Benthic Macroinvertebrates as Bioindicators of Water Quality in the Vilcanota River, Cusco-Peru

2.1 Study area and selection of sites

The Vilcanota Urubamba basin (73º47' and 73º 44' W, 14º39' and 10º09' S) is situated between the Cusco and Ucayali Department of Peru at an altitude of 2950 meters above sea level. This basin presents three main drainage axes: Vilcanota, Mapacho, and Yanatile Rivers. The Vilcanota River is located in the Calca district and flows from north to east of the city of Cusco, Peru. The average annual temperature fluctuates between 11 and 16℃, the maximum between 22 and 29℃, and the minimum between 7 and -4℃ during the winter [19]. Rainfall is regularly between December to March and drought between August to October.

2.2 Periods and sampling points

The sampling of benthic macroinvertebrates was carried out during the wet (November-December 2020 and January 2021) and dry (August to October 2021) seasons at four points: P1 (13º20'9.56'' S; 71º57'23.90'' W), P2 (13º19'51.50' S; 71º57'22.94'' W), P3 (13º19''39.66'' S; 71º57'17.39'' W), and P4 (13º19´33.84” S; 71º57´36.21” W) (Figure 1). The points represent four different groups of land use (Figure 2): P1 is located 50m upstream of the discharge of the leachate from the dump in the Campanachoc sector; P2 to 200m downstream of the discharge of leachate from the municipal dump (ETSA); P3 is located to 100m upstream of the suspension bridge Calca, where people throw their garbage; and P4 located at 50m from the mouth of the Qochoq River. In conclusion, all points influence anthropic activities and discharges of solid and liquid pollutants.

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Figure 1. Location of the sampling points along Vilcanota River, Cusco, Perú

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Figure 2. Collection points of substrate samples along the Vilcanota River, Calca district, Cusco, Peru

Source: Authors' photographs

2.3 Sampling and identification of benthonic macroinvertebrates

The benthic macroinvertebrates were collected following the methodology described in the book “Methods of Collection, identification, and Analysis of biological communities: plankton, periphyton, benthos (macroinvertebrates) and nekton (fish) in inland waters of Peru” [20]. Before sampling, the bottom was removed and covered an area of 6m². The samples were collected from downstream towards upstream to minimize the collection of drifted organisms. It was quantitatively carried out using a rectangular-shaped Surber net (50cm wide x 25cm high and 250µm pore size). Additionally, the organisms attached to stones, branches, leaves, and other objects that are in the place were collected. Collected macroinvertebrates were washed in the field and each sample separately was preserved in plastic bottles (500mL) using 70% ethanol [21, 22].

Simples were taken to the laboratory, where families were identified using the methodology described by Samanez et al. [20].

2.4 Data analysis

Descriptive analysis was carried out on the physicochemical parameters. Differences among sampling points were performed through the One-way ANOVA and post hoc Tukey Test. In addition, the Total Abundance (N) was calculated: total number of individuals collected at each sampling point; and Richness (S): total number of taxa recorded at each sampling point. Water quality was evaluated and compared using three biological indices: BMWP score, ABI, and Ephemeroptera, Plecoptera, and Trichoptera (EPT) index.

2.5 Biological Monitoring Working Party (BMWP) score

The BMWP is a simple, fast, and qualitative method (presence or absence) that allows to evaluation of the quality of the water using macroinvertebrates that only requires reaching the family level. The score ranges from 1 to 10 according to the tolerance of the different groups to organic pollution, with the most sensitive families receiving a score of 10, and the most tolerant only one. The sum of the scores of all families provides the BMWP total score (Eq. (1)) and its classification is given in Table 1.

$B M W P=T 1+T 2+T 3+\ldots T n$               (1)

where, T=tolerance level of each macroinvertebrate found, and the number corresponds to the family.

Table 1. BMWP and ABI index classification of water quality according to Ríos-Touma et al. [13], respectively

Class	Quality	BMWP Index	ABI Index	Meaning	Color
I	Good	˃150 101-120	˃70	Very clean waters Unpolluted waters	tab1_1.png
II	Acceptable	61-100	45-70	Effects of contamination are evident	tab1_2.png
III	Questionable	36-60	27-44	Waters moderately polluted	tab1_3.png
IV	Critical	16-35	11-26	Waters heavily polluted	tab1_4.png
V	Very critical	<15	˂11	Waters severely polluted	tab1_5.png

2.6 Andean biotic index (ABI)

The ABI method was calculated to assess the water quality and is based on the adaptations of the BMWP score for Andean areas with altitudes major than 2000 meters above sea level, being thus, widely used in Peruvian, Ecuadorian, and Colombian streams [13]. Its adaptation provides tolerance values for families of macroinvertebrates present in lotic environments whose tolerance values are from 1 (more tolerant) to 10 (more sensitive), and the total sum is the ABI score Eq. (2). The ABI classification is shown in Table 1.

$\mathrm{ABI}=\mathrm{Tl}+\mathrm{T} 2+\mathrm{T} 3 \ldots+\mathrm{Tn}$        (2)

where, T=tolerance level of each macroinvertebrate found, and the number corresponds to the family.

2.7 Ephemeroptera, Plecoptera, and Trichoptera (EPT) index

The EPT index is a measure of the percentage of Ephemeroptera (mayflies), Plecoptera (stoneflies), and Trichoptera (caddisflies) which estimates water quality by the relative abundance of these three orders of stream insects that have low tolerance to water pollution [23]. It is one of the methods more useful and effective of the macroinvertebrate indices, especially in lotic ecosystems [24]. This method has been successfully used in other studies that evaluated the river water quality [25-27]. The EPT value represents the sum of the taxa richness of these three orders and is found by dividing the number of EPT by the total number of individuals Eq. (3) at the sampling point and is classified according to Table 2.

ETP $=\left(\text { number of } \frac{\text { EPT }}{\text { number }} \text { of individuals }\right)^* 100$       (3)

where, the number of EPT refers to the total of individuals of orders Ephemeroptera, plecopteran, and trichopteran, and the number of individuals represents the total registered of other types of orders or families in total.

Table 2. ETP index classification of water quality according to Carrera and Fierro [28]

ETP Value (%)	Quality/Interpretation	Color
75-100%	Very Good	tab1_1.png
50-74%	Good	tab1_2.png
25-49%	Regular	tab1_3.png
0-24%	Bad	tab1_5.png

4.1 Physicochemical parameters

The physicochemical parameters showed relatively similar values (except the temperature and conductivity) for most sampling points and seasons.

In the temperature, both seasons did not exceed the MPL, indicating that the river complies with these specifications. A slight difference in mean temperature among sampling points and seasons (14.8±0.3℃ wet season and 16.4±0.3℃ dry season) was observed. Similar findings were reported by Malakane et al. [30] measured the temperature at seven sampling points from the Blyde River (South Africa). found a variation of 22.3℃ to 24.90℃. Likewise, Izatti and Retnaningdyah [31] in the Genjong River (Indonesia) found a temperature ranging from 16.7℃ to 23.7℃ at four different stations. Bonacina et al. [32] indicated that temperature is an abiotic factor that affects the structure and functioning of aquatic ecosystems and biological communities. Moreover, temperature influences the solubility of gases, pollutants, pH, electrical conductivity, density, and toxicity of chemicals. Higher temperatures promote Thus, an increase or decrease in temperature based on the season, climatic conditions, or changes in river flow could alter the number of biological communities. Likewise, changes in river flow of water [33]. In this work, the lowest temperature found may be related to a high altitude (colder air temperature) of the study area and to a decrease in the flow of water during the dry season.

The conductivity presented lower values in the wet season (218±14µS cm^-1) compared to the dry season (1100±66µS cm^-1). An increase or decrease in conductivity indicates pollution [34]. When fresh water is lost by evaporation the water level decreases, then the ions present become concentrated, and as a consequence, the conductivity increases [35]. This description may explain because a high conductivity level was found at all sampling points during the season. Besides, rivers less disturbed frequently present low conductivity, silt load, and turbidity [30]. The Vilcanota River showed moderate temperature, but higher levels of conductivity (202 to 1200µS cm^-1) compared to studies reported in the Sardinas River (Choco Andino, Ecuador) (4.4 to 22.0µS cm^-1) [36], and the Genjong River (Indonesia) (83.4 to 109µS cm^-1), but lower compared to Blyde River (2700 to 3660µS cm^-1) [30].

The pH level of the water in wetlands, lakes, and rivers is an environmental factor key that limits species distribution and life in aquatic habitats because affects most biological and chemical processes [37]. The EPA recommends a range of pH optima between 6.5 to 8 for the development of aquatic organisms. Thus, as our pH measured in both seasons ranged from 7.4 to 7.6, this may be categorized as normal to slightly basic, being suitable for the life of aquatic organisms [38].

All aquatic life depends on the availability of dissolved oxygen (free oxygen present in water) [39]. In the wet (5.4±0.2) and dry (5.1±0.1) seasons the values of O.D. were higher as recommended by the MPL (≥ 4mg L^-1), which indicates that this water can contain life. Fonseca and Salvador [40] reported a reduction of O.D. concentration in the water when the temperature was increased during the rainy season. In our study, the dry season presented higher temperatures (16.1℃ to 16.6℃) and a minor concentration of D.O (4.9 to 5.2mg L^-1) compared to the rainy season. Therefore, it was expected that in locations where the water temperature is higher, lower concentrations of D.O. would be found.

4.2 Macroinvertebrates

The Vilcanota River is one of the main rivers of the Cusco Region, which reaches the confluence of the Yanatile River, its section is impacted by various problems. Thus, it is necessary to carry out several actions of surveillance, monitoring, and control of the quality of water resources, to prevent, mitigate, and control impacts.

The number of macroinvertebrates found in the study area differed between seasons, being that the wet season (882 specimens) presented a major number of specimens compared to the dry season (749 specimens). Similar found were reported by Machado et al. [36] and Pascual et al. [16] reported significant changes in the composition of the macroinvertebrate community, with a greater number of specimens in the dry season than in the wet season, which coincides with our finding in the present study.

Based on the monitoring of macroinvertebrates, the family Insecta was the more representative in all sampling points and seasons. It was found that during the rainy season, Ceratopogonidae was dominant on P1 and P3, while hydropsychidae was dominant on P2 and P4 sampling points. Many species of Ceratopogonidae live in aquatic habitats such as ponds, freshwater marshes, swamps, lakes, and streams. During the rainy season are formed streams that favor the presence of this species, since during the dry period the presence of this species was not observed. Similarly, the hydropsychidae live in a wide range of lotic habitats from small streams to large rivers, lakes, and permanent or temporary ponds.

In the dry season, Chironomidae was dominant in all sampling points. The family Chironomidae is tolerant to water with low dissolved oxygen and a high concentration of conductivity [41, 42]. Our results reported lower levels of DO and higher values of conductivity, which would explain the presence of this family during the dry season. Likewise, several authors have associated its presence with a reduction in water quality and degradation of aquatic ecosystems [43, 44].

In rivers, lakes, creeks, and other freshwater habitats, insects are the most abundant and often exhibit a high diversity of macroinvertebrates in the benthic community [45]. Aquatic insects are derived from a variety of terrestrial ancestors and play a significant role in freshwater ecosystems because serve as food for other species and are used as water-quality condition indicators of both lotic and lentic systems [46]. Similar findings were reported by other authors where the major class was Insecta [30, 36, 47]. Thus, our findings are understudies previously published.

In function to the order, the macroinvertebrates presented the following sequence: Diptera ˃ Trichoptera ˃ Basommatophora (class Gastropod) ˃ Odonata, ˃ Hirudines (class Clitellata) ˃ Amphipoda (class Malacostraca) ˃ Haplotaxida (class Clitellata) ˃ Ephemeroptera ˃ Coleoptera ˃ Oligochaeta (class Clitellata) ˃ Hemiptera.

The Diptera order was represented by the family Ceratopogonidae (276 individual specimens-biting midges) during the wet season and the family Chironomidae (196 individual specimens) represented the dry season. The biting midges are considered an important vector of human and veterinary pathogens and their population is increasing due to that rainfall leads to more hatching of mosquito eggs [48], the reason why in the rainy season only this family was found.

The order Trichoptera (Caddisflies) are aquatic insects, and most species are moderately tolerant to water pollution. A greater number of families of Trichoptera were found in the wet season compared to the dry season. These macroinvertebrates often tend to adapt to changes that allow survive in water with low dissolved oxygen or turbid waters indicating that only these organisms can survive in this stream.

The order Basommatophora is abundant, widespread, and known to be very tolerant of pollution, especially in water polluted with organic pollution [49]. In this case, this family was widely distributed in both seasons, which indicates that the water of the Vilcanota River is polluted. Rivers, lakes, or ponds with good water quality usually showed a high number of Odonata species [50]. Here, the wet season showed 89 specimens, while the dry season 32 specimens. Thus, this finding suggested that the water quality from the Vilcanota River is not good. The order Ephemeroptera (mayflies) is considered a good indicator of water quality, although some specimens can tolerate a certain degree of pollution [51]. Here, only 52 specimens (38 and 15 specimens in wet and dry seasons, respectively) were found, which may indicate bad or regular water quality. The order Plecoptera lives mainly in temperate and cold areas and well-oxygenated water running, being used as indicators of high water quality due to that these are extremely sensitive to water pollution [52]. In this study no family of the order Plecoptera was found, indicating pollution in the Vilcanota River water.

It is important to note that in the habitats, some taxa may not be present because of a migration pattern, seasonal variation, or unstable habitats.

4.3 Water quality

The water quality evaluated in each sampling point with the indexes BMWP and ABI showed that the waters of the Vilcanota River were classified as questionable (moderately polluted) for all sampling points during the wet season and P1 during the dry season, while critical (heavily polluted) for P2, P3, and P4 of the dry season. Like all sampling points were located around urban sites, the water quality of Vilcanota River assessed through macroinvertebrates is responded to and discriminated by the sites, where multiple stressors occur. In our case, the sites studied have influence such as increasing urbanization, agriculture, cattle farming, and presence of municipal dumps, and the discharge of both solid and liquid pollutants on the Vilcanota River stream. Similar findings were reported by Castillejo et al. [53] in Ecuadorian Andean rivers.

Jerbes-Cobo et al. [54] indicated that ABI is primarily associated with variables such as DO, nitrite, and total solids during the rainy season. In this study, DO presents major values during the rainy season, indicating similar results and behavior. Likewise, high ABI values between 18 and 38 points indicate that these are influenced by the seasonality of precipitation and the flow of the body of water.

Besides, there is observed a decrease in water quality in the dry season compared to the wet season.

Similar findings were reported by Jacobsen and Encalada [55] who assessed eight bodies of water around the city of Ecuador and found high pollution during the dry season. Besides, the Alambi River basin (Ecuador) showed a decrease in the water quality index during the dry season [56]. Likewise, Pascual et al. [16] evaluated the water quality from the Rimac River and found a greater number of points polluted in the dry season than in the wet season.

These results of the ABI and BMWP index coincide with Coayla-Peñaloza [57] mention that its results were of regular generation in water quality and depends on different types of ecosystems of the place and the altitude, for which, the sandy bottoms are of great diversity, but the stony bottoms are of the great variety of supplies, in addition to the climatic conditions. They found a greater abundance of anthropic generations despite the wet season, their results showed greater sensitivity where they showed the taxonomic groups are some resistant to pollution.

The water quality of rivers is not static. Thus, river water quality changes constantly in response to environmental and human pressures, which display a dynamic variation over time [54].

As far as uniformity is concerned, all the points recorded in the Vilcanota River are given high values and polluted. Concerning the study carried out by Nuñez and Fragoso-Castilla [58] in the Ciénaga Mata de Palma River where the stations registered high values, which indicates that the population of aquatic macroinvertebrates is distributed homogeneously on the surface and bottom of the swamp. Espinosa et al. [59] stated that the distribution of aquatic macroinvertebrates in lentic ecosystems is mainly affected by the presence of floating vegetation, which constitutes a substrate and refuge for a great diversity of benthic communities.

Bad quality of the waters of the Vilcanota River then may be associated with the indiscriminate dumping of garbage and discharges of domestic and industrial wastewater as reported by the National Water Authority (ANA) and the Regional Government of Cusco [60]. Likewise, the ANA and local authorities of Cusco carried out actions to monitor and evaluate the quality of water and thus identified the main factors that affect the quality of the main river and its tributaries, as well as the main pressures that influence the watercourses that make it up. For this, a diagnosis study “Environmental Quality Index of Surface Water Resources (ICARHS)” was developed between 2012 to 2021, considering 143-point samplings through the Urubamba hydrographic units, where were measured physicochemical parameters (D.O., DQO, DBO₅,), trace elements (Pb, Mn, Zn, Fe, Al), and microbiological parameters (thermotolerant coliforms), revealing that 43 (30%) sampling points were classified as lousy, 54 (38%) as bad, 38 (27%) were qualified as regular and only 8 (6%) as good. According to our findings, the Vilcanota River had an important influence and pressure on anthropogenic activities along its course. Baytaşoğlu and Gözler [61] after the application of BMWP and ETP index concluded that urbanization, tourism, agricultural activities, and destruction of the river are the main responsible for bad water quality from Coruh River Basin (Turkey). In this study, we can say that all the reasons described above may have an effective role in the streams of the Vilcanota River.

The EPT index defines signs of bad quality overall in the dry season and regularly during the wet season, which are practically related to the small sample of specimens of EPT of families collected, and also to that was no found the order Plecoptera, which may explain the generation of pollution in the place by the discharged waters and generations of waste by the same community.