Evaluation the toxic effect of copper ions on the condition indices of benthic diatom Actinocyclus subtilis (W.Gregory) Ralfs 1861 in the experiment

Introduction. Pollution of marine coastal areas lead to the relevance of environmental monitoring including application of biotesting methods based on- the cultures of unicellular algae. Microalgae have different species-specific resistance to pollutants that expands application of different species as bioindicators of marine pollution.

The aim of the study was to determine the threshold concentration of copper ions (Cu²⁺) for the survival and increase in the cells number of benthic diatom Actinocyclus subtilis (W.Gregory) Ralfs 1861 (Bacillariophyta) under the wide range of toxicant concentrations during 10-day toxicological experiments.

Material and methods. The response of strain culture of the benthic diatom A. subtilis to various concentrations of copper sulfate (ranged from 16 to 1024 μg/l in terms of Cu²⁺ ions) was studied. In accordance with the previously developed protocol, the following indices were evaluated: alterations in the absolute number and proportion (%) of alive cells in the test-culture, as well as the specific growth rate in the number of A. subtilis cells at different concentrations of toxicant. Counting of alive and dead cells was carried out by micrographs taken for 12–15 random viewing fields under Nikon Eclipse inverted light microscope.

Results. It was found that in the control and at concentration of copper ions 16 μg/l, the increase in the absolute number of cells in culture is described by sigmoid response curve. At the control еhe exponential growth phase occurs on days 5–7 and at concentration of 16 µg/l on days 3–5 of the experiment. The threshold concentration of copper ions (32 μg/l) which is critical for the survival of A. subtilis was determined, which is 3–7 times lower than threshold level for other benthic diatom species. At concentration of 32 µg/l, the phases of acceleration and exponential growth on the abundance curve are absent. The proportion of living cells in the culture decreases to 80% of the control level on day 3 and to 39% by day 10. At Cu²⁺ concentrations of 64 µg/l and above, sharp inhibition and death of culture is observed as early as 1–3 days. A positive specific growth rate of A. subtilis culture was revealed in the period of 1–5 days at copper concentration of 16 and 32 µg/l, and at concentration of 64 µg/l and higher the culture dies off. Negative values of the specific growth rate for all concentrations of the toxicant within the period of 5–10 days were obtained.

Limitations. By the results of 10-day experiments the effect of 8 concentrations of copper sulfate on the culture of marine benthic diatom A. subtilis was studied. Three replicates in each concentration and exposure time were measured (1350 measurements in total), which is sufficient sampling for statistically reliable determination of the threshold values of copper ion toxicity for given test object.

Conclusion. Considering the results obtained, the benthic diatom A. subtilis is highly sensitive to copper ions impact and can be recommended as new test-object for toxicology, as well as for application in monitoring of marine water areas subject to technogenic pollution.

Compliance with ethical standards. The study does not require the decision of biomedical ethics committee or other documents, since all experiments were carried out on common unicellular non-toxic algae, which does not violate any prohibitions associated with damage to the ecological environment, the living space of bio-communities, and also does not lead to irreversible changes in the biological (genetic) nature and human health.

Contribution of the authors. All authors confirm that their authorship complies with the international ICMJE criteria (all authors made a significant contribution to the development of concept, research and preparation of the article, read and approved its final version before publication).

Gratitude. The authors express their gratitude to S.A. Trofimov and Yu.I. Litvin for their help in maintaining clone cultures and conducting experiments, as well as to V.N. Lishaev for microphotography on the Hitachi SU3500 SEM.

Conflict of interests. The authors declare no conflict of interests.

Acknowledgements. This study was carried out as a part of the State Assignment No. 121030100028-02 of the A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS in Benthos Ecology Dept.

Received: June 9, 2023 / Accepted: October 19, 2023 / Published: October 30, 2023