By Tina Anić, Enova’s biodiversity specialist for Environmental Impact Assessments
As part of the Western Balkans Investment Framework (WBIF) and the IPF8 Technical Assistance Programme, ENOVA is supporting the preparation of the Environmental and Social Impact Assessment (ESIA) for one of the most significant infrastructure projects currently underway in Bosnia and Herzegovina – the Konjic (Ovčari) – Prenj Tunnel – Mostar North section of Corridor Vc.
This project represents one of the most complex infrastructure developments in Bosnia and Herzegovina, with the 10.9 km long Prenj Tunnel as its central feature. This tunnel passes through a highly valuable, environmentally sensitive and complex karst mountain system, making biodiversity assessments a crucial aspect of the evaluation process. The mentioned landscapes of Mt. Prenj are found to be (potentially) characterised by underground water systems, subterranean habitats and highly specialised ecosystems.
As underground ecosystems are innately challenging to observe, yet are highly sensitive to environmental changes, an important question was raised during the ESIA process: could these underground environments represent a suitable home to the olm (Proteus anguinus), a rare and protected cave amphibian found only in the Dinaric karst?
Expert consultations, distribution maps and available data initially suggested that the presence of this species on Mt. Prenj is unlikely; however, due to its high sensitivity to environmental changes, its protected status necessitated the adoption of a precautionary approach. This required that the presence of this elusive species be either confirmed or reliably excluded before final conclusions on project impacts could be made.
Targeted environmental DNA (eDNA) analysis
To address this uncertainty, targeted environmental DNA (eDNA) analysis was selected through expert consultation as the appropriate and scientifically justified method. Given the difficulty of directly observing organisms within inaccessible underground habitats, this non-invasive method allows for the detection of genetic traces released into the environment which could help guide further assessments. Such traces in the case of the olm originate from shed skin cells containing DNA, transported through underground water systems, thereby making them detectable at connected surface springs.
So, what exactly is eDNA and why is it such a game-changer? Every living organism continuously sheds genetic material into its surroundings: fish leave traces of DNA in the water they swim through, amphibians release it from their skin, and even a single footprint in the mud can carry enough genetic information to identify a species. Scientists can collect water, soil or even air samples and use highly sensitive molecular techniques – the same kind used in forensic labs and medical diagnostics – to detect these invisible biological fingerprints.
Apply with care, controls and expert interpretation
The key advantages are hard to overstate: eDNA surveying is entirely non-invasive (no trapping, no handling, no harm to the animal or its habitat), it can detect species that are present in very low numbers long before a traditional survey would, and it can cover vast and otherwise inaccessible terrain. That said, the method is not without its challenges. Results reflect only conditions in one particular moment and place in which the sample was collected: DNA degrades quickly in warm or UV-exposed environments, water currents can transport genetic traces far from their source making precise localisation tricky, and the risk of laboratory contamination demands strict protocols. Like any powerful tool, eDNA works best when applied carefully, with appropriate controls and expert interpretation.
The focus of the research was on parts of the section where the tunnel enters the mountain massif, as this was the part of the project most likely to interact with underground water systems and potential habitats of the olm. Three main points were selected for sampling, based on hydrogeological data, dye tracing results and expert input: Gornja Bijela, Salakovac and Bosnjaci springs, all of which are directly connected to groundwater systems associated with the planned tunnel route.
The absence of detectable olm DNA in the sampled groundwater
Water samples were collected under carefully controlled conditions and filtered to capture environmental DNA. The extracted DNA was then analysed using advanced molecular techniques capable of detecting even the smallest fragments of species-specific genetic material. Each sample was tested through multiple parallel reactions with strict controls to ensure reliability, where two of three positive results indicate the presence of a species, and entirely negative reactions indicate its absence. This analysis was conducted in cooperation with the Tular Cave Laboratory in Slovenia, a leading specialised institution in Proteus research, and partner molecular laboratories in Ljubljana.
The results of the eDNA analysis exhibited no confirmed presence of Proteus anguinus in any of the analysed groundwater samples. All samples from Gornja Bijela, Salakovac and Bosnjaci springs tested negative, with one isolated inconclusive signal subsequently confirmed as an artefact through additional verification methods. These results indicated the absence of detectable olm DNA in the sampled groundwater at the time of testing, and its presence in the investigated area is unlikely.
eDNA increasingly recognised as a valuable tool within ESIA processes worldwide
Results of this analysis have been used to further refine the biodiversity impact assessment as part of the ESIA process and contribute to forming of associated mitigation measures. The Environmental and Social Management Plan, developed as part of the ESIA process, includes precautionary measures such as the requirement for eDNA testing in case of cavern discovery during construction and the involvement of qualified hydrogeologists and biologists to ensure timely response to potential impacts on groundwater systems.
Notably, this was not the first time eDNA methods were deployed to search for Proteus anguinus in Bosnia and Herzegovina. Previous eDNA surveys targeting the olm have been carried out in karst spring systems across the country, establishing important baseline data on its distribution within Dinaric groundwater networks. These earlier studies helped develop and validate species-specific molecular markers for Proteus detection in the region, making the methodology applied at Mt. Prenj all the more robust and scientifically grounded as it built on a growing body of local experience rather than starting from scratch.
The Prenj case helps illustrate why eDNA analysis is increasingly recognised as a valuable tool within ESIA processes worldwide. When a project intersects with sensitive habitats, be it a tunnel through karst, a dam on a biodiverse river, or a road through old-growth forest, traditional biodiversity surveys can be too slow, too disruptive, or simply impossible in inaccessible terrain. eDNA provides a rapid, cost-effective, and non-invasive alternative that can be used at the baseline, impact assessment, and monitoring stages of an ESIA alike.
Leading development finance institutions and environmental bodies are increasingly encouraging its integration into impact assessment frameworks, recognising that better biodiversity data leads to better decisions and, ultimately, better outcomes for both projects and the ecosystems they pass through.
