and Bacillariophyta or Diatoms (Table S3). The presence of Dinophytes (Tripos fus than 1 ind/mL) was only detected in February, and no cyanobacteria or Eugleno were found. The average Chlorophyta density of the locations in each month exceeded 600 ind/mL, decreasing over the time period (Table S3). In total, 12 popu of 12 genera of Chlorophytes were found (Table S5). The most notabl Dictyosphaerium, Coenocystis and Monoraphidium for their abundance and occ ( Figure 10). Diatoms do not show any trend over time (Table S3). Their density from 50 ± 40 ind/mL in December to 319 ± 263 ind/mL in November, with the ma value (972 ind/mL) being observed at P6, an intermediate point of the course estuary. This is the group of algae with the greatest diversity, and populations of 24 were found (Table S5). Aulacoseira granulata, Nitzschia sinuata and Surirella were th remarkable ( Figure 10). The CCA allowed us to discriminate a spatio-temporal pa the most abundant populations (Figure 10) with three possible associations: o appears in the rainy September, the month with the highest temperature but also levels of salinity and nutrients, and two other associations opposed to each oth more related to nutrients and sites located a long way from the sea, and the other the sites closest to the sea and related to salinity (Figure 11). The main algal groups that made up the MIC biovolume in ESG were Chlorophyta and Bacillariophyta or Diatoms (Table S3). The presence of Dinophytes (Tripos fusus, less than 1 ind/mL) was only detected in February, and no cyanobacteria or Euglenophytes were found. The average Chlorophyta density of the locations in each month barely exceeded 600 ind/mL, decreasing over the time period (Table S3). In total, 12 populations of 12 genera of Chlorophytes were found (Table S5). The most notable were Dictyosphaerium, Coenocystis and Monoraphidium for their abundance and occurrence ( Figure 10). Diatoms do not show any trend over time (Table S3). Their density ranged from 50 ± 40 ind/mL in December to 319 ± 263 ind/mL in November, with the maximum value (972 ind/mL) being observed at P6, an intermediate point of the course of the estuary. This is the group of algae with the greatest diversity, and populations of 24 genera were found (Table S5). Aulacoseira granulata, Nitzschia sinuata and Surirella were the most remarkable ( Figure 10). The CCA allowed us to discriminate a spatio-temporal pattern of the most abundant populations (Figure 10) with three possible associations: one that appears in the rainy September, the month with the highest temperature but also lowest levels of salinity and nutrients, and two other associations opposed to each other; one more related to nutrients and sites located a long way from the sea, and the other one to the sites closest to the sea and related to salinity ( Figure 11).   Figure 5. Ordination by CCA of EPR data was not significant (p < 0.001).   Figure 5. Ordination by CCA of EPR data was not significant (p < 0.001).

Discussion
In the EPR, features such as T, salinity, pH and dissolved oxygen do not vary spatially, and only respond to the hydrological season [6,19], with a higher water temperature until September and greater salinity, pH and DO in the dry season from Figure 11. CCA of samples, limnological variables and taxa obtained in Salinas Grandes. Explained variance is shown in each axis. Abbreviations as in Figure 5. Ordination by CCA of EPR data was not significant (p < 0.001).

Discussion
In the EPR, features such as T, salinity, pH and dissolved oxygen do not vary spatially, and only respond to the hydrological season [6,19], with a higher water temperature until September and greater salinity, pH and DO in the dry season from November to May. This same pattern is also found in the ESG, although in this estuary the effect of the dilution by the rains becomes more evident as we get closer to the estuary mouth. The concentrations of nutrients (mainly nitrate) obtained in the rainy season are much lower than those analyzed during the dry period; this indicates that the rains exert a dilution effect greater than a possible N contribution by runoff. In other estuaries, such as the Gulf of Nicoya, seasonal changes in nutrient concentrations (nitrate and dissolved orthophosphate), which were lower during the rainy season, were also observed [13], suggesting dilution by precipitation. This pattern is observed in both estuaries (EPR and ESG), and in both of them similar concentrations of N compounds are attained (i.e., approx. 8 mg/L of nitrate). During the dry period, the concentration of nitrates in the EPR is homogeneous. However, in the ESG the upper portion shows a higher concentration. The concentrations of orthophosphate in the ERP are very low, even undetectable on many occasions, so its high spatio-temporal irregularity warrants caution. However, in the ESG the concentration, which is ten times higher than that of the EPR, is lower in the rainy season and during the drought it is higher in the upper portion of the estuary. A spatio-temporal distribution similar to that found for nitrate. Therefore, we can suggest that we are dealing with estuaries in which the river has a greater effect than the ocean, and that in the rainy season, due to an increase in its flow rate into the sea, the compounds which the river obtains from the agricultural use in its catchment area are diluted.
In the EPR, the source of nutrients, mainly N compounds, is shrimp farming (Flores et al. 2007) which occupies 12% of the total area [6], practically up to the estuary mouth. Agriculture and livestock, which are present in its basin, occur on small farms for auto-consumption (peanuts, sugarcane, corn, chickens and pigs) and do not imply a great contribution of nutrients as they do not involve high levels of fertilization. However, in the ESG the nutrients, including phosphate, originate from the large areas devoted to corn cultivation (see these areas in Figure 1c). In fact, there are no changes in salinity that could explain changes in the behavior of the dissolved phosphorus [25], nor MIC growth, that could cause its decline [37]. The contribution of nutrients from these areas decreases towards the estuary mouth as the farm land gives way to the salt flats. There are some concerns about the possible nutrient supply from the snapper cages [47,48]. However, it can be stated that in none of the estuaries were differences in the nitrate concentration between the sites with and without cages, according to what had previously been observed in a snapper cage farm on the tropical coast of Mexico [14]. Furthermore, the average nitrate concentrations in the EPR and ESG (9.2 mg/L and 6.6 mg/L, respectively) were even lower than those of the aforementioned open-ocean floating cages (12.3 mg/L-15.8 mg/L; [14]). Phosphorous concentrations did not vary either between sites with or without cages, and their concentrations were low. The highest concentrations of phosphates were found in the sites furthest from the ESG coast during the dry months (on average 0.35 ± 0.09 mg/L), and the minimum value reported in the aforementioned study, which was observed at a site quite far from the cages, was 0.49 ± 0.42 mg/L [14]. So, it can be stated that the cages were not altering the concentrations of dissolved nutrients. Our data support the notion that the undeniable exudate from these sets of cages is minimized by the rains for 6 months, and by sufficient tidal circulation and flushing in these open waters to the sea, as also suggested by [14].
The MIC biovolume and estimated chlorophyll a concentration do not, at any time or in any place, exceed, in either of the two estuaries, the limit of 1 mm 3 /L (approx. 50 µgC/L) or 2.5 µg/L. These concentrations were lower than the minimum concentrations reported for the Gulf of Nicoya in the 2012 dry season (600 µg C/L and 3 µg Ch-a/L; [49]). Despite the differences between freshwater and estuarine systems, their similarities are sufficient for successful application of limnological modeling concepts to estuaries such the OECD model [50,51]. Therefore, with due caution, it can be stated that, according to [29] for the MIC biovolume, and the [30] for total chlorophyll a, the waters of both estuaries at all the sites, with or without snapper cages, and at any time of the hydrological year are oligotrophic. In addition, the spatial distribution of the biovolume of the phytoplankton seems to have a riverine rather than oceanic nutrient contribution, as already described, for example, for the Gulf of Nicoya [49], for a bar-built estuary in the northeastern Gulf of Mexico [52,53], or for the shallow low inflow estuary called Drakes Estero (California; [26]).
There is very little information on the dynamics of the taxonomic and functional groups of the MIC on the estuaries of the Pacific coast of Nicaragua (except for the specific works carried out by students of the University of Nicaragua). Reviewing the information on the most-studied tropical and subtropical Pacific coast estuaries, such as the externaloceanic or internal-fluvial zones of the Gulf of Nicoya (e.g., [13,39,54] or in the Gulf of Papagayo in Costa Rica [55], we can see that hydrology (river flow, flushing and tidal extension) and nutrients conditioned by hydrology are the main control factors of MIC groups. In the aforementioned estuaries, Chlorophytes are not relevant nor are they included in broad functional groups such as small flagellates (mainly Prasinophytes). Diatoms (centric and pennate) and Dinoflagellates are the groups that receive the most attention, followed by cyanobacteria, although the density of the latter is always low [13,54,55]. In the EPR and ESG, the composition of MIC assemblages varies throughout the hydrological year, but the lack of a longer time series prevents us from knowing if there is a temporal pattern of substitution, such as those described for temperate ecosystems [37]. In tropical and subtropical environments, it is recognized that the marked effect of the long rainy season functions as a great disturbance that reinitiates the dynamics of phytoplankton [20,56]. For now, we can suggest a pattern that takes into account the MIC variations in both studied estuaries. Diatoms do not show any trend over time, as observed in the Gulf of Nicoya [39]; and they were somewhat more abundant at two moments of the hydrological year: at the end of the rainy season (October to November), which is the period considered to be the transition to the dry season, and in the middle of the dry season (February). Chlorophytes, more typical of fresh water, were more abundant at the end of the dry season (April and September) and they decreased as the rainy season progressed. As for cyanobacteria, the most relevant thing is their scarcity, indicating that there are no large foci of eutrophication, or specific allochthonous contributions that may favor their bloom [8,57]. The dinoflagellates appear mainly at the time of transition from rain to drought and appeared throughout the estuary, therefore they do not seem to be linked to the most coastal zone as in the Gulf of Nicoya [13]. The densities reached by diatoms in the EPR and ESG are comparable to those mentioned for the interior of the Gulf of Nicoya (average 186 ± 159 ind/mL and 130 ± 23 ind/mL, respectively; [13]), but much lower than those that occur in its external area [39]. The density of toxic dinoflagellates is directly related to greater eutrophication of the coast [22,58] and a contribution of this coastal water to the estuary [13]. Thus, in the marine area of the Gulf of Nicoya, an annual average density of 59 ± 9 ind/mL has been described [39], and in the interior of the estuary, the density of dinoflagellates does not exceed 25 ind/mL [13]. Therefore, we can consider that the lack of eutrophication, and the fluvial nature of the ERP and ESG, protect them from the growth of these toxic dinoflagellates along their course, since they did not exceed 3 ind/mL. The presence and distribution of all these taxonomic groups reinforces the idea that the EPR and ESG have an even more marked fluvial character than other tropical estuaries dominated by freshwater influence in the Pacific [13] and Atlantic coasts, such as the estuary of the Paraíba do Sul River in Brazil [56].
Regarding the genera, and sometimes species, in EPR and ESG the most abundant and conspicuous populations of Chlorophytes were small freshwater (i.e., Dictyosphaerium, Coenocystis, Monoraphidium) planktonic organisms (nanoplankton), whose highest densities were found in the upper estuary; all this is consistent with the strategy of fast growing microalgae favored by higher river flow rates [56]. Among the diatoms there were purely planktonic genera such as Cyclotella and benthic organisms which are captured in the water column due to their detachment from the sediment by dragging. Diatoms were made up of genera mostly from brackish and marine environments. The species recognized as being from brackish environments (Nitzschia sinuata and Surirella) were the most conspicuous between September and November (rainy season and transition to drought) and more abundant in the central points of the estuary. In the period from January and February, which is the period of advanced drought, genera more typical of fresh waters appear, such as the centric diatom Aulacoseira granulata. Again we see that the drought does not propitiate a greater marine intrusion related to upwelling, nor a change towards assemblages of marine water species as described in those characterized as seasonal lowinflow estuaries adjacent to coastal upwelling [2]. The observed cyanobacteria populations that were observed were always filamentous specimens of the Oscillatoria and Anabaena genera. The former are planktonic organisms that are not N-fixers, whilst the latter are benthic N-fixers, which can be related to a drag from the benthos [59]. Both genera have potentially toxic brackish and freshwater species [60]; but the amounts in which they have been found are far from worrying [61]. In the ESG, the presence of Dinoflagellates of the species Tripos fusus was only detected occasionally during the drought period, as occurred in the Gulf of Papagayo (Costa Rica, [55]). The most abundant populations in the EPR belonged to the Noctiluca (i.e., Noctiluca cf. scintillans), Pyrodinium and Cochlodinium genera. Most of the populations found can be considered harmful marine environment algae (Table S4) and have been cited as common in tropical estuaries such as the Gulf of Mexico, and those in the tropical Pacific such as the Gulf of Nicoya, the Gulf of California [39,[62][63][64] or in the Gulf of Thailand in the South China Sea [65].

Conclusions
Our study shows that there are estuaries on the Pacific Coast of Nicaragua with excellent water quality (oligotrophy and absence of harmful organisms) due to their fluvial character. During the rainy season, there occurred a decrease in water compounds and microorganisms. In the dry period nutrients and salinity enhanced different assemblages of microscopic primary producers. Nutrient inputs from shrimp farms, farms, crops and snapper cages did not appear to cause eutrophication and cyanobacterial blooms were avoided. On the other hand, the river influence seemed to prevent the entry of harmful dinoflagellates from the marine area.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/w13121712/s1, Table S1: Name of sampled sites and their coordinates in Estero Padre Ramos and Estero Salinas Grande (Nicaragua). When in the site there are a cage of culture of Lutjanus spp. their installation data is indicated, Table S2: Results of repeated measures ANOVA (F and post-hoc Tukey's pairwise test) or H de Kruskal-Wallis (post-hoc Mann-Whitney's test) comparing values of physico-chemical variables between sampling data and between sampling sites. The lower-case letters indicate significant differences in pair-wise comparisons. Significant values when p < 0.001, Table S3: Mean and standard deviation of density (ind/mL) of main MIC groups in both studied estuaries (Estero de Padre Ramos and Estero de Salinas Grandes). Cyanobacteria and Dinophytes were undetectable in Estero Salinas Gandres, Table S4: Genera and some species of microalgae and Cyanobacteria observed in samples of Estuario de Padre Ramos. It is reported their reached maxima density and percentage, their occurrence as percentage of samples where they were found, their more probable functional group and habitat and their potential toxicity, Table S5: Genera and some species of microalgae and Cyanobacteria observed in samples of Estuario Salinas Grandes. It is reported their reached maxima density and percentage, their occurrence as percentage of samples where they were found, their more probable functional group and habitat and their potential toxicity.
Funding: This research work was developed with funds from the project "Strengthening capacities for the rearing and fattening of snapper (Lutjanus spp.) in floating cages in 8 Nicaraguan coastal communities", subsidized by the Spanish Agency for International Development Cooperation (AE-CID). In addition, the following NGOs, Fundación LIDER, Fauna and Flora International (FFI) and Foundation Ecology and Development (ECODES) subsidized the trips along the estuaries.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study will be made available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.