Biodiversity Specificity of the Mlava and Krupaja Springs - Proposal for Conservation Measures


Tijana Veličković1, Aleksandar Antonijević1, Ana Petrović1, Nataša Radojković1, Aleksandra Milošković1, Milena Radenković1, Vladica Simić1



1 University of Kragujevac, Faculty of Science, Radoja Domanovića 12, 34 000 Kragujevac, Serbia; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it



The springs of the Mlava and Krupaja Rivers are natural monuments representative of limestone springs from the aspects of biodiversity, level of importance and vulnerable habitats. Qualitative and quantitative analysis of the aquatic macroinvertebrate and fish communities are presented, as well as indicators of ecological status of the Mlava and Krupaja springs and the presence of endangered taxa in the hydrological systems of both springs. The aim of this work is to further research the specific biodiversity of the Mlava and Krupaja springs by analyzing their aquatic macroinvertebrate and fish communities and based on the obtained results make a proposal for conservation measures.

Keywords: aquatic macroinvertebrates, fishes, Mlava Spring, Krupaja Spring, diversity, conservation.



River springs make up boundary biotopes which are areas between groundwater biotopes and streams of biotopes, which is why it would be correct to say that the springs represent some of the most important habitats because of the living communities that inhabit them. Springs and especially their biological diversity have been poorly researched in Serbia (Markovic and Duli, 1998). The Homoljska Basin in Southeast Serbia is abundant with limestone springs, such as the springs of the Mlava and Krupaja Rivers. These springs are protected areas on the territory of Serbia and they are characterized as natural monuments (Kojadinović, 2013a; Kojadinović, 2013b).

The Mlava River Spring natural monument is located in Eastern Serbia, on the southern edge of the Žagubica Valley, in the suburbs of Beljanica Mountain (Kojadinović, 2013a). From a hydrological point of view, the Mlava River Spring is a typical karst limestone spring. The water from the spring flows into the Tisnica River. Approximately 140 m from the boil a dam was constructed, which diverts the largest part of the spring water flow towards a closed canal, which channels the water towards a large trout pond in Žagubica. After the pond flow finally makes its way to the upper flow of the Mlava River. On the left side of the spring, there is a motel, which is a site of uncontrolled restocking of rainbow trout into the spring (Kojadinović, 2013a).

The Krupaja Spring was naturally more likely to have the characteristic features of a spiked spring, however, by creating an artificial stone dam some 30 m from the cave of the spring, a lake was formed with an average depth of 1.7 m. Thus, the appearance and characteristics of a limestone spring were obtained. Further, due to the decrease in inclination of the trough, a large source was formed from which the water slowly flows into the Krupaja River. The springs have an average depth of 0.7 m.

Below the spring, there are residential facilities with a privately owned mill and privately owned land within the protected area. In addition to usage of a significant share of the spring water supply, water is also used to supply the rainbow trout fish pond located below the spring and for operating the mill and generator for the production of electricity (Kojadinović, 2013b).

The aim of this work is to further research the specific biodiversity of the Mlava and Krupaja springs by analyzing aquatic macroinvertebrate and fish communities in these springs and based on the results make a proposal for conservation measures.


Materials and Methods

The research began in September 2014 and it was carried out in 2015, 2016 and 2017, during the same period of the year. It included qualitative and quantitative analysis of aquatic macroinvertebrate groups. Fish fauna was also analyzed. The research of the aquatic ecosystems of the Mlava and the Krupaja springs had been conducted using standard hydrobiological methods. Samplings of aquatic macroinvertebrates were completed on 5 representative sampling sites on both springs using the Ekman Grab (surface area 225 cm2) or benthological hand net (25x25 cm, 500 μm mesh size). Aquatic macroinvertebrates were sampled from the coastal part of the springs and along the entire watercourse. In the Mlava Spring the deepest sampling point was 10 m. Conservation of samples was performed using 80% ethanol solution and they were transported to the laboratory for determination.

Identification of aquatic macroinvertebrates was performed using identification keys (Mann and Watson, 1964; Brinkhurst and Jamieson, 1971; Elliott et al. 1988; Wallace et al. 1990; Maccan and Douglas Cooper, 1994; Pescador et al. 1995; Nilsson, 1997). The collected biological material was used in assessing the ecological status of the Mlava and Krupaja springs, using the biological parameters within the Asterix ver 4.0 software package (Perlodes, 2013) with special emphasis on the saprobic index (Zelinka & Marvan method), the BMWP (Biological Monitoring Working Party) score, the diversity index (Shannon-Weaver method), number of taxa and the percentage share of Oligochaeta (Tubificidae) in the aquatic macroinvertebrates community.

The presence of globally endangered species was determined in accordance with the IUCN Red List of Endangered Species (IUCN, 2014). The presence of species endangered at the national level was established according to the Ordinance to Designate and Protect the Strictly Protected and Protected Species of Wild Plants, Animals and Fungi of the Republic of Serbia (Anonymus, 2010).

During field work on the Mlava and Krupaja springs, fish fauna were sampled with the standardized electro-fishing method using an electrofisher "Aquatech" IG1300 (2.6 kW, 80 – 470 V). Fish were sampled on five sites chosen based upon the best possible coverage of all parts of both springs. Identification of sampled fish was carried out directly in the field, immediately after catch. After the analysis all fishes were returned to the water.


Results and Discussion

The results of the structure of the aquatic macroinvertebrates groups, from all the sampling periods are shown in Table 1.




The quantitative structure of the aquatic macroinvertebrates of the Mlava and the Krupaja springs are characterized by the dominant presence of an amphipod crustacean from the family Gammaridae, species from genus Gammarus. The population of this genus is numerous both in the very springs and in the watercourses of the rivers. In addition to the amphipod crustacean, on the spring of the Mlava River a large population of beetle larvae (Coleoptera) was observed, primarily the cold stenotherm species E. angustatus, including a Gastropoda representative A. floviatilis, a Trichoptera B. minuteus species, as well as Turbellaria species D. lacteum and D. lugubris. The large number of tolerant species of Oligochaeta from the Tubificidae family have been noted in the Krupaja Spring, which indicates the presence of organic pollution that mostly comes from the rainbow trout fish pond which is located next to the spring.

By analyzing the qualitative-quantitative structure of the macroinvertebrates community at the sampling sites in the spring of Mlava River, the largest number of taxa were recorded in the group Mollusca (A. fluviatilis, R. pereger, Pisidium sp., Bythinella sp. and Lymnaea sp.). The species of amphipod crustacean from the family Gammaridae, G. balcanicus stands out as the dominant species in the Mlava Spring. The population of this species is numerous in all observed sites of the Mlava Spring. Thereafter, the species from the group Diptera (fam. Chironomidae) M. gr. praecox as well as from the Mollusca group: Pisidium sp. and A. fluviatilis.

The research carried out in 2016 confirmed both the presence of species from the Trichoptera group, B. minutus species, as well as the Turbellaria group D. lugubris, D. lacteum and D. gonochephalus, found in the Mlava River and in the Krupaja Spring which were recorded in 2014 and 2015. The presence of species from the group Oligochaeta (fam. Tubicidae) was recorded in both springs during this period indicating a moderate presence of organic pollution.

For the purpose of more precise determination of the ecological status of the waters of the Mlava and Krupaja springs, an analysis of the macroinvertebrates community was performed using the "Asterix" software program. The package contains numerous methods that determine water quality, elements of the structure of the community and ecological status of water from which only the most crucial ones are shown in this paper. The results of parameters of particular importance for evaluation of water quality are shown in Table 2 with the emphasis of the most important ones for each year and with reflection on their main changes through the years. Based on the analysis performed in 2015, it was determined that the ecological status of the Mlava Spring is somewhat better compared to the Krupaja Spring. The worst impact of organic loads was observed immediately downstream from the rainbow trout fish pond. Pollution loads of the Krupaja was observed, including floating fish food residues and fish feces from the rainbow trout fish pond, as well as the remains of fish which are disposed off into the river from the restaurant located next to the spring. The effects are reflected in the reduction of species diversity and a greater presence of tolerant species from the family Tubificidae.




The most important parameters for assessing the water quality of the Mlava Spring and the Krupaja Spring were given the following results. The saprobic index in 2015, according to the Zelinka & Marvan method, both in the Mlava and Krupaja corresponds to the III class of water quality (α-mesosaprobic water). During 2017, the saprobic index according to Zelinka & Marvan shows that water of the Mlava Spring belongs to II class, and for the Krupaja Spring corresponds to the III class of water quality.

Based on the BMWP score, the water quality in both springs belongs to the II class of water (β-mesosaprobic water class). The index of diversity (the Shannon-Weaver method) in the Mlava and Krupaja springs, corresponds to the II class of water or β-mesosaprobic water class.

In addition to the analysis of ecological status, the presence of rare and endangered species of aquatic macroinvertebrates in the hydrological systems of the springs were analyzed, and the results are shown in Table 3.




In this protected area, globally threatened species of aquatic macroinvertebrates species, according to the categorization of the International Union for Conservation of Nature (IUCN, 2014) and the Berne Convention (Council of Europe, 1979) are not present. At the national level, the presence of two endangered species from the Trichoptera group is constant: B. minutus as a threatened and strictly protected species and M. testacea as a vulnerable and protected species. The presence of a B. minutus population and the fact that there is a population of this nationally endangered species in the Mlava Spring, is of greatest importance from a conservation aspect. In addition to the presence of endangered species in the Krupaja Spring, the presence of Oligochaeta of the genus Trichodrilus (Trichodrilus sp.) was recorded. It is known that the species of Oligochaeta of the genus Trichodrilus are mainly endemic. A more precise determination in the later period of this species will be of conservation importance for the Krupaja Spring Reservoir. Research carried out in 2016 confirmed the presence of these species in the Mlava and Krupaja springs.

All the above mentioned implies that these are sensitive ecosystems which are inhabited by a large number of highly specialized species for such habitat conditions.

Also, for further conservation of habitats of both springs, the study of aquatic macroinvertebrates of deep parts of the springs is of extraordinary significance. At this time due to technical reasons, this research was not possible, because it is necessary to engage professional divers.

On the basis of detailed studies of the systematic characteristics of the entire fish fauna inhabiting the Mlava and Krupaja springs, it has been established that these biotopes are being inhabited by eight different species of fish from five families. Research on the diversity of the Mlava Spring revealed only two species, Cottus gobio Linnaeus, 1758 from the Cottidae family and Oncorynchus mykiss (Walbaum, 1792) from the Salmonidae family (Table 4).




Three species belonging to the Cyprinidae family were recorded in the Krupaja Spring: Barbus balcanicus Kotlik (Tsigenopulos, Rab & Berrebi, 2002), Leuciscus cephalus (Linnaeus, 1758) and Phoxinus phoxinus (Linnaeus, 1758). There are also, Salmo trutta Linnaeus, 1758 and O. mykiss from Salmonidae family. Also, C. gobio from the Cottidae family and Barbatula barbatula (Linnaeus, 1758) belonging to the Nemacheilidae family. While the only record of Eudontomyzon mariae (Berg, 1931) in the Krupaja Spring was observed in 2017 (Table 5).




The most dominant fish species, both in number and biomass, observed throughout the entire sampling period of four years in the Mlava Spring, is O. mykiss, which is an allochthonous species for the ecosystems of Serbia. This site has a huge anthropogenic influence mainly caused by the needs of restaurants on the shore of the Mlava Spring. The ecological conditions of this habitat correspond to the requirements of salmonids, however, the input of the allochthonous rainbow trout has no ecological justification. The autochthonous species present in this habitat is C. gobio, which has a negligible share in the total biomass of the sample.

During 2014 and 2015 the presence of allochthonous fish species was not recorded in the Krupaja Spring. P. phoxinus, a small sized fish and inhabitant of the upper streams of the river, exhibits the highest numbers. Leuciscus cephalus also has a dominant biomass. The brown trout (S. trutta), which is a representative of the salmonids in the ichthyofauna of the Krupa Spring, also has a significant share in the total biomass.

During 2016, a certain change in the structure of the fish fauna of the Krupaja Spring and its surroundings is observed. Firstly, changes in the qualitative and quantitative composition of the fish fund are observed. During 2016 and 2017 a significant share of cyprinids in relation to salmonids has increased. Most notable is the dominance of biomass of L. cephalus. The presence of rainbow trout, which originates from the fish pond, was also recorded, in addition to a smaller proportion of brown trout.

As a conservation measure, biological monitoring is proposed which should be comprehensive, with separate key indicator species. In addition to biological monitoring, hydrological, physical, chemical and microbiological monitoring, which has already been established, should be continued in the coming period.

Catchment and removal of allochthonous rainbow trout from the Mlava Spring should also be considered the highest priority. The catch can (with the special permission of the Ministry) be done by boom and net tools or electro-bribes. After the catch of rainbow trout, the spring should be inhabited by a genetically pure population of brown trout, preferably from the Mlava River itself. The quantities and age classes of the brown trout that would inhabit the Mlava Spring may be given after more detailed research. Research in 2017, indicates the current problem of bringing an allochthonous fish species such as rainbow trout into the Mlava Spring. This problem should be solved in a short period of time. To begin with, the entry of new quantities of rainbow trout in the Mlava Spring must be forbidden, and the removal of the previously introduced individuals should be performed. Contrary to this, research in 2017 suggests that new quantities of rainbow trout have been introduced into the stream (it is observed that fish clearly differ in size). Also, imported rainbow trout is fed with pelleted food and food waste from the restaurant, which increases the amount of organic matter.

In order to preserve brown trout populations in the springs, but also to popularize the importance of this autochthonous fish species in general, future plans are being made to cultivate this species in reprocenters in the following period. In addition to the above priorities for the conservation of the biological natural values of protected areas, and especially vulnerable aquatic ecosystems, it is also necessary to eliminate the smallest possible source of water pollution.

Also, it is necessary to perform the control and correctness of the drainage pipes and septic tanks of the sewage system on the Mlava and Krupa springs.

In order to promote the natural values of protected areas, it is of particular importance that the results of current and future biological research are presented to the public in a popular way, primarily in the area of protected areas.



This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Project No. 173025).



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