During 2025, nine research projects are expected to be implemented as part of the Youth Ecologist Support Program “Dr. Ljupcho Melovski.” Below we present short summaries of the studies.
Ecological Approach to Extracting Bioactive Compounds from Hypericum perforatum Using Deep Eutectic Solvents
Lead researcher: Anastasija Hadžismileva
The concept of green chemistry is based on 12 principles established in 1998, of which the most important for chemical research are: the design of safe chemicals and the use of safe solvents. Since the choice of solvent is crucial for all chemical processes, and given the toxicity and hazards of classical organic solvents (e.g., methanol), there is a growing need to develop new, alternative, and safe reagents. Natural deep eutectic solvents (NADES) are biodegradable, environmentally friendly, and sustainable reagents used to extract bioactive compounds from various plant species and natural products. Hypericum perforatum L., known as St. John’s Wort, is a medicinal plant rich in various groups of bioactive compounds such as flavonoids, naphthodianthrones, phenolic acids, xanthones, procyanidins, and tannins. Extracting these compounds using ecological solvents would allow their direct application in in vivo studies, which is particularly important for determining their pharmacological activity.
Research objectives
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Natural deep eutectic solvents (NADES) have greater efficiency in extracting polyphenolic compounds compared to classical organic solvents. (H1)
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Is there a correlation between the physical properties of NADES and their extraction capacity? (P1)
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Is there a qualitative/quantitative difference in the chemical profile of extracted compounds when different NADES are used compared to organic solvents? (P2)
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What is the type and content of chemical compounds in samples from wild-growing Hypericum perforatum and its in vitro cultures? (P3)
Methodology
This project will synthesize NADES, then characterize them by determining several physical properties. They will be used to extract polyphenolic compounds from Hypericum perforatum and its in vitro cultures. The extraction procedure and chromatographic methods for extract analysis will be optimized.
Scientific contribution (ecology)
Applying green chemistry in scientific research contributes to environmental protection by reducing waste, pollution, and the “carbon footprint.” Developing safe and efficient chemicals is key to ecological sustainability. Extracts with a higher yield of bioactive compounds and their direct application will enable more efficient pharmacological studies.
Micronucleus Test in Tradescantia (Trad-MCN) as an Indicator of the Genotoxic Potential of Air Pollution in Skopje
Lead researcher: Ivan Arsov
Аs a global threat, air pollution respectively influences the daily life in Skopje as one of the bigger European cities. According to AQI (www.iqair.com) in 2021, Macedonia held the 34th place with an average concentration in PM2.5 of 25.4 which is over 5-7 over the limit according to who (www.who.int). Air pollution poses a challenge as a cause to several illnesses, including cancer. However, other living organisms are prone to the genotoxicity from the damaging effects from massive urbanization. A genotoxin is a chemical agent that causes DNA or chromosome damage. A successful test-model for genotoxicity is Tradescantia Micronucleus Test. According to various results using the Trad-MCN test, it has been confirmed that a mutagenic effect occurs in plants due to air pollution. As a result this research represents an essential tool in mutagenic potential screening in nature.
Research objectives
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• Air pollution in Skopje contains genotoxic components that increase the mutagenic potential which leads to micronucleus production in pollen grains. (H1)
• Exposure to air pollution results in chromosomal material loss and other nuclear abnormalities that are not micronucleus occurrence (H2)
Methodology
The methodology consists of preparation, transport and setting Tradescantia sp. seedlings at specific hotspots in Skopje, setting one negative control outside of Skopje, analysis of microscopic images of pollen grain cells, statistical analysis and finding correlation between genetic material and air pollution.
Scientific contribution (ecology)
This research will help in understanding the mutagenic effects caused by air pollution as an abiotic stressor to plants. It also serves a great foundation for future studies linked to genetic abnormalities in plants as a consequence of abiotic stress.
Determining the State of Indirect Pesticide Contamination of Wild Flora in the Prespa Region
Lead researcher: Jovana Danova
Excessive use of pesticides in agriculture poses a serious ecological risk. About 98% of the sprayed pesticides affect non-target species, and 80-90% of them evaporate into the environment and spread several hours after application. Pesticide residues are spread in soils, river waters, lakes, and are also found in the animal life of lakes. Increasing evidence of their presence in drinking water and food chain products is being reported. Regions such as Prespa, with intensive agricultural activities, are exposed to an increased risk of contamination.
The Law on Phytopharmaceuticals prescribes rules for pesticide use, their application methods, disposal practices, and gives special attention to protecting the aquatic environment and drinking water. Despite regulations, unofficial surveys show that farmers are not aware of the harmful effects, and they report higher pesticide consumption than the normal prescribed/required amounts. Despite this, they report a decrease in crop yields, which is most likely due to pesticide resistance.
Research objectives
- Question 1 (Q1): Is there indirect contamination of wild flora in the surroundings of Lake Prespa and on Golem Grad Island?
- Question 2 (Q2): Is there indirect contamination on Golem Grad Island, a location where pesticides are not actively used?
- Question 3 (Q3): Are there differences between the various locations and samples regarding the type of pesticides and their quantitative presence?
Methodology
• Sites for collecting representative plant material will be identified.
• Laboratory analyses will assess the possible presence of pesticide residues.
• A risk analysis will be conducted based on the maximum permitted pesticide concentrations.
• The results will be disseminated to raise public awareness and protect the environment.
Scientific contribution (ecology)
For the first time, indirect pesticide contamination of wild flora in the vicinity of Lake Prespa and Golem Grad island will be determined. With risk analysis the presence of pesticide residues in selected samples according to permited limits will be assessed, contributing to environmental protection.
Ecological Conditions, Status, and Importance of Pterocephalus perennis for Jablanica Mountain
Lead researcher: Mila Stojanovska
During the 2024 Biology Students’ Research Society’s summer research expedition on Mount Jablanica, the plant Pterocephalus perennis was observed for the first time in Macedonia.
- perennis (from the Dipsacaceae family) is an endemic sub-mediterranean plant that forms dense, woody mats on rocky limestone slopes, generally on altitudes from 800 to 2000 m. Flowering rather late from July to early September. The most important characteristic of this genus is its conspicuous calyx, formed by numerous plumose bristles. The closest distribution points of this plant are in the village Gjergjevicë, in Tomorri National Park in Albania and in Pindos National Park in Greece.
Considering the distance of the nearest points and the specific conditions which this species needs to thrive, this project opens questions about why and how it has reached Mount Jablanica.
Research objectives
• How big is the population of Pterocephalus perennis on mountain Jablanica? (Q1)
• What is the population status of P. perennis according to IUCN criteria? (Q2)
• Are the populations of Pterocephalus perennis on Mount Jablanica morphologically different from other populations recorded in literature4? (Q3)
• The plant P. perennis on Mount Jablanica is present in habitats with a specific soil type, such as lithosols. (H1)
Methodology
1. Field research to determine the condition of the population of Pterocephalus perennis.
2. Taking soil samples for further lab analysis.
3. Mapping the potential distribution (through GIS) and determining its status in accordance with IUCN guidelines.
4. Morphometric analysis and description of the species.
5. Raising awareness about the species.
Scientific contribution (ecology)
This study will provide the first data on the specific ecological preferences of Pterocephalus perennis, along with information on its total areal and the ecological factors influencing its distribution. Of secondary importance will also be the contribution to the valorization of the Jablanica Mountain for a future protected area, due to the endemic character of P. perennis.
The Salamanders of Jablanica and Bistra — Population Analysis of Triturus macedonicus, Ichthyosaura alpestris, and Bombina variegata
Lead researcher: Filip Krsteski
The alpine and Macedonian crested newt migrate to their aquatic breeding centres in spring, reproduce, and then return to their terrestrial phase, making observation difficult. These newts rarely travel more than 150 meters from their breeding centres. In contrast, the yellow-bellied toad inhabits ephemerent water bodies throughout the active season (spring and summer). Both the Macedonian crested newt and yellow-bellied toad are listed in Annex II of the Habitats Directive, while the Macedonian crested newt is classified as vulnerable and the alpine newt as endangered on the national Red Lists. As such, their protection is a priority at both national and European levels. Nevertheless, their distribution is still not fully understood, and basic population data is lacking. While all three species often share wetland habitats like ephemeral ponds and slow-moving streams, the alpine newt is found only in the highlands above the forest belts of Shar Mountain, Bistra, Deshat, and Jablanica.
Research objectives
➔ How big is the population of the alpine newt and yellow-bellied toad at the Tonivoda site on the mountain Bistra? (П1)
➔ The Macedonian newt inhabits the Tonivoda site. (Х1)
➔ How big is the population of the alpine newt and yellow-bellied toad at the lakes Podgorecko and Labunishko as well as surrounding puddles? (П2)
➔ There is no difference in the estimated population size of the Macedonian crested newt and the alpine newt between seasons (spring-summer). (Х2)
➔ How big is the population of the yellow-bellied toad at the Jankov kamen site? (Х3)
➔ The Macedonian crested newts and the alpine newts which inhabit various puddles within the sites of Tonivoda and Labunishki puddles with lake Podgorecko are not separate populations but part of one metapopulation. (X4)
Methodology
Several well-known sites on the mountains Bistra and Jablanica will be visited and specimens of the target species will be captured and recorded. Capture-mark-recapture (CMR) methodology will be used to calculate the population through photographs and subsequent processing of the photographs in the free software I3S Pattern+10.
MicroPlant – Ethnobotanical Perspectives on Microbial Diversity in Medicinal Plants in North Macedonia
Project Lead: Mihail Zhupan
Endophytic and epiphytic microorganisms play a vital role in plant health, enhancing nutrient absorption, increasing stress tolerance, and potentially contributing to medicinal properties¹. Although their biodiversity in medicinal plants has been well studied globally², similar research and data in North Macedonia remain limited. In particular, little is known about how ecological factors such as altitude, geographical diversity, and environmental cleanliness influence the composition of microbial communities. The plants Verbascum pulverulentum, Onopordum acanthium, and Cichorium intybus, which are widely distributed across the Galičica and Šar Mountains, represent an ideal model for studying these interactions. Despite established evidence of the microbiome’s sensitivity to ecological gradients, these regions remain underexplored. Investigating the symbiosis between these plants and microorganisms is essential for understanding understudied ecosystems and conserving medicinal plants and their associated microflora³, thereby contributing to the global mapping of microbial biodiversity.
Research Objective
• How do plant species and ecological factors—such as altitude, geographic location (Galichica vs. Shar Mountain), and clean environmental conditions—affect the presence, distribution, and survival of endophytic and epiphytic bacterial species in medicinal plants? (Q1)
• Epiphytic bacterial communities in hoary mullein (Verbascum pulverulentum), cotton thistle (Onopordum acanthium), and chicory (Cichorium intybus) are more diverse than their respective endophytic communities. (H1)
Methodology
Plant samples will be collected from the mountains Galichica and Shar Mountain. Laboratory work includes sterilization, culturing under controlled conditions for growth, isolation of endophytic and epiphytic bacteria, inoculation on selective media, and preliminary identification through colony morphology and Gram staining. Further biochemical identification will follow as needed⁴, ⁵.
Scientific Contribution (Ecology)
This project will reveal how ecological factors such as altitude, geographic isolation, and environmental cleanliness influence microbial diversity and survival in medicinal plants. It will also provide the systematic overview of the endophytic and epiphytic bacterial flora of these plants in North Macedonia, enriching the country’s ecological and microbiological database.
HydroVSoil – Microbial Diversity Along the Vardar River: Investigating the Link Between Soil and Water Ecosystems
Project Lead: Hristo Dimitrievski
The water quality of the Vardar River has been researched several times over the years, but it has been insufficient in terms of water classification and microbiological biodiversity along the river’s course in the soils surrounding it (latest report from the Public Health Institute, 2019). Anthropogenic activities increase the discharge of various pollutants into the aquatic environment, which causes the deterioration of the ecosystem, the surroundings, and the environment in general. The source mountain waters of the Vardar River consistently show low level of water pollution with diatom flora (species) dominated by taxa confirmed to be intolerant to pollution. Downstream of the river area, starting from the city of Skopje and extending beyond the city of Veles, the organically polluted part of the river is characterized by the inflow of wastewater, making the river completely lifeless. The biodiversity of microorganisms, which are also affected by environmental changes, can provide biological responses to ecological disturbances not only in the aquatic but also in the surrounding soil ecosystem.
Research Objective
- Are the differences in microbiological diversity in the aquatic and soil ecosystems at selected locations along the Vardar River directly connected? (Q1)
- Pollution of the Vardar River along its course affects the microbiological diversity in the aquatic ecosystem. (H1)
- Microorganisms from fecal contamination found in the Vardar River in the Skopje, Veles, and Valandovo regions can also be found in the fertile soils near the Vardar River. (H2)
Methodology
Three different locations will be selected for water and soil sample collection for the isolation of microorganisms. By studying the biodiversity and characterization of the isolated microorganisms, the connection between the two ecosystems (aquatic and soil) will be examined. An educational informational lecture will also be conducted.
Scientific Contribution (Ecology)
Through this research, we will gain information on whether and how fecal contamination of the river indirectly affects the soil pollution as an ecosystem. The study of microbiological diversity, the role and significance of river contamination in Macedonia, and its impact on the surrounding arable soil areas is very under-researched.
Essential Oils as an Environmentally Friendly Alternative for Plant Protection: Antimicrobial Potential of Laboratory-Derived Hydrodistillate from Lavandula Species
Project Lead: Jovan Karoski
Essential oils of plant origin represent an excellent alternative to synthetic pesticides as they reduce toxic impacts on food, water, and soil. The concept of organic farming is becoming increasingly prevalent worldwide, and sustainable agriculture is gaining greater recognition, with essential oils considered a safer alternative. Green chemistry contributes to reducing the toxic effects of synthetic pesticides and the development of new, natural, biodegradable pesticides, such as plant extracts, essential oils, and other natural substances categorized as “green pesticides.” Essential oils have been proven to inhibit the growth of pathogenic microorganisms while simultaneously enhancing agricultural production. Notably, organic food production increases with the use of essential oils. Essential oils play a key role in the natural defense of plants against insects and pathogenic microorganisms. Therefore, their use as fungicides and pesticides should be considered a sustainable alternative in agriculture.
Research Objective
- Can essential oils be used as an environmentally friendly alternative for protecting plants from pathogenic microorganisms? (P1)
- Essential oils exhibit antimicrobial activity. (H1)
- Essential oils are safe and have a relatively low potential for microbial resistance development. (H2)
- Essential oils should be used in low concentrations. (H3)
Methodology
The research will examine all aspects of the entire process of hydrodistillation, as well as the application of appropriate methods for testing antimicrobial activity, including the diffusion test method and the microdilution test method.
Scientific Contribution (Ecology)
This project aims to explore lavender as a source of bioactive compounds, despite its limited use due to its chemical composition. Since synthetic chemicals have negative effects and are not biodegradable, plant essential oils are considered a sustainable alternative to pesticides.
Carbon Sequestration in the Monospitovsko Blato Wetland According to Moisture and Conservation Gradient
Project Lead: Nikoleta Georgievska
Wetlands are defined as ecosystems that depend on continuous or periodic soil saturation with water (Mitsch & Gosselink, 2015). They encompass a diverse range of ecosystems ranging from swamps and marshes to peatlands, which are characterized by heterogeneity in their role in the carbon cycle (Sütünç, 2024). They slow down the decomposition process through anoxic soil conditions, leading to the accumulation of large amounts of organic carbon. This makes them important carbon sinks in the atmosphere with a significant impact on greenhouse gas reduction (Li et al., 2024). Their drainage can lead to degradation, and thus the release of CO2 (Maltby & Barker, 2009).
Wetlands in Macedonia have been under continuous pressure for a long time, among which anthropogenic activities and climate change are the most pronounced and lead to their endangerment and disappearance (Jovanovski et al., 2018).
Monospitovsko Blato, located about ten kilometers northeast of Strumica, is one of the largest wetlands in Macedonia, rich in unique flora and fauna. Although it was declared a natural monument in 1986, the need for its revitalization and the introduction of active protection is still emphasized precisely for the above reasons (Melovski et al., 2008).
Research Objective
The main objective of this research is to determine the carbon sequestration capacity of Monospitovsko Blato depending on the habitat preservation and the moisture gradient.
- What is the carbon sequestration rate in Monospitovsko Blato? (Q1)
- What historical changes has Monospitovsko Blato gone through? (Q2)
- Carbon accumulation has varied over the years. (H1)
- The current state of the wetland is correlated with the moisture index. (H2)
- The moisture gradient directly affects the carbon accumulation of the wetland. (H3)
Methodology
- Field collection of about ten soil profiles from three selected locations along a moisture gradient and their morphometric analysis
- Collection of soil material and data on basic physical and chemical properties (temperature, conductivity, moisture)
- Determination of carbon content by dry combustion
- Statistical analysis of the correlation between CCS, soil moisture and water and wetness index
Scientific Contribution (Ecology)
This research will contribute to a better understanding of the impact of wetland modification on its carbon sequestration. The results obtained will enable further formulation of strategies for the protection of this ecosystem, but will also help in assessing its carbon storage potential in relation to climate change. In this way, Macedonia will be able to better assess greenhouse gas emissions. The aim is for this to be only a basis for further research and the implementation of preventive and restoration measures for Monospitovsko Blato. In addition, this methodology and method of research can be undertaken to examine other wetlands, but the results obtained from this research cannot be extrapolated to them due to the uniqueness of each ecosystem and specific, individual factors that are in a different correlation for each of them.
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