Clam Enzymes Reveal Hidden Pollution Stress Along the Bulgarian Black Sea Coast

On the sandy shallows of the Bulgarian Black Sea coast, a small bivalve is quietly keeping score of environmental degradation. New research published in the journal BioRisk shows that the wedge clam Donax trunculus, one of the most common inhabitants of the region’s shallow sandy seabed, carries in its soft tissues a detailed biochemical record of the pollution and seasonal pressures acting on its habitat. By measuring the activity of antioxidant enzymes and the concentration of glutathione in clam tissues collected from nine coastal localities, researchers found clear, measurable differences between sites — differences that track tourism intensity, proximity to settlements, and other local environmental pressures.

The study focused on oxidative stress, a fundamental cellular phenomenon that occurs when the balance between reactive oxygen species and the molecules that neutralize them tips toward damage. Pollutants such as heavy metals, polycyclic aromatic hydrocarbons, microplastics, and pesticides — all of which enter the marine environment through industrial and municipal wastewater, agricultural runoff, tourism, and atmospheric deposition — stimulate the production of reactive oxygen species in living cells. Aerobic organisms respond by activating an antioxidant defense system, a suite of enzymes and small molecules that intercept and neutralize these reactive species before they can damage proteins, lipids, and DNA. When that defense falters, cellular impairment follows, with consequences that can cascade up to the level of whole ecosystems.

Because bivalves are filter feeders that bioaccumulate and concentrate pollutants in their tissues, they serve as natural sentinels for marine contamination. Comparative studies have shown that bivalves possess a highly active antioxidant enzyme complex and a substantially larger pool of non-enzymatic antioxidants than vertebrates, making them particularly informative subjects for biomonitoring. The wedge clam is especially well suited to this role in the Black Sea: it lives in the shallow sandy sediments where most pollutants are deposited, and it forms a basic component of the coastal food web, so its physiological condition has direct implications for ecosystem health.

The research team sampled adult clams, measuring 23 to 35 millimeters in shell length, from their natural sandy habitats along the Bulgarian coast in two seasons — June and September of 2020. Samples were gathered manually or obtained from commercial providers and stored at minus 80 degrees Celsius until analysis. In the laboratory, soft tissues were homogenized in phosphate buffer and centrifuged to obtain fractions suitable for biochemical assay. The researchers then measured the activity of four key antioxidant enzymes — catalase, superoxide dismutase, glutathione peroxidase, and glutathione-S-transferase — along with the concentration of total glutathione, the non-enzymatic antioxidant that serves as a co-substrate for several of these enzymes.

The results revealed a striking pattern of spatial and seasonal variability. In summer samples, glutathione levels and the activities of superoxide dismutase and catalase showed no statistically significant differences among localities, but glutathione peroxidase and glutathione-S-transferase activities did. The lowest glutathione peroxidase activity was measured in clams from Shkorpilovtsi, while the highest glutathione-S-transferase activity appeared in samples from Slanchev Bryag — a resort locality experiencing intensive tourism — with the lowest values recorded at Ahtopol. In autumn, the overall picture shifted: glutathione, superoxide dismutase, and glutathione peroxidase values were generally higher than in summer, a rise the authors attribute to the activation of the clams’ antioxidant defenses after the summer tourist season, when pollution loads at heavily visited sites are greatest.

The glutathione-S-transferase finding is particularly significant. This enzyme is the major phase II detoxification enzyme, catalyzing the conjugation of xenobiotic compounds with glutathione to make them more water-soluble and easier to eliminate from the body. It also contributes to antioxidant protection through peroxidase activity and can regenerate proteins that have been modified by oxidative stress. Previous studies have documented increased glutathione-S-transferase activity and expression in bivalves exposed to polycyclic aromatic hydrocarbons, polychlorinated biphenyls, DDT, dioxins, metals, and microplastics. Because the enzyme shows strong positive correlations with toxicant burdens and relatively low seasonal variability, it is widely regarded as a reliable biomarker of pollutant exposure — and indeed, in this study, no seasonal changes in its activity were found at any single locality.

Beyond enzyme activities, the researchers calculated a condition index for the clams — the ratio of dry soft tissue weight to shell length — as an indicator of individual physiological health. Summer samples showed relatively similar condition indices across localities, with significantly lower values at Slanchev Bryag and Dyuni. Autumn samples were more heterogeneous: clams from Ahtopol and Kabakum had markedly lower condition indices, while the remaining autumn localities scored significantly higher than all summer sites. A factorial analysis of variance showed that locality alone had a highly significant effect on condition index, whereas season alone did not — but the interaction between the two factors was highly significant. In other words, the state of the environment at a particular site plays the leading role in clam health, with seasonal effects subordinate to local conditions.

Multiple regression analysis added a further layer of insight. When the condition index was treated as the dependent variable and the five antioxidant measures as predictors, only catalase and glutathione-S-transferase emerged as statistically significant predictors of clam health. This suggests that these two enzymes could serve as practical early-warning indicators of environmental risk for wedge clam populations, allowing researchers to detect stress before it manifests as measurable declines in tissue condition or population health.

The study’s broader message is one of cautious reassurance mixed with clear warning. The elevated enzyme activities observed across the sampled localities indicate that the wedge clams’ antioxidant protection systems were activated in response to environmental pressure — evidence that the animals were successfully coping with induced oxidative stress and adapting to local conditions, thereby helping to maintain ecosystem health. At the same time, the combination of high detoxification enzyme activity and low condition index at tourism-intensive sites such as Slanchev Bryag points to significant biological risk for marine life in those areas. The authors emphasize that further work is needed to establish antioxidant enzymes as fully reliable bioindicators of ecosystem change, but the present findings mark an important step toward using the humble wedge clam as a living gauge of coastal environmental quality along the Black Sea.

Subject of Research: Antioxidant defense biomarkers in wedge clams as indicators of environmental stress on the Bulgarian Black Sea coast

Article Title: State of the antioxidant defense system in wedge clams from Bulgarian Black Sea as a measure of resistance to environmental impacts

Article References: Georgieva, A., Tsvetanova, E., Chipev, N., & Alexandrova, A. (2022). State of the antioxidant defense system in wedge clams from Bulgarian Black Sea as a measure of resistance to environmental impacts. BioRisk, 17, 169-178. https://doi.org/10.3897/biorisk.17.77384

Image Credits: AI Generated

DOI: 10.3897/biorisk.17.77384

Keywords: wedge clam, Donax trunculus, oxidative stress, antioxidant enzymes, biomarkers, Black Sea, marine pollution, glutathione-S-transferase, biomonitoring, condition index, ecosystem health, bivalves