Using bacteria and benthic macroinvertebrates as water quality parameters in Mat River, Albania

d

Density of each family

D

Total amount of densities

EC

Electrical conductivity

EC1-EC5

Electrical conductivity in each sampling site

EPT

Ephemeroptera-Plecoptera-Trichoptera Index

F

Faecal coliforms

F1–F5

Faecal coliforms in each sampling site

FBI

Family biotic index

HT

Heterotrophic bacteria

HT1–HT5

Heterotrophs in each sampling site

MPN

Most probable number

SS

Sampling site

St. Dev

Standard deviation

TC

Total coliforms

TC1–TC5

Total coliforms in each sampling site

Temp

Temperature

Temp1–Temp5

Temperature in each sampling site

TV

Tolerance value

Water gives life to our planet and water resources are used for various purposes. The human population growth accompanied by the increased growth of agricultural and industrial activities has influenced indirectly or directly the quality of water resources. Pollution of surface and underground water is a concern because they are the sources of drinking water and the habitat of many organisms. Water pollution sources are of natural or anthropogenic origin. The main cause of surface water pollution is the discharge of urban, agricultural, and industrial wastes, without any kind of preliminary treatment (Kirschner et al. 2009). In Albania, the pollution of surface waters is mainly attributed to urban discharges, which contain organic matter, soluble phosphorus, and nitrogen substances, that promote the eutrophication process, pathogenic bacteria and viruses, heavy metals, etc. (Hamzaraj et al. 2014; Selamaj et al. 2016). Detection of pollution sources is essential for proper watershed management to maintain water traits according to quality goals.

Water quality is a network of physical, chemical, and biological variables that affect each other. The assessment of surface water quality through continuous monitoring is very important for maintaining ecosystem health (Páll et al. 2013). This can be done through the evaluation of a variety of water quality parameters, but in our study, we are focused on microbial indicators of faecal pollution and benthic macroinvertebrates. Microbial indicators such as faecal coliforms, total coliforms (TC), and faecal streptococci are normally used in determining bacterial contamination in water. Total coliforms include bacteria whose natural habitat is most often the digestive tract of warm-blooded animals. Since their habitat specificity is weak, some coliforms are present in the environment without any source of faecal contamination. Faecal coliform bacteria, a subset of TC, are natural inhabitants of the digestive tract of humans and warm-blooded animals; they are therefore good indicators of the contamination of the natural environment by faeces. As faecal coliforms are generally found in the same environment with many pathogenic microorganisms, they are suitable indicators for the incidence of disease-causing microbes (Bensig et al. 2014). Microbiological contamination with faecal bacteria due to anthropogenic activity is crucial throughout the rivers. The concentration of HT bacteria is another parameter indirectly related to water quality. HT bacteria are a diverse group of bacteria that obtain their energy by consuming organic matter. They are commonly found in various aquatic environments, including rivers, lakes, and groundwater (Allena et al. 2004). The presence and diversity of HT bacteria can indicate the overall microbial health of water systems. HT bacteria respond to changes in nutrient levels, particularly organic matter, and nitrogen compounds. High concentrations of organic pollutants, such as sewage or agricultural runoff, can lead to an increase in HT bacteria populations. Monitoring changes in bacterial abundance can provide insights into nutrient pollution levels and potential risks to water quality (Khattab & Merkel 2012).

Benthic macroinvertebrates are very important for the proper functioning of river ecosystems. They constitute essential elements in river monitoring processes (Balderas et al. 2016). Among the benthic macroinvertebrates, insects are of particular importance as their relative abundance indicates water quality and the health of the river ecosystem. The impact of abiotic factors and pollution is rapidly reflected in the composition of the benthic macroinvertebrate community (Tampo et al. 2021).

The aim of this study was to provide information on the water quality of the Mat River through the evaluation of some biological parameters. The data collected will help policymakers formulate management strategies for controlling and reducing water pollution.

The sampling and the tests were performed following APHA (1998) International Standard Methods. Water samples were collected using sterilized glass bottles, at a depth of 30–50 cm from the water surface and 30–100 cm from the riverbank. The water samplers were placed in cool boxes and analysed within 24 h. The water temperature, pH, and conductivity were measured in situ by using a portable multi-parameter instrument (WTW multiline IDS 3630). The number of coliforms was determined according to the Most Probable Number method based on the preliminary test (Lactose Broth, incubation at 35 ± 0.5 °C for 48 h) and the confirmatory test (Brilliant Green Medium, incubation at 35 ± 0.5 °C for 24–48 h, and EC Broth, incubation at 44.5 °C for 24–48 h). The MPN index was calculated by using the MPN statistical tables and is expressed as the number of colony-forming units per 100 mL (CFU/100 mL). The number of HT bacteria was determined by counting colonies on plates with Plate Count Agar, cultivated with 0.1 mL sample after three series dilutions, and incubated at 22 °C in a biological thermostat for 72 h. The concentration of HT bacteria is expressed as colony-forming units per 1 mL (CFU/mL). Classification of faecal and organic pollution of rivers according to Kavka et al. (2006) is used for the assessment of pollution level in Mat River (Table 2).

Benthic macroinvertebrates were collected according to Campaioli et al. (1994) and Lenat (1988), with a benthic net of the kick net type with 0.5 mm holes. The benthic net was positioned vertically in the river water, opposite the flow direction and at a depth of 45–60 cm. The riverbed was excavated by foot at about 1 m from the net. Three samples were collected for each sampling site. For each sample, the material from the net was cleaned in a tray and after the large objects were removed, the material was transferred to a 300 mL bottle. Then, 70% alcohol was added to each bottle. The specimen selected from the field sampling was determined in the laboratory based on the determination criteria of Campaioli et al. (1994), Bode et al. (1996), and Edington & Hildrew (2005).

Density, TV, EPT, and EPT-Biotic index (Tables 3 and 4) are calculated for every sample (Schmiedt et al. 1998). The EPT-Biotic index value is calculated based on the number of families of three major orders of invertebrates E – Ephemeroptera, P – Plecoptera, and T – Trichoptera, which are sensitive to water pollution and any other nonpoint pollution (Table 3).

The data of bacterial and physical–chemical parameters were statistically analysed by descriptive statistics to investigate the level and the variation of the data. Normality distribution was tested by Kolmogorov–Smirnov test, confirmed at p > 0.05, and the probability plot of lognormal distribution was confirmed at the same p level. Cluster analysis of the whole dataset was used to evaluate the similarity between different parameters, based on Euclidean distance (Michalik 2008). The statistical data analysis was performed using MINITAB 21 software package.

The microbiological parameters in Mat River water showed important spatial and temporal fluctuations. High concentrations of faecal coliforms were recorded in sampling sites 4 and 5 from June to October 2018 (1,600–5,200 CFU/100 mL), while low values were recorded in sampling sites 1 and 2 during all monitoring periods (30–520 CFU/100 mL) (Table 5). These were expected results according to the geographical position of sampling sites. The first sampling site was chosen in the uppermost part of the river course, further from the inhabited areas and with no anthropogenic impact. Referring to Directive 2006/7/EEC of the European Union on water quality and to the classification system by Kavka et al. (2006), and based on our study, we can say that at this point the water of the Mat River is of ‘very good’ quality throughout the year. Going down the river course (SS4 and SS5) the level of faecal coliforms in water increases greatly not only due to a cumulative effect but more importantly, due to more inhabited areas in the river catchment. As far as temporal fluctuations are concerned, we can say that in spring and winter, the water quality of the river fluctuated in classes I and II (according to the classification system by Kavka et al. (2006), Table 2), which means between little and moderate faecal pollution. Summer and autumn constitute that period of the year when, because of high temperatures (in summer) and increased water flows due to rainfall (in autumn), the concentration of faecal coliforms in water also increases. This is accompanied by a deterioration of the river water quality in these seasons.

In addition to the level of faecal coliforms in water, the level of HT bacteria was also determined, which are defined as microorganisms that require organic carbon for growth (Table 5). The highest concentration of HT bacteria at each sampling site was found in July 2018. The climate in the Mat Valley is approximately the average of the climate of the whole of Albania. It is related to the Mediterranean climate zone, which is characterized by hot and dry summers and relatively mild winters. High temperatures in July could be one reason for such a high concentration of HT bacteria. But, the high availability of organic substrates during the hot season due to increased biological activity in water overall, runoff, or decomposition processes may also affect the concentration of HT bacteria.

Lower concentrations were found in sampling site SS1. Small fluctuations of HT were observed in SS2, 3, 4, and 5. The concentration of HT bacteria usually corresponds to contamination by organic matter (Kohl 1975). Thus, referring to the classification of the level of organic pollution in river waters according to Kavka et al. (2006) we can say that the average values of the concentration of HT bacteria in the five sampling sites generally show an average organic pollution (class II). However, it should be noted that approximately half of the analysed samples indicated the presence in water of a small amount of easily degradable organic matter (class I).

The results of the statistical analysis of physical–chemical parameters performed using descriptive statistics are shown in Table 6. Temperature and conductivity are stable and show low variance (CV < 25%). Relatively small differences between median and mean values resulted in all monitoring sampling sites indicating that these parameters are less influenced during sampling periods. Kolmogorov–Smirnov test confirmed at p > 0.05 was used for testing normal distribution, and a probability plot of lognormal distribution confirmed at the same p level was also used.

The electrical conductivity of the water samples showed small fluctuation and was in the range of natural freshwater (0.5–1,500 μS/cm) (Rodier et al. 2009). The EC values ranged from 247 to 318 μs/cm, with a maximal variability of 7% that indicates a stable EC at all river water catchments (Figure 6). EC of water depends on the content and concentration of dissolved ionized salts in the water system and is a useful indicator of water salinity or total salt content (Rusydi 2018). The lowest conductivity was observed at sampling site SS1, while the highest one was at SS5. It depends on water temperature and discharge conditions in these sites. SS1 is positioned in the vicinity of spring water, in an area of relatively high elevation, low water temperature, and far from urban areas, while SS5 is positioned in the western lowland with higher temperature and in the vicinity of urban areas.

Unlike physicochemical parameters, which provide a snapshot of the state of a water body, benthic macroinvertebrates provide the overall integrative measure of the health of a stream and can sometimes adequately identify impaired waters (USEPA 2005; Bonada et al. 2006; Kenney et al. 2009). During the monitoring period, 964 individuals, belonging to 5 orders and 15 families of the class Insecta, were determined for the 5 sampling sites along the Mat River (Table 7).

The order with the largest number of individuals is Ephemeroptera with 577 individuals. The family with the largest number of individuals is the family Heptageniidae with 271 individuals. The family found in all sampling sites is the family Baetidae of the order Ephemeroptera with 248 individuals, while the family with the largest number of individuals Heptageniidae is absent only for the fifth sampling site of the river. The family Potamonthidae of this order is found only in the first, second, and third river sampling sites.

Representatives of the Plecoptera order (43 individuals in total) again dominate the first sampling site. In the other sampling sites, they have a very small number of individuals or are completely absent. The family of this order with the largest number of individuals is the family Perlodidae with 32 individuals (Table 7).

Even the representatives of the Trichoptera order (95 individuals in total) have a narrow distribution, concentrating on the upper course of the river, except for the Hydropsychidae family which was found in five sampling sites. The other two families Rhyacophilidae and Philopotamidae are present only in the first and second sampling sites (Table 7).

Representatives of the Diptera order (229 individuals in total) are found in all the sampling sites, and the Chironomidae family is the most represented family of this order with 150 individuals, while the Simuliidae family has 30 individuals (Table 7).

For the Odonata order, we found only representatives of the Gomphidae family in the first, second and third sampling sites with a total of 14 individuals. It is absent in sampling sites four and five (Table 7). The sampling site with the largest number of individuals and families is the first one with 541 individuals and 14 families. SS5 has the smallest number of individuals and families, 88 individuals and 6 families. In this sampling site, the families with the largest number of species are those of the Diptera order.

During this study, 11 families of the EPT group were found in all sampling sites. To calculate the EPT Index (Bode et al. 1996) we determined for each sampling site the number of EPT families (E – Ephemeroptera, P – Plecoptera and T – Trichoptera), as families with a high sensitivity to pollution. For SS1, the EPT Index has a value of 11, SS2 has a value of 10, SS3 has a value of 6, SS4 has a value of 5, and SS5 has a value of 3.

According to the water quality classification (Fritz 1975; McGonigle 2000) presented in Table 3 and the EPT Index value, the water in SS1 is of very good quality, in sampling sites 2 and 3, the water is of good quality, and in sampling sites 4 and 5, the water is clean (Table 8).

FBI values were calculated for each sampling site based on the data presented in Table 7.

The FBI values presented in Table 9 show that SS1 is the sampling site with the best water quality. The water quality decreases descending towards the mouth of the river. SS2 and SS3 have good water quality with slight organic pollution, while the water in SS4 and SS5 has significant organic pollution.

In cluster 1 are grouped together all benthic macroinvertebrates, except the families Tabanidae, Chironomidae, Simuliidae, faecal coliforms, and temperature are grouped together in cluster 2. Benthic macroinvertebrates have different levels of tolerance for water pollution. Therefore, the presence or absence of more or less tolerant organisms is used to evaluate the level of pollution in a river. The resistance to pollution of tolerant benthic macroinvertebrates can be explained by their lifestyle and feeding strategy (Henriques-Oliveira et al. 2003). For example, chironomid larvae are opportunistic omnivores. Although they can feed on algae, detritus, and associated microorganisms, macrophytes, wood debris, and invertebrates, most species present a low degree of selectivity, feeding on what is available. According to many studies, the families Tabanidae and Simuliidae of the Diptera order include species tolerant to organic water pollution (Al-Shami et al. 2010). On the other hand, the presence of faecal coliforms in water indicates faecal contamination of water and a certain level of water pollution. This justifies the grouping into the same cluster of the above parameters, which is consistent with Tampo et al. (2021), according to which Diptera are significantly correlated with faecal coliforms and TC. Clusters 2 and 3 are linked together with high similarity (66%) between them, supporting the correlation of these variables with organic pollution. The concentrations of HT bacteria correlate commonly to organic pollution (Kavka et al. 2006). Our data of HT plate count show on average moderate organic pollution of the river, but with values that increase slightly from the upper reaches to the lower reaches of the river. While, based on the abundance of Tabanidae, Chironomidae, and Simuliidae from the Diptera order, the level of organic pollution goes from ‘Organic pollution unlikely’ in SS1 to ‘Substantial pollution likely’ in SS5. In cluster 4 are grouped together pH values of all sampling sites, which is probably derived from the dissolution of substrate minerals of the area mostly as limestone rich in Ca and Mg by leaching them in river water (Roche et al. 2019). The cluster analysis dendrogram shows a low similarity of 37% between clusters 1 and 4. This could be due to the importance of pH in the ecology of aquatic benthic macroinvertebrates as revealed by various studies (Tampo et al. 2021).

The water quality of the Mat River in Albania was evaluated by the combination of chemical–physical and microbiological parameters and benthic macroinvertebrates as biological indicators of stream water quality from the river spring to the end flows into the Adriatic Sea. Descriptive statistic data and spatial and temporal distribution graphs of microbiological and physical–chemical data are presented and analysed. The levels of the measured parameters and the respective relationships between them were examined from the data obtained from five sampling sites spread out along the entire length of the river.

Microbiological parameters revealed significant geographic and temporal variations, with higher faecal levels in the summer and autumn than in the winter and spring. As it was expected, the bacterial load is higher near urban areas, and the pollution increases with the course of the river. HT bacteria in all sampling sites generally showed average organic pollution (class II). The Family Biotic Index values of macroinvertebrates and microbiological indicators revealed that the river spring (SS1) had the best water quality. The water quality falls towards the mouth of the river and the downstream sampling sites. SS2 and SS3 have good water quality with slight organic pollution, while SS4 and SS5 show significant organic pollution. The cluster analysis based on our data reveals the possibility to use each bioindicator for the assessment of water quality. Having the possibility to use different water quality indicator parameters increases the possibility to perform more monitoring studies, according to human and laboratory capacities. We recommend regular monitoring of the Mat River because knowing the level of water pollution helps state authorities to take appropriate measures to have surface water quality in accordance with national and international standards.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.