Romania 2022 CURE Details

Students will be assigned one of two CUREs related to water issues affecting the Danube Delta. Course-based Undergraduate Research Experiences (CUREs) are designed to engage an entire class in a research question within the context of the course itself.

Each CURE is described in detail on this page.

Land Use Patterns and Nutrient Pollution in Aquatic Ecosystems of the Danube Delta

(Dr. Mihaela Verga, Dr. Irina Zarafu, Drf. Andreea Andra-Toparceanu, Dr. Andreea Marin, Dr. Sorin Avramescu, Dr. Petre Ionita, Dr. Carmen Postolache, Dr. Florina Botez, Drd. Maria Iasmina Moza)

Introduction: Large River deltas are characterized by complicated land use/land cover (LULC) dynamics driven by intertwined natural and anthropogenic processes. Understanding spatio-temporal LULC dynamics associated with multiple drivers can help mitigate the adverse ecological impacts resulting from human activities (Ma et al., 2019) since more than 80 million people are directly impacting the Danube River along its course (Gasparotti, 2014) and the anthropic pressures encompass agriculture, industry, mining and forestry. As an interface between marine, freshwater, and terrestrial ecosystems, Danube Delta is facing many degradation risks, such as wetland shrinking, environmental pollution and coastal erosion (Gâștescu, 1993, Gomez-Baggethun et al., 2019)  and Danube Basin is under continuous pressure from nutrient pollution being the primary tributary of Black Sea pollution (Galatchi and Tudor, 2006). All these highly impact Danube’s ecological status.

Study sites: Shallow upstream lakes within the Danube Delta or Small Braila Island

Aim: Assess land use patterns and their environmental impact (according to the Water Frame Directive guidelines) of specific water bodies, and quantify aqueous and sediment-based nutrients, especially phosphorus and nitrogenous forms.

Objectives: Analyze the interactions and relationships between human activities and the environment to identify and delineate different land use (LU) and land cover (LC) categories and patterns within the Danube Delta over the last three decades; to determine the shift in LU-LC categories through spatial comparison of the LU-LC maps produced; and to analyse water samples from shallow lakes of the Danube Delta and/or upstream Delta (Small Braila Island) in order to compare the aquatic ecosystems in term of P, N, and C forms and establish the nutrient enrichment status in relation with the human activities and land use pattern versus the buffer capacity of the Delta in reduction of nutrient pollution.

Methods: Field inventory and field mapping; collection and processing of statistical data using official documents (e.g., The National Cadaster Register, the Cadastral Map of Romania, the Statistic Yearbook of Tulcea County which certifies the LU, National Corine Land Cover Database-CLC database); images and surveys (e.g., The LU / LC area frame statistical survey, abbreviated LUCAS, a European field survey program that provides harmonized and comparable statistics on LU and LC across the whole of EU`s territory); LU-LC changes/mapping and analysis; water and sediment sampling methods; in situ measurements (using a multiparameter device able to measure pH, oxygen, conductivity, and temperature); sampling design, storage, and labelling; sampling extraction and analysis; database organization; data interpretation and visualization; ecosystems ecological status classification.

Skills: Field organization; LU and LC survey and sampling design; mapping techniques using GIS to generate landscape metrics from spatial data; experimental design; proper equipment use and handling (sediment corer, water sampling device, filtering system, multiparameter analyser); reagents preparation; data analysis including statistical evaluation; scientific reporting; and oral presentation.

Possible partners: University of Bucharest – Sfantu Gheorghe field station; ARBDD (The Danube Delta Biosphere Reserve Authority); Tulcea County Council; local municipalities; INS (National Institute of Statistics); ICPA (National Institute of Research-Development for Pedology, Agrochemistry and Environmental Protection); Institute of Biology - Bucharest (Sulina field station); National Institute for Danube Delta Research – Tulcea; National Institute of Research and Development for Biological Sciences– Bucharest; University of Bucharest - Braila field station.

CURE 1 References:

1. Galatchi, L.D. & Tudor, M., 2006. Europe as a source of pollution–the main factor for the eutrophication of the Danube Delta and Black Sea. In Chemicals as Intentional and Accidental Global Environmental Threats (pp. 57-63). Springer, Dordrecht.

2. Gasparotti, C., 2014. The main factors of water pollution in Danube River basin. Euro Economica, 33(01), pp.91-106.

3. Gâștescu, P., 1993. The Danube Delta: Geographical characteristics and ecological recovery, GeoJournal, 29:57-67.

4. Gomez-Baggethun, E., Tudor, M., Doroftei,M., Covaliov, S., Năstase, A., Onănă, D.-F. & Mierlă, M., 2019. Changes in ecosystem services from wetland loss and restoration: An ecosystems assessment of the Danube Delta (1960-2010), Ecosystem Services, 39. Gomoiu, M.T., 1996, Facts and remarks on the Danube Delta, Danube Delta – Black Sea system under global changes impact, Bucharest-Constanța / Romania: National Institute of Marine Geology and Geo-ecology, pp 70-82.

5. Ma, T., Li, X., Bai, J. & Cui, B, 2019. Tracking three decades of land use and land cover transformation trajectories in China’s large river deltas, Land Degrad. Dev, 30:799-810.

Water Quality Indicators, Microplastics Pollution and Waste Management in the Danube Delta Biosphere Reserve

Dr. Carmen Chifiriuc, Dr. Carmen Postolache, Dr. Florina Botez, Dr. Corina Bradu, Dr. Sorin Avramescu , Dr. Irina Gheorghe,  Drd. Maria Iasmina Moza, Drd. Darmina Niță, Drd. Valentin Dinu, Dr. Mihaela Verga, Dr. Mioara Clius, Dr. Irina Zarafu, Dr. Andreea Marin, Dr. Petre Ionita,
Dr. Luminita Marutescu, Dr. Gratiela Gradisteanu

Introduction: Waste, especially plastic, affects the quality of the environment and the health of an ecosystem. Millions of tons of plastic waste go to the ocean every year, both from developing countries with insufficient solid waste infrastructure and from high-income (EU) countries (Borrelle et al., 2020, Law et al., 2020, Lau et al., 2020). To reduce the mass of waste generated and to mitigate the damage caused by the global pollution requires immediate, concerted, and vigorous action, such as integrated waste management systems (Galatchi & Tudor, 2007). Waste management includes the activities of collecting, transporting, treating, recycling, and storing waste to reduce its effect on human health, the environment, or the appearance of a habitat, as well as saving natural resources by reusing recyclable parts (Orlescu & Costescu, 2013). In Romania, the waste management activity is regulated by Law No 211/2011, which transposes several Council of Europe directives (*). Given the ecosystem significance of the Danube Delta and the fragility of its natural systems, it is important to integrate a sustainable waste management system to reduce the environmental waste impact and to maintain a low level of pollution (Păunică, 2014, Săgeată et al., 2016).

Study sites: UAT (administrative-territorial units) from the Danube Delta or Small Braila Island and upstream river.

Aim: Describe the water quality using bioindicators and nutrients overload and assess the occurrence and extent of microplastic (MP) contamination as well as the status of waste management in the Danube Delta Biosphere Reserve.

Objectives: 1) analyze water quality indicators to establish the ecosystem status, 2) evaluate the water and sediment microbial diversity and metabolic activity, 3) identify MPs types and concentration in water and sediments and 4) identify the sources and types of waste and legal regulations regarding waste management.

Methods: Sampling methodology* for water, sediment, and soil; design for replicates; sample storage and preparation; labelling; water filtration; sample extraction; in situ parameters measurements; samples extraction and laboratory physico-chemical and microbiological analyses by culture dependent and culture-independent methods for water and soil quality; database organization; data interpretation and visualization; scientific reporting; and oral presentation, field inventory and field mapping; collection and processing of statistical data using surveys, interviews, and desk research; spatial distribution mapping of waste flow.

*The aquatic samples will be collected from previously established sampling points using Net-based sampling devices and different mesh sizes will be used for filtration of grab samples; water pumping will be applied when necessary to ensure the sampling of larger water volumes. Water sediments sampling will be performed with a box corer. The collected aquatic and sediment samples will be first analysed by physico-chemical and microbiological standard analyses according to the international Water framework directive (WFD) 2000/EC-60 recommendations, then monitored for the presence of MPs (in this purpose MPs will be separated using zinc chloride solution and then identified by wet oxidation coupled with a basic enzymatic protocol). For culture-dependent microbiological investigations the samples will be transported at 4oC and processed within a maximum of 24 hours after reaching the laboratory. The physiological profile of the epiplastic versus planktonic aquatic communities will be evaluated with the EcoPlates from Biolog™. In the analysis of water microbiota, a special focus will be given to Clostridium perfringens anaerobic sporulated species that could serve as a useful predictor for the presence of stress factors in a certain ecosystem. For culture-independent microbiological investigations the samples will be preserved at -70oC until DNA extraction will be performed. For the analysis of the epiplastic microbial community diversity, microbial DNA will be isolated from water samples (filter membranes) and sediment using commercial kits and further used for 16S and 18S ribosomal RNA genes amplification and sequencing.

Skills: organizing sampling trip; appropriate equipment use; sampling by or without filtration; field troubleshooting; labelling; sampling storage and preparation for extraction and preparation for analyses**; harvesting on different media; achieving serial dilutions; inoculating/reading the EcoPlates; DNA extraction from water/sediment samples; PCR; sequencing and bioinformatic analysis; aseptic lab techniques; laboratory skills in sample preliminary preparation and MPs identification; survey and sampling design (waste categories selection, parameters of interest), appropriate sampling (frame, method, size); organizing field-research; mapping techniques (waste flow mapping: preparation and mapping of waste generation and fractions on-site); field and lab prep; collecting, recording and analyzing information and data; field troubleshooting; scientific reporting; and oral presentation.

**The soil samples will be sieved to a maximum particle size of 2 mm and stored at −7 °C.  250 mg portions of soil will undergo DNA extraction using the PowerSoil DNA isolation kit (MOBIO Laboratories Inc., Carlsbad, CA) according to the manufacturer's instructions (Lau et al., 2020); water and soil microbial diversity and activity will be investigated and analysed by EcoPlates. The method will provide a characterization of the diversity of community-level physiological profiles (Rutgers et al., 2016; Feigl et al., 2017; Jiang et al., 2017). This method was used for the characterization of the ecological status of environmental samples (Agata et al., 2014), such as sediments (Lopes et al., 2016), wastewater (Zhang et al., 2014), activated sludge (Paixão et al., 2007), and soils (Rutgers et al., 2016; Al-Dhabaan and Bakhali, 2017).

Possible partners: University of Bucharest – Sfantu Gheorghe field station; National Institute for Research and Development in Chemistry and Petrochemistry (ICECHIM) -Bucharest; Institute of Biology - Bucharest (Sulina field station): National Institute for Danube Delta Research – Tulcea; National Institute of Research and Development for Biological Sciences– Bucharest; University of Bucharest - Braila field station; Politehnica University Bucharest; Institute of Biochemistry, Romanian Academy – Bucharest; Tulcea County Council; local municipalities; ARBDD (The Danube Delta Biosphere Reserve Authority); APM Tulcea (Agency for Environmental Protection Tulcea); ANAR (National Authority Romanian Water); National Institute for Materials Physics; Institute for Pedologic and Agrochemical Research.

CURE 2 References:

1. Agata, G., Magdalena, F., & Karolina, O. (2014). The application of the biolog ecoplate approach in ecotoxicological evaluation of dairy sewage sludge. Appl. Biochem. Biotechnol. 174, 1434–1443. doi: 10.1007/s12010-014-1131-8

2. Al-Dhabaan, F. A. M., & Bakhali, A. H. (2017). Analysis of the bacterial strains using biolog plates in the contaminated soil from Riyadh community. Saudi J. Biol. Sci. 24, 901–906. doi: 10.1016/j.sjbs.2016.01.043

3. Borrelle, S. B., Ringma J., Law, K. L., Monnahan, C. C., Lebreton, L., McGivern, A., Murphy, E., Jambeck, J., Leonard, G. H., Hilleary, M. A., Eriksen, M., Possingham, H. P., De Frond, H., Gerber, L. R., Polidoro, B., Tahir, A., Bernard, M., Mallos N., Barnes M., & Rochman, C. M., (2020) Predicted growth in plastic waste exceeds efforts to mitigate plastic pollution, Science, Vol. 369, Issue 6510, pp. 1515-1518, DOI: 10.1126/science.aba3656

4. Feigl, V., Ujaczki,É., Vaszita, E., & Molnár, M. (2017). Influence of red mud on soil microbial communities: application and comprehensive evaluation of the biolog ecoplate approach as a tool in soil microbiological studies. Sci. Total Environ. 595, 903–911. doi: 10.1016/j.scitotenv.2017.03.266

5. Galatchi, L.-D. & Tudor, M., 2007. Europe as a source of pollution – The main factor for the eutrophication of the Danube Delta and Black Sea. In: Simeonov, L., Chirila, E. (eds) Chemicals as International and Accidental Global Environmental Threats. NATO Security through Science Series. Springer, Dordrecht.

6. Jiang, L. L., Han, G. M., Lan, Y., Liu, S. N., Gao, J. P., Yang, X., et al. (2017). Corn cob biochar increases soil culturable bacterial abundance without enhancing their capacities in utilizing carbon sources in Biolog eco-plates. J. Integr. Agric. 16, 713–724. doi: 10.1016/S2095-3119(16)61338-2

7. Lau, W. W. Y., Shiran, Y., Bailey, R. M., Stuchtey, M. R., Koskella, J., Velis, C. A., Godfrey, L., Boucher, J., Murphy, M. B., Thompson, R. C., Jankowska, E., Castillo, A. C., Pilditch, T.D., Dixon, B., Koerselman, L., Kosior, E., Favoino, E., Gutberlet, J., Baulch, S., Atreya, M. E., Fischer, D., He, K. K., Petit, M. M., Sumaila, U. R., Neil, E., Bernhofen, M. V., Lawrence, K., & Palardy, J. E., (2020) Evaluating scenarios toward zero plastic pollution, Science, Vol. 369, Issue 6510, pp. 1455-1461, DOI: 10.1126/science.aba9475.

8. Law, K. L., Starr, N.,. Siegler, T. R, Jambeck, J. R., Mallos, N. J, & Leonard G. H. (2020) The United States’ contribution of plastic waste to land and ocean, Science Advances, Vol. 6, no. 44, eabd0288, DOI: 10.1126/sciadv.abd0288

9. Orlescu, C.M. & Costescu, I-A., 2013. Solid waste management in Romania: Current and future issues, Environmental engineering and management journal, 12(5):891-899.

10. Paixão S.M., Sàágua M.C., Tenreiro R., & Anselmo A.M., 2007. Assessing microbial communities for a metabolic profile similar to activated sludge. Water Environ. Res. 79: 536-546. doi: 10.2175/106143006X123148

11. Păunică, M., 2014, Economic benefits of the infrastructure projects implemented in the Reservation of the Danube Delta Biosphere, Theoretical and Applied Economics Volume XXI, No. 11(600), pp. 95-104

12. Rutgers, M., Wouterse, M., Drost, S. M., Breure, A. M., Mulder, C., Stone, D., et al. (2016). Monitoring soil bacteria with community-level physiological profiles using Biolog ECO-plates in the Netherlands and Europe. Appl. Soil Ecol. 97, 23–35. doi: 10.1016/j.apsoil.2015.06.007

13. Săgeata, R., Damian, N. & Mitrică, B., 2016. Waste management system in the riparian towns of the Romanian Danube Sector, Urbanism.Arhitectura.Construcții, Vol. 7, Issue 3:199-212.

14. Zhang, T. Y., Wu, Y. H., Zhuang, L. L., Wang, X. X., & Hu, H. Y. (2014). Screening heterotrophic microalgal strains by using the Biolog method for biofuel production from organic wastewater. Algal Res. 6, 175–179. doi: 10.1016/j.algal.2014.10.003