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Article

Environmental DNA Metabarcoding as a Tool for Fast Fish Assessment in Post-Cleanup Activities: Example from Two Urban Lakes in Zagreb, Croatia

by
Matej Vucić
1,*,†,
Thomas Baudry
2,3,†,
Dušan Jelić
4,
Ana Galov
1,
Željko Pavlinec
5,
Lana Jelić
6,
Biljana Janev Hutinec
6,
Göran Klobučar
1,
Goran Slivšek
7 and
Frédéric Grandjean
2
1
Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia
2
Laboratoire Ecologie et Biologie des Interactions, Université de Poitiers, UMR CNRS 7267, Equipe Ecologie Evolution Symbiose, 86000 Poitiers, France
3
Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, HR-10000 Zagreb, Croatia
4
BIOTA Ltd., Maksimirska Cesta 129/5, HR-10000 Zagreb, Croatia
5
Aquarium Pula Ltd., Verudela 33, HR-52100 Pula, Croatia
6
Public Institution for Management of Natural Values of City of Zagreb—Zagreb City Nature, Maksimirski perivoj 1, HR-10000 Zagreb, Croatia
7
Centre for Applied Bioanthropology, Institute for Anthropological Research, Gajeva ulica 32, HR-10000 Zagreb, Croatia
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fishes 2025, 10(8), 375; https://doi.org/10.3390/fishes10080375
Submission received: 13 June 2025 / Revised: 17 July 2025 / Accepted: 24 July 2025 / Published: 4 August 2025
(This article belongs to the Special Issue Mapping and Monitoring Aquatic Biodiversity in Hotspot Habitat Areas Using Environmental DNA (eDNA))
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Abstract

This study evaluated the effectiveness of eDNA metabarcoding in assessing fish communities in two urban lakes (First Lake and Second Lake) in Zagreb, Croatia, following IAS removal. Water samples were collected in April and June 2024 and analyzed using MiFish primers targeting the 12S rRNA gene. The results indicated that the cleanup efforts were largely successful, as several IAS previously recorded in these lakes were not detected (Ameiurus melas, Lepomis gibbosus, and Hypophthalmichthys spp.). However, some others persisted in low relative abundances, such as grass carp (Ctenopharyngodon idella), topmouth gudgeon (Pseudorasbora parva), and prussian/crucian carp (Carassius sp.). Species composition differed between lakes, with common carp (Cyprinus carpio) dominating Maksimir First Lake, while chub (Squalius cephalus) was prevalent in Maksimir Second Lake. Unexpected eDNA signals from salmonid and exotic species suggest potential input from upstream sources, human activity, or the nearby Zoo Garden. These findings underscore the utility of eDNA metabarcoding in biodiversity monitoring and highlight the need for continuous surveillance and adaptive management strategies to ensure long-term IAS control.
Keywords:
invasive species; eDNA; management; removal; urban ecosystem
Key Contribution: The manuscript showcases a pioneering use of eDNA metabarcoding in European urban freshwater ecosystems, revealing the successful removal of several invasive species following cleanup efforts in urban lakes, while also uncovering persistent low-level invasives and unexpected exotic DNA signals—underscoring the method’s sensitivity and the need for continuous bio-surveillance.

1. Introduction

Freshwater fish represent 40% of global fish diversity and 25% of all vertebrate species [1,2,3], and they are among the most endangered animal groups [4], with 26% of them threatened with extinction [5]. The fragility of freshwater ecosystems is particularly concerning, as they are especially impacted by non-native and invasive alien species (IAS), mainly introduced by shipping, aquarists, or aquaculture [6,7,8,9,10,11,12]. The adverse effects of IAS on native species, endemic, or endangered ichthyofauna are multiple, encompassing ecological, evolutionary, and economic dimensions, for example, habitat degradation, competition for predation, food, and space, hybridization, the transmission of parasites and pathogens, and alterations to food niches [13,14,15,16,17]. Despite significant progress in understanding the ecological consequences of IAS, assessments of their economic impacts remain geographically and taxonomically limited, often restricted to direct costs associated with a few high-profile species or habitats [18,19]. Recent advances in integrative monitoring approaches, such as the use of environmental DNA (eDNA) metabarcoding and Geographic Information Systems (GIS), have considerably improved early detection and spatial mapping of IAS in freshwater environments [20,21,22]. These tools are now recognized as essential components of modern surveillance strategies, offering scalable and non-invasive alternatives to traditional biomonitoring. Europe represents a significant center for migration, tourism, and trade [23], and biological invasions have become so frequent that colonization by successive IAS, a process called over-invasion, is now widespread [24,25,26].
Croatian freshwater ichthyofauna is one of the most diverse in Europe, with 137 species [27], out of which 25 species are considered IAS [16]. They were introduced intentionally or unintentionally from different parts of the world for aquaculture, recreational fishing, biological control, or as a part of a pet trade [16]. IAS are also recognized as a threat from a legal perspective. The laws that deal with the issue of IAS in Croatia are as follows: (i) The Act on the Prevention and Spread of Alien and Invasive Alien Species and their Management (Official Gazette 15/2018), (ii) the Act on Freshwater Fisheries (Official Gazette 14/2014), and (iii) the Act on Aquaculture (Official Gazette 130/2017). Since Croatia’s accession to the European Union, regulations dealing with IAS (Regulation No. 1143/2014 and Regulation No. 1141/2016) have also been in force and contain a list of IAS of concern at the European Union level (the so-called Union list). The list currently includes 88 species, 28 of which have been recorded in the Republic of Croatia, including four freshwater fish—the stone moroko (Pseudorasbora parva), the amur sleeper (Perccottus glenii), the pumpkinseed (Lepomis gibbosus), and the Prussian carp (Carassius gibelio) [16]. Effective management, control, and eradication of IAS freshwater fish are challenging and almost impossible [28]. Most important approaches in controlling IAS are early detection and continuous monitoring of freshwater fish communities [16,29].
Innovative monitoring methods are essential for accurately capturing species diversity, particularly rare and invasive species, while also assessing the environmental factors influencing their presence and distribution [30]. These approaches are critical for advancing our understanding of aquatic ecosystems and ensuring effective biodiversity conservation through precise and comprehensive data collection [31,32]. Molecular methods have emerged in recent years, and the application of environmental DNA (eDNA) has gained popularity due to its reported reliability, speed, and cost-effectiveness in monitoring the distribution of various aquatic organisms [33,34]. Based on the detection of DNA shed in the environment by species (i.e., feces, skin tissue, mucus, etc.) [35], eDNA methodology offers quick insights into species presence, with minimal habitat disturbance. Inversely, traditional survey methods, such as nets, traps, seines, and electrofishing, are frequently labor-intensive, invasive, dependent on taxonomic expertise [36,37], and present some biases when it comes to detecting low-abundance species presence [38,39,40,41]. eDNA has now proven its efficiency and has been developed for many species across all taxonomic groups [42], especially cryptic, rare, endemic, and threatened species [26,43], as well as invasive species in the early stages of invasion [26].
Maksimir Park is one of the largest and oldest parks in Zagreb, the capital of Croatia [44,45]. At the end of the 18th and the beginning of the 19th century, the Maksimir lakes were created artificially within Maksimir Park. All five lakes are utilized for recreational purposes in addition to their scenic and landscape value [44,45]. Due to its exceptional natural value, Maksimir Park is today protected under the Nature Protection Act (Official Gazette 80/13) as a monument of park architecture and under the Act on the Protection and Preservation of Cultural Heritage (Official Gazette 69/99, 151/03, 157/03, 100/04, 87/09, 88/10, 61/11, 25/12, 136/12, 157/13, 152/14, and 98/15) as a cultural heritage site [36]. Nevertheless, it faces several fish IAS, whose primary vectors are intentional and unintentional imports from aquaculture, fishkeeping, and the inadvertent escape of certain species from the adjacent Zoo Garden.
This study explored the efficacy of eDNA metabarcoding in assessing fish biodiversity and their relative abundance in Maksimir First Lake and Maksimir Second Lake, located in Maksimir Park (Zagreb, Croatia), following cleanup operations, including IAS removal, conducted in 2021–2022. The assessment was conducted during two different sampling periods (April and June) using the MiFish primers [46], which have proven reliable in detecting a broad spectrum of fish species across various ecosystems [47,48].

2. Materials and Methods

2.1. Study Area and Sampling

As mentioned, Maksimir Park is one of the largest parks in Zagreb, encompassing five lakes (Figure 1) supplied with water from streams that spring in the Medvednica Mountain Nature Park. These lakes are presently utilized for boating and as fishponds in addition to their scenic and landscape value (Figure 1). The Bliznec stream, which empties straight into the Second and Fifth Lakes, provides most of the water for the artificially formed lakes. In addition, the lakes are occasionally irrigated by the Mirni Dol and Dahlia precipitation streams [44]. All five lakes are characterized by a diverse fish population, similar in composition, comprising both native and IAS [49,50]. All of the water that flows into the Second Lake from the Bliznec steam then goes into the First Lake, since they are connected through a small concrete overflow, which enables the water to come into the First Lake, but not the other way due to the ~1 m elevation difference. The First Lake drains into a drainage canal that takes the water into the Sava River. The First and Second Lakes are regularly drained and cleaned to address the accumulation of organic debris and the proliferation of IAS, probably due to their connection to one another and with the nearby Zoo Garden (Figure 1). Cleaning operations included the complete drainage of the lakes, the removal of all fish species, and the cleanup of accumulated organic matter from the bottom [50]. The most recent cleaning efforts for the two lakes were conducted in 2021–2022, during which almost 7000 native fish (17 species) ([27], Table S1), 2000 indigenous shellfish, and 200 native freshwater crayfish were relocated to adjacent lakes, while all non-native and invasive species were removed [44]. Data on the population composition and abundance of fish removed from the First and Second Lake (Table S1) and fish used for repopulation of the Second Lake (Table S2) served as a basis for comparison with the eDNA data.
Environmental DNA sampling was conducted at the First Lake (Maksimir 1) and the Second Lake (Maksimir 2) at one point each during two sampling periods: April 2024 and June 2024 (Table 1, Figure 1). These sampling points were selected based on water flow dynamics: in the Second Lake near its outlet into the First Lake, and in the First Lake where water from the Zoo Garden enters. The sampling protocol followed the methods described by Vucić et al. (2023) [43] and Baudry et al. (2022) [42]. Briefly, 1.5 L pre-sterilized (with 50% bleach solution, for 15 min and then rinsed with distilled water) containers were used. Before each sampling period, they were rinsed three times with water from the site and then the water was taken by submerging the bottle below the water surface until it was filled. At each location and during each sampling period, three field replicates were collected, as described above, in separate 1.5 L containers, totaling 12 containers.

2.2. Lab Analysis

2.2.1. eDNA Filtration

The collected water samples were immediately transported in a cooler box to the laboratory, where water filtration was performed using 1.2 µm pore-sized sterile nitrocellulose filters (Sartorius®, Göttingen, Germany; 47 mm diameter) and an electric vacuum pump (Rocker Lafil300 Oil-Free Pump, Rocker Scientific, New Taipei City, Taiwan) equipped with a 1 L filtration unit (Nalgene™, Rochester, NY, USA), following the protocols of Baudry et al. (2021) [42] and Vucić et al. (2023) [43]. For each station and sampling period, a control sample (1 L of tap water) was filtered as well as each of the three collected water samples (independent field replicates). Filtration was performed until the filter became clogged, with 1000 to 1200 mL of lake water filtered. Each filter was stored individually in molecular-grade absolute alcohol (96%) and labelled. Between different stations, the pump and filtration unit were sterilized by rinsing with a 50% bleach solution and absolute alcohol to prevent cross-contamination between stations. Samples were stored at −20 °C until further laboratory processing.

2.2.2. eDNA Isolation

Environmental DNA isolation was performed using the Macherey-Nagel NucleoSpin Tissue Kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s protocol with modifications as described by Baudry et al. (2021) [42] and Vucić et al. (2023) [43]. Briefly, a quarter of each filter was cut and shredded using sterilized scissors and left to air-dry until the ethanol evaporated. The filter pieces were transferred to a 2 mL tube, to which 450 µL of T1 buffer and 50 µL of proteinase K were added to submerge the filter pieces. The mixture was then vortexed before incubation overnight at 56 °C. The isolation process continued with the addition of 500 µL of B3 buffer and 500 µL of absolute ethanol (100%), after which the mixture was transferred onto the spin columns. Further steps followed the manufacturer’s protocol, until elution in a final volume of 60 µL, to concentrate the eDNA. The samples were then frozen at −20 °C until further processing.

2.2.3. eDNA Amplification

eDNA amplifications were performed using MiFish-U-F-GTCGGTAAAACTCGTGCCAGC and MiFish-U-R-CATAGTGGGGTATCTAATCCCAGTTTG primers targeting a 175 bp fragment of the 12S rRNA mitochondrial gene [38], based on their applicability and resolution for assessing fish communities [46,51].
PCR amplifications were conducted in a sterile room that was decontaminated nightly using UV light exposure. Each eDNA sample underwent PCR amplification in triplicate (later pooled for sequencing), resulting in nine PCR triplicates per station and sampling period, for a total of 36 PCR reactions. This combination has proven to be satisfactory for maximal species detection and cost-efficiency [52]. Each PCR reaction was performed in a final volume of 25 µL, comprising the following: 12.5 µL of KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland), 5 µL of each indexed primer (final concentration of 0.2 µM), and 2.5 µL of DNA template. The same index was used for the nine PCR triplicates for each station and sampling period, resulting in a total of four indices. A negative control was included (i.e., no-template DNA) to monitor for potential contamination during amplification, along with three positive mock controls. These mock controls consisted of DNA extracted from individual organisms, mixed in known concentrations, acting as positive controls, and, once sequenced, used to fine-tune the bioinformatic pipeline (see, for instance, Baudry et al., under review).
The amplification protocol consisted of an initial activation step at 95 °C for 3 min, followed by 35 cycles of denaturation at 98 °C for 30 s, annealing at 65 °C for 30 s, and extension at 72 °C for 30 s. A final elongation step was run at 72 °C for 5 min.
PCR products were visualized on 1.5% agarose gels and subsequently sent to the INRAE-PGTB sequencing platform (Bordeaux, France) for quality assessment using a TapeStation system (Agilent, Santa Clara, CA, USA) and sequencing of the pooled PCR products, which was performed on an Illumina NextSeq 2000 (San Diego, CA, USA) with a 2 × 150 bp kit.

2.3. Data Analysis

2.3.1. Llumina Sequencing Handling

The dada2 pipeline (v1.30.0; [53]) implemented in R (v4.3.2; [54]) was used to handle reads generated via Illumina sequencing. First, primers were trimmed, reads containing Ns were pre-filtered, and their quality profiles were carefully assessed. Based on these profiles and the expected read lengths, sequences were filtered and trimmed accordingly. Reads were then dereplicated and merged into paired-end sequences using the error correction model implemented in dada2, and chimeric sequences were removed from the final sequence table. Finally, taxonomic assignments were performed with MOTHUR [55] on a newly curated database (MIDORI2; [56]), supplemented and curated again (i.e., remove sequences containing ambiguities (Ns) or of poor quality—length or homopolymer; see Baudry et al., under revision). Taxonomic assignments were run on the above-mentioned double-curated database using the following parameters: method = wang, iters = 1000, and cutoff = 75.
To minimize potential false positives, contaminants, or sequencing artefacts, additional filtering steps were applied. Variants with fewer than 10 reads in a sample were excluded from the analysis. Then, variant depths (number of reads assigned to a particular taxon) were converted to relative abundances, and those accounting for less than 0.1% of the total reads in a sample were removed from further analyses.

2.3.2. Statistical Analysis

All statistical and graphical analyses were performed in the R environment (R v4.3.2; [54]).
First, the species relative abundance was plotted for each of the two lakes and each sampling period, using the geom_bar function in ggplot2 [57]. Based on this, the dissimilarity between lakes and the sampling period was tested, using the Bray–Curtis index implemented in phyloseq and ape packages [58]. Non-Metric Multidimensional Scaling (NMDS) was then performed using the metaMDS function in vegan [59], with number of dimensions k = 2 and iter = 100 to ensure convergence. Finally, the envfit function (vegan package) was used to determine the goodness of fit (statistically reported as r2 and p-values) of each vector (species) and abiotic parameter (lake and sampling period).

3. Results

3.1. Bioinformatics and Data-Set Cleanup

In total, 5,808,980 reads were generated from the 12 biological samples (mean 484,081.67 ± 72,518.01). After data filtering, taxonomic assignment, and curation, the average sample read count per sample reached 363,744.83 ± 109,173.01 and so, 1,091,234.5 ± 290,290.46 reads per lake (station) and period. These reads were assigned to 163 genetic variants related to 25 different taxonomic units (species or unassigned at the species level).

3.2. Post Cleanup Results and Biodiversity Assessment

Most of the IAS removed during cleanup operations [44] were not recorded with eDNA metabarcoding, such as black bullhead catfish (Ameiurus melas), sunfish (Lepomis gibbosus), and silver carp (Hypophthalmichthys spp.). Absence of the European eel (A. anguilla), a non-native species in this inland continental region but naturally encountered in Adriatic watershed, was also shown by eDNA analysis (Table S1). The only IAS recorded after the cleanup, but with very low relative abundance, were grass carp (Ctenopharyngodon idella) (April—0%; June—3.3%) and stone moroko (Pseudorasbora parva) (April—3.9%; June—2.2%), both detected in Maksimir First Lake (Figure 2A).
Although visual inspection of the ordination (Figure 2B) suggested some temporal variability and site-specific differences between the lakes, these patterns were not supported by statistical significance. Lake identity explained a greater, but non-significant, proportion of the variation (r2 = 0.85, p = 0.33) than temporal differences (between April and June) (r2 = 0.115, p = 0.67) (Figure 2B). The NMDS axes were strongly correlated to both Cyprinus carpio and Squalius cephalus (both r2 values of 0.99 and p = 0.04). Among other species, only Pseudorasbora parva and the Gobionidae sp. seemed to make a significant contribution to community structuring, in a marginal way (both r2 value of 0.98 and p = 0.08333).
The common carp (Cyprinus carpio) emerged as the dominant species in Maksimir First Lake, accounting for 81.6% of the relative abundance in April and 47.7% in June. However, this species was far less abundant in Maksimir Second Lake, where its relative abundance remained below 1% in both months (Figure 2A). On the other hand, the European perch (Perca fluviatilis) showed consistent and significant presence across both lakes, particularly in Maksimir Second Lake, where it represented 36.6% and 31.4% of the relative abundance in April and June, respectively (Figure 2A). Variability was also notable for the rudd (Scardinius erythrophthalmus), with it being prominent in Maksimir First Lake during June, contributing 9.3% to the total, while its representation in Maksimir Second Lake was comparatively lower. In contrast, chub (Squalius cephalus) dominated Maksimir Second Lake, representing over 42% of the relative abundance in both April and June, yet was almost absent in Maksimir First Lake (Figure 2A).
The presence of unclassified variants at the genus level was recorded (i.e., Gobionidae sp., Rutilus sp., Squalius sp., and Knipowitschia sp.) (Figure 2A). Furthermore, eDNA signals from salmonid species (Salmo sp. and Oncorhynchys mykiss) were detected, as well as signals from exotic species belonging to the Haemulidae, Cichlidae, and Caranx families (Figure 2A) (see all species detection and relative abundance in Table S3).

4. Discussion

This study represents the first application of eDNA metabarcoding in Croatia for fish community assessment, with the primary focus on IAS removal during cleanup efforts. The initial results of the cleanup are promising, as most of the species previously removed from these lakes were not detected, indicating the success of the efforts. However, some concerning findings emerged, particularly the continued presence of certain IAS, highlighting the challenges of monitoring and managing invasive populations in freshwater ecosystems, as well as inconsistencies in taxonomic assignments. These points are discussed below, but the results emphasize the need for ongoing and robust monitoring strategies to ensure the long-term success of such environmental restoration projects and to further refine the management of aquatic biodiversity.
The eDNA metabarcoding results indicated that the 2021–2022 cleanup efforts in Maksimir First and Second Lakes were largely successful. Several IAS that were previously present, such as black bullhead (A. melas), sunfish (L. gibbosus), and silver carp (Hypophthalmichthys spp.), were not detected in our study. This suggests that removal efforts effectively reduced or eliminated these species from the lakes. While the absence of certain invasive species suggests successful eradication, the continued presence of P. parva and C. idella in Maksimir First Lake is concerning. These species accounted for nearly 5.5% of the total relative abundance in June 2024, indicating either residual populations that survived the cleanup or new introductions after removal. Several factors may contribute to their persistence, including (i) the survival of individuals in refugia during cleanup efforts, (ii) unintentional reintroductions through human activities, such as aquarium releases or stocking for aquaculture, or (iii) upstream sources and the Bliznec stream contributing to the dispersal of IAS and their DNA input into the lakes. Unintentional introductions through human activities may be influenced by the vicinity of the large urban area, the City of Zagreb, and nearby lakes used for recreational angling, both of which are globally recognized as contributing to IAS dispersal [60,61,62,63]. Indeed, urban areas, with their high human population densities and associated activities, are particularly vulnerable to invasions [16,64,65].
Although statistical analyses did not show significant differences in fish species composition and abundance between Maksimir First and Second Lakes, some notable trends were observed. For instance, C. carpio (common carp) was dominant in the First Lake, particularly in April (81.6% relative abundance), but declined in June (47.7%), while its abundance was almost negligible in the Second Lake. Conversely, S. cephalus (chub) was the dominant species in the Second Lake, accounting for over 42% of the relative abundance in both sampling periods. Several factors may influence species composition and these site-specific differences. The difference in carp abundance is due to restocking activities in the First Lake (Table S2), where C. carpio was reintroduced in the Second Lake only with six specimens (Public Institution for Management of Natural Values of City of Zagreb, pers. comm.). Additionally, upstream migration from the First Lake is not possible due to the presence of a concrete overflow, with the Second Lake positioned higher than the First Lake. Furthermore, hydrological connections, such as the existence of direct connection with the Zoo and First Lake, and the Bliznec stream feeding the Second Lake, may introduce distinct species or alter community composition, thus explaining the high relative abundance of chub, making it the dominant species in the Second Lake. Previous research on ichthyofauna of Bliznec showed that the chub is the most abundant fish species in this stream with more than 75% share in species composition [66]. Lowland streams and lakes are the natural habitat of S. cephalus, and the presence of smaller streams is crucial for its spawning migration in faster-flowing water [67]. Except chub, perch (P. fluviatilis), gudgeon (Gobio gobio), and Prussian/crucian carp (Carassius sp.) are present in the Bliznec stream [66], thus sharing 100% species composition with Maksimir Second Lake and proving the importance of these lakes for urban freshwater ichthyofauna biodiversity.
The detection of eDNA signals from salmonid species (Salmo sp., Oncorhynchus mykiss) and exotic species from the Haemulidae, Cichlidae, and Caranx families was unexpected. There are a few possible explanations for these findings. First, salmonids are of high interest in recreational fishing activities and fishkeeping, which is known to be one of the most common causes of unrecorded IAS introductions [68,69]. Additionally, the release of unwanted aquarium fish into open waters actively contributes to non-native species dispersal [70,71]. Furthermore, the nearby Zoo Garden may influence unintentional releases of non-native species into Maksimir First Lake, although data on zoos acting as vectors for the spread of non-native and invasive species is scarce. Indeed, Cassey & Hogg (2015) [72] concluded that the risk of introduction by alien species from zoos is minimal compared to the other ‘backyard’ and illicit sources of private species possession and commerce. Finally, some IAS detected in eDNA metabarcoding may have originated from animal feed in the Zoo Garden (e.g., S. salar, O. mykiss, C. auratus/gibelio, and P. parva) (Zagreb Zoo, pers. comm.; [73]), persisting in the form of degraded eDNA and explaining why they were classified at the genus level.
Lack of taxonomic resolution was also encountered with variants of Gobionidae sp., Rutilus sp., Squalius sp., and Knipowitschia sp., which were classified at the family level. Such observations are not uncommon in metabarcoding analysis and typically result from the absence of reference sequences in public genetic databases [74,75]. This issue is particularly relevant in regions with high endemicity, where local reference sequence libraries are often necessary to improve taxonomic classification [76]. Their absence hinders large-scale application and accurate species detection [76,77]. Croatia falls into this category, recognized as a European hotspot for freshwater ichthyofauna, with 137 fish species, including 49 endemic species [27]. This highlights the need for increased efforts in species surveys and sequencing to fill gaps in reference databases at regional and/or national scales.
To conclude, the findings of this study highlight the need for continuous monitoring and adaptive management strategies to ensure the long-term success of cleanup efforts. Several key recommendations emerged from this research: (i) conducting pre- and post-cleanup eDNA sampling to track species presence and assess removal effectiveness, (ii) assessing water inflows and investigating the role of streams, lakes, and other water inputs in introducing external eDNA signals, (iii) evaluating of the Zoo Garden’s influence and examining the potential role of the zoos and botanical gardens in the spread of non-native and invasive alien species. By implementing these recommendations, future restoration projects can be more effective in preserving aquatic biodiversity and mitigating the impact of invasive species. Given the ecological threat posed by invasive species, targeted management strategies should be developed to address their presence and prevent future reintroductions. Environmental DNA has once again demonstrated its efficacy as a valuable instrument for the rapid and accurate assessment and monitoring of aquatic systems. Its applicability is particularly evident in urban settings, where anthropogenic activities can facilitate the introduction of non-native and invasive alien species, thereby disrupting aquatic systems that are essential for maintaining urban biodiversity.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/fishes10080375/s1. Table S1: Number of specimens per species caught by electrofishing during cleaning activities in Maksimir First Lake (1) and Maksimir Second Lake (2). Maksimir Second Lake was cleaned in three steps (dates of the cleaning activities shown below lake names). IAS marked by *. Source: Ćaleta et al. (2021) [78]; Table S2: Number of specimens of native species taken from Lake 3, Lake 4 and Lake 5 and relocated to Maksimir Second Lake, after clean-up operation. Source: Ćaleta et al. (2021) [78]; Table S3: Relative abundance for each species, reported by eDNA metabarcoding performed on Maksimir First Lake (1) and Second Lake (2) in April and June samplings. IAS marked by *.

Author Contributions

Conceptualization, M.V., T.B. and F.G.; methodology, M.V., T.B., A.G. and Ž.P.; software, T.B. and Ž.P.; validation, M.V., T.B., D.J., G.K. and Ž.P.; formal analysis, M.V., T.B., Ž.P., A.G. and G.K.; investigation, M.V., T.B., D.J., L.J. and B.J.H.; resources, M.V., T.B., A.G., G.K., F.G. and B.J.H.; data curation, M.V., T.B. and G.S.; writing—original draft preparation, M.V. and T.B.; writing—review and editing, M.V., T.B., D.J., A.G., Ž.P., L.J., B.J.H., G.K., G.S. and F.G.; visualization, M.V. and T.B.; supervision, A.G., G.K. and F.G.; project administration, D.J., L.J. and B.J.H.; funding acquisition, F.G., L.J. and B.J.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors upon request.

Acknowledgments

The authors want to thank the Centre National de la Recherche Scientifique (CNRS) and the University of Poitiers for the lab facilities used during T.B.’s post-doctoral contract funded by the Office Français de la Biodiversité (OFB), and the PGTB (doi:10.15454/1.5572396583599417E12) sequencing platform, with the help of Préscillia Alves-Gomes and Erwan Guichoux.

Conflicts of Interest

The author Dušan Jelić is employed by BIOTA Ltd. and the author Željko Pavlinec is employed by Aquarium Pula Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

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Fishes 10 00375 g001
Figure 1. Map of Maksimir Park, Zagreb, Croatia, with Maksimir Lakes and sampling localities.
Figure 1. Map of Maksimir Park, Zagreb, Croatia, with Maksimir Lakes and sampling localities.
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Figure 2. Graphical representation of (A) the relative abundance of species detected via eDNA metabarcoding in Maksimir First Lake (Maksimir 1) and Second Lake (Maksimir 2) in April and June and (B) the Non-Metric Multidimensional Scaling (NMDS) plotting the dissimilarities of community assemblages depending on lakes and sampling period, based on the Bray–Curtis index. These results are based on the 1,091,234.5 ± 290,290.46 reads obtained per lake and period, after data cleaning.
Figure 2. Graphical representation of (A) the relative abundance of species detected via eDNA metabarcoding in Maksimir First Lake (Maksimir 1) and Second Lake (Maksimir 2) in April and June and (B) the Non-Metric Multidimensional Scaling (NMDS) plotting the dissimilarities of community assemblages depending on lakes and sampling period, based on the Bray–Curtis index. These results are based on the 1,091,234.5 ± 290,290.46 reads obtained per lake and period, after data cleaning.
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Table 1. General information on the First and Second Maksimir Lakes.
Table 1. General information on the First and Second Maksimir Lakes.
NameSurface Area (km2)DepthXY
First LakeMaksimir 10.0171.4 m16.0212645.8226
Second LakeMaksimir 20.00571.5 m16.0186745.8211
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Vucić, M.; Baudry, T.; Jelić, D.; Galov, A.; Pavlinec, Ž.; Jelić, L.; Hutinec, B.J.; Klobučar, G.; Slivšek, G.; Grandjean, F. Environmental DNA Metabarcoding as a Tool for Fast Fish Assessment in Post-Cleanup Activities: Example from Two Urban Lakes in Zagreb, Croatia. Fishes 2025, 10, 375. https://doi.org/10.3390/fishes10080375

AMA Style

Vucić M, Baudry T, Jelić D, Galov A, Pavlinec Ž, Jelić L, Hutinec BJ, Klobučar G, Slivšek G, Grandjean F. Environmental DNA Metabarcoding as a Tool for Fast Fish Assessment in Post-Cleanup Activities: Example from Two Urban Lakes in Zagreb, Croatia. Fishes. 2025; 10(8):375. https://doi.org/10.3390/fishes10080375

Chicago/Turabian Style

Vucić, Matej, Thomas Baudry, Dušan Jelić, Ana Galov, Željko Pavlinec, Lana Jelić, Biljana Janev Hutinec, Göran Klobučar, Goran Slivšek, and Frédéric Grandjean. 2025. "Environmental DNA Metabarcoding as a Tool for Fast Fish Assessment in Post-Cleanup Activities: Example from Two Urban Lakes in Zagreb, Croatia" Fishes 10, no. 8: 375. https://doi.org/10.3390/fishes10080375

APA Style

Vucić, M., Baudry, T., Jelić, D., Galov, A., Pavlinec, Ž., Jelić, L., Hutinec, B. J., Klobučar, G., Slivšek, G., & Grandjean, F. (2025). Environmental DNA Metabarcoding as a Tool for Fast Fish Assessment in Post-Cleanup Activities: Example from Two Urban Lakes in Zagreb, Croatia. Fishes, 10(8), 375. https://doi.org/10.3390/fishes10080375

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