Acid Phosphatase Activity in Freshwater Ecosystems of Western Cuba and its Relationship with Water Quality

Enzyme activity plays an important role in the functioning of aquatic ecosystems. It is sensitive to changes in environmental conditions such as pH, temperature, and nutrient concentration. The objective of this work was to determine the acid phosphatase activity (AcPA) in the Almendares and San Juan rivers (western Cuba) and its relationship with physicochemical and microbiological indicators. For this purpose, AcPA, temperature, pH, total dissolved solids, electrical conductivity, dissolved oxygen, concentration of nitrates, nitrites, ammonium, phosphates, total heterotrophs, enterococci, Escherichia coli, thermotolerant coliforms, chlorophyll a and chemical oxygen demand (COD) were determined at three sampling stations on the Almendares River and at three sampling stations on the San Juan River. In addition, the nutrient pollution index (NPI) and the N:P ratio were calculated. In both ecosystems, spatio-temporal variability was observed in the enzymatic activity. In the Almendares River (polluted ecosystem), AcPA was positively correlated with nitrate concentration and COD. While in the San Juan River (slightly contaminated ecosystem) the AcPA correlated negatively with the pH and NPI and positively with the concentrations of total heterotrophs, Escherichia coli, chlorophyll a and the N:P ratio. These results show the impact of anthropogenic pollution on AcPA in freshwater ecosystems with a tropical climate. Mann-Whitney U linear Statistical analyzes in the


Introduction
The extracellular enzymes produced by microorganisms play an important role in the biogeochemical cycles of nutrients in aquatic ecosystems, contributing to the remineralization of organic matter, speci cally macromolecular debris (Arnosti et al. 2014;Egli and Janssen 2018). For the degradation of macromolecular debris into assimilable substrates, heterotrophic microorganisms release enzymes into the environment through active secretion or cell lysis (Arnosti et al. 2011;Sinsabaugh and Shah 2012). Among the enzymes produced by microorganisms are phosphatases, which release soluble inorganic phosphate from organophosphates, making it available to most organisms (Jackson et al. 2013).
There are three groups of hydrolytic enzymes responsible for the hydrolysis of organic phosphorus, phosphosterases (mono and diesterase), nucleotidases, and nucleases (exo and endonuclease) (Chróst and Siuda 2002;Torres et al. 2017). Phosphomonoesterases (PMEase) are non-speci c enzymes that hydrolyze simple phosphomonoesters and can be produced by different microorganisms. Furthermore, depending on the pH at which they exhibit their maximum activity, they can be divided into two groups: acidic (pH 2.6-6.8) and alkaline (7.6-10) (Torres et al. 2017). Although it is suggested that both enzymes are regulated by the availability of orthophosphate, acid PMEase (hereinafter acid phosphatases) are usually considered constitutive enzymes whose synthesis is related to phosphorus concentration and demand within the cell (Siuda 1984; Jasson et al. 1988). In freshwater ecosystems, acid phosphatases (AcP) have been less studied compared to alkaline phosphatases, possibly due to the high number of systems with neutral pH where the preservation of AcP enzyme activity could be affected (Siuda 1984). Due to this, the activity of AcP has hardly been investigated in freshwater ecosystems with different trophic states where the pH is neutral or slightly alkaline. Studies carried out in freshwater lakes with different pH in Japan (Tabata et al. 1988), showed that in systems with neutral pH the activities of acid and alkaline phosphatases were similar. Therefore, both enzymes show activity in these systems although they do not exhibit their maximum activity (Jasson et al. 1988).
On the other hand, research on the factors that affect phosphatase activity in freshwater ecosystems have focused more on the analysis of the factors that affect the activity of alkaline phosphatase in the water column and sediment and the activity of acid phosphatase (AcPA) in sediments of acidotrophic freshwater ecosystems (Huang and Morris 2003;Torres et al. 2017) and little attention has been paid to AcPA in the water column of ecosystems with neutral or slightly alkaline pH. Taking these aspects into account, we hypothesize that in contaminated freshwater ecosystems where the pH is neutral or slightly alkaline, AcPA will be more in uenced by polluting factors (eg, phosphate concentration, organic matter) compared to less impacted systems where AcPA will depend on natural conditions (eg: temperature, pH, phytoplankton activity).
The objective of this study is to determine the acid phosphatase activity (AcPA) in the Almendares and San Juan rivers (western Cuba) and its relationship with physicochemical and microbiological indicators.  (Romeu et al. 2015a). The samples were taken in the morning at a distance of one meter from the shore and 15 cm deep. The samples were transferred to the laboratory in sterile plastic bottles of 500 mL in a refrigerator (4°C) and were processed in a period of less than 12 h (AFNOR 2009).

Determination of physicochemical and microbiological indicators
Determination of temperature, pH, electrical conductivity, total dissolved solids (TDS), dissolved oxygen, nutrients (PO 4 3

Calculation of the nutrient pollution index
The nutrient pollution index (NPI) was calculated according to Isiuku and Enyoh (2020), using the formula:

NPI in Almendares and San Juan rivers
The nutrient pollution index for each sampling station of the Almendares and San Juan rivers, as well as the annual mean in each river is presented in Table 1.
All the sampling stations of the Almendares River are considered very high polluted. The Paila station presented the highest pollution values followed by Puente de Río and Río Cristal. The values in red represent NP1 > 6 (very high polluted), values NPI < 1 (no polluted), NPI de 1 ≤ 3 (moderately polluted), NPI > 3 ≤ 6 (considerable polluted) according to [16].
In the case of the San Juan River, the Presa El San Juan and Baños del San Juan sampling stations only in the month of April were considered very high polluted and considerable polluted, respectively. Presa El Palmar station in June, Presa San Juan in February, June and October and the Baños del San Juan station in June were considered no polluted. The rest of the time, the Presa El Palmar and Baños del San Juan stations were considered moderately polluted. In a general sense, the Almendares River is classi ed as a very high polluted ecosystem and the San Juan River as a moderately polluted ecosystem.  3.3. Acid phosphatase activity (AcPA) and its relationship with physicochemical and microbiological indicators of water quality

N:P ratio in Almendares and San Juan rivers
In Almendares River, AcPA showed signi cant differences between the sampling months in each sampling station and between the river sampling stations in each of the sampled months (Fig. 1). AcPA values were found between 0 and 0.025 µmol.mL ¹min ¹. AcPA positively correlated with the COD (p < 0.01) and with the concentration of nitrates (p < 0.05) in the Almendares River, but it did not signi cantly correlate with the rest of the physicochemical and microbiological indicators of water quality, the NPI and the N: P ratio (Table 3). In San Juan River, AcPA showed signi cant differences between the sampling months in each sampling station and between the river sampling stations in the months of February, June and October (Fig. 2). AcPA values were found between 0 and 0.018 µmol.mL ¹min ¹. In a general sense, enzymatic activity was AcPA positively correlated with chlorophyll a (p < 0.05), the N: P ratio (p < 0.05), the concentration of E. coli (p < 0.05) and the concentration of total heterotrophs (p < 0.01) and negatively correlated with the pH (p < 0.01) and the NPI (p < 0.05) ( Table 3).
When the AcPA values obtained in the Almendares and San Juan rivers during the dry and rainy periods were compared (Fig. 3), it was observed that the highest values were obtained in the Almendares River in both periods (p < 0.05). However, the existence of signi cant differences between the dry period and the rainy period was only evidenced in the San Juan River (p = 0.000329).

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 rati ed as a very high polluted ecosystem (Table 1) In the case of the San Juan River, the NPI indicated that this is a moderately polluted ecosystem (Table 1) 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.

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 signi cant differences between the sampling sites and the sampling months ( Figs. 1 and 2 In the Almendares River, despite obtaining the highest activity values in October, no signi cant 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 occulation of ne suspended matter in large particles, and on the other hand, the action of the tides also causes the resuspension of the particles 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

Conclusions
Our results show that in freshwater ecosystems with neutral or slightly alkaline pH, acid phosphatase activity is present and also plays an important role in recycling organic matter in these environments. Furthermore, in contaminated freshwater systems this activity is more in uenced by polluting factors (eg, phosphate concentration, organic matter) compared to less impacted systems where it depends on natural conditions (eg: temperature, pH, phytoplankton activity); so acid phosphatase activity in water column could be a good indicator of the anthropogenic impacts on microbial functioning in freshwater ecosystems. Future research should be done to assess whether the observations in this article can be generalized to other aquatic systems. In addition, metaproteomics and metagenomics studies could make it possible to analyze the response of the microbial community to the degradation of organic matter and eutrophication in the freshwater ecosystems in western Cuba.

Declarations
Con icts of Interest: The authors declare no con icts of interest.
Data Availability Statement: Data related to physicochemical and microbiological indicators of water quality from February-June 2017 can be found as supplementary material in https://doi.org/10.1007/s11270-020-04909-z. The rest of the data is within the present article, including supplementary information of water quality on October 2017.
Code availability: Not applicable