Plastic pollution in marine environments poses a new global threat. Microplastics (MPs) can bioaccumulate in marine organisms, leading to oxidative stress (OS). This study investigates MP accumulation and associated OS responses in six invertebrate species (Bivalvia, Gastropoda, and Malacostraca) and three key fish species of the Bulgarian Black Sea ecosystems. The target hydrobionts were collected from nine representative coastal habitats of the northern and southern aquatory. MPs were quantified microscopically, and OS biomarkers (lipid peroxidation, glutathione, and antioxidant enzymes) were analyzed spectrometrically in fish liver and gills and invertebrate soft tissues (STs). The specific OS (SOS) index was calculated as a composite indicator of the ecological impact, incl. MP effects. The results revealed species-specific MP bioaccumulation, with the highest concentrations in Palaemon adspersus, Rathke (1837) (0.99 ± 1.09 particles/g ST) and the least abundance in Bittium reticulatum (da Costa, 1778) (0.0033 ± 0.0025 particles/g ST). In Sprattus sprattus (Linnaeus, 1758), the highest accumulation of MPs was present (2.01 ± 2.56 particles/g muscle). The correlation analyses demonstrated a significant association between MP counts and catalase activity in all examined species. The SOS index varied among species, reflecting different stress responses, and this indicated that OS levels were linked to ecological conditions of the habitat and the species-specific antioxidant defense potential to overcome multiple stressors. These findings confirmed the importance of environmental conditions, including MP pollution and the evolutionarily developed capacity of marine organisms to tolerate and adapt to environmental stress. This study emphasizes the need for novel approaches in monitoring MPs and OS to better assess potential ecological risks.

Plastic pollution in marine environments poses a new global threat. Microplastics (MPs) can bioaccumulate in marine organisms, leading to oxidative stress (OS). This study investigates MPs accumulation and associated OS responses in six invertebrate species (Bivalvia, Gastropoda, Malacostraca) and three key fish species of the Bulgarian Black Sea ecosystems. Hydrobionts were collected from representative coastal habitats, MPs were quantified microscopically, and OS biomarkers (lipid peroxidation, glutathione, antioxidant enzymes) were analyzed spectrometrically in fish liver and gills, and invertebrate soft tissues. Results revealed species-specific MPs bioaccumulation, with Palaemon adspersus, Rathke (1837) and Sprattus sprattus (Linnaeus, 1758) having the highest concentrations per gram soft tissue and muscle, respectively. The alien Pacific oyster exhibited higher MPs loads than the native species. OS biomarkers varied markedly among species, reflecting different stress responses. Cluster analyses indicated that MPs were mainly grouped with catalase activity in all species. The specific OS (SOS) index showed that OS levels were linked to both habitat conditions and species-specific antioxidant defense. These findings confirmed the importance of environmental conditions, including MPs pollution, and evolutionarily developed adaptation capacity of organisms for tolerance and resilience to induced stress. The study emphasizes the need for monitoring MPs and OS to better assess the ecological risk.

Fish diversity is widely recognized as a key indicator of aquatic ecosystem health and integrity but is increasingly threatened by anthropogenic pressures and biological invasions. Consequently, reliable and efficient methods for monitoring fish communities are essential for biodiversity conservation and ecosystem-based management. This pilot study represents the first application of environmental DNA (eDNA) metabarcoding to assess the taxonomic diversity of coastal fish assemblages in Varna Bay during the summer season. Information on coastal fish diversity along the Bulgarian Black Sea coast remains limited, as shallow coastal habitats are difficult to sample effectively using conventional fishing gears. In June 2025, water samples were collected from five coastal sites in Varna Bay. MiFish universal primers targeting the mitochondrial 12S rRNA gene region and the Illumina MiSeq platform were used. Raw demultiplexed sequencing reads were subjected to quality assessment using FastQC v0.12.1, and primers were removed using Cutadapt v4.0. Amplicon sequence variants (ASVs) were inferred using the DADA2 pipeline v1.32.0 in the R environment. Raw paired-end reads were quality filtered using filterAndTrim, discarding reads containing ambiguous bases (maxN=0) and reads with expected errors greater than two (maxEE=2). Reads were truncated at 160 bp (truncLen=c(160,160)) and additionally truncated when quality scores dropped below Q2 (truncQ=2); PhiX sequences were removed. Error rates were estimated from the data and ASVs were inferred using the DADA2 error-modeling algorithm. Forward and reverse reads were merged requiring a minimum overlap of 12 bp and no mismatches (minOverlap=12, maxMismatch=0). Chimeric sequences were removed using the consensus method implemented in removeBimeraDenovo. The resulting non-chimeric ASVs were used for downstream analyses. Taxonomic assignment was conducted using the BLAST+ tool (blastn, v2.16.0) with a top-hit approach against the MitoFish v2026.02 database. Sequences assigned to fish species were further verified using the NCBI BLAST web interface. A similarity threshold of ≥97% was used for taxonomic assignment. A total of 18 fish species were identified, whereas only a single species was reported in commercial catches by the Executive Agency for Fisheries and Aquaculture (EAFA) from the same area and sampling period. The detected fish assemblage included commercially important species that use the area as feeding and spawning grounds, such as European anchovy, horse mackerel, mullets, spiny dogfish, big-scale sand smelt, flathead grey mullet, golden grey mullet, and gobies. It also included non-commercial species of high ecological importance that are rarely detected by conventional monitoring methods, including blennies, pipefishes, Atlantic stargazer, and wrasses, as well as relatively rare species such as Mediterranean sand smelt and Atlantic horse mackerel. These findings underscore the need for comprehensive biodiversity baselines and long-term monitoring of coastal fish assemblages in the region. Despite its potential, several challenges (e.g., biases related to sampling, DNA extraction, amplification primers and conditions, bioinformatic analyses, reference databases, and the lack of standardization) require further improvement. However, the study demonstrate that environmental DNA (eDNA) metabarcoding is a sensitive and effective tool for assessing fish biodiversity in coastal marine ecosystems, highlighting its strong potential to support biodiversity monitoring, conservation planning, and evidence-based marine resource management.

Plastic pollution has emerged as one of the most pervasive environmental challenges, with microplastics (MPs) widely distributed across marine ecosystems worldwide. This study aimed to assess the uptake of MPs by key fish and invertebrate species from different locations in the coastal zone of the Bulgarian Black Sea. Fish were collected during routine monitoring surveys in September–November 2024, while invertebrates were obtained via scuba diving. The presence of MPs in fish stomachs and invertebrate soft tissues, and their polymer composition, shape and size were analyzed using an Agilent 8700 LDIR Chemical Imaging System. Potential biological effects of ingested MPs were evaluated by an integrated Specific Oxidative Stress (SOS) index. The results revealed MP uptake levels comparable to those reported globally. Small-sized particles (<50 µm) with rounded shapes were most abundant across studied taxa. Polymer composition varied considerably depending on species and sampling region, indicating differences in exposure sources and environmental conditions. Oxidative stress levels in both fish and invertebrates showed substantial interspecific variation, and clear differences between the northern and southern region of the Bulgarian Black Sea. Overall, elevated uptake of MPs appears to contribute to oxidative stress in marine organisms, potentially affecting their health status, resilience, and adaptive capacity, as reflected by increased SOS index values.