Periodic Reporting for period 1 - App-Marie (Studying the combined effect of microplastics, pesticides and global warming on the sea urchin: a multidisciplinary approach to protect marine ecosystems)
Reporting period: 2022-04-01 to 2024-03-31
Human activities severely impacted the marine environment, threatening ecosystems and human health. Plastic pollution and agricultural contamination are major contributors. Plastics account for about 80% of marine litter, with global production raising from 1.7 to 299 million tonnes between the 1950s and 2014. The COVID-19 pandemic worsened the situation by increasing plastic demand. Microplastics (MP), particles smaller than 5 mm, proliferated, posing significant risks to marine life and ecosystems. By 2050, MPs were predicted to outnumber fish in the oceans, increasing the incidence of lethal entanglement and ingestion by sea animals by 40%. MP also absorb toxic chemicals, which enter into the food chain, raising exposure risks. Agricultural products, such as organophosphorus pesticides (OPs), also contaminate the oceans. Chlorpyrifos, a widely used OP, cause neurotoxic effects in non-target organisms. Alongside plastics and toxins, global warming and ocean acidification further threaten marine life. Surface and coastal water temperatures are expected to rise by 1.8-4°C by the century's end, while ocean pH might drop by 0.2-0.5 units in the next 60 years, exacerbating stress on ecosystems and increasing contaminant toxicity. Despite efforts to mitigate these impacts, information on the combined effects of multiple stressors on marine life remains scarce. App-MariE pioneered the study of the joint impact of MP and an OP, Chlorpyrifos, on the sentinel species P. lividus under climate change scenarios. P. lividus, a key component of marine ecosystems, was chosen due to its simplicity, low cost, and regulatory advantages for bioassays. The project aimed to standardize bioassays to evaluate the complex effects of multiple stressors and link early life stage effects to adult impacts (Objective 1). Additionally, App-MariE sought to understand the molecular mechanisms triggered by pollutant combinations and their broader implications across species, providing insights for predicting ecosystem impacts (Objective 2). The project proposed a multidisciplinary approach to manage data and foster cooperation between researchers, industries, and governments (Objective 3).
As a result of App-MariE, the traditional sea urchin embryo test was improved by incorporating data on morphology, gene expression, and informatics applications, making it more predictive and capable of providing detailed information about the toxicity of pollutant mixtures. Additionally, the project demonstrated that research should focus on studying the impact of pollutants within mixtures, which is more realistic than evaluating each contaminant or stressor individually. Finally, the integration of applied informatics and standardized operating procedures (SOPs) for data analysis made the sea urchin embryo test easier to perform and more reproducible. This will enable others, even those without prior experience, to analyze pollutant mixtures using the App-MariE approach.
As a result of App-MariE, the traditional sea urchin embryo test was improved by incorporating data on morphology, gene expression, and informatics applications, making it more predictive and capable of providing detailed information about the toxicity of pollutant mixtures. Additionally, the project demonstrated that research should focus on studying the impact of pollutants within mixtures, which is more realistic than evaluating each contaminant or stressor individually. Finally, the integration of applied informatics and standardized operating procedures (SOPs) for data analysis made the sea urchin embryo test easier to perform and more reproducible. This will enable others, even those without prior experience, to analyze pollutant mixtures using the App-MariE approach.
Experimental work with P. lividus larvae was conducted to evaluate the viability of using 24-well plates for bioassays. A protocol is being developed to outline the usage, advantages, and limitations of this method for acute bioassays (48 hours). Acute bioassays were performed using various supports (culture plates, vials, bottles) to assess the effects of CPF, MP, MP+CPF, Ocean Warming (OW), and Ocean Acidification (OA) on P. lividus pluteus larvae, with growth and morphology as key endpoints (paper published). The molecular effects were investigated through RNA-seq analysis and candidate gene markers for OW effects were identified. Additionally, biological processes and molecular functions altered by the pollutant mixtures were identified, including those related to growth, development, and detoxification mechanisms. The enzymatic activity of commonly used toxicological biomarkers (AChE, GST, GRx) was measured (paper published), along with the effects on lipid and protein content in larvae. This data on enzymatic activities related to detoxification and lipid metabolism was compared with gene expression obtained from the RNA-seq analysis. As a result, certain gene isoforms were identified as potential predictors of the corresponding enzyme activity, making them candidates for toxicity markers. A SOP has been finalized, and two apps -Growth Calculator and Morpheus- were developed to analyze the growth and morphology of 48-hour pluteus larvae. These apps allow for the rapid assessment of the apical effects of stressors. The Growth Calculator also features a section for analyzing growth curves and determining EC10 and EC50 concentrations, providing valuable insights for scientists and stakeholders. Once the molecular data is fully analyzed and published, the apps will be upgraded to include real-time analysis of gene expression data, enabling the identification of molecular markers.
These results were disseminated among the scientific community through two international meetings (PRIMO 22nd meeting. Nantes, France; SETAC Europe 33rd Annual Meeting. Dublin, Ireland) and four original articles:
1. "Developmental and biochemical markers of the impact of pollutant mixtures under the effect of Global Climate Change." Chemosphere, vol. 358, 142162, 2024. Pergamon. https://doi.org/10.1016/j.chemosphere.2024.142162(opens in new window)
2. "The effect of environmental stressors on growth in fish and its endocrine control." Frontiers in Endocrinology, vol. 14, 1109461, 2023. Frontiers Media SA. https://doi.org/10.3389/fendo.2023.1109461(opens in new window)
3. "Global climate change increases the impact of pollutant mixtures in the model species Paracentrotus lividus." Science of the Total Environment, vol. 893, 164837, 2023. Elsevier. https://doi.org/10.1016/j.scitotenv.2023.164837(opens in new window)
4. "Impact of microplastics and ocean acidification on critical stages of sea urchin (Paracentrotus lividus) early development." Chemosphere, vol. 301, 134783, 2022. Pergamon. https://doi.org/10.1016/j.chemosphere.2022.134783(opens in new window)
Two additional articles are in preparation: one on RNAseq molecular data analysis, and a review article on the use of the apps and SOPs for toxicity analysis in pollutant mixtures using the Sea Urchin Embryo Test.
These results were disseminated among the scientific community through two international meetings (PRIMO 22nd meeting. Nantes, France; SETAC Europe 33rd Annual Meeting. Dublin, Ireland) and four original articles:
1. "Developmental and biochemical markers of the impact of pollutant mixtures under the effect of Global Climate Change." Chemosphere, vol. 358, 142162, 2024. Pergamon. https://doi.org/10.1016/j.chemosphere.2024.142162(opens in new window)
2. "The effect of environmental stressors on growth in fish and its endocrine control." Frontiers in Endocrinology, vol. 14, 1109461, 2023. Frontiers Media SA. https://doi.org/10.3389/fendo.2023.1109461(opens in new window)
3. "Global climate change increases the impact of pollutant mixtures in the model species Paracentrotus lividus." Science of the Total Environment, vol. 893, 164837, 2023. Elsevier. https://doi.org/10.1016/j.scitotenv.2023.164837(opens in new window)
4. "Impact of microplastics and ocean acidification on critical stages of sea urchin (Paracentrotus lividus) early development." Chemosphere, vol. 301, 134783, 2022. Pergamon. https://doi.org/10.1016/j.chemosphere.2022.134783(opens in new window)
Two additional articles are in preparation: one on RNAseq molecular data analysis, and a review article on the use of the apps and SOPs for toxicity analysis in pollutant mixtures using the Sea Urchin Embryo Test.
App-MariE project has advanced significantly beyond the state of the art in several areas: 1) Improved the traditional sea urchin embryo test by integrating morphological, gene expression, and bioinformatics data, making it more predictive and informative for assessing pollutant mixtures. 2) Analyzed the combined effects of pollutants under realistic environmental conditions, identifying key biological processes and molecular functions affected by OW and OA. 3) Used RNA-seq to identify gene markers and linked them to enzymatic activities, introducing potential new toxicity markers. 4) Developed the Growth Calculator and Morpheus apps for quick assessment of growth and morphology in response to stressors, with ongoing enhancements to include real-time gene expression analysis. 5) Finalized SOPs and created accessible tools, making advanced methodologies available to a broader audience, including those with limited experience. 6) Dissemination: Results have been widely shared through international conferences and publications, influencing the field of marine ecotoxicology.