The main goal of the project is to evaluate the effects generated by microplastics on aquatic organisms – freshwater fish, given that laboratory investigations of the interactions of fish and microplastics are less common than those on bivalves and little knowledge on levels of microplastics in small sized freshwater fish exists. A variety of experimental designs will be used to evaluate the impacts of microplastics on freshwater fish.

OBJECTIVE 1 (WP1) 2022 – Identification and characterization screening of microplastics

Activity 1.1 – Development of analytical / screening methods for microplastics identification and characterization.

The activity involves the implementation in current laboratory practice of some methods for microplastics identification, characterization and quantification in synthetic solutions, test solutions and fish organs. Different microplastics will be tested (in accordance with their incidence in water respectively polyethylene, polystyrene, polyethylene, etc.) in different sizes (<5 mm) and density.

The qualitative and quantitative identification and characterization of microplastics will be perform using infrared spectrometry, microscopy (ex. scanning electron microscopy – SEM) and gravimetric methods.

OBJECTIVE 2 (WP2) 2023 – Assessment of ecotoxicological effects of microplastics on fish

The ecotoxicological tests will be performed on fish (Cyprinus carpio, with health and origin certificate) purchased from a recognized Romanian Fisheries Research and Development Station. The fish will be acclimatized in laboratory conditions within the Aquatic Biobase of the National Research and Development Institute for Industrial Ecology (ECOIND, Bucharest, Romania), accredited for acute toxicity studies. Fish maintenance and experimental tests will be performing in accordance with the Guide for the Use and Care of Laboratory Animals and Organization for Economic Co-operation and Development (OECD) recommendation related to the animal suffering and to reduce the number of animals used in tests. The in vivo study will be supervised by the Commission of Ethics and Professional Deontology of ECOIND.

Activity 2.1 – Assessment of acute / chronic toxicity and bioaccumulation of microplastics in fish

Acute (5 days) and chronic (60 days) tests with fish will be perform in accordance with OECD test guideline no. 203 and OECD test guideline no. 305. In acute tests different microplastics sizes and densities will be used according to environmental incidence of this contaminants in fresh water. The acute toxicity concentration (micropalstic density) lethal for 50% of tested fish (LC50) will be established base on mortality effects. Chronic tests will be performed in dynamic conditions, according to acute results obtained for the most toxic microplastics. The batch fish tested and the control batch will be monitored for the identification of behavior changes, symptoms, mortality, biometrical data, growth coefficients, food utilization and biomass. The effects of chronic exposure observed in case of tested batch will be compared with the control batch. After each exposure time, an established number of individs test fish will be sacrifice and their organs will be collected, weighing and frozen at -80°C for biochemical analyses. Also parts of organs will be used in histopathological investigations. In addition, the collected samples will be prepared for microscopy analyses using the staining in order to identify the presence of microplastics in fish tissues.

Also within this work package, will be performed analysis for the investigation of changes in the intestine microbiota of fish exposed to microplastics.

Activity 2.2 – Biochemical analyses to assess the enzymatic activities involve in anti-stress mechanisms, reproduction and nervous systems.

Microplastics are emerging persistent contaminants and for these reason we expected to find these particles in unchanged forms blocking some physiological processes such as respiration and digestive / excretion function or reproduction and nervous systems. In this way will be analyzed: 1) Hepatosomatic and transaminases activity for liver metabolism; 2) The antioxidant proteins expression (enzyme involve in oxidative stress process) such as Superoxide dismutase, Catalase, Glutathion reductase, Glutathione peroxidase, Lactate dehydrogenase, Glucozo-6-Phosphat dehydrogenase, etc. and lipid peroxidation (using malondialdehyde control); 3) Gonadosomatic index and vittelogenin proteins will be assess to evaluate the effects on fish reproduction system; 4) Acetylcholinesterase inhibition will be assessing for the microplastics action on nervous system; 5) The upregulation of enzyme ethoxyresorufin-O-deethylase (EROD) wil be analyzed for the rapidly measure the of this protein in response to exposure to microplastics using a fish EROD antibody. All enzymatic methods are spectrometric techniques using enzymatic kinetic protocol for the calculation of enzymatic activities. All of the enzymatic activities will be normalized to protein concentration in order to be expressed in terms of Units of activity/mg of protein and as percentage from control. The analyzes will be performed for more than 3-5 individuals in replicates for statistical changes differences.

Activity 2.3 – The evaluation of biomarkers expression at translational level induced by microplastics using molecular techniques

The stress induced by microplastics on aquatic organisms (fish) will be evaluated by proteins expression. The separation of proteins based on their molecular weight will be done by SDS-PAGE electrophoresis. Post-translational modifications of proteins can lead to a different relative mobility (i.e. a band shift).

OBJECTIVE 3 (WP3) 2024 Mechanisms of action of microplastics in fish organs

Activity 3.1 – Identification and characterization of histopathological changes in fish organs exposed to microplastics. 

The histological changes induced by microplastics exposure of Cyprinus carpio will be evaluated through hematoxylin & eosin staining. The organs tissues (liver, gill, kidney, intestine, gonad, etc.) of each individual contaminated fish will be microscopic investigate for the incidence of structural changes specific for each type of organ. The observations will be quantified and statistically evaluated.

Activity 3.2 –  Dissemination of relevant results

Dissemination and increasing visibility will be the main purposes of this activity, thus all means of communication, dissemination or further exploitation will be used. We propose 2 scientific publications in journals indexed in international databases (Q1 / Q2 category); 3 scientific public / academic communications. Dissemination will take into account intellectual property and ethical issues.

WP1

Results of Phase I – 2022

Phase 1/2022 was completed by a study, respectively "Analytical study / screening methods for the identification and characterization of microplastics" which implicitly lead to the preparation of the Interim Report 1.

1 communication / BDI paper: MICROPLASTICS – ECOTOXICOLOGICAL EFFECTS AND MECHANISMS OF ACTION IN CYPRINUS CARPIO FISH (MICROPLASFISH), Stefania Gheorghe, Catalina Stoica, Alina Banciu, Alina Constantin, Anda Tenea, Dorian Neidoni - poster, at the International Symposium "Environment and Industry" organized by INCD ECOND Bucharest on 29.09.2022, https://www.simiecoind.ro/wp-content/uploads/2022/09/RJEEC-9.-Gheorghe-S.-Poster-SIMI-2022.pdf ;

1 Editorial "MICROPLASTICS - ECOTOXICOLOGICAL EFFECTS AND MECHANISMS OF ACTION IN CYPRINUS CARPIO FISH (MICROPLASFISH)" - Stefania Gheorghe, Catalina Stoica, Alina Banciu, Alina Constantin, Anda Tenea, Dorian Neidoni in the Romanian Journal of Ecology & Environmental Chemistry (RJEEC), https://www.incdecoind.ro/wp-content/uploads/2022/11/Editorial-MicroPlasFish-Gheorghe-Stefania.pdf;

Project web page: https://www.incdecoind.ro/en/microplastice-efecte-ecotoxicologice-si-mecanisme-de-actiune-in-pesti-microplasfish

Other displays of the project in specialized networks:

Researchgate: https://www.researchgate.net/publication/365196585_Microplastics_-Ecotoxicological_effects_and_mechanisms_of_action_in_Cyprinus_carpio_fish_MicroPlasFish

Brainmap: https://www.brainmap.ro/public-profile-82877716

WP2

Phase 2/2023 was completed by the next studies:

• Study concerning the ecotoxicity of microplastics
• Study on impact of microplastics on the antioxidant enzymatic system
• Study on biomarkers expression at translational level induced by microplastics

In this phase the next dissemination was perform:

1. Gheorghe, S.; Stoica, C.; Harabagiu, A.M.; Neidoni, D.; Mighiu, E.D.; Bumbac, C.; Ionescu, I.A.; Pantazi, A.; Enache, L.B.; Enachescu, M. Laboratory assessment for determining microplastics in freshwater systems – characterization and identification along Somesul Mic River. https://www.preprints.org/manuscript/202311.1467/v1, under review at Water (ISI 3.4 – Q2).
2. Stefania Gheorghe, Catalina Stoica, Anca Maria Harabagiu, Nile red staining of polyethylene and polystyrene in Daphnia magna tests, Romanian Journal of Ecology & Environmental Chemistry, ISSN-L: 2668-5418 – accepted for publication.
3. Gheorghe Stefania, Catalina Stoica, Neidoni Dorian, Bumbac Costel, Harabagiu Anca-Maria, Mighiu Emanuel Daniel, Microplastic pollution from the urban rivers: sampling, isolation, characterization and identification, 10th International Conference on Environmental Management, Engineering, Planning and Economics (CEMEPE) & SECOTOX Conference, Greece 05- 09.06. 2023 – oral presentation.
4. Stefania Gheorghe, Catalina Stoica, Anca Maria Harabagiu, Nile red staining of polyethylene and polystyrene in Daphnia magna tests, International Symposium” The Environment and the industry” – poster, https://www.simiecoind.ro/wp-content/uploads/2023/10/RJEEC-1.-Gheorghe-S.pdf.
5. Catalina Stoica, Gheorghe Stefania, Neidoni Dorian, Bumbac Costel, Occurrence of microplastics in freshwater systems: visual identification methods, 10th International Conference on Environmental Management, Engineering, Planning and Economics (CEMEPE) & SECOTOX Conference, Greece 05- 09.06. 2023 –poster;
6. G. Tenea, C. Dinu, F.L. Chiriac, G.G. Vasile, S. Gheorghe, G.V. Serban, L.E. Barbulescu, Adsorption mechanism of Cadmium on polypropylene microplastics in synthetic solutions, ICCE 2023 Venetia – poster.

WP3

Phase 2/2024 was completed by the next study:

• Identification and characterization of histopathological changes in fish organs exposed to microplastics

In this phase the next dissemination was perform:

1. Anda-Gabriela Tenea, Cristina Dinu, Paul Alexandru Rus, Ioana Alexandra Ionescu, Stefania Gheorghe, Vasile Ion Iancu, Gabriela Geanina Vasile, Luoana Florentina Pascu, Florentina Laura Chiriac,  Exploring adsorption dynamics of heavy metals onto varied commercial microplastic substrates: isothermal models and kinetics analysis, Heliyon-D-24-07014,  https://doi.org/10.1016/j.heliyon.2024.e35364.

2. Stefania Gheorghe, Metode calitative de determinare a micoplasticelor in probe de apa de suprafata, Editorial Revista Magurele nr. 9, pp.37, iunie 2024. https://magurelesciencepark.ro/wp-content/uploads/2024/06/20.06.MSP-9-digital.pdf

3. Gheorghe Stefania, Stoica Catalina, Anca Maria Harabagiu, Ecotoxicological effects of polystyrene particles on Cyprinus carpio – laboratory assessment, The 1^st International Electronic Conference on Toxics 20-22 martie 2024 (prezentare - on-line) 2c9c5ed178a0d41d28392d4795c182b7.pdf (sciforum.net) --> Certificate of Participation
4. Stefania Gheorghe, Stoica Catalina, Anca Maria Harabagiu, Laura Novac, Alina Banciu, Dorian Neidoni, Effects of Chronic Exposure of Polyethylene on Common Carp (Cyprinus Carpio) and the Impact on Enzymatic Biomarkers, 38th BARCELONA International Conference on “Chemical, Biological and Environmental Sciences” (BCBES-24) April 24-26, 2024 Barcelona (Spain) – poster
5. Catalina Stoica, Stefania Gheorghe, Alina Banciu, Mihai Nita-Lazar, Monitoring the microplastics impacts on Cyprinus carpio by biomarkers expression at translational level, 38th BARCELONA International Conference on “Chemical, Biological and Environmental Sciences” (BCBES-24) April 24-26, 2024 Barcelona (Spain) – poster

Prize:

Poster Best Paper  - Catalina Stoica, Monitoring the microplastics impacts on Cyprinus carpio by biomarkers expression at translational level, 38th BARCELONA International Conference on “Chemical, Biological and Environmental Sciences” (BCBES-24) April 24-26, 2024 Barcelona (Spain)