4.1. NPI and N:P ratio in Almendares and San Juan rivers
The Nutrient Pollution Index (NPI) takes into account the possible summative effects of nitrate and phosphate concentration on environmental health, allowing the estimation of water quality (Isiuku and Enyoh 2020). According to the NPI values obtained in the present study, the Almendares River is ratified as a very high polluted ecosystem (Table 1), which is in accordance with previous research carried out in this ecosystem using different physicochemical and microbiological indicators and the water quality index (Arpajón et al. 2011; Romeu et al. 2015b; Izquierdo et al. 2020) (Online Resource 3). The values obtained in this ecosystem are higher than those obtained by Isiuku and Enyoh (2020) in water bodies of southeastern Nigeria. The Almendares River is located in the province of Havana and receives little or no treatment wastewater of domestic and industrial origin from the city that contributes to the pollution of the ecosystem (Romeu et al. 2015b). The high phosphate concentrations determined in the Almendares River [see Izquierdo et al. (2020) and Online Resource 3 for values of physicochemical and microbiological indicators] contributed to the high NPI values obtained in this study.
In the case of the San Juan River, the NPI indicated that this is a moderately polluted ecosystem (Table 1), which is in correspondence with previous studies carried out in this river (Izquierdo et al. 2020; Larrea et al. 2021). The San Juan River is located in the province of Artemisa, in the “Sierra del Rosario” Biosphere Reserve, where it does not receive the same anthropogenic impact as the Almendares River because it is in a rural area where there is no presence of industries. In April, the highest NPI values were obtained at the Presa el San Juan and Baños del San Juan stations, classifying them as very high polluted and considerably polluted respectively. This result is in correspondence with the water quality index values reported by Izquierdo et al. (2020) for these two stations. The month of April constitutes one of the months that are within the dry season in Cuba. In particular, the province of Artemisa (western Cuba) was one of the most affected areas with the greatest deficit in accumulated rainfall according to the spatial analysis carried out by the Institute of Meteorology (INSMET 2018) in the dry season. Drought and increased temperature are factors that can cause the concentration of nutrients in water bodies (Roy et al. 2014) and thus affect water quality.
According to Redfield (1958), during phytoplankton growth, the N:P ratio should be equal to 16: 1. The N:P values obtained in the present study in the Almendares and San Juan rivers are not in correspondence with that observed by Redfield. In the Almendares River, phosphate concentrations are higher than nitrate concentrations, with a limitation due to nitrate. In the San Juan River, the opposite occurs, detecting a phosphate limitation (Table 2). The nitrate limitation conditions found in the Almendares River are associated with the anthropogenic activities carried out in this ecosystem, the low concentrations of dissolved oxygen and high concentrations of phosphate (Izquierdo et al. 2020) and possibly due to denitrification (Isiuku and Enyoh 2020; Wemedo et al. 2021). All of which leads to the eutrophication conditions that this river presents (Arpajón et al. 2011). In the San Juan River, the limitation by phosphate implies higher concentrations of nitrate, less addition of phosphates from polluting sources and therefore, less risk of eutrophication and better water quality (Izquierdo et al. 2020; Larrea et al. 2021).
These results show the usefulness of the NIP and the N: P ratio in evaluating water quality in aquatic ecosystems and should be used in conjunction with the water quality index to provide more complete information on the characteristics of the surface waters.
4.2. Acid phosphatase activity detected in the waters of Almendares and San Juan rivers
Acid phosphatase activity (AcPA) during the study period in the Almendares and San Juan rivers showed significant differences between the sampling sites and the sampling months (Figs. 1 and 2), showing spatial-temporal variability in both ecosystems. These results are in agreement with studies carried out in different aquatic systems, such as lakes (Tabata et al. 1988; Patel et al. 2018), rivers (Pandey and Yadav 2017; Patel et al. 2018), estuaries (Labry et al. 2016), mangroves (Luo et al. 2017) and seas (Ivancic et al. 2009), where the phosphatases activity (acid and/or alkaline) in the water column or sediment has been analyzed. In both, the Almendares River and the San Juan River, the highest AcPA values were obtained in the month of October in almost all the stations. This month marks the end of the rainy season in Cuba and was characterized by the presence of abundant rainfall (INSMET 2018). In addition, in the San Juan River the highest activities were found in the rainy season compared to the dry season (Fig. 3), which is in correspondence with the results reported by Giraldo et al. (2014) who obtained the highest phosphatase values in the rainy period in the Aburra-Medellín River in Colombia. Different researchers have suggested that during the rainy season the extracellular enzymatic activity increases as a result of the dilution of nutrients, which become less available to the microorganisms present in the waters. These microorganisms must synthesize enzymes to obtain nutrients from the organic matter available (Tiquia 2011; Giraldo et al. 2014; Jaramillo et al. 2016). On the other hand, in dry periods, there is an accumulation of nutrients that can be used by heterotrophic microorganisms, producing a decrease in the enzymatic activity (Cunha et al. 2010; Tiquia 2011).
In the Almendares River, despite obtaining the highest activity values in October, no significant differences were observed between the rainy season and the dry season, which may be due to the high levels of contamination by organic matter that reach the river from the discharges of domestic and industrial origin and from animal husbandry, throughout the year (Arpajón et al. 2011; Romeu et al. 2015b). In addition, AcPA positively correlated with COD (Table 3), which is an indicator of organic matter. This suggests that high levels of contamination maintain high levels of enzymatic activity, which is in correspondence with the results obtained by Jaramillo et al. (2016) who found that there was a correlation between COD and phosphatase activity in the Aburra-Medellín River in Colombia. Previously, it has been reported that contamination with sewage and sediments can constitute a source of alkaline phosphatase activity (Zhou et al. 2004; Nedoma et al. 2006) and could also constitute a source of AcPA considering that many microorganisms present in wastewater can produce both enzymes, as was demonstrated by Larrea et al. (2018) in isolates from Almendares River. The Puente de Hierro station on the Almedares River showed the highest AcPA values, which correlated with the COD and the nitrate concentration (Table 3). This station is located in the river estuary where a greater arrival of nutrients occurs with the entry of the tides and with the discharge of the freshwater ecosystem. In addition, the mixture of fresh river water with sea water causes the flocculation of fine suspended matter in large particles, and on the other hand, the action of the tides also causes the resuspension of the particles (Sigee 2005); contributing to the increase of organic matter and therefore to the increase in enzymatic activity. High values of alkaline phosphatase activity in the presence of high concentrations of organic matter and high concentrations of phosphates in estuaries have been fundamentally attributed to the presence of particles associated bacteria according to studies carried out in the Aulne and Elorn estuaries in the Northeast France (Labry et al. 2016); conditions that occurred in all the stations of the Almendares River.
The fact that the highest AcPA values have been obtained in the Almendares River with respect to the San Juan River may be due to the high values of organic matter found in the Almendres River and the slightly alkaline pH observed in the San Juan River (Izquierdo et al. 2020) (Online Resource 3). Particularly, an increase in enzyme activity in the water column has been detected in streams with more intensive human land use (Williams et al. 2012) as is the case of Almendares River. In the San Juan River, pH correlated negatively with the AcPA, where fundamentally, in the months of February and April values of pH > 8 were obtained (Izquierdo et al. 2020). At these pH values, the activity of acid phosphatases is practically null because their activity occurs at pH 2.6–6.8 (Torres et al. 2017). In the San Juan River, a negative correlation between the AcPA and the nutrient pollution index was also observed. The San Juan River is an ecosystem located in the Sierra del Rosario Biosphere Reserve, which is not exposed to the same pollutant load as the Almendares River because it is in a rural environment. In addition, this Reserve constitutes a mountainous ecosystem where abundant rainfall occurs that tends to dilute the pollutants present in the waters. Thus, the microorganisms present in the river waters, faced with a lower availability of nutrients, must synthesize extracellular enzymes to obtain the nutrients from the available organic matter (Cunha et al. 2010).
On the other hand, in the San Juan River, the AcPA positively correlated with chlorophyll a, the concentration of Escherichia coli, the concentration of total heterotrophs and the N: P ratio. This result suggests that both bacteria and phytoplankton present in this ecosystem are responsible for the production of acid phosphatase activity under phosphate-limited conditions, which is in correspondence with previous studies that have been carried out in different aquatic systems where the activity of alkaline or acid phosphatases has been determined (Yiyong et al. 2002; Nedoma et al. 2006; Labry et al. 2016; Li et al. 2019).