Ecological Safety of Coastal and Shelf Zones of Sea Ecological Safety of Coastal and Shelf Zones of Sea 2413-5577 20230110 Research Article Effect of Bicomponent ZnO-ZnFe₂O₄ Nanoparticles on Mediterranean Mussel (Mytilus galloprovincialis) Hemocytes under in vitro Conditions Podolskaya M. S. podolskaya_m99@bk.ru Tkachuk A. A. aatkachuk86@gmail.com Andreyeva A. Yu. andreevaal@gmail.com Kladchenko E. S. Kladchenko_Ekaterina@bk.ru Chelebieva E. S. elina.chelebieva@gmail.com Mosunov A. A. aamosunov@sevsu.ru A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS Sevastopol Russia Sevastopol State University Sevastopol Russia 24 03 2023 2023 1 124 136 09 08 2022 01 02 2023 05 12 2022 Copyright © 2023, M. S. Podolskaya, A. A. Tkachuk, A. Yu. Andreyeva, E. S. Kladchenko, E. S. Chelebieva, A. A. Mosunov 2023 M. S. Podolskaya, A. A. Tkachuk, A. Yu. Andreyeva, E. S. Kladchenko, E. S. Chelebieva, A. A. Mosunov https://creativecommons.org/licenses/by-nc/4.0/ https://ecological-safety.ru/en/repository/issues/2023/01/10/

The present work investigates the toxic effect of bicomponent ZnO-ZnFe₂O₄ nanoparticles, which are the main active component of the domestic antifouling coating, on marker indicators of Mediterranean mussel (Mytilus galloprovincialis) hemolymph cells (hemocytes) under in vitro experimental conditions. The following indicators were evaluated: mortality, cellular composition and production of reactive oxygen species. In the experiment, hemocytes were incubated for 1 and 2 hours in 1 mL of sterile seawater containing nanoparticles of different concentrations: 0.03, 0.3 and 3 mg/mL. The data were analyzed using the flowing cytometry. It was shown that ZnО-ZnFe₂O₄ nanoparticles had an effect on the cellular composition of the hemolymph: the proportion of agranulocytes decreased and hour exposure to 0.03 mg/mL nanoparticles reduced the level of production of reactive oxygen species by 2.5 times compared to the control (p ≤ 0.05). Incubation of hemocytes with a maximum concentration of nanoparticles (3 mg/mL) led to cell death within 1 hour after exposure. No acute toxic effects on hemocytes with the use of 0.03 mg/mL and 0.3 mg/mL of zinc oxide and zinc ferrite nanoparticles were observed.

 nanoparticles Mediterranean mussel hemocytes reactive oxygen species The synthesis of nanoparticles (ZnO-ZnFe₂O₄) was carried out under RSF project no. 21-13-00498 "Environmentally safe and highly effective antifouling coatings based on bicomponent metal nanoparticles and their oxides". Assessment of the toxicity of nanoparticles on the body of mussels (analysis of hemocyte parameters) was carried out at the expense of the state task of FRC IBSS No. 121102500161-4 "Patterns of the organization of the immune system of commercial hydrobionts and the study of the influence of environmental factors on the functioning of their protective systems".

About the authors:

Maria S. Podolskaya, Acting Junior Research Associate, A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS (2 Nakhimov Av., Sevastopol, 299011, Russian Federation), ORCID ID: 0000-0001-8185-3985, podolskaya_m99@bk.ru

Anastasia A. Tkachuk, Junior Research Associate, A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS (2 Nakhimov Av., Sevastopol, 299011, Russian Federation), ORCID ID: 0000-0002-4017-7164, aatkachuk86@gmail.com

Alexandra Yu. Andreyeva, Head of the Laboratory of Ecological Immunology of Hydrobionts, A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS (2 Nakhimov Av., Sevastopol, 299011, Russian Federation), Ph.D. (Biol.), ORCID ID: 0000-0001-7845-0165, ResearcherID: Z-6151-2019, Scopus AuthorID: 57191916535, andreevaal@gmail.com

Ekaterina S. Kladchenko, Research Associate, A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS (2 Nakhimov Av., Sevastopol, 299011, Russian Federation), ORCID ID: 0000-0001-9476-6573, ResearcherID: U-7749-2019, Scopus AuthorID: 57205560752, Kladchenko_Ekaterina@bk.ru

Elina S. Chelebieva, Senior Research Associate, A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS (2 Nakhimov Av., Sevastopol, 299011, Russian Federation), Ph.D. (Biol.), ORCID ID: 0000-0002-7662-2573, ResearcherID: H-3359-2014, Scopus AuthorID: 56549997100, elina.chelebieva@gmail.com

Andrey A. Mosunov, Leading Research Associate, Sevastopol State University (33 Universitetskaya St., Sevastopol, 299053, Russian Federation), Ph.D. (Phys.-Math.), ORCID ID: 0000-0002-1390-5832, ResearcherID: R-5570-2016, Scopus AuthorID: 36802573200, aamosunov@sevsu.ru

Contribution of the authors:

Maria S. Podolskaya – preparation of the text of the article, conducting experimental studies, qualitative data analysis and interpretation, processing and description of the study results, preparation of graphic materials, review of literature on the study topic

Anastasia A. Tkachuk – formalized data analysis, literature review on the study topic, conducting experimental studies, text editing

Alexandra Y. Andreyeva – formulation and research task statement, development of the concept, development of methods for conducting experimental studies, formulation of conclusions, editing of the text

Ekaterina S. Kladchenko – preparation of graphic materials, conducting experimental studies, editing text

Elina S. Chelebieva – conducting experimental studies, text editing

Andrey A. Mosunov – synthesis of nanoparticles, text editing

All the authors have read and approved the final manuscript.

Article text not included.

REFERENCES ZvyagintsevA.Y. PoltarukhaO.P. MaslennikovS.I. Fouling on Technical Water Supply Marine Systems and Protection Method Analysis of Fouling on Water Conduits (Analytical Review) Water: Chemistry and Ecology 2015 1 30 51 (in Russian) AbacharaevM.M. AbacharaevI.M. Perspective Developments on Marine Fouling Control Vestnik of Astrakhan State Technical University. Series: Marine engineering and technologies 2011 3 7 9 (in Russian) ZvyagintsevA.Yu. Marine Fouling in the North-West Part of Pacific Ocean Vladivostok Dalnauka 2005 432 (in Russian) NurioğluA.G. EstevesA.C.C. de WithG. Non-Toxic, Non-Biocide-Release Antifouling Coatings Based on Molecular Structure Design for Marine Applications Journal of Materials Chemistry B 2015 3 32 6547 6570 10.1039/C5TB00232J Castristi-CathariosJ. AlambritisG. MiliouH. CotouE. ZouganelisG. Comparative Toxicity of "Tin Free" Self-Polishing Copolymer Antifouling Paints and Their Inhibitor Effects on Larval Development of a Non-Target Organism Materials Sciences and Applications 2014 5 158 169 10.4236/msa.2014.53026 MeadorJ.P. Predicting the Fate and Effects of Tributyltin in Marine Systems Reviews of Environmental Contamination and Toxicology New York Springer 2000 166 1 48 LeggM. YücelM.K. Garcia de CarellanI. KappatosV. SelcukC. GanT.H. Acoustic Methods for Biofouling Control: A Review Ocean Engineering 2015 103 237 247 10.1016/j.oceaneng.2015.04.070 ShanmugasundaramT. RadhakrishnanM. GopikrishnanV. PazhanimuruganR. BalagurunathanR. A Study of the Bactericidal, Anti-Biofouling, Cytotoxic and Antioxidant Properties of Actinobacterially Synthesised Silver Nanoparticles Colloids and Surfaces B: Biointerfaces 2013 111 680 687 10.1016/j.colsurfb.2013.06.045 LozhkomoevA.S. BakinaO.V. GlazkovaE.A. SvarovskayaN.V. LernerM.I. Patterns of the Formation of Antimicrobial Micro/Nanocomposites during the Oxidation of Bimetallic Al/Zn Nanoparticles Russian Journal of Physical Chemistry A 2018 92 12 2530 2534 10.1134/S0036024418120270 PalzaH. Antimicrobial Polymers with Metal Nanoparticles International Journal of Molecular Sciences 2015 16 1 2099 2116 10.3390/ijms16012099 HoshiyarN. GrayS. HanH. BaoG. The Effect of Nanoparticle Size on in vivo Pharmacokinetics and Cellular Interaction Nanomedicine 2016 11 6 673 692 10.2217/nnm.16.5 HuW. CullotyS. DarmodyG. LynchS. DavenportJ. Ramirez-GarciaS. DawsonK.A. LynchI. BlascoJ. Toxicity of Copper Oxide Nanoparticles in the Blue Mussel, Mytilus edulis: a Redox Proteomic Investigation Chemosphere 2014 108 289 299 10.1016/j.chemosphere.2014.01.054 XiaT. KovochichM. BrantJ. HotzeM. SenpfJ. OberleyT. SioutasC. YehJ.I. WiesnerA.E. Comparison of the Abilities of Ambient and Manufactured Nanoparticles to Induce Cellular Toxicity according to an Oxidative Stress Paradigm Nano Letters 2006 6 8 1794 1807 10.1021/nl061025k HsinY.-H. ChenC.-F. HuangS. ShihT.-S. LaiP.-S. ChuehP.J. The Apoptotic Effect of Nanosilver is Mediated by a ROS-and JNK-Dependent Mechanism Involving the Mitochondrial Pathway in NIH3T3 Cells Toxicology Letters 2008 179 3 130 139 10.1016/j.toxlet.2008.04.015 SvarovskayaN.V. BakinaO.V. GlazkovaE.A. LernerM.I. LozhkomoevA.S. SerovaA.N. KhorobrayaE.G. Evaluation of the Toxicity of Nanostructural Aluminium Oxihydroxide with the Help of Hydrobions Svarovskaya Chemistry for Sustainable Development 2013 21 4 411 414 ZhuX. WangJ. ZhangX. ChangY. ChenY. Trophic Transfer of TiO₂ Nanoparticles from Daphnia to Zebrafish in a Simplified Freshwater Food Chain Chemosphere 2010 79 9 928 933 10.1016/j.chemosphere.2010.03.022 MillerR.J. LenihanH.S. MullerE.B. TsengN. HannaS.K. KellerA.A. Impacts of Metal Oxide Nanoparticles on Marine Phytoplankton Environmental Science and Technology 2010 44 19 7329 7334 10.1021/es100247x CongY. JinF. WangJ. MuJ. The Embryotoxicity of ZnO Nanoparticles to Marine Medaka, Oryzias melastigma Aquatic Toxicology 2017 185 11 18 10.1016/j.aquatox.2017.01.006 AllahverdiyevA.M. AbamorE.S. BagirovaM. RafailovichM. Antimicrobial Effects of TiO₂ and Ag₂O Nanoparticles against Drug-Resistant Bacteria and Leishmania Parasites Future Microbiology 2011 6 8 933 940 10.2217/fmb.11.78 MosunovA.A. EvstigneevV.P. Nanoparticles in Marine Antifouling Coatings: a Case Study Journal of Physics: Conference Series 2021 2094 2 022041 10.1088/1742-6596/2094/2/022041 BakinaO. GlazkovaE. RodkevichN. MosunovA. ChzhouV. LernerM. Electroexplosive Synthesis of Composite ZnO/ZnFe₂O₄/Zn Nanoparticles with Photocatalytic and Antibacterial Activity Materials Science in Semiconductor Processing 2022 152 107076 10.1016/j.mssp.2022.107076 CanesiL. CiacciC. BettiM. FabbriR. CanonicoB. FantinatiA. MarcominiA. PojanaG. Immunotoxicity of Carbon Black Nanoparticles to Blue Mussel Hemocytes Environment International 2008 34 8 1114 1119 10.1016/j.envint.2008.04.002 AndreyevaA.Y. KladchenkoE.S. KukharevaT.A. SakhonE.G. Analysis of Cell Cycle and Morphological and Functional Abnormalities of Mytilus galloprovincialis Lam., 1819 (Bivalvia) Hemocytes from Coastal Ecosystems near Sevastopol, Crimea Inland Water Biology 2019 12 2 96 103 10.1134/S1995082919060038 CanesiL. GalloG. GavioliM. PruzzoC. Bacteria-Hemocyte Interactions and Phagocytosis in Marine Bivalves Microscopy Research and Technique 2002 57 6 469 476 10.1002/jemt.10100 KoutsogiannakiS. KaloyianniM. Signaling Molecules Involved in Immune Responses in Mussels Invertebrate Survival Journal 2010 7 1 11 21 TiscarP.G. MoscaF. Defense Mechanisms in Farmed Marine Molluscs Veterinary Research Communications 2004 28 suppl. 1 57 62 10.1023/B:VERC.0000045379.78547.23 JovanovićB. PalićD. Immunotoxicology of Non-Functionalized Engineered Nanoparticles in Aquatic Organisms with Special Emphasis on Fish-Review of Current Knowledge, Gap Identification, and Call for Further Research Aquatic Toxicology 2012 118 141 151 10.1016/j.aquatox.2012.04.005 ChalevT.E.A. GallowayJ.F. GraczykT.K. Pilot Study on Effects of Nanoparticle Exposure on Crassostrea virginica Hemocyte Phagocytosis Marine Pollution Bulletin 2012 64 10 2251 2253 10.1016/j.marpolbul.2012.06.026 DayemA.A. HossainM.K. LeeS.B. KimK. SahaS.K. YangG.-M. ChoiH.Y. ChoS.-G. The Role of Reactive Oxygen Species (ROS) in the Biological Activities of Metallic Nanoparticles International Journal of Molecular Sciences 2017 18 1 120 10.3390/ijms18010120 SokolovaI.M. Energy-Limited Tolerance to Stress as a Conceptual Framework to Integrate the Effects of Multiple Stressors Integrative and Comparative Biology 2013 53 4 597 608 10.1093/icb/ict028 BarmoC. CiacciC. CanonicoB. FabbriR. CorteseK. BalbiT. MarcominiA. PojanaG. GalloG. In Vivo Effects of n-TiO₂ on Digestive Gland and Immune Function of the Marine Bivalve Mytilus galloprovincialis Aquatic Toxicology 2013 132-133 9 18 10.1016/j.aquatox.2013.01.014 DonaghyL. LambertC. ChoiK.-S. SoudantP. Hemocytes of the Carpet Shell Clam (Ruditapes decussatus) and the Manila Clam (Ruditapes philippinarum): Current Knowledge and Future Prospects Aquaculture 2009 297 1-4 10 24 10.1016/j.aquaculture.2009.09.003 StarkovA.A. The Role of Mitochondria in Reactive Oxygen Species Metabolism and Signaling Annals of the New York Academy of Sciences 2008 1147 1 37 52 10.1196/annals.1427.015 WangT. HuangX. JiangX. HuM. HuangW. WangY. Differential in vivo Hemocyte Responses to Nano Titanium Dioxide in Mussels: Effects of Particle Size Aquatic Toxicology 2019 212 28 36 10.1016/j.aquatox.2019.04.012 DaviesL.C. RiceC.M. McVicarD.W. WeissJ.M. Diversity and Environmental Adaptation of Phagocytic Cell Metabolism Journal of Leukocyte Biology 2019 105 1 37 48 10.1002/JLB.4R016518-195R CanesiL. CiacciC. GalloG. MarcominiA. PojanaG. In vitro Effects of Suspensions of Selected Nanoparticles (C60 fullerene, TiO₂, SiO₂) on Mytilus Hemocytes Aquatic Toxicology 2010 96 2 151 158 10.1016/j.aquatox.2009.10.017