Abstract of the 2nd International Online Conference on Toxics ^†

The 2nd International Online Conference on Toxics (IOCTO 2025) successfully showcased essential advancements across the entire spectrum of toxicology, from environmental exposure modelling to molecular mechanisms and novel ecotoxicological applications. The event highlighted the urgency and complexity of addressing chemical risks in an era dominated by emerging, persistent, and mixed pollutants.

Discussions emphasized the crucial need to bridge fundamental toxicological research with practical risk management strategies, propelling the field toward effective public health and environmental safeguards. Collectively, IOCTO 2025 demonstrated the transformative impact of rigorous, multidisciplinary toxicological and pharmacological research in identifying, characterizing, and mitigating chemical threats to humans and ecosystems.

S1. Exposure Pathways—Air and Water Contamination

This session provided critical insights into the transport and fate of toxic substances via air and water. Presentations focused on advanced monitoring technologies, multi-pathway exposure assessments, and the predictive modelling of contaminant distribution, including fine particulate matter and emerging substances like PFAS. Attendees gained a deeper understanding of the complex exposure scenarios driven by urbanization and climate change and discussed actionable strategies for real-time sensing and pollution mitigation aligned with planetary health goals.

S2. Molecular Stressors—Nanoparticles, Mixtures, and Persistent Pollutants

Talks centred on the significant challenges posed by emerging pollutants, including nanoparticles and persistent chemicals, highlighting their unique physicochemical properties and environmental behaviour. Discussions focused on the complex issues of hazard identification, ecological risk assessment, and toxicity mechanisms of mixed chemical exposures. Participants explored cutting-edge findings and breakthroughs necessary for establishing early warning systems and contributing to sustainable solutions for mitigating the widespread risks of artificial chemicals in the environment.

S3. Cutting-Edge Approaches in Pharmacology & Toxicology

This session showcased innovative findings and methodologies in toxicological investigation, with a specific emphasis on chemicals affecting the Central Nervous System (CNS), Peripheral Nervous System (PNS), and various vital organs (liver, kidney, lung, heart). Key topics included the toxicological effects of pharmaceuticals, chemotherapeutic medications, and a wide array of drugs of abuse (e.g., opioids, psychedelics, cannabis, alcohol). The presentations provided insight into drug-induced neurotoxicity and organ injury, advancing experimental methods for assessing off-target effects.

S4. Pesticides, Pollutants, and Health Risk

Discussions in this session illustrated the intricate relationships between exposure to pesticides and various pollutants and the resulting health risks to humans and ecosystems. Researchers presented the latest findings on contaminant sources, exposure pathways, and toxicological mechanisms. Key themes included the development and application of biomarkers for exposure and effect monitoring, alongside methodologies for comprehensive risk assessment and the strategies required for effective mitigation and regulation of these widespread environmental contaminants.

S5. Exploring Non-Model Species for Ecotoxicology

The concluding session examined the pivotal role of non-model species in expanding the ecological relevance of toxicological testing. Presentations covered the necessity of moving beyond standardized laboratory tests to include field-collected species in in situ bioassays and micro/mesocosms. Insights into high-throughput screening, assessing impacts across multiple levels of biological organization (molecular, behavioural, community structure), and using invertebrates as surrogate vertebrate models emphasized the need for more reliable and repeatable methods for assessing toxic and endocrine effects under ecologically relevant conditions.

1. Session: Exposure Pathways—Air and Water Contamination

1.1. La₂Ti₂O₇ Doped with Pt, Obtained by the Sol–Gel Method, with Photocatalytic Activity in the Oxidative Degradation of Ethanol

• Elena-Alexandra Ilie (Săndulescu) ¹, Luminita Predoana ¹, Crina Anastasescu ¹, Silviu Preda ¹, Veronica Bratan ², Daniela C. Culita ¹, Ioan Balint ¹ and Maria Zaharescu ¹

¹ 

“Ilie Murgulescu” Institute of Physical Chemistry, Romanian Academy, 060021 Bucharest, Romania

² 

Chemical Sciences Department, Romanian Academy, 010071 Bucharest, Romania

• Introduction and Aim: Perovskites are materials with increasing applications in a variety of domains, including catalysis and photocatalysis The oxidation of organic compounds by sunlight is significant for several reasons, including its low cost, its ability to purify air and water and its potential as an alternative to the selective synthesis of high-value oxygenated compounds. The aim of this work is to study the synthesis of La₂Ti₂O₇ perovskite through the sol–gel method and to test its photocatalytic activity in oxidative ethanol degradation under simulated solar light.
• Methods: Materials used: lanthanum (III) nitrate hexahydrated [La(NO₃)₃ × 6H₂O] and titanium (IV) isopropoxide [Ti[OCH(CH₃)₂]₄] were used as precursors, glacial acetic acid (CH₃COOH) and isopropanol (i-C₃H₇OH) were used as solvents, and PtCl₄ was used as a dopant. The reaction products of gas phase oxidation processes were analyzed by gas phase chromatography (GC-TCD and GC-FID).
• Results: The structural and morphological comparison of nanopowders was accomplished by Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) coupled with EDX, transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray fluorescence (XRF), ultraviolet–visible spectroscopy (UV–Vis), and specific surface area and porosity analysis (BET). Their photocatalytic activity was evaluated by the oxidative photodegradation of ethanol under simulated solar irradiation. The highest conversion of ethanol (29.75%) was obtained in the case of the Pt-doped sample.
• Conclusions: Nanopowders of La₂Ti₂O₇ (LTA) were prepared by the sol–gel method. The effect of the dopant on the properties of the Pt-doped powder was evaluated. The samples’ morphology revealed nanoparticle aggregates with well-defined mesopores. The photocatalytic activity in terms of ethanol conversion and selectivity to CO₂ was increased by the addition of platinum to the LTA catalyst. Consequently, these powders demonstrate potential applications for air depollution technologies.

1.2. Optimization and Validation of a Stir Bar Sorptive Extraction–HPLC–FLD Method for Monitoring PAHs in Water

• Eduardo Lage ^1,2, Maria José José Alves ^1,2, Cosme Moura ^3,4 and Juliana Garcia ^1,5

¹ 

AquaValor—Centre for the Valorisation and Transfer of Water Technology—Association, 5400-342 Chaves, Portugal

² 

Research Centre for Active Living & Wellbeing, Polytechnic Institute of Bragança, 5300-253 Bragança, Portugal

³ 

Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal

⁴ 

Institute of Molecular Sciences Chemistry Research Centre, University of Porto (IMS/CIQUP), 4169-007 Porto, Portugal

⁵ 

LiveWell—Research Centre for Active Living & Wellbeing, Polytechnic Institute of Bragança, 5300-253 Bragança, Portugal

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants with known mutagenic and carcinogenic properties. They are commonly introduced into aquatic environments through industrial activities, fossil fuel combustion, and residential heating. Given their low concentrations and environmental relevance, the development of sensitive, selective, and cost-effective methods for their determination in water matrices is of critical importance. This study presents an optimized and validated analytical methodology for the simultaneous quantification of six priority PAHs—fluoranthene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, benzo[ghi]perylene, and indeno [1,2,3-cd]pyrene—in drinking water, groundwater, and surface water. The method employs stir bar sorptive extraction (SBSE) followed by high-performance liquid chromatography with fluorescence detection (HPLC–FLD). Key extraction parameters, including sample temperature, extraction time, stirring rate, matrix composition, and desorption time, were systematically optimized. The method demonstrated excellent linearity (R² > 0.998) across all analytes. Limits of detection ranged from 0.04 to 0.18 ng/L, while limits of quantification ranged from 0.13 to 0.61 ng/L. Recovery values were within 82–108%, with intra- and inter-day precision (RSD) below 10%. The total chromatographic runtime was 24 min, followed by a 2 min post-run. Measurement uncertainty was also evaluated according to ISO/IEC 17025 guidelines[1]. The validated SBSE–HPLC–FLD method provides a reliable, sensitive, and efficient approach for the monitoring of PAHs in environmental water matrices, contributing to improved risk assessment and regulatory compliance.

• Reference

• ISO/AWI 21807; Nanotechnologies—Vocabulary—Core terms. International Organization for Standardization (ISO): Geneva, Switzerland,2017

1.3. Plastic Particle Migration in Bottled Water: Emerging Health Risks Associated with Long-Term Storage

• Codau Natanael

• Faculty of Electrical Engineering and Computer Science, Ștefan cel Mare University of Suceava, 7210229 Suceava, Romania

The widespread reliance on bottled water has prompted increasing scrutiny regarding its chemical and particulate content, particularly under prolonged storage and variable environmental conditions. Recent investigations reveal that bottled water may contain hundreds of thousands of plastic particles per liter, predominantly in the nanometric range. These particles, often invisible to the naked eye, originate primarily from the degradation of common packaging materials such as polyethylene terephthalate (PET) and polycarbonate. Their small size and high surface-area-to-volume ratio make them difficult to detect, filter, and neutralize, posing a growing challenge to both scientific monitoring and public health protection. Prolonged exposure to elevated temperatures—such as those encountered during transport or storage—can accelerate the leaching process, increasing the release of micro- and nanoplastics, along with hazardous chemical additives like bisphenol A (BPA), antimony, and phthalates. These substances are known endocrine disruptors with the potential to interfere with hormonal regulation, immune function, and developmental processes. An expanding body of toxicological evidence associates chronic exposure to these contaminants with a range of health problems, including hormonal imbalances, neurodevelopmental disorders, metabolic disturbances, and increased cancer risk. Alarming recent findings report the presence of nanoplastics in human blood, placenta, and other biological tissues, raising urgent concerns about their bioaccumulation and long-term systemic effects. In response, emerging regulatory frameworks—such as new European Union directives and WHO guidelines—aim to limit exposure and enhance transparency. This abstract advocates for a comprehensive approach involving safer packaging alternatives, advancement in detection and filtration technologies, and heightened consumer education as pillars of an effective public health strategy to reduce the potential risks associated with bottled water consumption.

1.4. Abundance, Characterization, and Risk Assessment of Microplastics in the Payra River, Bangladesh

• Abdullah Bin Firoz ¹, Salma Begum ² and Md Abdullah Yousuf Al Harun ²

¹ 

Institute of Water and Flood Management (IWFM), Bangladesh University of Engineering and Technology (BUET), Dhaka 1000, Bangladesh

² 

Environmental Science Discipline, Life Science School, Khulna University, Khulna 9208, Bangladesh

Microplastics (5 mm) are persistent emerging contaminants now ubiquitous in all environmental matrices, posing serious ecological and health risks due to their toxic effects, their ability to act as vectors of toxic pollutants, and their integration into food webs. Despite extensive global research on freshwater microplastic contamination, only a few rivers in Bangladesh have been studied, with no prior research conducted on rivers in the Barishal region, which experience increased anthropogenic pressure and have ecological significance. Hence, this research was carried out to provide the first overview of microplastic distribution in the region. A total of 30 surface water samples were collected from ten locations along the length of the river during the pre-monsoon season and analyzed using a microscope and FTIR. The results revealed microplastic abundance ranging from 93.33 ± 8.08 particles/L to 196 ± 11.89 particles/L, with downstream sites exhibiting significantly higher concentrations. Fragments and fibers were the predominant shapes, with red and navy blue as the most common colors. The majority of particles fell under a 100–500 µm size range, followed by smaller particles (100 µm), indicating a potential for greater pollutant adsorption and bioavailability. Six polymer types were identified: PE (34%), PP (22%), PETE (16%), PS (8%), PVC, and nylons (both at 10%). These polymers indicate sources such as municipal waste, industrial discharge, plastic bags, bottles, pipes, packaging materials, and fishing nets. Risk assessment through indices revealed an overall moderate pollution scenario where Polymer Hazard Index (PHI) values ranged from 7 to 4236.6 (Category II to IV) and ERI_river = 145.86. PLIi and PLI_river values were >1, indicating that all sites were significantly polluted. This study provides baseline microplastic contamination data for the Payra River and is crucial for future research and evidence-based policymaking to ensure sustainable riverine ecosystem management.

1.5. Eco-Treatment of Microplastics: Laccase Enzyme-Based Biodegradation

• Sílvia D. Martinho ¹, Luís Gonçalves ¹, Virgínia Cruz Fernandes ^1,2,3, Sónia A. Figueiredo ¹, Malin Hultberg ⁴ and Cristina Delerue Matos ¹

¹ 

REQUIMTE/LAQV—ISEP, Polytechnic of Porto, 4249-015 Porto, Portugal

² 

Chemical and Biomolecular Sciences, ESS, Polytechnic of Porto, 4200-072 Porto, Portugal

³ 

RISE-Health, Center for Research in Translational Health and Medical Biotechnology (TBIO), ESS, Polytechnic of Porto, 4200-072 Porto, Portugal

⁴ 

Department of Biosystems and Technology, Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden

Microplastics (MPs), defined as plastic particles smaller than 5 mm, are persistent and widespread in both aquatic and terrestrial ecosystems owing to the mismanagement of plastic waste [1,2]. These particles undergo aging through environmental weathering, increasing their toxicity and capacity to adsorb pollutants [3,4]. These particles can cause physical stress, inflammation, oxidative damage, and immune responses, potentially contributing to diseases such as cancer, metabolic disorders, and neurodevelopmental conditions [5]. In response to growing concerns, the revised EU Urban Wastewater Treatment Directive highlights the need to reduce the release of target micropollutants, including MPs, into the environment [6]. In this study, the potential of biodegradation using the enzyme laccase was investigated as an eco-friendly and sustainable approach for their removal from wastewater. According to previous studies, laccase can break polymer bonds, potentially reducing environmental persistence and toxicity [7]. Three of the most common types of MPs were tested: low-density polyethylene (LDPE, 300 and 600 µm), polyethylene terephthalate (PET, 300 µm), and polyamide nylon 6 (PA6, 20 µm). Laccase concentrations ranging from 50 to 200 U/L were applied over a 7-day incubation period, and enzyme activity was determined using the ABTS oxidation method. MP samples were analyzed using FTIR and Raman spectroscopy to assess their structural modifications. The results indicated potential surface alterations evidenced by increased fluorescence in the Raman spectra; however, further optimization is required to enhance the degradation of specific functional groups and improve biodegradation efficiency.

• References

• Gao, Y.; Gao, W.; Liu, Y.; Zou, D.; Li, Y.; Lin, Y.; Zhao, J. A comprehensive review of microplastic aging: Laboratory simulations, physicochemical properties, adsorption mechanisms, and environmental impacts [J]. Science of the Total Environment, 2024, 957(177427).
• Wang, L.; Zhang, J.; Huang, W.; He, Y. Laboratory simulated aging methods, mechanisms and characteristic changes of microplastics: A review. Chemosphere 2023, 315, 136701.
• Martinho, S.D. Toxic Effects of Micro- and Nanoplastics: Environment, Food and Human Health; Wiley: Hoboken, NJ, USA, 2024.
• Binda, G Kalčíková, G.; Allan, I.; Hurley, R.; Rødland, E.; Spanu, D.; Nizzetto, L. Microplastic aging processes: Environmental relevance and analytical implications. TrAC Trends Anal. Chem. 2024, 172. 117566.
• Molina, E.; Benede, S. Is There Evidence of Health Risks from Exposure to Micro- and Nanoplastics in Foods? Front. Nutr. 2022, 9, 910094.
• Directive (EU) 2024/3019 of the European Parliament and of the Council of 27 November 2024 concerning urban wastewater treatment (recast). https://eur-lex.europa.eu/eli/dir/2024/3019/oj/eng (accessed on 1 July 2025)
• Ramamurthy, K.; Thomas, N.; Gopi, S.; Sudhakaran, G.; Haridevamuthu, B.; Namasivayam, K.; Arockiaraj, J. Is Laccase derived from Pleurotus ostreatus effective in microplastic degradation? A critical review of current progress, challenges, and future prospects. Int. J. Biol. Macromol. 2024, 276, 133971.

1.6. Evaluation of Spatio-Temporal Patterns of Nutrient-Related Water Quality Parameters: A Case Study in Greater Houston Area, Texas

• Reynaldo Raya and Dongmin Sun

• Department of Environmental Science, University of Houston, Clear Lake, Houston, TX 77058, USA

This study analyzed spatial and temporal patterns of surface water quality, specifically focusing on nutrient-related parameters in the Greater Houston Area (GHA), Texas. It addressed two key questions: (1) How have population growth and changes in land use over the past four decades influenced water quality over time and across space? (2) Have ongoing pollution control efforts effectively maintained water quality over the past 40 years? We analyzed water quality data from 20 stations in GHA, focusing on total nitrogen, total phosphorus, dissolved oxygen, total solids, fecal coliform, and pH levels. The Mann–Kendall test and Sen’s slope estimator were utilized to detect long-term trends. The results show that nutrient pollution is increasingly becoming a significant issue in the GHA. Our spatial analysis reveals that water quality is inversely correlated with population density and the percentage of impervious land cover. This suggests areas with higher population density are more prone to water quality degradation. Mann–Kendall trend analysis indicates a strong increasing trend in fecal coliform, total nitrogen, and total phosphorus at stations situated in heavily urbanized counties. In contrast, these parameters remain stable in less populated counties, despite rapid population growth. The results demonstrate that the ongoing pollution control efforts have not been equally effective across the study area. These findings highlight the challenges in densely populated counties and underscore the necessity of sustained water pollution control and long-term monitoring efforts.

1.7. How Polluted Is the Black Sea?: A Pilot Assessment of Heavy Metals, Toxic Elements, and Persistent Organic Pollutants in Surface Waters, Sediments, and Mussels

• Vesela Yancheva ¹, Stela Stoyanova ², Borislava Todorova ¹, Elenka Georgieva ², Krisztián Nyeste ³ and László Antal ³

¹ 

Department of Ecology and Environmental Conservation, Faculty of Biology, Paisii Hilendarski University of Plovdiv, 4000 Plovdiv, Bulgaria

² 

Department of Developmental Biology, Faculty of Biology, Paisii Hilendarski University of Plovdiv, 4000 Plovdiv, Bulgaria

³ 

Department of Hydrobiology, Faculty of Science and Technology, Institute of Biology and Ecology, University of Debrecen, H-4032 Debrecen, Hungary

The present study presents, for the first time, results from comprehensive chemical analyses concerning heavy metals, toxic elements, various polychlorinated diphenyl ethers (PCBs), congeners, polycyclic aromatic hydrocarbons (PAHs), and pesticides in sediments, surface waters, and mussels from the Bulgarian Black Sea. In the samples from most of the investigated locations, iron, zinc, PCB-28, and PAHs (benz(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(ghi)perylene, and chrysene) were detected in the waters; arsenic, copper, iron, and zinc in the sediments; and arsenic, copper, cadmium, iron, lead, zinc, and PAHs (acenaphtene, acenaphthylene, anthracene, bezno(b)fluoranthene, chrysene, dibenzo(a,h)anthracene, fluoranthene, fluorene, naphthalene, phenanthrene, and pyrene) in the mussels. No pesticides were detected in any of the analyzed matrices. According to Commission Regulation (EU) 2023/915 of 25 April 2023 on the maximum levels for certain contaminants in food and repealing Regulation (EC) No. 1881/2006 [1], there are now human health hazards in terms of consuming bivalve mollusks because the levels of the contaminants that we investigated and are also included in the regulation—Cd, Pb, Hg, ∑PAHs (bezno(a)anthracene, bezno(a)pyrene, bezno(b)fluoranthene, and chrysene)—were lower than the maximum permissible levels set by law. The findings suggest that despite the detectable contaminants, the levels in mussels remain below thresholds deemed hazardous to human health. Yet, a set of biomarkers should be assessed in future in order to determine the effects on mussels which seem to live in a chronically contaminated aquatic environment, even though the contaminant levels are lower than the ones set in Directive 2000/60/EC [2] of the European Parliament and of the Council of 23 October 2000.

• Acknowledgments: This study is financed by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project number BG-RRP-2.004-0001-C01.

• Reference

• Regulation (EU) 2023/915; Commission Regulation (EU) 2023/915 of 25 April 2023 setting maximum levels for certain contaminants in foodstuffs and repealing Regulation (EC) No 1881/2006. Official Journal of the European Union (OJEU): Brussels, Belgium, 2023.
• Directive 2000/60/EC; Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy (Water Framework Directive). Official Journal of the European Union (OJEU): Brussels, Belgium, 2000.

1.8. Invisible Invaders: Tracing the Journey and Threats of Microplastics in Aquatic Ecosystems

• Jana Rammal ¹, Dalia El Badan ^2,3, Zaher. Abdel Baki ⁴, Akram Hijazi ¹ and Wassim Rammal ¹

¹ 

Environmental Sciences Research and Analysis Platform (EDST-PRASE), Beirut 6573/14, Lebanon

² 

Department of Biological Sciences, Faculty of Science, Beirut Arab University, Beirut 11-5020, Lebanon

³ 

Department of Botany and Microbiology, Faculty of Science, Alexandria University, Alexandria 21568, Egypt

⁴ 

College of Engineering and Technology, American University of the Middle East, Egaila 54200, Kuwait

Microplastics, or plastic fragments smaller than 5 mm in diameter, are widespread water contaminants globally, raising international alarm due to their persistence, mobility, and toxicity. Microplastics enter aquatic systems from primary (microbeads and plastic pellets) and secondary (fragmentation of larger plastics) sources through runoff, wastewater effluent, and atmospheric transport. Having invaded the environment, they are conveyed by flows and lodged in sediments or in food webs. The present review integrates recent findings concerning their mode of behavior within the environment, their biological consequences, and health implications for mankind. Microplastics are transportation media for unwanted chemicals—persistent organic pollutants and heavy metals—that amplify their effects in the environment. Microplastics are widely ingested by aquatic life forms, precipitating obstructive disorder in the gut, dwarfing growth, inflammation, and trophic transfer. Human exposure is increasingly documented through seafood consumption, drinking water, and inhalation, though the long-term effects are largely uncharacterized. There is no international standardization of existing monitoring techniques, and thus comparative analysis and regulation are problematic. This review delineates the essential research requirements and calls for interdisciplinary collaboration, improved analytical tools, and policy changes. Mitigation of microplastic pollution is no longer an option—it is a requirement to safeguard ecosystem resilience and public health against a silent but increasing environmental hazard.

1.9. Microbiota Shifts in Vicia faba Exposed to Microcystin-Contaminated Water

• El Mahdi Redouane ^1,2, Andrés Núñez ^3,4, Wafa Achouak ⁵, Mohamed Barakat ⁵, José Carlos Martins ⁶, Anoop Alex ^6,7, Ana M. García ³, Richard Mugani ^1,8, Alexandre Campos ⁶, Majida Lahrouni ¹, Khalid Oufdou ¹, Vitor Vasconcelos ^6,7 and Brahim Oudra ¹

¹ 

Water, Biodiversity and Climate Change Laboratory, Phycology Biotechnology and Environmental Toxicology Research Unit, Faculty of Sciences Semlalia Marrakech, Cadi Ayyad University, Marrakech 40000, Morocco

² 

UMR-I 02 INERIS-URCA-ULH SEBIO, Stress Environnementaux et BIOsurveillance des Milieux Aquatiques, Université de Reims Champagne-Ardenne, Campus du Moulin de la Housse, 51687 Reims, France

³ 

Escuela Técnica Superior de Ingenieros Industriales, Universidad Politécnica de Madrid (ETSII-UPM), 28006 Madrid, Spain

⁴ 

Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain

⁵ 

BIAM-LEMIRE, Laboratory of Microbial Ecology of the Rhizosphere, UMR 7265, Aix-Marseille Université, CEA, CNRS, 13115 Saint Paul-Lez-Durance, France

⁶ 

CIIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leixões, 4450-208 Matosinhos, Portugal

⁷ 

Department of Biology, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal

⁸ 

National Institute of Public Health, Ministry of Health and Fight Against AIDS, Bujumbura 0001, Burundi

Microcystins (MCs), common toxins produced during cyanobacterial blooms, are frequently found in water sources used for irrigation, leading to their introduction into agricultural environments. While MCs are well-known for their toxic effects on plant growth and physiology, their influence on plant-associated microbial communities remains underexplored. To address this gap, we studied how exposure to 100 µg/L of MCs affects the bacterial microbiota inhabiting bulk soil (BS), root-adhering soil (RAS), and root tissue (RT) in Vicia faba using a greenhouse pot experiment. Our findings revealed that MC exposure significantly altered the microbial community structure, co-occurrence patterns, and assembly mechanisms, with the most pronounced changes observed in RT microbiota, followed by BS and then RAS. Notably, MCs caused a marked decline in several Actinobacteriota taxa within RT, particularly members of the genus Streptomyces, which are commonly associated with plant health. In contrast, the abundance of certain Proteobacteria, including Methylobacillus, Methylotenera, and Paucibacter, genera potentially involved in MC degradation, increased under MC treatment. Network analysis showed a reduction in microbial co-occurrence complexity in MC-exposed conditions compared to the control. Moreover, community assembly in RT appeared to be predominantly governed by deterministic processes, while both deterministic and stochastic processes influenced BS and RAS microbiota, with a stronger deterministic trend overall. These results suggest that MCs may reshape the microbiota structure in the soil–plant system by reducing bacterial taxa with potential phytobeneficial traits and increasing other taxa with a potential capacity to degrade MCs.

1.10. Microplastic Contamination in Reservoirs: An Invisible Threat to Ecosystem and Water Quality Integrity

• Catarina Guimarães ¹, Ivo Pinto ^1,2,3,4 and Sara C. Antunes ^1,2

¹ 

Department of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre S/N, 4169-007 Porto, Portugal

² 

Interdisciplinary Center for Marine and Environmental Research, University of Porto, Leixões Cruise Terminal, Avenida General Norton de Matos, S/N, 4450-208 Matosinhos, Portugal

³ 

Abel Salazar Institute of Biomedical Sciences, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal

⁴ 

Multidisciplinary Biomedical Research Unit—Abel Salazar Institute of Biomedical Sciences, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal

Reservoirs are essential aquatic ecosystems that provide drinking water and support various human activities. However, they are increasingly threatened by plastic pollution, posing risks to both environmental and human health. Despite this, the evaluation of the Ecological Potential (EP) of reservoirs under the Water Framework Directive (WFD) does not yet account for microplastics (MPs). This study aimed to quantify and characterize MPs in two Portuguese reservoirs (Rabagão and Aguieira), focusing on their relation to the water quality and anthropogenic pressures. For this purpose, throughout 2023, sub-surface water samples were collected to assess the EP of the reservoirs, based on WFD metrics, and to identify MPs by size, color, type, and chemical composition. Furthermore, the study areas were characterized by land use, soil occupation, and anthropogenic pressures to help identify potential sources of MPs. A total of 5862 MPs were observed in Rabagão and 1658 in Aguieira, with the highest concentrations recorded near the dam, in both reservoirs. For both reservoirs, the predominance of fibers between 0.1 and 0.5 mm was observed, and blue, black, and gray were the most common colors. Chemical composition analysis using ATR-FTIR identified polyethylene as the predominant polymer present in the samples from both reservoirs. Rabagão reservoir exhibited aquaculture as the main anthropogenic pressure, and a Good EP classification, while Aguieira exhibited greater anthropogenic impact (e.g., wastewater discharges, recreational activities, intensive forestry) and a Moderate EP classification. Overall, an apparent discrepancy was observed between the water quality of the reservoirs and the concentration of MPs, suggesting that a good ecological classification, based on existing metrics, does not necessarily indicate low microplastic pollution. This emphasizes that incorporating MPs monitoring into water quality assessments can provide important complementary information, particularly for reservoirs of notable environmental and social significance.

1.11. Polymer Inclusion Membrane for Passive Sampling of Zn(II) in Water

• Marin Senila, Lacrimioara Senila and Eniko Kovacs

• Research Institute for Analytical Instrumentation, INCDO-INOE, 400293 Cluj-Napoca, Romania

Heavy metals, due to their toxicity, are known to cause various environmental and health issues; therefore, their monitoring is essential. Polymer inclusion membranes (PIMs) are innovative materials that can be utilized in passive sampling (PS) tools for monitoring heavy metals. Passive sampling is an integrative technique that has been developed over recent years and serves as a promising complementary tool for grab sampling. Its principle is based on the passive diffusion of contaminants through a membrane and their accumulation in a receiving phase. The concentration of pollutants in the studied system can thus be inferred from the amount of pollutant recovered from the binding phase. However, commercially available tools for passive sampling are quite limited. This work reports a preliminary application of a polymer inclusion membrane for potential integration into passive sampling tools. The PIM was prepared using 50% poly(vinyl chloride) (PVC) as the base polymer, 40% di(2-ethylhexyl) phosphoric acid (D2EHPA) as the carrier, and 10% dioctyl phthalate (DOP) as the plasticizer. Transport experiments through the PIM were conducted in a homemade permeation cell, using a Zn(II) solution at a concentration of 20 mg L⁻¹ as the donor phase. The acceptor solution was 0.1 M HCl. The results indicated good performance regarding the transport of Zn(II) ions, with over 80% of the analyte being transported across the membrane.

1.12. Research on the Presence of Selenium in the Bodies of Dogs and Its Effect on Animals of This Species

• Wioleta Sowińska ¹ and Liliana Rytel ²

¹ 

District Veterinary Inspectorate, 13-300 Nowe Miasto Lubawskie, Poland

² 

Department of Internal Medicine with Clinic, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland

Purpose and justification of the research: Selenium is an essential trace element for dogs, affecting the proper functioning of the body. Both its deficiency and excess carry the risk of serious health problems. The presentation presents the results of the presence of selenium in the bodies of dogs by analyzing the results of tests in the dog’s fur and the effect of this element on the organisms of this species. The purpose of this study is to analyze the results of the presence of selenium in the fur of dogs collected for testing from one hundred individuals living in the Warmian–Masurian Voivodeship.

Materials and methods: Hair samples were analyzed using inductively coupled plasma mass spectrometry (ICP-MS). Hair samples were collected in April 2022 from 100 adult dogs of both sexes, different breeds, ages, and body conditions. All animals were domestic dogs that went for walks with their owners three to six times a day and had a similar diet. All dogs lived in the Iława and Nowe Miasto districts of the Warmian–Masurian Voivodeship. Hair samples were collected from the abdomen, as close to the skin as possible.

Results and conclusions: The results indicate the presence of selenium in the fur of all dogs tested, significantly exceeding the detection limit, with the lowest result being 0.20 µg/g and the highest 1.37 µg/g. The average content of this chemical element in the fur of the dogs tested was 0.58 µg/g.

The results obtained show that selenium is present in all dogs’ bodies, and its level may depend on both its supply in the diet as well as its presence in the dogs’ environment. In addition, this study indicates that the analysis of dog hair samples is useful for examining the level of selenium in the bodies of these animals.

2. Session: Molecular Stressors—Nanoparticles, Mixtures, and Persistent Pollutants

2.1. Cigarette- and E-Cigarette-Derived Pollutants Impact the Physiology of Daphnids

• Izabela Malgorzata Antepowicz, Flavia Melati Chiappara, Emma Rowan, Anne Leung and Konstantinos Grintzalis

• School of Biotechnology, Dublin City University, D09 V209 Dublin, Ireland

Not only is smoking a main contributing cause of several diseases with implications in human health, but as the most popular habit with global consumption, it has severe implications and impact on the ecosystem, something that remains uncontrolled. Smoking leaves behind cigarette filter butts, which take up to fifteen years to decompose and can concentrate a great number of toxic compounds. These compounds leach out into the aqueous environment, polluting surface waters. Pollution assessment is based on the detection of chemicals and pollutants in the environment to estimate quality; however, such measurements are merely descriptive and provide, to their limit of detection, a report of the panel of pollutants present without any mechanistic information for their action. Therefore, water chemistry methods cannot predict the appearance of specific pollutants early. This is the reason why, in recent years, environmental risk assessment and monitoring are moving towards effect-based methods and novel approach methodologies as more sensitive tools for risk assessment. These approaches use biological systems and molecular information as new metrics for monitoring. Focusing on the freshwater ecosystem, the water flea is a sentinel organism used extensively in ecotoxicology. Combining phenotypic and holistic approaches, we screened for the molecular effects of a cigarette and an e-cigarette on daphnids following the analysis of filter extracts to assess their chemical composition. We identified perturbations in the physiology and metabolism of daphnids with feeding, enzyme activities, mortality and survival, and metabolic perturbations. Aiming to elevate the water flea as an equivalent to “a canary in the coal mine”, we intend to identify the presence of cigarette pollutants in the environment in actual river samples.

2.2. Molecular Mechanisms of Foodborne Titanium Dioxide Nanoparticle-Induced Intestinal Toxicity: A Multi-Omics and Adverse Outcome Pathway Analysis

• Min Zhang and Wei Mu

• School of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

• Introduction: Titanium dioxide (TiO₂) is frequently used as a whitening agent (E171). Although historically classified as “generally recognized as safewhile China and the United States maintain permissible limits. E171 ranges between 200 and 300 nm in diameter, while smaller nanoparticles are more likely to be absorbed by intestina”, the European Food Safety Authority (EFSA) suspended its food additive approval due to unresolved concerns (EFSA Panel, 2021)[1], l epithelial cells. Based on bioinformatics, we studied the molecular mechanism of the TiO₂-induced intestinal damage process.
• Methods: The Comparative Toxicogenomics Database (CTD) identified 160 colitis- and 258 colorectal cancer-associated genes modulated by TiO₂, and Cytoscape was used to make a PPI network diagram. Functional enrichment analysis (GO/KEGG) revealed key pathways, cross-validated with transcriptomic datasets from TiO₂-exposed enteritis (GSE92563) and colorectal tumor models (GSE109520). We screened the AOP-wiki database and constructed a new pathway network of factors. In addition, we used MECE and CT26 cells separately co-cultured with E171 and TiO₂-10 nm for 48 h for gene sequencing to validate our predictive model.
• Results: In the intestinal inflammation model, TiO₂ exposure initially activates the JNK and MAPK signaling pathways, causing changes in the cell membrane (invagination, endocytosis), cell death, inflammation, and hypoxia, leading to inflammatory bowel disease. For colorectal cancer, cytokines (Stat1, Pik3cd, Cxcr4, etc.) were activated, leading to extracellular matrix response and apoptosis, resulting in colorectal cancer and migration. Meanwhile, we analyzed the existing AOPs with high relevance to the intestinal damage mechanism of TiO₂ (AOP392, AOP451, and AOP303) and constructed a new AOP. In cell sequencing, we also enriched the relevant pathways, which pointed to different factors in the two types of cells.
• Conclusions: We introduced these regulatory cascades as new MIE and KE into the AOP framework, outlining the precise mechanism by which TiO₂ exposure induces colorectal damage and providing new evidence for food safety issues.

• Reference

• EFSA 6585; Scientific opinion on the safety assessment of titanium dioxide (E 171) as a food additive. European Food Safety Authority (EFSA): Parma, Italy, 2021. https://www.efsa.europa.eu/sites/default/files/2021-05/6585.pdf#:~:text=Based%20on%20all%20the%20evidence%20available%2C%20a,safe%20when%20used%20as%20a%20food%20additive (Accessed on 1 July 2021).

2.3. Advancing Nanotoxicology: High-Throughput Screening for Assessing the Toxicity of Nanoparticle Mixtures

• Newton Neogi, Kristi Priya Choudhury, Sabbir Hossain, MD. Golam Sazid and Ibrahim Hossain

• Department of Environmental Research, Nano Research Centre, Sylhet 3114, Bangladesh

The widespread application of nanoparticles (NPs) in fields ranging from consumer products to industrial processes has led to increased concerns about their potential toxic effects on human health and the environment. While traditional toxicological studies often evaluate the effects of individual nanoparticles, real-world exposure scenarios typically involve mixtures of nanoparticles, where interactions between particles can significantly alter their toxicological profiles. This study focuses on addressing this critical gap by employing high-throughput screening (HTS) to evaluate the combined effects of nanoparticle mixtures under various exposure conditions. Our research investigated how metal oxide nanoparticles can provide advancements in commercial applications thanks to their cytotoxic, genotoxic, and oxidative stress-inducing effects. By leveraging HTS platforms, we rapidly screened multiple mixture ratios and exposure durations using human lung epithelial cells and zebrafish embryos as model systems. The results revealed a range of interactions, from synergistic effects, where the combined toxicity exceeded the sum of individual toxicities, to antagonistic effects, where toxicity was mitigated. Mechanistic analyses showed that oxidative stress and metal ion release were key drivers of toxicity, particularly in ZnO-dominant mixtures. This study highlights the importance of integrating HTS into nanotoxicology research to provide a more comprehensive understanding of nanoparticle mixtures’ behavior. The large datasets generated through HTS enable predictive modeling, allowing researchers to anticipate toxicological outcomes and guide the development of safer nanomaterials. Furthermore, the findings emphasize the need for regulatory frameworks to incorporate mixture effects into nanoparticle risk assessments, moving beyond the current single-particle-focused approaches.

2.4. Biochemical and Transcriptomic Effects of Polystyrene Nanoplastics in Juvenile Amphiprion ocellaris

• Manuela Piccardo ¹, Rosa Maria Sepe ², Lucia Pittura ³, Pasquale De Luca ⁴, Monia Renzi ¹, Stefania Gorbi ³, Alberto Pallavicini ¹, Laurence Besseau ⁵, Vincent Laudet ⁶, Mirko Mutalipassi ^7,8, Paolo Sordino ⁹ and Antonio Terlizzi ⁷

¹ 

Department of Life Sciences, University of Trieste, Via Giorgieri, 10, 34127 Trieste, Italy

² 

Department of Marine Biotechnology, Stazione Zoologica Anton Dohrn, Via F. Acton, 55, 80133 Naples, Italy

³ 

Department of Life Sciences and Environment, Marche Polytechnic University, Via Brecce Bianche, 60131 Ancona, Italy

⁴ 

Department of Research Infrastructures for Marine Biological Resources, Stazione Zoologica Anton Dohrn, Villa Comunale, 80126 Naples, Italy

⁵ 

BIOM, Observatoire Océanologique, CNRS, Sorbonne Université, 66650 Banyuls-sur-Mer, France

⁶ 

Okinawa Institute of Science and Technology, Marine Eco-Evo-Devo Unit, 1919-1 Tancha, Onna son, Okinawa 904-0495, Japan

⁷ 

Department of Integrated Marine Ecology, Stazione Zoologica Anton Dohrn, Villa Comunale, 80126 Naples, Italy

⁸ 

National Biodiversity Future Center, Piazza Marina 61, 90133 Palermo, Italy

⁹ 

Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Sicily Marine Centre, Contrada Porticatello, 29, 98167 Messina, Italy

Nanoplastics (NPs) pose significant ecological risks due to their small size and ability to penetrate biological tissues across all trophic levels. However, their biological effects remain incompletely understood, particularly in tropical reef ecosystems. This study investigated the effects of short-term (7-day) exposure to carboxyl-modified polystyrene nanoparticles (PS-NPs, 100 nm) on juvenile clownfish (Amphiprion ocellaris), using two concentrations: low (20 µg/L, environmentally relevant) and high (2000 µg/L). A multidisciplinary approach combining biochemical and transcriptomic analyses was used to assess toxicity. Biochemical assays revealed limited changes in oxidative stress biomarkers (CAT, GR, GST, and TOSCA assays). However, the Integrated Biomarker Response index (IBRv2i) suggested compromised fish health, a finding supported by transcriptomic data. Gene expression analysis showed that both concentrations induced significant changes, with shared differentially expressed genes (DEGs) mostly upregulated, indicating a core molecular response. Despite this, each treatment also elicited unique transcriptional signatures. GO enrichment analysis of the high-concentration group highlighted processes related to muscle contraction, extracellular matrix organization, and oxidative stress responses, suggesting structural and physiological remodeling. In contrast, the low-concentration group showed enrichment for sensory system development, particularly visual function, as well as neurodevelopment and metabolic processes. These results indicate that even low NP concentrations can trigger substantial molecular alterations, although the nature of the biological response varies by exposure level. While high exposure is associated with stress adaptation and tissue restructuring, low exposure predominantly affects neural and sensory pathways. In conclusion, this study demonstrates that PS-NP exposure can alter gene expression and physiological state in juvenile reef fish, even at environmentally relevant concentrations. The distinct molecular profiles observed at different concentrations underscore the importance of exposure level in determining affected biological systems and potential long-term impacts of nanoplastic pollution in marine environments.

2.5. Exposure to Diesel Exhaust Nanoparticles: Depression-like Behavior Induced by Systemic Inflammation and Brain-Derived Neurotrophic Factors

• Mojtaba Ehsanifar ¹ and Mehravar Rafati ²

¹ 

Department of Environmental Health, School of Medicine, Torbat Jam Faculty of Medical Sciences, Torbat Jam 9571877484, Iran

² 

Department of Medical Physics and Radiology, School of Allied Medical Sciences, Kashan University of Medical Sciences, Kashan 87159-73471, Iran

The impact of particulate matter (PM) on health is an ongoing concern, with PM2.5 pollution being implicated in a broad spectrum of negative health outcomes, including cardiovascular and respiratory diseases, metabolic disorders, and various mood disorders. Despite significant research efforts, the exact mechanisms through which these particles exert their harmful effects remain largely elusive. In our current study, we utilized a sophisticated small animal whole-body inhalation exposure system to closely examine diesel engine exhaust particles (DEPs), aiming to replicate real-world atmospheric conditions as accurately as possible. The focus of our investigation was to scrutinize potential depression-like symptoms in mice subjected to long-term exposure to these particles, with insights into the molecular underpinnings offered via cutting-edge RNA sequencing techniques. In this study, we utilized male BALB/C mice that were systematically exposed to DEPs over varied durations. Behavioral assessments and pathological analyses of mice exposed to DEPs for 6, 8, and 10 weeks were conducted and juxtaposed with those from unexposed control groups. The findings revealed a discernible onset of depression-like behaviors in mice after 6, 8 and 10 weeks of DEP exposure, identifiable through behavioral testing and microscopic tissue examinations. Furthermore, RNA sequencing results suggested that the depression-like behaviors observed in DEP-exposed mice might be intricately tied to the modulation of both pro-inflammatory and anti-inflammatory cytokines, along with alterations in the BDNF pathways in brain regions such as the hippocampus and olfactory bulb. This evidence underscores the notion that exposure to DEPs could potentially instigate depressive patterns in a controlled murine model.

2.6. Nanoparticle-Induced Oxidative Stress: Mechanisms and Implications for Human Health and Environmental Safety

• Newton Neogi, Kristi Priya Choudhury, Ibrahim Hossain, Sabbir Hossain and MD. Golam Sazid

• Department of Environmental Research, Nano Research Centre, Sylhet 3114, Bangladesh

Nanoparticles (NPs), which possess unique physicochemical qualities such as large surface area and reactivity, have brought about a revolution in a variety of sectors, including medicine and electronics. The growing ubiquity of these substances, on the other hand, has given rise to worries over the toxicological effects they have on human health and ecosystems. The condition known as oxidative stress, which is caused by an imbalance between the formation of reactive oxygen species (ROS) and antioxidant defenses, is one of the key processes that contribute to the toxicity of NPs. An excessive amount of ROS may cause damage to cellular components such as lipids, proteins, and DNA, which can result in detrimental consequences such as inflammation, apoptosis, and the development of cancer. NP-induced oxidative stress is investigated in this work, which focuses on the molecular mechanisms that are responsible for it. These processes include mitochondrial dysfunction, catalytic redox cycling, and the release of metal ions from particle disintegration. In addition, we investigate how the features of NPs, such as their size, shape, surface charge, and composition, affect their capacity to produce ROS. Additionally, the consequences of oxidative stress for both acute and chronic health effects are examined, in addition to the role that it plays in the toxicity of the environment. The use of antioxidants and alterations to the surface of NPs are two examples of mitigation measures that are discussed in this article. The findings of this study highlight the significance of gaining knowledge of the processes behind oxidative stress to ensure the safe design and deployment of nanoparticles.

2.7. Phytoremediation of Cobalt Nanoparticles by Lemna minor: Insights into Uptake Mechanisms and Toxicity Responses

• Marcelo Pedrosa Gomes ¹, Ana Carolina Malin ¹, Raizza Zorman Marques ¹, Maritana Mela Prodocimo ², Leonardo César de Moraes ³, Cleber Cunha Figueredo ³ and Philippe Juneau ⁴

¹ 

Laboratório de Fisiologia de Plantas sob Estresse, Departamento de Botânica, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba 81531-900, Brazil

² 

Departamento de Biologia Celular, Universidade Federal do Paraná, Curitiba 81531-980, Brazil

³ 

Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Curitiba 81531-980, Brazil

⁴ 

Ecotoxicology of Aquatic Microorganisms Laboratory, GRIL, EcotoQ, TOXEN, Department of Biological Sciences, Université du Québec à Montréal, Succ. Centre-ville, H3C 3P8 Montréal, Canada

The increasing use of cobalt nanoparticles (CoNPs) in industrial and biomedical applications raises concerns about their environmental fate and impact on aquatic ecosystems. In this study, we evaluated the phytotoxicity and phytoremediation potential of CoNPs using Lemna minor, a macrophyte known for its metal accumulation capacity. Specifically, we investigated CoNP-induced oxidative stress, physiological responses, and nanoparticle uptake mechanisms. L. minor plants were exposed to CoNPs at concentrations ranging from 0.1 to 20,000 µg L⁻¹ for seven days. Growth rates, photosynthesis, and respiration were measured, while oxidative stress markers (H₂O₂ and MDA) and antioxidant enzyme activities (SOD, APX, and CAT) were analyzed. Cobalt accumulation in plant tissues was quantified using ICP-MS, and transmission electron microscopy (TEM) was used to assess ultrastructural changes and intracellular cobalt localization. Our results showed that CoNP exposure led to significant oxidative stress and metabolic impairment at concentrations ≥1000 µg L⁻¹, reducing photosynthesis by 85% and growth by 77%. TEM analyses revealed that CoNPs were internalized by L. minor and localized in chloroplasts, causing thylakoid disorganization and plastoglobuli accumulation. Interestingly, CoNPs were found in different subcellular locations than Co²⁺ ions, suggesting distinct toxicity mechanisms. Despite these effects, L. minor removed over 99% of CoNPs and Co²⁺ from the medium, highlighting its potential for nanoparticle remediation. This study provides novel insights into the uptake and impact of CoNPs in aquatic plants, reinforcing the role of L. minor as an effective phytoremediator in nanoparticle-contaminated environments. Future research should explore long-term accumulation dynamics and optimize phytoremediation strategies for sustainable nanoparticle management.

2.8. Scientific Validation of a Cortical Neurosphere Model for Developmental Neurotoxicity Assessment and Evaluation of Chronic PFAS Mixture Exposure

• Narimane Kebieche ¹, Seungae Yim ¹, Claude Lambert ², Farzana Liakath Ali ³ and Rachid Soulimani ⁴

¹ 

Food Neurotoxicology and Bioactivity Unit, LCOMS laboratory, University of Lorraine, 57070 Metz, France

² 

Immunology Laboratory, University Hospital of Saint-Étienne, 42055 CEDEX 2 Saint-Étienne, France

³ 

Department of Obstetrics and Gynecology, University of Chicago, 60637 Chicago, USA

⁴ 

Neurotoxicologie Alimentaire et Bioactivité, LCOMS laboratory, University of Lorraine, 57070 Metz, France

• Developmental neurotoxicity (DNT) is increasingly recognized as a public health concern due to evidence linking environmental exposures to neurodevelopmental disorders. Traditional in vivo approaches are resource-intensive, highlighting the need for alternative in vitro models. In this context, 3D neurosphere cultures derived from neural stem cells (NSCs) provide a promising platform to evaluate key neurodevelopmental processes and their disruption by toxicants. This study aimed to develop and characterize a 3D neurosphere model of cortical differentiation and assess its response to chronic exposure to the pesticide chlorpyrifos (CPF) and a representative mixture of per- and polyfluoroalkyl substances (PFASs). After 21 days of spontaneous differentiation, the model expressed genes corresponding to multiple neurodevelopmental endpoints—neurogenesis, gliogenesis, synaptogenesis, and neuronal signaling—as confirmed by means of qPCR and flow cytometry. Additional phenotypic endpoints included cell proliferation, neurosphere size, radial migration, and cytotoxicity (XTT assay). To assess the model’s sensitivity to environmental toxicants, neurospheres were chronically exposed from Day 0 of differentiation, targeting a critical developmental window. The toxicants tested were CPF (125 μM and 250 μM) and a PFAS mixture at concentrations relevant to human serum exposure. CPF significantly reduced cell viability, inhibited sphere growth and radial migration, and disrupted gene expression across neurodevelopmental pathways, including the downregulation of genes involved in neuronal and glial differentiation and synaptogenesis, while upregulating early neurogenesis and signaling markers, particularly NTRK1. Furthermore, cytometry revealed CPF-induced alterations in the cell cycle profile and increased expression of proliferation markers. PFAS exposure, though not overtly cytotoxic, caused significant transcriptional disturbances in glial and neuronal genes, suggesting early interference with developmental programming even at non-cytotoxic, environmentally relevant levels. This neurosphere model recapitulates key stages of brain development and enables integrated molecular and functional DNT analysis. Its responsiveness to CPF and PFAS supports its relevance as an in vitro platform for chemical safety assessment.

2.9. Toxicity Evaluation of Solid Lipid Nanocarriers Using In Vitro and In Vivo Approaches

• Beatriz Cristão ^1,2, Paula Martins-Lopes ^1,2, Filomena Adega ^2,3, Helena Rodrigues ⁴, Marlene Lúcio ⁵ and Carla M. Lopes ^6,7

¹ 

DNA & RNA Sensing Lab, University of Trás-os-Montes and Alto Douro, Genetics and Biotechnology Department, Blocos Laboratoriais Ed, 5000-801 Vila Real, Portugal

² 

BioISI—Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisboa, 1749-016 Lisboa, Portugal

³ 

CAG—Laboratory of Cytogenomics and Animal Genomics, Department of Genetics and Biotechnology, University of Trás-os-Montes e Alto Douro, 5000-801 Vila Real, Portugal

⁴ 

LAQV, REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal

⁵ 

Centre of Molecular and Environmental Biology (CBMA), Laboratório para Materiais e Tecnologias Emergentes, Centre of Physics, University of Minho and Porto, Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.

⁶ 

Biomedical and Health Sciences Research Unit, RISE HEALTH—UFP, I3ID—Instituto de Investigação, Inovação e Desenvolvimento, Faculdade Ciências da Saúde, Universidade Fernando Pessoa

⁷ 

Associate Laboratory i4HB–Institute for Health and Bioeconomy, UCIBIO–Applied Molecular Biosciences Unit, MEDTECH, Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal

Nanotechnology has been extensively used in the development of drug delivery systems, with various benefits on the protection, control release, and improvement of drug delivery to target sites. Among nanocarriers, lipid-based ones, such as solid lipid nanoparticles (SLNs), have been found to be biocompatible and theoretically safe for pharmaceutical application. Even so, studies evaluating the possible toxicity of nanocarriers have been dismissed and are lacking in the literature. Therefore, our study aimed to evaluate the toxicity of SLNs and their components using in vitro and in vivo studies. The in vitro studies were carried out in a normal human fibroblast cell line. The formulations of positively charged SLNs (SLNs⁺) exhibited greater cytotoxicity compared to negatively charged ones (SLNs⁻), especially at higher concentrations (10, 20, and 100 µg/mL), suggesting that this response is probably due to the higher interaction of SLNs⁺ with cell membranes. The SLN components (Precirol^® ATO 5, Tween^® 80, and benzalkonium chloride) were also studied in their higher concentration within the formulation. Benzalkonium chloride was the only component that exhibited toxicity in the in vitro studies. The in vivo assays were performed using Drosophila melanogaster with SLN⁺ at 100 µg/mL and their components. The toxicity was studied taking into consideration chronic and periodic exposure, and the parameters assessed were egg number, hatched flies, and sex. Considering chronic exposure, no statistical differences were found considering these parameters. When F₁ flies, submitted to chronical exposure, were crossed with non-treated flies, the effect on gender was evident when the different components were tested. The discrepancy between the in vitro and in vivo results suggests that while SLNs demonstrate toxicity in cell cultures, their effects are less pronounced in whole organisms, likely due to biological factors such as metabolism and detoxification mechanisms, highlighting the need for comprehensive in vivo testing to assess the true impact of nanocarriers.

2.10. When Nanoplastics Meet Neurons: Ihe Impact of Functionalized Polystyrene Nanoplastics on Human Neuroblastoma Cells

• Carolina Mota ¹, Ana Margarida Araújo ¹, Maria Enea ², Eulália Pereira ², Ana Reis Mendes ¹, Rui Fernandes ³, Sofia Pacheco ³, Marlene Lúcio ^4,5, Carla Martins Lopes ^6,7, Isabel M.P.L.V.O. Ferreira ¹ and Márcia Carvalho ^1,6,8

¹ 

Bromatology and Hydrology Laboratory, LAQV/REQUIMTE, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal

² 

Department of Chemistry and Biochemistry, LAQV/REQUIMTE, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal

³ 

HEMS-Histology and Electron Microscopy, Instituto de Investigação e Inovação em Saúde (i3S), University of Porto, 4200-135 Porto, Portugal

⁴ 

Department of Physics, CF-UM-UP—Physics Center of the Universities of Minho and Porto, University of Minho, 4710-057 Braga, Portugal

⁵ 

CBMA—Center for Molecular and Environmental Biology, University of Minho, 4710-057 Braga, Portugal

⁶ 

FFP-I3ID, FP-BHS, Faculty of Health Sciences, Fernando Pessoa University, 4249-030 Porto, Portugal

⁷ 

UCIBIO-Applied Molecular Biosciences Unit, MedTech-Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal

⁸ 

RISE-Health, Faculty of Health Sciences, Fernando Pessoa University, Fernando Pessoa Teaching and Culture Foundation, 4249-030 Porto, Portugal

Nanoplastics (NPs) have emerged as a growing environmental and health concern due to their widespread presence in various environmental matrices, including food, water, and air. They are inadvertently ingested, inhaled, or absorbed through the skin, raising concerns about their potential to interact with cellular systems, including the nervous system. Despite increasing awareness, their neurotoxic potential, particularly the effects of functionalized NPs, remains poorly understood. This study provides novel insights into the neurotoxicity of four types of polystyrene nanoplastic (PS-NPs)—plain PS-NPs (50 nm and 100 nm) and amine- and carboxyl-functionalized PS-NPs (100 nm)—on human SH-SY5Y cells. Cells were exposed to NP concentrations ranging from 1 to 500 µg/mL for 24 and 48 h, with NP characterization and stability assessments conducted in culture media prior to toxicity evaluations (cell viability, ROS/RNS production, NP internalization, morphological and ultrastructural changes). In-depth toxicity assessments revealed that functionalized NPs, particularly amine-modified ones, induced greater cytotoxicity than their plain counterparts. Cellular viability assays demonstrated a concentration- and time-dependent reduction, with significant cytotoxicity observed at 200–500 µg/mL. ROS/RNS production was markedly elevated in plain 100 nm and amine-functionalized NPs at concentrations of 200–500 µg/mL, with oxidative stress intensifying over time. Transmission electron microscopy uncovered distinct subcellular damage patterns, including endoplasmic reticulum dilation, mitochondrial impairment, and Golgi fragmentation, correlating with NP size, concentration and functionalization. Notably, functionalized NPs exhibited greater cellular uptake, with amine-modified NPs showing the highest internalization. Further mechanistic analyses revealed that PS-NPs induced apoptosis, autophagy, and lysosomal dysfunction, with functionalized variants exhibiting more pronounced effects. These findings underscore the critical influence of NP functionalization on neurotoxicity and highlight the urgent need for further investigation into their potential health implications, particularly concerning human exposure and neurodegenerative risk.

• Acknowledgments: This work received support from PT national funds through the UID/50006 project and FCT/MCTES through the SALIVA+ project (DOI 705 10.54499/2022.08978.PTDC).

3. Session: Cutting-Edge Approaches in Pharmacology and Toxicology

3.1. Molecular and Behavioral Effects of Atorvastatin Exposure in Zebrafish (Danio rerio): Insights into Statin Toxicity at Environmentally Relevant Levels

• Phyllis Wah, Christopher Martyniuk, Hailey Skaggs, Ciara Saccente, Emma Ivantsova, Lev Avidan and Cole English

• Center for Environmental and Human Toxicology, Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611, USA

Atorvastatin, a widely used cholesterol-lowering statin, functions by inhibiting 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. While its environmental presence has been documented, its toxicity to aquatic organisms remains poorly understood. In this study, we conducted RNA-seq and measured sublethal developmental and locomotor-related endpoints in zebrafish to identify toxicity mechanisms related to environmentally relevant exposures to atorvastatin. Zebrafish were exposed to atorvastatin continuously, and their water was renewed every 24 h, with a 90% change in the proportion of fresh chemical in the water every day. No significant impacts on survival, hatching success, or deformities were observed across treatment groups. However, the amount of reactive oxygen species (ROS) was increased at lower doses, and locomotor activity was significantly reduced at 100 µg/L. RNA-seq analysis revealed dose-specific transcriptomic responses. At 1 µg/L, differentially expressed genes included collagen type I, alpha 1a, fatty acid desaturase 2, and prolactin, implicating pathways such as B-cell leukemia signaling, CD8+ T-cell activation, and NF-κB regulation. At 100 µg/L, genes such as solute carrier family 12 (potassium/chloride transporter) and G-Protein subunit alpha z were affected, linked to pathways like Th17-cell activation and oxytocin signaling. These findings suggest that atorvastatin can alter neurodevelopmental and immune-related pathways even at low concentrations, without causing overt morphological defects. Data from this study are expected to contribute to our understanding of atorvastatin toxicity to fish in their early stages and can support efforts to assess the risks of statin drugs in aquatic ecosystems.

3.2. Advanced Analytical Method for Determination of Aflatoxins in Chili Powder Using LC-MS/MS

• Manisha Dhanshetty ¹, Gajanan Vishnu Mali ² and Marco Garcia Vaquero ¹

¹ 

School of Agriculture and Food Science, University College Dublin, Belfield, D04 V1W8 Dublin, Ireland

² 

Department of Microbiology, Bharati Vidyapeeth (Deemed to be University), Yashwantrao Mohite College, Erandwane, Pune 411 038, Maharashtra, India

Chili powder, a widely used spice, is commonly contaminated with aflatoxins (AFs) posing significant health risks. AFs, particularly Aflatoxin B1, a highly carcinogenic mycotoxin produced by Aspergillus species, pose serious health risks, especially in agricultural commodities like chili, where contamination must be closely monitored. Traditional detection methods such as direct chromatographical analysis using HPLC often face challenges due to the complex matrix of chili powder. Due to the lack of a validated method for the direct detection of AFs in chili powder, it was essential to develop one for effective monitoring and to support trade. In this study, we established and validated a sensitive, accurate, and precise analytical method capable of detecting AFs (AFB1, AFB2, AFG1, AFG2) in chili powder. The optimized sample preparation involved the QuEChERS method followed by analysis through liquid chromatography with tandem mass spectrometry (LC–MS/MS). This direct analysis provided the quantification limits (LOQs) of 0.25 ng/g for each AF well below the maximum levels set by the European Commission. Recoveries at and above the LOQ levels lie in the range of 80–108%, with RSD below 8%. The QuEChERS extraction protocol was compared with the Immuno affinity clean up method. Market sample analysis showed AFB1 levels with precision RSDs below 6%. Given its high efficiency and compliance with regulatory standards, this method is suitable for routine AF and OTA testing in chili powder.

3.3. Beyond the Blotter: Forensic Identification of Novel LSD Analogs via GC–QqQ–MS and UHPLC–QqQ–MS/MS

• Kaja Tusiewicz ¹, Olga Wachełko ², Karolina Nowak ³, Marcin Zawadzki ⁴ and Paweł Szpot ⁵

¹ 

Department of Forensic Medicine, Wroclaw Medical University, 50-345 Wroclaw, Poland

² 

Institute of Toxicology Research, 55-093 Borowa, Poland

³ 

Faculty of Medicine, Department of Pharmacology, University of Opole, 45-052 Opole, Poland

⁴ 

Faculty of Medicine, Department of Social Sciences and Infectious Diseases, Wroclaw University of Science and Technology, 50-370 Wroclaw, Poland

⁵ 

Department of Forensic Medicine, Wroclaw Medical University, 50-345 Wroclaw, Poland

• Introduction: The proliferation of novel LSD analogs, often synthesized to circumvent existing legal regulations, presents a growing challenge for both forensic toxicology and public health. These so-called “designer psychedelics” often remain undetectable by routine screening methods and may exist in isomeric forms with different pharmacological profiles. As their availability rises, particularly via online markets, so does the urgency for accurate analytical methodologies that can distinguish known analogs and anticipate those yet to appear.
• Methods: Two complementary analytical strategies were employed: GC–QqQ–MS and UV spectroscopy for analytical standards, and UHPLC–QqQ–MS/MS for biological matrices. The GC–QqQ–MS method was optimized for the separation and identification of 13 LSD analogs, including structural isomers, with attention paid in particular to solvent influence on compound stability. In parallel, the UHPLC–QqQ–MS/MS protocol was used to develop a comprehensive method for the determination of extremely low concentrations of analytes in biological material, as well as to investigate their stability over time under various storage conditions.
• Results: GC–QqQ–MS with EI effectively differentiated critical isomer pairs (e.g., LSD vs. MiPLA, 1P-LSD vs. 1P-MiPLA) by unique ion fragmentation patterns and chromatographic separation. Solvent studies confirmed that methanol induces degradation in several analogs, whereas diethyl ether and acetone preserve compound integrity. The UHPLC–QqQ–MS/MS method demonstrated exceptional sensitivity (LOQ 0.5 pg/mL) and robustness, detecting analogs in forensic case samples and confirming degradation pathways, particularly for N1-substituted compounds converting to LSD or MiPLA.
• Conclusions: This study highlights the necessity of robust, sensitive analytical methods to accurately identify LSD analogs and their isomers. Given their possible instability, ongoing method development is essential for reliable forensic interpretation and early detection of emerging substances.

3.4. Molecular and Metabolic Profiles of Individual and Combined Nanoplastic–BPA Exposure Assessed by FTIR Spectroscopy

• Carina Ladeira ^1,2,3, Daniela Tomás ⁴ and Cecília Calado ^5,6

¹ 

H&TRC—Health & Technology Research Center, Escola Superior de Tecnologia da Saúde de Lisboa (ESTeSL), Polytechnic University of Lisbon, Avenida D. João II, lote 4.69.01, Parque das Nações, 1990-096 Lisboa, Portugal

² 

Public Health Research Centre, NOVA National School of Public Health, Universidade NOVA de Lisboa, 1150-105 Lisbon, Portugal

³ 

Comprehensive Health Research Center (CHRC), 1169-056 Lisbon, Portugal

⁴ 

Lisbon School of Health Technology (ESTeSL), Polytechnic Institute of Lisbon, Avenida D. João II, lot 4.69.01, Parque das Nações, 1990-096 Lisbon, Portugal

⁵ 

ISEL—Higher Institute of Engineering of Lisbon, Polytechnic Institute of Lisbon, R. Conselheiro Emidio Navarro 1, 1959-007 Lisbon, Portugal

⁶ 

CIMOSM—Research Center in Modeling and Optimization of Multifunctional Systems, ISEL—Higher Institute of Engineering of Lisbon, Polytechnic Institute of Lisbon, R. Conselheiro Emidio Navarro 1, 1959-007 Lisbon, Portugal

The excessive use of plastic has raised concerns about exposure to nanoplastics (NPs) and bisphenol A (BPA) and their impacts on human health. This study aimed to assess a new method of detecting the in vitro effects of NPs and BPA that is sensitive yet simpler and faster compared to cytogenetic techniques such as cytokinesis-blocked micronucleus (CBMN) assay.

Individual and combined exposure of BPA and polystyrene nanoparticles (PS-NPs) was evaluated using the GP202 gastric cell line. The culture media, designated as 0 h after exposure and 24 h after the exposure, and cells 24 h after exposure, were analyzed by Fourier-transform infrared (FTIR) spectroscopy. CBMN did not reveal statistically significant differences in exposure to NPs and BPA compared with controls, though a trend towards an increase in MN, nucleoplasmic bridges (NBPs), and nuclear buds (NBUDs) was observed with combined exposure to PS-NPs and BPA. A univariate analysis of several FTIR spectral bands was performed after various spectral preprocessing methods, including baseline correction, baseline correction with normalization, second derivative, and second derivative with normalization. Several statistically different bands were observed, with significance at 1%, 5%, and 10%, both in multiple comparisons between exposure conditions and pairwise comparisons between exposure conditions. These changes were noted in spectra obtained from culture media and directly from cells. The FTIR spectrum represents the molecular profile of the culture media and cells, reflecting the metabolic state of the cells. Thus, FTIR spectroscopy proves to be a highly sensitive, rapid, and straightforward technique for detecting metabolic changes in gastric cell lines when exposed to NPs and BPA, even in conditions where no genotoxic changes are detected, as observed by CBMN.

3.5. The Determination of 2-Phenylethylamine Derivatives in MGG-Stained and Unstained Blood Smears: A Cutting-Edge Approach in Contemporary Toxicology

• Karolina Nowak ¹, Paweł Szpot ² and Marcin Zawadzki ^3,4

¹ 

Department of Pharmacology, Faculty of Medicine, University of Opole, 48 Oleska Street, 45052 Opole, Poland

² 

Department of Forensic Medicine, Faculty of Medicine, Wroclaw Medical University, 4 J. Mikulicza-Radeckiego Street, 50345 Wroclaw, Poland

³ 

Department of Social Sciences and Infectious Diseases, Faculty of Medicine, Wroclaw University of Science and Technology, 27 Wybrzeze Wyspianskiego Street, 50370 Wroclaw, Poland

⁴ 

Institute of Toxicology Research, 45 Kasztanowa Street, 55093 Borowa, Poland

• Introduction: Complete blood count (CBC) is one of the fundamental tests for assessing a patient’s condition. Despite widespread automation in hematology, manual blood smear analysis remains essential. This study aimed to evaluate the use of manual blood smears, both unstained and May–Grünwald–Giemsa (MGG)-stained, as a novel alternative biological matrix for xenobiotic determination.
• Methods: Analytical standards of selected 2-phenylethylamine derivatives were added to K₂EDTA whole blood, followed by manual smear preparation. Half of the smears were stained using the MGG technique. The developed method involved transferring the matrix from the slide into a test tube, followed by extraction with an n-hexane/ethyl acetate mixture under alkaline conditions. Analyses were conducted using UHPLC-ESI-QqQ-MS/MS. In unstained smears, xenobiotics were monitored across concentrations ranging from pg/mL to µg/mL. Total hemoglobin was evaluated at low (critical), moderate, and normal levels. The method was further applied to evaluate xenobiotic stability in blood smears stored at room temperature, the typical storage condition for such samples.
• Results: The study demonstrated that unstained blood smears can serve as a valuable alternative matrix for xenobiotic determination, enabling detection not only in poisoning cases (toxic/lethal concentrations) but also in exposure assessments, with a limit of quantification down to the ng/mL range. Despite substantial differences in total hemoglobin concentration, effective xenobiotic detection was achieved. In MGG-stained smears, however, staining steps reduced detection capabilities, and a qualitative interpretation approach is recommended.
• Conclusions: The results indicate that small blood volumes are sufficient for both qualitative and quantitative toxicological analyses, particularly in unstained smears. Furthermore, the ability to detect substances at trace levels highlights the necessity for cautious interpretation due to contamination risk. The findings regarding unstained blood smears, including stability results, may provide valuable support for toxicological examinations of blood traces on other glass surfaces.

4. Session: Pesticides, Pollutants, and Health Risk

4.1. A Green In-Syringe Microextraction Approach Using Deep Eutectic Solvents for the Determination of Pesticides in Environmental Water by GC-MS

• Seyed Mosayeb Daryanavard ¹, Sahar Shokouh ¹, Masoud Memar ² and Adrián de la Fuente Ballesteros ³

¹ 

Department of Chemistry, Faculty of Science, University of Hormozgan, Bandar Abbas 79158-93155, Iran

² 

Food Health Research Center, Hormozgan University of Medical Sciences, Bandar Abbas 79149-64468, Iran

³ 

I. U. CINQUIMA, Analytical Chemistry Group (TESEA), Faculty of Sciences, University of Valladolid, 47011 Valladolid, Spain

The pervasive presence of pesticide residues in aquatic environments poses significant risks to both ecosystems and human health. In response to the growing need for environmentally sustainable analytical techniques, we present a novel in-syringe dispersive liquid–liquid microextraction (IS-DLLME) method utilizing a natural deep eutectic solvent (DES) for the extraction of selected pesticides from water samples.

In this study, the DES was synthesized from choline chloride and a hydrogen bond donor, offering a biodegradable and non-volatile alternative to traditional organic solvents. The microextraction was carried out inside a standard plastic syringe, eliminating the need for additional equipment and minimizing solvent consumption. Following extraction, analytes were separated and quantified using gas chromatography–mass spectrometry (GC-MS).

The method demonstrated excellent analytical performance, with extraction recoveries ranging from 86% to 97%, relative standard deviations below 4.5%, and limits of detection in the low µg/L range. The linearity was confirmed over a broad concentration range, and matrix effects were negligible. Application to environmental water samples confirmed the method’s robustness and applicability to real-world analysis.

This work highlights the potential of combining IS-DLLME with DESs for rapid and sustainable monitoring of trace contaminants in water. The integration of green chemistry principles into sample preparation not only enhances analytical performance but also supports safer and more eco-conscious laboratory practices.

4.2. Evaluating the Combined Effects of Pesticides and Heavy Metals on Human and Honey Bee Health: A One Health Perspective

• Mai Awad

• Biology Department, School of Science, Technology, Engineering, and Mathematics (STEM), Adams State University, Alamosa, CO 81101, USA

The synergistic impact of pesticides and heavy metal exposure poses significant risks to both human health and honey bee populations, necessitating a comprehensive One Health assessment. This study integrates findings from recent research to evaluate the combined effects of these environmental contaminants. Pesticides and heavy metals, which are prevalent in agricultural and industrial settings, exhibit enhanced toxicity in co-exposure, leading to severe physiological and ecological consequences. This paper will specifically explore the mechanisms and outcomes of combined exposure and toxicity, providing case examples from both experimental and field-based studies. For humans, this synergistic toxicity manifests in increased oxidative stress, disruption of endocrine functions, and heightened risks of chronic diseases, including cancer, cardiovascular diseases, and neurodegenerative disorders. Honey bees, who are crucial pollinators in ecosystems, suffer from impaired neural functions, reduced foraging efficiency, and increased mortality rates, which can lead to significant declines in pollination services and biodiversity. The One Health approach underscores the interconnectedness of human, animal, and environmental health, advocating for integrated strategies to mitigate these risks. By addressing combined exposure to pesticides and heavy metals, this assessment aims to inform policy decisions and promote sustainable practices that safeguard health across species and ecosystems. Ultimately, this holistic perspective emphasizes the need for collaborative efforts among scientists, policymakers, and stakeholders to develop effective interventions that protect both human and environmental health. Such collaborative efforts are essential for ensuring the resilience and sustainability of ecosystems, which, in turn, support human wellbeing and biodiversity.

4.3. Exposure Assessment Outside and Inside Firefighters’ Respiratory Protection System: Levels of PAHs and Associated In Vitro Toxicity

• Joana Teixeira ^1,2, Maria João Bessa ³, Cristina Delerue-Matos ², Bruno Sarmento ^3,4, Alice Santos-Silva ¹, Francisca Rodrigues ⁵ and Marta Oliveira ⁵

¹ 

UCIBIO i4HB, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal

² 

REQUIMTE/LAQV, Institute of Engineering, Polytechnic Institute of Porto, 4200-072 Porto, Portugal

³ 

UNIPRO—Oral Pathology and Rehabilitation Research Unit, University Institute of Health Sciences (IUCS), CESPU, 4585-116 Gandra, Portugal

⁴ 

i3S—Institute for Research and Innovation in Health, University of Porto, 4200-135 Porto, Portugal

⁵ 

REQUIMTE/LAQV, School of Engineering, Polytechnic Institute of Porto, 4200-072 Porto, Portugal

Structural firefighting exposes firefighters to intense physical and thermal stress, and emissions of health-burden compounds [e.g., particulate matter and polycyclic aromatic hydrocarbons (PAHs)]. The use of a self-contained breathing apparatus (SCBA) is mandatory during structure fires, although in the overhaul phase, firefighters often remove it. The characterization of inhaled PAHs during structure fires remains scarce, particularly for European firefighters. This study assessed, for the first time, the levels of PAH in the air of firefighters outside and inside the SCBA during training firefighting activities and at fire stations on regular working days. The control group included 13 male volunteer firefighters without active participation in firefighting in the previous 7 days, while the exposed group included 31 professional sapper firefighters. Outside PAHs were monitored with pre-treated polyurethane foam (PUF), placed in the personal protective equipment, while a cotton disk was placed inside the SCBA. PAHs were quantified by high-performance liquid chromatography. Total PAH levels in PUFs of exposed firefighters were higher than PUFs from the control group (11.5–28.0 µg/m³ versus 1.41 µg/m³) and significantly higher than levels detected inside the SCBA (1.96–10.40 ng/m³). Regarding carcinogenic PAHs, levels quantified outside the SCBA were higher for exposed firefighters than the control group (5.80–17.2 µg/m³ versus 0.84 µg/m³) and superior to the inside SCBA’s levels (0.77–0.93 ng/m³) (p 0.005). International occupational guidelines were not exceeded. The inside–outside ratios (1) suggested that ambient PAHs contributed to the levels quantified inside the SCBA. Extracts collected outside and inside the SCBA caused cytotoxicity in human alveolar and bronchial cell lines. Moreover, the collected samples were applied to an in vitro co-culture air–blood barrier model to assess permeability. Carcinogenic benzo(a)pyrene (100%) permeated the model. Overall, the negative effects of fire emissions on human respiratory cell lines were confirmed, suggesting that fire pollutants contribute to firefighters’ respiratory health burden.

4.4. A Comprehensive Multi-Class Method for the Determination of Pesticide Residues and Selected Mycotoxins in Nuts Using Liquid Chromatography with Tandem Mass Spectrometry: Single Laboratory Validation

• Raviraj Chandrakant Shinde ^1,2 and Pandit Shiragave ¹

¹ 

Department of Agrochemicals and Pest Management, Devchand College, Shivaji University, Kolhapur 416416, India

² 

Spectralytic Scientific India PVT. LTD. (PerkinElmer), Mumbai 400072, India

• Highlights

The simultaneous analysis of pesticide residues and mycotoxins in nuts is reported for the first time. The acetonitrile-based sample preparation provided satisfactory extraction. The two-step cleanup strategy effectively minimized the matrix effects. The performance of the method complied with the national (FSSAI) and international regulatory requirements and guidelines.

For the first time, a multiresidue analytical method is reported for the simultaneous analysis of multi-class pesticides and mycotoxins in nuts (groundnut, almond, cashew, and pistachio). The optimized sample preparation method involved comminution with water, extraction by acetonitrile, and cleanup using dispersive solid-phase extraction with a C₁₈ sorbent followed by a freezing step at −80 °C. The final analysis was carried out using liquid chromatography coupled with tandem mass spectrometry. The method’s performance was evaluated on 208 multi-class pesticides and mycotoxins [aflatoxins and ochratoxin A]. The strategies minimized the matrix effects and achieved suitable LOQs for checking regulatory compliance. The findings indicated satisfactory recoveries (70–110%) at an LOQ of 0.01 mg/kg and higher levels, with a repeatability of 16% RSD. Notably, the extraction and analysis using a single chromatographic run provided satisfactory selectivity, sensitivity, quantification, and accuracy (recovery and precision) for target compounds during single laboratory studies. The developed method complied with the regulatory requirements as well as international analytical quality control guidelines.

4.5. A Study Evaluating the Performance of the GHS Mixtures Equation for Predicting the Acute Oral Toxicity (LD₅₀) of Spray Adjuvants

• Phuong Hang Le, Xiyuan Cheng, Neelima Verma and Shelley DuTeaux

• Department of Pesticide Regulation, California Environmental Protection Agency, Sacramento, CA 95812, USA

Spray adjuvants are products used with pesticides to improve their application or effectiveness. While exempt from federal pesticide registration by the US Environmental Protection Agency (US EPA), California law requires their registration as pesticides before sale or use. The California Department of Pesticide Regulation evaluated the suitability and acceptability of the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS) Mixtures Equation for predicting the acute oral toxicity of spray adjuvants as an alternative to requiring in vivo animal toxicity testing. The GHS Mixtures Equation estimates the acute oral toxicity (LD₅₀) of a mixture by combining individual LD₅₀ values of component ingredients. This study compared the results of the acute oral toxicity categories derived from the GHS Mixtures Equation with toxicity categories determined from in vivo oral studies for 51 mixtures. The results showed that the availability of LD₅₀ values for the relevant ingredients in the mixture limited the equation’s predictivity. The equation predicted the LD₅₀ values for 67% (N = 34) of the dataset. When these datasets were further compared against the US EPA toxicity classification system, the GHS Mixtures Equation results achieved 71% concordance, with toxicity either over-classified (6%) or under-classified (22%) by one category. Most discordant results were for spray adjuvant mixtures with LD₅₀ > 2000 mg/kg (limit test), particularly when 20% or more of the components had unknown LD₅₀s (N = 5). Finally, when compared against the US Occupational Safety and Health Administration’s GHS classification system, our results showed 82% concordance with 15% over-classified and 3% under-classified by one toxicity category (N = 34). Overall, the findings indicate that the GHS Mixtures Equation can reliably predict acute oral toxicity in low-toxicity mixtures, provided that all active ingredients have known LD₅₀s and that fewer than 20% of the components have unknown LD₅₀s.

4.6. Apicultural Contaminants as Emerging Food Safety Hazards: The Case of Acaricides

• Adrián Fuente-Ballesteros ¹, Zehra Hajrulai-Musliu ², Mila Arapcheska ³, Iveta Pugajeva ⁴, Ana M. Ares ¹, José Bernal ¹, Anton Gradišek ⁵ and Maj Smerkol ⁵

¹ 

Analytical Chemistry Group (TESEA), Faculty of Sciences, University of Valladolid, 47011 Valladolid, Spain

² 

Faculty of Veterinary Medicine—Skopje, University “Ss. Cyril and Methodius”—Skopje, 1000 Skopje, North Macedonia

³ 

Faculty of Biotechnical Sciences—Bitola, University “St. Kliment Ohridski”, 7000 Bitola, North Macedonia

⁴ 

Animal Health and Environment BIOR, Institute of Food Safety, LV-1076 Riga, Latvia

⁵ 

Department of Intelligent Systems, Jožef Stefan Institute, SI-1000 Ljubljana, Slovenia

Bee products are widely consumed and valued for their nutritional and therapeutic properties. However, these products are increasingly at risk of contamination due to the routine use of acaricides in apiculture to control Varroa destructor, a major parasitic threat to honey bee colonies. When improperly applied or overused, these chemical treatments can leave residues in bee-derived foods, representing an emerging hazard to food safety and consumer health. This study presents a comprehensive review of acaricide residues in bee products across Europe and Turkey, emphasizing their potential impact on human exposure. The literature review covered a six-year period (2019–2024) and included original research articles published in English, retrieved from Scopus, Web of Science, and PubMed. Selection criteria included studies analyzing more than 15 samples, using validated analytical methods and covering a broad range of bee-related matrices (pollen, bee bread, honey, beeswax, royal jelly, and propolis). The review focused on residues from seven pesticide families, including neonicotinoids, fungicides, herbicides, acaricides, insecticides, inert ingredients, and glyphosate-related compounds. The data were compiled in collaboration with over 20 international researchers. The most frequently reported residues were fluvalinate, coumaphos, amitraz, and chlorpyrifos, frequently found in beeswax compared to other bee products. These compounds, some of which are persistent and bioaccumulative, may pose chronic toxicological risks, including endocrine disruption and neurotoxicity. This work emphasizes the need for continuous monitoring and risk assessment of apicultural contaminants and highlights the importance of integrating food safety considerations into pest management strategies in beekeeping.

4.7. Arterial Damage Associated with Chronic Exposure to Glyphosate and Dichlorophenoxyacetic Acid: A Study in Rats

• Gisele Alborghetti Nai ¹, Fernanda Maria Bottino Vizzotto Toreto ², Maria Eduarda Silva Souza ³, Gabriela Hernandes Ribeiro ³ and Renata Calciolari Rossi ⁴

¹ 

Department of Pathology/Medical College of Presidente Prudente, Universidade do Oeste Paulista (UNOESTE), Presidente Prudente—SP 19050-680, Brazil

² 

Department of surgery of the Medical College, Universidade do Oeste Paulista (UNOESTE), Presidente Prudente—SP 19050-680, Brazil

³ 

Medical College of Presidente Prudente, Universidade do Oeste Paulista (UNOESTE), Presidente Prudente—SP 19050-680, Brazil

⁴ 

Graduate Program on Environment and Regional Development, Universidade do Oeste Paulista (UNOESTE), Presidente Prudente—SP 19050-680, Brazil

• Introduction: Atherosclerosis is a chronic inflammatory disease which can culminate in significant cardiovascular manifestations. Some pesticides have been implicated in atherogenesis. Glyphosate and dichlorophenoxyacetic acid (2,4-D) are the most widely used herbicides in crops worldwide. The objective of this study was to compare the potential for arterial damage from chronic inhalation and oral exposure to the herbicide glyphosate and 2,4-D in rats. Methods: This study was approved by the Animal Use Ethics Committee of the proposing institution (Protocol 6724). Seventy adult male Wistar rats were distributed into 14 groups: 2 control groups (exposed to distilled water via inhalation and oral route), 6 groups exposed to glyphosate, and 6 exposed to 2,4-D (n = 10/group). The animals were exposed to three doses of each herbicide via inhalation (inhalation groups) and in the diet (oral groups): low concentration, 3.71 × 10⁻³ g of active ingredient per hectare (g.a.i./ha); medium concentration, 6.19 × 10⁻³ g.a.i./ha; and high concentration, 9.28 × 10⁻³ g.a.i./ha. The experiment lasted six months. The aorta was collected for histological analysis. Results: Fatty streaks were observed only in animals exposed to herbicides (p 0.0001), with no difference regarding the route of exposure (oral or inhalation) (p > 0.05). Animals exposed to GBH had twice as many cases of cholesterol streaks as those exposed to 2,4-D (p 0.05). There was no significant difference in the thickness of the aorta between those exposed and those not exposed (p > 0.05). Animals exposed to 2,4-D showed a greater fractal dimension of the nuclei when compared to animals exposed to GBH and those in the control group (p 0.05). Conclusions: Both herbicides have atherogenic potential, but this is greater with exposure to GBH. Animals exposed to 2,4-D have the largest nuclear fractal dimension, showing that this herbicide causes greater nuclear reactivity of the aortic wall.

4.8. Assessing Variability of Heavy Metal Concentrations in Follicular Fluid: Insights from Novel ICP-MS/MS Methodology and Previously Published Studies

• Núria Ferrer-Cortés ¹, Andrea López-Botella ^1,2, María José Gómez-Torres ^1,3, José -Luis Todolí-Torró ², Raquel Sánchez-Romero ², Sergio Rogel ⁴ and Jon Aizpurua ^3,4

¹ 

Biotechnology Department, Faculty of Sciences, University of Alicante, 03690 Alicante, Spain

² 

Department of Analytical Chemistry, Nutrition and Food Sciences, University of Alicante, 03690 Alicante, Spain

³ 

Catedra Human Fertility, University of Alicante, Alicante, Carretera de San Vicente s/n, P.O. Box 99, 03080 Alicante, Spain

⁴ 

IVF Spain Treatment Clinic, Avenida de Ansaldo 13, 03540 Alicante, Spain

• Introduction: Environmental heavy metals have been associated with female infertility. Follicular fluid (FF), which envelops the oocyte, offers insights into element composition. Nevertheless, standardized studies on its trace elements remain limited. Due to its direct contact with the oocyte, FF is a critical medium through which heavy metal concentrations can significantly impact female fertility. Last year, our group attempted to detect 22 analytes (Ba, Be, Bi, Cd, Ce, Co, Cr, La, Li, Mn, Mo, Ni, Pb, Rb, Sb, Sn, Sr, Ti, Tl, U, V, Zn) in four FF samples from patients undergoing in vitro fertilization, using ICP-MS/MS without sample digestion. Consequently, a novel and reliable methodology was developed. This study aimed to compare our results with previous reports, analyze their similarities and differences, and identify possible causes.
• Methods: An exhaustive literature search was conducted using adequate keywords. Relevant data were extracted for comparison, considering the type of matrix analyzed, the method used, and the elements detected. Finally, these were compared with those obtained by our group in a database.
• Results: Of our 22 analytes, values were available for 16 of them, as the remaining ones were not detected (Be, Ce, Cd, La, Pb, and U). Comparable values for 13 of the 16 elements were found in the literature, as there is no published data on FF regarding Bi, Sb, and Rb. Some of the comparisons showed very similar data, but most presented considerable variations.
• Conclusions: Although there is similarity in some of the data compared, great variability still exists. This may be due to different factors, such as the methodology used in each case, lifestyle habits, occupational exposure, place of residence, pathologies, or demographic factors. Further study in this field is necessary in order to homogenize conditions and obtain more robust results of analysis regarding potentially toxic elements.

4.9. Assessment of Endocrine-Disrupting Chemicals in Companion Animals

• Inês Boaventura ¹, Sara Sousa ², Tiago Bordeira Gaspar ^1,3,4,5,6, Inês Borges ⁴, Cristina Delerue-Matos ², Paula Soares ^5,6 and Valentina Fernandes Domingues ²

¹ 

Department of Veterinary Sciences, Vasco da Gama University School, 3020-210 Coimbra, Portugal

² 

REQUIMTE/LAQV-ISEP, Polytechnic of Porto, 4249-015 Porto, Portugal

³ 

Department of Veterinary Sciences, Vasco da Gama Research Centre (CIVG), Vasco da Gama University School, 3020-210 Coimbra, Portugal

⁴ 

Veterinary Clinical Laboratory, Leça do Balio, 4465-671 Matosinhos, Portugal

⁵ 

Institute for Research and Innovation in Health (i3S), University of Porto, 4200-135 Porto, Portugal

⁶ 

Institute of Molecular Pathology and Immunology, University of Porto (IPATIMUP), 4200-135 Porto, Portugal

Endocrine-disrupting chemicals (EDCs) can interfere with hormone signaling, posing a significant risk to animal and human health. Usually, EDCs are environmental pollutants and, due to their lipophilic nature, can accumulate in fat tissues, leading to chronic exposure and serious health issues, such as endocrine-related neoplasia. Companion animals, particularly dogs and cats, are frequently exposed to EDCs at higher rates than their owners, owing to their proximity to the ground and their behaviors.

Despite increasing concern regarding EDC exposure, most biomonitoring studies to date have focused on humans and livestock, with limited attention given to pets. This study aimed to assess the presence of EDCs in the adipose tissue of dogs and cats diagnosed with hormone-sensitive neoplasms. Biological samples were collected from animals with and without neoplasia (i.e., a control group) at veterinary care centers across the north of Portugal. Clinical and pathological data, along with lifestyle information obtained through in-person questionnaires, were also gathered. A broad range of persistent and emerging EDCs were quantified using gas chromatography after a single ultrasound-assisted extraction (UAE) method was performed. Statistical data analysis was conducted to explore associations between EDC exposure and various clinical/pathological characteristics or animal habits. The findings from this research could provide valuable insights into the risks posed to pets by EDCs and support the development of improved strategies for the prevention, diagnosis, and treatment of endocrine-related diseases in companion animals.

• Acknowledgements: This work received support and help from FCT/MCTES (LA/P/0008/2020-DOI 10.54499/LA/P/0008/2020, UIDP/50006/2020-DOI10.54499/UIDP/50006/2020 and UIDB/50006/2020-DOI 10.54499/UIDB/50006/2020), through national funds.

4.10. Assessment of Potentially Toxic Elements in the Peritoneal Fluid of Patients with Ovarian Endometrioma, Peritoneal Endometriosis, and Deeply Infiltrative Endometriosis: Differences Between Entities

• Andrea López-Botella ^1,2, Raquel Sánchez-Romero ², José -Luis Todolí-Torró ², Irene Velasco ^1,3, Maribel Acién ^3,4 and María José Gómez-Torres ¹

¹ 

Biotechnology Department, Faculty of Sciences, University of Alicante, 03690 Alicante, Spain

² 

Department of Analytical Chemistry, Nutrition and Food Sciences, University of Alicante, 03690 Alicante, Spain

³ 

Service of Obstetrics and Gynecology, Unit of Human Reproduction, FISABIO—San Juan University Hospital, Carretera Alicante-Valencia s/n, 03550 Alicante, Spain

⁴ 

Gynecology Division, Faculty of Medicine, Miguel Hernández University, Elche, Spain

• Introduction: Endometriosis is a chronic gynecological disease affecting up to 7–10% of menstruating individuals worldwide. To date, its etiopathogenesis remains largely unknown, with environmental factors such as potentially toxic elements (PTEs) being contributors.
• Methods: Peritoneal fluid (PF) samples were collected from 13 patients with superficial peritoneal endometriosis (SUP), 13 with ovarian endometrioma (OMA), 4 with deeply infiltrative endometriosis (DEEP), and 13 non-endometriosis controls. The samples were collected between June 2020 and September 2022 from patients who underwent laparoscopic procedures within the Gynecology Service of a University Hospital in San Juan (Spain). The group classification was based on surgical findings and histopathological reports. After sample processing, the PF was stored in the dark at −20 °C until analysis by inductively coupled plasma tandem mass spectrometry (Agilent 8900 ICP-MS/MS). Statistical analyses were performed using IBM SPSS Statistics version 28.0 (IBM, Armonk, NY, USA). Elemental ratios were calculated by normalizing the average concentration of each element to that of the control group.
• Results: The concentration of Fe was significantly elevated in the OMA group compared to the control group (p = 0.005), with mean concentrations of 8172 ± 13,361 and 967 ± 1524 µg L⁻¹, respectively. No statistically significant differences were found when comparing the elemental concentrations in SUP and DEEP with those in the control group. When examining the elemental ratios (threshold of >1.5), elevated levels were observed as follows: the levels of Fe and Co were above average in SUP, OMA, and DEEP; the level of Ba was above average in OMA and SUP; the level of Pb was above average in SUP and DEEP; the levels of Cd and Mn were above average in OMA; and finally, the levels of Cu and Zn were above average in DEEP. Distinct patterns of Spearman correlations between the elements were identified depending on the specific endometriosis entity.
• Conclusions: These findings suggest that multielemental profiles in peritoneal fluid vary according to the specific endometriosis entity.

4.11. Associations Between Occupational Exposure to Endocrine-Disrupting Chemicals and Sperm Quality

• Hafsat Bulama, Allan Pacey, Martie Van Tongeren and Andrew Povey

• Faculty of Biology, Medicine and Health, The University of Manchester, Oxford Road, Manchester M13 9PL, UK

Declining male fertility rates have become a significant global health concern, with potential implications for individual reproductive health and broader societal demographics. Increasing evidence from animal and human studies suggests a strong link between occupational exposure to endocrine-disrupting chemicals (EDCs) and impaired sperm quality. EDCs are exogenous substances that interfere with the normal function of the endocrine system, potentially disrupting hormonal balance and reproductive processes. Occupational settings, particularly in industries like agriculture, manufacturing, and waste management, often involve exposure to complex mixtures of these EDCs, raising significant concerns about their cumulative impact on male reproductive health. The aim of this study is to determine the associations between occupational exposure to EDCs and sperm quality using data from the CHAPS-UK study. This was a multi-center case-referent study of male infertility that recruited men who were part of couples attempting conception without success for at least 12 months. The study involved 2249 men who were recruited over the period of 37 months, between January 1999 and February 2002. Risk factors associated with low sperm motility and adverse sperm morphology were identified, including glycol ether exposure. Exposure to potential EDCs will be estimated for these men and their mothers using a previously developed job exposure matrix, and the associations between exposure and sperm quality (concentration, motility, and morphology) will be determined.

In addition to synthesizing the existing knowledge on this topic, this study seeks to bridge critical gaps in understanding the reproductive toxicity of EDCs. The findings are expected to provide valuable insights into preventing and mitigating EDC-related health risks, guiding regulatory policies and public health interventions to reduce exposure and protect reproductive health.

4.12. Dietary Exposure to Arsenic in the Cuban Population: Results from a Total Diet Study

• Luisbel González Pérez de Medina ¹, Ociel Muñoz-Fariña ² and Yenisleidys Fernández Guerrero ³

¹ 

Laboratorio de Biomateriales, Departamento de Ingeniería Química, Facultad de Ingeniería, Universidad de Concepción, Concepción 4030000, Chile

² 

Instituto de Ciencia y Tecnología de los Alimentos, Facultad de Ciencias Agrarias y Alimentarias, Universidad Austral de Chile, Valdivia 5090000, Chile

³ 

Doctorado en Ingeniería, Universidad del Bío-Bío, Concepción 4051381, Chile

Arsenic (As), particularly in its inorganic form (iAs), is a toxic metalloid commonly found in food and associated with various health risks. This study assessed dietary exposure to total arsenic (t-As) and estimated inorganic arsenic intake in the Cuban adult population using a Total Diet Study (TDS). A 24 h dietary recall survey was conducted with 450 individuals from different regions of Cuba. A total of 107 commonly consumed food items were purchased, prepared as typically consumed, and grouped into 17 categories. The samples were digested and analyzed by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) to quantify t-As. Estimated Daily Intake (EDI), Target Hazard Quotient (THQ), and Carcinogenic Risk (CR) were calculated. The highest concentrations of t-As were detected in the fish and seafood group (0.288 µg/g), followed by rice (0.032 µg/g) and vegetables (0.018 µg/g). The estimated intake of t-As was 54.6 µg/day. Assuming that 10% corresponds to iAs, the intake of inorganic arsenic was estimated at 5.46 µg/day. The THQ for iAs was 0.22, suggesting no significant non-carcinogenic risk. However, the CR for iAs was 1.0 × 10⁻⁴, indicating a potential lifetime cancer risk for 10 individuals per 100,000, which falls within the acceptable risk range. In conclusion, while total arsenic intake in the Cuban population complies with national limits, the presence of iAs may still pose a low but noteworthy cancer risk. Continued monitoring and public health interventions are recommended to minimize exposure through dietary sources.

4.13. Effective Pesticide Degradation and Toxicity Control Through Selective Oxidation of Permanganate

• Dean Song

• Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, China

In recent years, many new pesticides have been developed to meet the growing agricultural demands driven by global population growth. Unfortunately, this has led to their frequent detection in aquatic ecosystems, posing risks to non-target organisms and human health through environmental pollution and dietary exposure. Therefore, there is an urgent requirement to develop effective technologies to remove these pesticides from the environment. However, due to the complexity of pesticide structures, their degradation products are commonly unknown and may result in new environmental risks.

Permanganate is a strong oxidant and plays a vital role in drinking water purification, groundwater remediation, and wastewater treatment. It does not introduce new pollutants, and its reduction product, manganese dioxide, can also act as a coagulant to further remove pollutants.

In this study, we used experimental and theoretical methods to explore how potassium permanganate degrades pesticides. The reaction between permanganate and quinclorac was first studied, showing a bimolecular reaction mode, which is stable in a wide pH range. The UPLC-Q-TOF-MS analysis showed that the initial product was mono-hydroxylated quinclorac, which was formed by permanganate oxidation at the benzene ring and could be further oxidized to a less toxic catechol structure.

It is then hypothesized that permanganate oxidation is selective for certain functional groups, better than non-selective oxidants like hydroxyl radical. Then, the imidacloprid degradation by permanganate was further studied. The reaction was stable and efficient near neutral pH and different ion strengths. Background substances like humic acid and chloride ions in water barely affected degradation. UPLC-Q-TOF-MS analysis showed that the main pathway was C-H bond hydroxylation on the imidazole ring, finally leading to the breakage of the imidazole ring. Toxicity analysis with the ECOSAR program showed that the degradation products of imidacloprid by permanganate oxidation were less toxic to aquatic organisms.

4.14. Environmental Exposure Assessment Among Infant via Diapered Urine Analysis: A Challenge of Methodology

• Thanawadee Chantong ^1,2, Tippawan Prapamontol ¹, Kriengkrai Srithanaviboonchai ², Parinya Panuwet ³ and Dana Boyd Barr ³

¹ 

Environment and Health Research Group, Research Institute for Health Sciences (RIHES), Chiang Mai University, Chiang Mai 50200, Thailand

² 

School of Health Sciences Research (SHSR), Research Institute for Health Sciences (RIHES), Chiang Mai University, Chiang Mai 50200, Thailand

³ 

Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta, GA 30329, USA

• Introduction

• Assessment of environmental exposure using biological samples such as urine is non-invasive. However, most infants wear disposable diapers. Therefore, extraction of urine from diapers is a challenging step before laboratory analysis. This study aims to develop and validate a method for detecting ten hydroxylated polycyclic aromatic hydrocarbons (OHPAHs) in diapered urine.

• Methods

Ten individual adult urine samples (≈100 mL) were employed for the experiment. Thirty millimeters of urine was passed through the diaper, and the diaper urine (DU) was extracted with 50 mL of 50 g/L calcium chloride (CaCl₂) solution. The calcium ion in the DU extract was measured using atomic absorption spectroscopy (AAS). Then, a 10 mL aliquot of extracted DU along with normal individual urine (U) was acidified to pH 5.0 and hydrolyzed with β-glucuronidase, and the extract was cleaned up using solid-phase extraction (C18 OH). Ten OHPAH metabolites (2-hydroxynaphthalene (2-OHNap), 1-hydroxynaphthalene (1-OHNap), 2+3-hydroxyfluorene (2+3-OHFlu), 2-hydroxyphenanthrene (2-OHPhe), 3-hydroxyphenanthrene (3-OHPhe), 9-hydroxyphenanthrene (9-OHPhe), 1-hydroxyphenanthrene (1-OHPhe), 4-hydroxyphenanthrene (4-OHPhe), and 1-hydroxypyrene (1-OHP)) were quantified using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) with deuterated internal standards (1-OHP-d9 and 1-OHNap-d7) to ensure accuracy. The detected OHPAH concentrations of DU and U were compared and discussed.

• Results

We found that the recovery rates of OHPAH metabolites from DU compared to U ranged from approximately 34 to 94%, with the highest recoveries observed for 4-OHPhe (94%) and 1-OHPhe (92%), followed by 1-OHP (87%), 2-OHPhe (68%), 2+3-OHFlu (47%), 3-OHPhe (41%), 2-OHNap (40%), 9-OHPhe (36%), and 1-OHNap (34%).

• Conclusions

This study developed and validated a method for detecting OHPAH metabolites in infant diaper urine, with recovery rates ranging from 34% to 94%. Lower recoveries for some metabolites suggest losses during extraction from diaper, as well as interference of high calcium levels in diaper-extracted urine with enzymatic hydrolysis. Further study is warranted to address these challenges.

4.15. Exploring the Association Between Exposure to Multiple Toxic Metals and Endometriosis Risk Using Bayesian Kernel Machine Regression: Evidence from Peritoneal Fluid

• Andrea López-Botella ^1,2, Raquel Sánchez-Romero ², José -Luis Todolí-Torró ², Irene Velasco ^1,3, María José Gómez-Torres ¹ and Maribel Acién ^3,4

¹ 

Biotechnology Department, Faculty of Sciences, University of Alicante, 03690 Alicante, Spain

² 

Department of Analytical Chemistry, Nutrition and Food Sciences, University of Alicante, 03690 Alicante, Spain

³ 

Unit of Human Reproduction, Service of Obstetrics and Gynecology, FISABIO, 46026 Valencia, Spain

⁴ 

Gynecology Division, Faculty of Medicine, Miguel Hernández University, Elche, Spain

• Introduction: Endometriosis is a chronic gynecological disorder affecting approximately 7–10% of menstruating individuals worldwide. Despite its high prevalence, the etiopathogenesis of endometriosis remains largely unclear. Emerging evidence suggests that environmental contaminants such as cadmium (Cd), nickel (Ni), and lead (Pb) may contribute to its development.
• Methods: Peritoneal fluid (PF) samples were obtained from 50 subjects (25 controls and 25 patients with endometriosis) attending the Gynecology Service of San Juan University Hospital in Spain between June 2020 and September 2022. Samples were processed and analyzed by inductively coupled plasma tandem mass spectrometry (Agilent 8900 ICP-MS/MS). Statistical analyses were conducted using IBM SPSS Statistics version 28.0 (IBM, Armonk, NY, USA), and Bayesian Kernel Machine Regression (BKMR) was performed in R (version 4.4.3, package “bkmr” and “ggplot2”) to provide new insights into the effects of Cd, Pb, and Ni on endometriosis risk.
• Results: Cadmium levels were significantly higher in the endometriosis group compared to the control group (0.71 ± 0.94 vs. 0.25 ± 0.22 µg/L, p = 0.009). The BKMR model using PF concentrations of Cd, Ni, and Pb revealed a positive trend in endometriosis risk with increasing metal exposure. However, this combined effect was not statistically significant, and none of the metals showed an independent association with the disease.
• Conclusions: Although no statistically significant associations were observed, the positive trend in the combined effect of Cd, Ni, and Pb suggests a potential role of metal mixtures in endometriosis pathogenesis. This lack of statistical significance may be related to sample size limitations and inherent variability in peritoneal fluid composition. Nevertheless, the observed trend is consistent with the previous literature on the endocrine-disrupting properties of these metals. These findings highlight the importance of considering environmental mixtures in research on reproductive health and gynecological diseases and emphasize the need for larger studies to better understand these associations.

4.16. Genotoxicity and Cytotoxicity Induced by Water and Sediment Samples from River Systems in Vicia faba Root Cells

• Hugo Ahuactzi-Cortes ¹, Juana Sánchez-Alarcón ^2,3,4, Josefat Gregorio-Jorge ⁵, Mirta Milić ⁶ and Rafael Valencia Quintana ^2,3,4

¹ 

Facultad de Agrobiología, Universidad Autónoma de Tlaxcala, Tlaxcala C.P. 90120, Mexico

² 

Laboratorio “Rafael Villalobos-Pietrini” de Toxicología Genómica y Química Ambiental, Facultad de Agrobiología, Universidad Autónoma de Tlaxcala, Tlaxcala C.P. 90120, Mexico

³ 

Red Temática “La Toxicidad de los Plaguicidas”, Consejo Nacional de Ciencia y Tecnología-Universidad Autónoma de Nayarit, Mexico 63155

⁴ 

CA Ambiente y Genética UATLX-CA-223, Facultad de Agrobiología, Universidad Autónoma de Tlaxcala, Tlaxcala 90000, Mexico

⁵ 

Consejo Nacional de Ciencia y Tecnología—Comisión Nacional del Agua, Ciudad de México 03940, Mexico

⁶ 

Division of Toxicology, Institute for Medical Research and Occupational Health, Ksaverska Cesta 2, 10000 Zagreb, Croatia

In the context of the Anthropocene, river ecosystems have been profoundly altered by the intensification of anthropogenic activities that modify their physical, chemical, and biological characteristics. The continuous discharge of urban, industrial, and agricultural effluents has generated a substantial deterioration in water quality, compromising biodiversity and ecosystem functions. Globally, it is estimated that approximately 50% of major river systems are polluted; in Mexico, this figure reaches nearly 60%, underscoring the urgency of implementing bioindicator tools for environmental assessment and monitoring. The objective of this study was to evaluate the genotoxic and cytotoxic potential of water and sediment samples from the Briones and Negros rivers (Tlaxcala, Mexico) by applying the micronucleus (MN) test and the comet assay in Vicia faba cells. Spot sampling was carried out during the rainy and dry seasons at five representative sites. The results showed statistically significant increases (p 0.05) in MN frequency and DNA fragmentation, as well as a reduction in the mitotic index (MI) in most of the samples evaluated compared to negative controls. Marked spatial and temporal variability was observed, suggesting differential exposure to toxic agents. The presence of compounds such as polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides, and heavy metals is postulated as a possible cause of the observed effects. These findings confirm the existence of contaminants with genotoxic and cytotoxic potential in the studied water bodies, which represent not only a threat to the ecological integrity of aquatic ecosystems but also a latent risk to human health. The implementation of comprehensive genotoxicological monitoring programs is recommended, as well as the development of strategies for ecological restoration and mitigation of anthropogenic impacts.

4.17. Growth Response and Phytoremediation of Copper and Chromium in Dumpsite Leachate by Common Duckweed (Lemna minor L.): A Case Study of a Market Dumpsite in Maiduguri, Nigeria

• Musa Adamu Ibrahim ¹, Muhammad Kabir Usman ^1,2, Yusha’u Usman Muhammad ¹, Musa Ibrahim ¹, Ahmed Abbator ³, Ibrahim Galadima Dagona ⁴, Mohammed Abdulhamid Umar ³ and Mustapha Saidu ⁵

¹ 

Department of Biology, University of Maiduguri, Maiduguri 600234, Nigeria

² 

Department of Environmental Science, Sharda University, Greater Noida 201310, India

³ 

Department of Botany, University of Maiduguri, Maiduguri 600234, Nigeria

⁴ 

Nigeria Agricultural Quarantine Service (NAQS) Northeast Zonal Office, Maiduguri 600001, Nigeria

⁵ 

Department of Biology, University of St Andrews, St Andrews KY16 9ST, UK

Water safety in low- and middle-income countries is an issue of serious concern globally. Heavy metals in leachates from residential dumpsites and other businesses affect water quality due to their non-biodegradability and toxicity. Copper (Cu) and chromium (Cr) in water pose a danger to local ecosystems and public health. This study determined the growth response and phytoremediation of Cu and Cr in market dumpsite leachate using a floating aquatic plant, common duckweed (Lemna minor), as a locally applicable, cost-effective, and eco-friendly solution. The plant was exposed to different concentrations of leachate for 14 days. Growth and the physicochemical parameters were measured. Our results showed a significant reduction in Co, Cr, electrical conductivity (EC), total dissolved solids (TDSs), salinity, oxidation–reduction potential (ORP), and dissolved oxygen (DO) at a 50% v/v concentration of the leachate. The pH increased from slightly alkaline to more alkaline at the same concentration. The number of fronds, biomass, relative growth rate, and chlorophyll contents decreased significantly at 100% v/v. This finding indicates that L. minor can tolerate and remediate 50% v/v of dumpsite leachate. Further studies are recommended to evaluate other environmental and eco-physiological factors to improve the phytoremediation of leachate using this plant in Maiduguri metropolis and beyond.

4.18. Influence of Urban Pollution and Smoking in Human Salivary Nitrate Concentrations: Negative Result

• Vanda Lopes de Andrade ^1,2,3,4, Bianca Delagado Saraiva ¹, Gonçalo Fialho Oliveira ¹, Jéssica Magalhães Gravata ¹, Martim Gomes Ribeiro ¹, Nuno Gaudêncio Barreiros ¹, Nuno Fialho da Mota ¹, Salvador Rego Duarte ¹ and Vanessa Maurício Jordão ¹

¹ 

School of Agriculture, Santarem Polytechnic University, Quinta do Galinheiro-S. Pedro, 2001-904 Santarém, Portugal

² 

Life Quality Research Centre (CIEQV), Santarem Polytechnic University, Complexo Andaluz, Apartado 279, 2001-904 Santarém, Portugal

³ 

Research Centre for Natural Resources, Environment and Society (CERNAS), Santarem Polytechnic University, Quinta do Galinheiro-S. Pedro, 2001-904 Santarém, Portugal

⁴ 

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal

Urban runoff is a major source of nitrate pollution in rivers, posing significant risks to environmental health and public safety, especially in densely populated cities. Additionally, due to the aging process used to make cigars, cigar tobacco contains high concentrations of nitrogen compounds, including nitrates. When ingested, nitrate can react with amines and amides to form N-nitroso compounds (NOCs), which may cause cancer in humans. It is known that salivary glands actively concentrate plasma nitrate into saliva, leading to salivary nitrate levels about 10 times higher than in plasma. Considering that urban or smoker populations might have higher nitrate exposures, we hypothesized that such exposure could be detected through increased salivary nitrate levels. A total of 21 saliva samples were collected in Portugal, comprising 11 samples from rural residents (3 non-smokers and 8 smokers) and 10 from urban residents in Lisboa (5 non-smokers and 5 smokers). After centrifugation and dilution, nitrate levels were determined based on a previously established method using UV spectrophotometry. Mann–Whitney tests were performed to assess differences between groups. Data analysis by rural/urban residence showed no significant difference (p > 0.05), with similar salivary nitrate levels: 4.36 ± 0.28 and 4.40 ± 0.26 mg/L in rural and urban populations, respectively. Similar results (p > 0.05) were found when considering smoking as a factor, with 4.45 ± 0.36 mg/L in non-smokers and 4.42 ± 0.46 in smokers. These findings suggest that salivary nitrates do not reflect nitrate exposure due to urban living or smoking, or that neither factor substantially increases nitrate body burden. As this study is preliminary, further research with a larger sample is needed for clarification.

4.19. Ion Chromatographic Profiling of Major Anions in Sweet Corn Genotypes: Implications for Food Safety and Health Risks

• Edward Muntean ¹, Tania Mihaiescu ² and Rajmund Michalski ³

¹ 

Department of Food Science, Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine, 400372 Cluj Napoca, Romania

² 

Department of Environmental Engineering and Protection, Faculty of Agriculture, University of Agricultural Sciences and Veterinary Medicine, 400372 Cluj Napoca, Romania

³ 

Department of Waste Management and Environmental Analyzes, Institute of Environmental Engineering—Polish Academy of Science, 41-819 Zabrze, Poland

Sweet corn is an important crop and a high-demand commodity, generating substantial revenue in both its fresh and processed forms; it holds significant value in human nutrition due to its rich content of carbohydrates, dietary fiber, vitamins, and essential minerals, serving as a good source of energy while supporting digestive health. Monitoring the major inorganic anions in sweet corn kernels is essential for evaluating food safety compliance, quality attributes, and agricultural impact; while anions such as phosphate, sulfate, and chloride contribute to nutritional benefits, elevated levels of nitrate and nitrite pose potential health risks. Consequently, monitoring these anions is critical for consumer safety, supports quality control during processing, and provides insights into soil fertility and fertilizer efficiency. This study aimed to determine the major inorganic anions in twelve sweet corn genotypes from the University of Agricultural Sciences and Veterinary Medicine Cluj Napoca. Ion chromatographic analysis was performed using a Shimadzu instrument with non-suppressed conductivity detection, equipped with an Allsep Anion 7u column, achieving simultaneous determination of chloride, nitrite, nitrate, phosphate, and sulfate in under 15 min. Minimal sample preparation involved blending of representative kernels in ultrapure water, followed by membrane filtration. The results revealed distinct chromatographic fingerprints among genotypes: phosphate concentrations reached 58.65 mg/kg, chloride concentrations reached 27 mg/kg, sulfate concentrations reached 14.23 mg/kg, and nitrate concentrations reached 7.15 mg/kg, while nitrite was detected in only three samples, with a maximum concentration of 0.41 mg/kg. These findings highlight significant variability in anion content across sweet corn genotypes, demonstrating that ion chromatography is a reliable and efficient tool for assessing sweet corn’s nutritional quality and safety. By identifying potential food hazards and promoting environmental sustainability, these findings support improved agricultural practices and breeding programs and the development of functional foods that cater to health-conscious consumers.

4.20. Multiple Physiological Alterations in a Cosmopolitan Fish, Cyprinus Carpio Exposed to Malathion

• Amandeep Singh Dhot ¹, Shanthanagouda A. Holeyappa ^1,2, Neelam Bansal ³, Meera D. Ansal ⁴, Sethi R.S ⁵ and Naveenkumar BT ¹

¹ 

Department of Aquatic Environment, College of Fisheries, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141 004, India

² 

Inland Fisheries Unit, Zonal Agricultural and Horticultural Research Station, Keladi Shivappa Nayaka University of Agricultural and Horticultural and Sciences, Shivamogga 577 204, India

³ 

Department of Veterinary Anatomy, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141 004, India

⁴ 

Department of Aquaculture, College of Fisheries, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141 004, India

⁵ 

College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141 004, India

Organophosphates (OPs), commonly used pesticides to increase agricultural production with minimal labor efforts, unknowingly affect non-target animals. Amongst OPs, malathion (MAL) is neuro- and immunotoxic and widely used. This investigation aimed to understand the multiple physiological responses in common carp exposed to MAL. In this study, acute toxicity test showed 6.75 mg/L as LC50 in 96 h, following which, a separate sublethal toxicity test was conducted (28 d), having environmentally realistic (2.618 μg/L) and higher concentrations (0.675 & 1.35 mg/L). In higher exposures, selected transcripts such as acetylcholinesterase (Ache) and component complement-3 (c3) levels significantly decreased. On the other hand, hematological parameters [(total erythrocytes (TEC), hemoglobin (Hb), hematocrit (Ht), Mean Corpuscular Hemoglobin (MCH), and Mean Cell Hemoglobin Concentration (MCHC)] decreased with an increase in MAL levels except for total leucocytes (TEC), which increased significantly (P0.05). Further, histomorphology of the brain revealed marked alterations such as degenerated neurons, pyknosis, vacuolization, increased fibrous components, and focal necrosis with MAL exposure. On the other hand, gills showed hyperplasia, telangiectasia, and fused lamellae, the kidneys revealed damaged glomeruli, increased lumen, and exudates, and the liver exhibited a shrunken central vein, thickened bile duct, and dilated portal vein following exposure. Collectively, the study revealed that MAL is neuro- and immunotoxic and damages the microarchitecture of the tissues. The non-judicious application of MAL leads to harmful effects; hence, usage should be monitored regularly.

4.21. Pesticide Residues as a Converging Threat to Ecosystem Integrity and Public Health

• Alexandra Andreea Botnaru ¹, Paula Cristina Morariu ², Elena Carmen Bogdan ³, Branco Adrian Morariu ⁴ and Ionela Daniela Morariu ¹

¹ 

Department of Environmental and Food Chemistry, “Grigore T. Popa” University of Medicine and Pharmacy, 700115 Iasi, Romania

² 

Department of Internal Medicine, “Grigore T. Popa” University of Medicine and Pharmacy, 700115 Iasi, Romania

³ 

Faculty of Medicine and Biological Sciences, “Ștefan cel Mare” University of Suceava, 720229 Suceava, Romania

⁴ 

Faculty of General Medicine, “Grigore T. Popa” University of Medicine and Pharmacy, 700115 Iasi, Romania

• Introduction: Pesticides, while integral to modern agricultural practices, pose substantial risks to both environmental integrity and human health. Their extensive application results in the pollution of air, water, soil, flora, and animal feed, resulting in bioaccumulation within the food chain. Furthermore, research indicates that pesticide residues are among the most prevalent dietary pollutants. Methods: A comprehensive literature search was conducted across four major databases—PubMed, Google Scholar, NIH, and ScienceDirect. Relevant and recent studies focusing on pesticide residues in food products and their environmental and health impacts were selected. Results: Numerous studies have confirmed that persistent pesticide residues are contributing to reduced soil biodiversity, water contamination, and the decline in non-target species such as pollinators, aquatic organisms, and mammals. Furthermore, pesticides disrupt beneficial soil microorganisms, affect food safety, and contribute to long-term environmental degradation through bioaccumulation and chemical stability. Human exposure generally occurs through ingestion, inhalation, and skin contact, especially via the intake of contaminated food and water. Pesticides are linked to several health effects, including acute poisoning, endocrine disruption, reproductive dysfunction, immunosuppression, neurological illnesses, and various types of cancer. Conclusions: Achieving meaningful progress requires a concerted effort from all stakeholders—governments must implement robust regulatory frameworks and incentives, farmers should embrace agroecological strategies such as integrated pest management and crop diversification, and consumers need to support sustainably grown foods while minimizing waste. By fostering collaboration across sectors, we can move toward resilient food systems that prioritize environmental sustainability and human health.

4.22. Physicochemical Alterations of LDPE Microplastics During Simulated Gastrointestinal Digestion

• Mariana Lamas ¹, Francisca Rodrigues ¹, Marta Oliveira ¹, Paulo C. Costa ^2,3 and Virgínia Cruz Fernandes ^1,4,5

¹ 

REQUIMTE/LAQV, ISEP, Polytechnic of Porto, Rua Dr. António Bernardino de Almeida, 4249-015 Porto, Portugal

² 

REQUIMTE/UCIBIO, MedTech-Laboratory of Pharmaceutical Technology, Department of Drug Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal

³ 

Associate Laboratory i4HB, Institute for Health and Bioeconomy, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal

⁴ 

Chemical and Biomolecular Sciences, School of Health, Polytechnic of Porto, Rua Dr. António Bernardino de Almeida 400, 4200-072 Porto, Portugal

⁵ 

RISE-Health, Center for Translational Health and Medical Biotechnology Research (TBIO), ESS, Polytechnic of Porto, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal

Microplastics (MPs) are widespread environmental contaminants that are increasingly detected in food chains and human tissues. While traditionally considered chemically inert, recent studies suggest that MPs may undergo surface and chemical transformations upon exposure to physiological environments. The human gastrointestinal (GI) tract presents dynamic conditions, including enzymatic activity, pH shifts, bile salts, and microbial interactions, which can modify the structure and behavior of MPs. These digestion-related transformations may influence MPs’ bioavailability, toxicity, and ability to interact with biological systems.

This study investigated the surface-level transformations and chemical composition of low-density polyethylene (LDPE), a polymer commonly found in food packaging, during simulated gastrointestinal digestion. LDPE samples were subjected to a standardized in vitro digestion protocol that mimicked oral, gastric, and intestinal phases. Samples were collected at each stage and analyzed using Fourier-transform infrared spectroscopy (FTIR) to assess the surface chemical changes.

The FTIR spectra revealed consistent differences between the virgin and digested LDPE samples. The digested samples exhibited lower overall transmittance, suggesting surface alterations. The reduction in the intensity of the CH₂ rocking peak (~719 cm⁻¹) indicated a potential decrease in crystallinity. The methyl group peak (~1379 cm⁻¹) was absent, and the CH₂ bending region (~1460 cm⁻¹) showed slight shifts and intensity changes, possibly linked to the degradation or structural rearrangement of the polymer chain. Additionally, a new band appeared at approximately 2070 cm⁻¹ in the oral and gastric phases, which may be associated with degradation products or contaminant residues.

These findings demonstrate that gastrointestinal conditions can alter the physicochemical properties of LDPE microplastics, particularly at the surface level. Such modifications could enhance their ability to adsorb or transport toxic substances, potentially increasing biological interactions and health risks. Understanding these transformations is critical for the accurate toxicological risk assessment of ingested MPs.

4.23. Potential of Wood-Based Activated Carbon to Mitigate Toxic Element Uptake in Spinach for Safe Consumption

• Oana Cadar ¹, Eniko Kovacs ¹, Erika Levei ¹, Anamaria Iulia Torok ¹, Jimena Castro Gutierrez ² and Vanessa Fierro ²

¹ 

Research Institute for Analytical Instrumentation Subsidiary, INCDO INOE 2000, 400305 Cluj-Napoca, Romania

² 

CNRS-Centre National de la Recherche Scientifique, Université de Lorraine-IJL, 88000 Épinal, France

Toxic elements (TEs) are characterized by high toxicity, even at low concentrations, and have the potential to induce various harmful effects in living organisms. Pollution with TEs constitutes a major concern in the agricultural sector, as this decreases crop growth, yield, and food quality. Soil amendments derived from agricultural byproducts and waste can improve physicochemical properties, fertility, and plant growth in TE-contaminated soils by providing nutrients and can reduce the TEs’ bioavailability. Due to its high surface area, porosity, and adsorption capacity, activated carbon emerges as a promising solution for removing TEs, increasing soil nutrient levels, and improving soil water holding capacity and aeration, consequently improving crop nutrition. A pot experiment was conducted to study the effect of wood-based activated carbon on the growth of spinach and the bioavailability of Cd and Cr in soils artificially contaminated at a concentration level of 10 mg/kg. Mixtures of TE-contaminated soil and wood-based activated carbon at 0% (control), 0% (contaminated control), 1.0%, 1.5%, and 2% were prepared in plastic pots and used as substrates for cultivating 15-day-old nursery spinach (Spinacia oleracea L. var. Matador) plants. The utilization of 2% wood-based activated carbon significantly increased shoot weight, as well as soil pH and organic matter content. The application of 2% wood-based activated carbon also reduced TE concentrations by 35% for Cd and 55% for Cr in shoots, and by 45% for Cd and 50% for Cr in roots, respectively, compared to the contaminated control group. Amendment with 2% wood-based activated carbon can immobilize Cd and Cr in soil and increase the chlorophyll and carotenoid contents in spinach grown in soils artificially contaminated with Cd and Cr. However, further research is needed to identify the optimal application rate and long-term effects of wood-based activated carbon amendments to enhance soil quality and promote plant growth in contaminated soils.

4.24. The Association Between Organophosphate Insecticide Metabolites (DAPs) and COPD: Multi Model Epidemiological Analysis and Network Toxicology Analysis

• Zhen Ma, Qingqing Cao, Qian Gao, Pengcheng Yuan, Xin Li and Wenqian Huo

• Department of Occupational and Environmental Health, College of Public Health, Zhengzhou University, Zhengzhou, China

• Background and Aims

In the general population, there is little epidemiological evidence linking exposure to organophosphate pesticides (OPs) to lung function. The aim of this study is to explore the relationship between urinary OP metabolites and chronic obstructive pulmonary disease (COPD).

• Materials and methods

A total of 9291 participants (including 2598 patients with chronic obstructive pulmonary disease) were included in the National Health and Nutrition Examination Survey from 1999 to 2012 and from 2015 to 2017. We used weighted logistic regression, combined with quantile g calculation (Qg) and weighted quantile sum (WQS) regression, to evaluate the relationship between individual or mixed exposure to OP and COPD, and further explored the molecular mechanism of increased COPD risk caused by OP exposure through bioinformatics analysis, providing ideas for the prevention and treatment of COPD.

• Result

Dimethyl phosphate (DMPT) (OR = 1.15, 95% CI: 1.01, 1.22), diethyl thiophosphate (DEPT) (OR = 1.29, 95% CI: 1.21, 1.38), dimethyl dithiophosphate diester (DMDTP) (OR = 1.25, 95% CI: −1.20, 1.29), and diethyl dithiophosphate (DEDTP) (OR = 1.54, 95% CI: 1.40, 1.70) are significantly associated with an increased risk of COPD. The QG results showed that mixed OP exposure significantly increased the risk of COPD (OR = 1.58, 95% CI: 1.47, 1.70, p 0.001), and the weights of DMPT, DEPT, DMDTP, and DEDTP were relatively high. In the WQS model, co-exposure to the OP mixture is significantly associated with an increased risk of COPD (OR = 1.8; 95% CI: 1.67, 1.94). The COPD target genes associated with OP exposure are mainly enriched in pathways related to inflammation, infection, metabolism, and hormone regulation.

• Conclusions

Exposure to OPs increases the risk of COPD through a mixed effect, emphasizing the significant impact of OPs and the need to prioritize the regulation of high-risk components such as DMPT, DEPT, DMDTP, and DEDTP.

4.25. The Ecotoxicological Effects of Metformin Under Global Warming Scenarios: Daphnia magna and Danio rerio Insights

• Marta Rojais ^1,2, Sara C. Antunes ^1,3 and Sara Rodrigues ^1,3

¹ 

Biology Department, Sciences Faculty, University of Porto, Rua do Campo Alegre S/N, 4169-007 Porto, Portugal

² 

School of Medicine and Biomedical Sciences, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal

³ 

Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos S/N, 4550-208 Matosinhos, Portugal

• Background: Diabetes Mellitus (DM) is a major global health issue, affecting over 500 million people. Metformin (MET), an antihyperglycemic drug prescribed for Type II DM treatment, is frequently detected in aquatic environments due to its widespread use and poor removal capacity from wastewater. Concomitantly, freshwater ecosystems are also threatened by other environmental stressors, including global warming, making it necessary to understand the interactions between anthropogenic and climate-related pressures. This study aimed to assess the ecotoxicological effects of metformin on Daphnia magna and Danio rerio under rising temperature scenarios. Methods: D. magna acute immobilization (48 h; 0.00–120 mg MET/L; 20 ± 1 °C) and D. rerio Fish Embryo Acute Toxicity (FET) (96 h; 0.00–3000 mg MET/L; 26 ± 1 °C) assays were performed following OECD guidelines. D. magna feeding inhibition assays (24 h; 0.00–80 mg MET/L; 20 ± 1 °C) were conducted following previous works (standard temperature). The same bioassays were also performed with a 4 °C temperature increase (projections by the IPCC until 2100), i.e., 24 ± 1 °C for D. magna and 30 ± 1 °C for D. rerio. Results: At standard temperature, MET induced acute toxicity in D. magna (LC₅₀ (48 h) = 70.64 mg/L; EC₅₀ (48 h) = 53.67 mg/L) and reduced feeding rate (EC₅₀ = 54.18 mg/L). In D. rerio, MET had no significant effects on mortality and hatching but induced malformations (scoliosis and edema). MET exposure induced oxidative stress and neurotoxicity in D. magna and altered the homeostasis of the antioxidant and detoxification enzymes in D. rerio larvae. Increased temperature influenced MET’s toxic effects, affecting individual and sub-individual responses. Conclusions: This study showed that increased temperatures (based on global warming projections) influence MET toxicity at the individual and sub-individual level, with D. magna showing higher sensitivity. The combined effects of rising temperatures and MET pollution underscore the vulnerability of freshwater environments.

4.26. The Hidden Threat of Combined Stressors: The Influence of Temperature and pH on Antibiotic Toxicity in Danio rerio

• Bárbara S. Diogo ^1,2,3, Sara Rodrigues ^2,3 and Sara C. Antunes ^2,3

¹ 

Abel Salazar Institute of Biomedical Sciences, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal

² 

Interdisciplinary Center for Marine and Environmental Research, University of Porto, Leixões Cruise Terminal, Avenida General Norton de Matos, S/N, 4450-208 Matosinhos, Portugal

³ 

Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal

• Background: Aquatic ecosystems are increasingly subjected to overlapping stressors, including rising temperatures, pH variations, and antibiotic contamination (e.g., sulfamethoxazole—SMX, trimethoprim—TRIM, and their mixture—MIX). These abiotic factors may interact in complex ways, altering antibiotic toxicity and impairing key biological processes in aquatic organisms. This study aims to contribute significant data to bridge the gap regarding the combined impacts of environmental stressors, particularly in a climate change context, underscoring the need for integrative studies. Methods: We evaluated the chronic effects of environmentally relevant concentrations of SMX (150 µg/L), TRIM (30 µg/L), and their mixture (150 µg SMX/L + 30 µg TRIM/L) on Danio rerio after exposure to various (i) temperatures (26, 28, and 32 °C) and (ii) pH values (6.5, 7.5, and 9.0). A multi-biomarker approach was applied to assess antibiotic ecotoxicity and the biological health status of D. rerio. Results: Temperature and pH variations affected the antibiotic’s toxicity to D. rerio. At 28 °C, SMX and MIX exhibited moderate toxicity, inducing significant biological alterations (neurotoxicity and DNA damage), while TRIM showed only slight toxicity, mainly altering antioxidant/detoxification defenses. At 32 °C, MIX emerged as the most toxic compound, causing genotoxic and histopathological damage. In terms of the influence of pH, SMX had a greater impact under low-pH conditions (pH 6.5 and 7.5), leading to oxidative stress, lipid peroxidation, and DNA damage. In contrast, TRIM and MIX showed increased toxicity at neutral-to-alkaline pH (7.5 and 9.0), with notable impairment of antioxidant defenses, as well as DNA and histopathological injuries. Conclusions: The results highlight the urgent need for integrated studies that address chemical pollutants and climate change-related stressors. Ignoring these combined pressures could lead to long-term impacts on aquatic biodiversity and ecosystem stability. These findings reinforce the importance of comprehensive risk assessments that consider the potential amplification of pharmaceutical toxicity under changing environmental conditions.

4.27. The Potential for Fetal Lead Exposure in Utero Due to Contamination of Cow’s Milk or Soy-Based Beverages Sold on the Portuguese Market

• Vanda Lopes de Andrade ^1,2,3,4, Ana Paula Marreilha ¹, Iolanda Ribeiro ⁵ and Maria Luísa Mateus ¹

¹ 

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal

² 

School of Agriculture, Santarem Polytechnic University, Quinta do Galinheiro-S. Pedro, 2001-904 Santarém, Portugal

³ 

Life Quality Research Centre (CIEQV), Santarem Polytechnic University, Complexo Andaluz, Apartado 279, 2001-904 Santarém, Portugal

⁴ 

Research Centre for Natural Resources, Environment and Society (CERNAS), Santarem Polytechnic University, Quinta do Galinheiro-S. Pedro, 2001-904 Santarém, Portugal

⁵ 

Faculty of Sciences, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal

Pollution with lead (Pb) constitutes a primary route for the contamination of foods like cow’s milk (CM) or soya beverages (SBs), which are frequently consumed on a daily basis. Even at low exposure levels, Pb is toxic; no level is safe, with effects seen at blood Pb concentrations of just 3.5 µg/dL, which is the current Blood Lead Reference Value of the Centers for Disease Control and Prevention. In alignment with this value, the US Food and Drug Administration (FDA) establishes an Interim Reference Level (IRL) for dietary Pb of 8.8 μg/day for females of childbearing age (FCBAs), considering the potential for fetal Pb exposure. The objective of this work was to assess whether the exposure to Pb of FCBAs through the consumption of CM or SB brands available on the Portuguese market was higher than the FDA’s IRL. The lead levels of 14 CM and 14 SB brands were determined by Atomic Absorption Spectrophotometry. Three CM or SB consumption scenarios were considered: the estimated adult CM consumption in Portugal and the minimum and maximum recommended CM consumption (MinC and MaxC). For the estimated CM intake, totaling 0.1 L/day, all the analyzed samples exceeded the FDA’s IRL. However, for MinC (0.5 L/day), 42 and 21% of the CM and SB brands, respectively, surpassed the IRL; in this scenario, the mean Pb exposure from the 14 analyzed CM brands was 9.66 µg/day, and the mean Pb exposure from the 14 SB brands was 6.72 µg/day. Regarding MaxC (0.75 L/day), 64 and 42% of the analyzed CM and SB brands, respectively, surpassed the FDA’s IRL; the mean Pb exposure from the CM brands was 14.49 µg/day, and from the SB brands, it was 10.08 µg/day. Although CM constitutes a higher contributor to dietary Pb exposure than SBs, greater efforts must be made to control the exposure of FCBAs to Pb through the consumption of both food products.

4.28. The Toxicological Effects of α-Cypermethrin on Placental Oxidative Balance in a Rat Model

• Suzana Žunec, Dubravka Rašić and Anja Katić

• Division of Toxicology, Institute for Medical Research and Occupational Health, Zagreb, Croatia

α-Cypermethrin, a synthetic pyrethroid, is highly lipophilic, allowing it to accumulate in fatty tissues, the liver, and the brain, and potentially cross the placental barrier. Although α-cypermethrin primarily acts by disrupting neuronal sodium channel function, its potential to induce oxidative stress highlights an additional pathway of toxicity. Pyrethroids are nowadays recognized as potential endocrine disruptors, with oxidative stress suggested as a mechanism linking exposure to adverse health outcomes. This study aimed to investigate the effects of α-cypermethrin exposure on oxidative stress induction by analyzing relevant biomarkers (malondialdehyde, MDA; reactive oxygen species, ROS; reduced glutathione, GSH; and activities of catalase, CAT, superoxide dismutase, SOD, and glutathione peroxidase, GPx) in the placentas of pregnant rats exposed to 1, 10, or 19 mg/kg/day throughout gestation. A dose-dependent increase in ROS production and lipid peroxidation, as indicated by elevated MDA levels, was observed. These elevated MDA concentrations likely resulted from the lipophilic nature of α-cypermethrin, which facilitates its penetration into cell membranes, promoting lipid peroxidation. GSH levels remained unchanged across all exposure groups. Although CAT and GPx activities decreased, SOD activity was consistently elevated in all samples, indicating compensatory activation of the antioxidant defense system. The most pronounced disruption of oxidative–antioxidant balance occurred at the lowest tested dose (1 mg/kg). These findings suggest that α-cypermethrin induces oxidative stress in sensitive tissues such as the placenta, potentially contributing to disruption of the endocrine system.

4.29. Volatile Organic Compounds in Indoor Air of Residences of Fungus-Related Allergic Airway Disease Patients

• Yoshika Sekine ¹, Daisuke Sato ², Yoshiki Shiraishi ³, Kazuhiro Harada ⁴, Fumitoshi Ogino ⁴, Tsuyoshi Oguma ⁵ and Koichiro Asano ⁵

¹ 

Department of Chemistry, School of Science, Tokai University, Tokyo 151-8677, Japan

² 

Graduate School of Science, Tokai University, Tokyo 151-8677, Japan

³ 

Department of Clinical Pharmacology, Tokai University School of Medicine, Isehara 259-1193, Japan

⁴ 

Research and Development, Duskin Co., Ltd., Suita 564-0051, Osaka, Japan

⁵ 

Division of Pulmonary Medicine, Department of Medicine, Tokai University School of Medicine, Isehara, Japan

• Introduction: Allergic bronchopulmonary mycosis (ABPM) is an allergic airway disorder caused by fungal colonization within the respiratory tract. Although pharmacological treatment can lead to temporary remission, the recurrence rate remains high. Therefore, controlling fungal contamination in residential environments is considered essential for both the prevention and management of ABPM and/or other fungus-related allergic airway diseases. The aim of this study was to assess the state of fungal contamination in indoor environments through the observation of volatile organic compounds (VOCs). Methods: Two survey visits were conducted during the autumn seasons of 2020 and 2021 at 17 residences of patients diagnosed with fungus-related allergic airway diseases. VOCs in the indoor air of living rooms were collected using a passive flux sampler over a 24 h period in principle. Following collection, 23 types of VOCs were quantified by gas chromatography–mass spectrometry and converted to air concentrations using theoretical sampling rates. These VOC concentrations were then compared with the fungal contamination inside air conditioners installed in the respective living rooms. Results: Indoor air concentrations of 23 VOCs—considered to originate from building materials, microorganisms, and the human body—were obtained. No consistent trends were observed in the levels or compositions of VOCs among the 17 residences. However, geosmin concentrations in indoor air showed significant correlations with fungal contamination detected on the heat exchanger (r = 0.47, p = 0.006) and vents (r = 0.37, p = 0.034) of the air conditioners used in these residences. Conclusions: Geosmin in indoor air may serve as an indicator of fungal contamination within air conditioner units, which are often not visibly contaminated.

5. Session: Exploring Non-Model Species for Ecotoxicology

5.1. Assessing the Impact of Air Pollution on the Associated Outcomes of Bird Species in North China Plain

• Qingyang Liu

• College of Ecology and Environment, Nanjing Forestry University, Nanjing 210037, China

• Background and Objective: Air pollution in the North China Plain frequently exceeds WHO limits and is linked to human respiratory diseases, but its effects on birds remain understudied. This study examines the impacts of air pollutants (AQI, CO, O3, SO2, smoke, and particulate matter) on bird health and behavior in the region, focusing on House sparrows (Passer domesticus) and Rock pigeons (Columba livia). This study includes data from the literature review in December 2023, using Clarivate Analytics.
• Methods: Data on average air pollution levels, respiratory disease incidence, and behavioral changes were collected from publicly available sources spanning from 2011 to 2023. The study included data from 12 bird colonies and approximately 500 individual birds across the North China Plain. These comprised both migratory and resident species, with a focus on sparrows and pigeons due to their prevalence in the region. The primary species analyzed were House sparrows (Passer domesticus) and Rock pigeons (Columba livia). Behavioral changes were assessed using direct observational studies that were in controlled and natural environments to record alterations in foraging, vocalization, and mobility, respectively. GPS tracking for pigeons was used to monitor flight patterns and homing speed under varying AQI conditions. Respiratory health was evaluated through post-mortem examinations of deceased birds (e.g., lung tissue analysis for lesions or particulate matter accumulation) and non-invasive biomarkers (e.g., nasal swabs and blood tests for inflammatory markers) in live specimens.
• Key Findings: High fine particle concentrations were linked to acute respiratory infections and lung damage in sparrows. AQI significantly affected pigeon behavior, with 415 pigeons returning home faster under severe pollution (AQI 144).
• Conclusions: Air pollutants are strongly associated with respiratory diseases and behavioral shifts in birds. Mitigating air pollution is critical for avian conservation in the region.

5.2. Biodegradation of Dianix Yellow Brown Azo Dye by Paramecium jenningsi Isolated from Industrial Wastewater

• Tuba Arooj, Itrat Zahra and Farah Rauf Shakoori

• Institute of Zoology, University of the Punjab, Quaid-i-Azam Campus, Lahore, Pakistan

• Introduction:

Azo dyes are widely used in textile industry processes. Due to their persistent and recalcitrant nature, they are classified as xenobiotic compounds resistant to biodegradation. However, recent studies suggest that under specific environmental conditions, certain microorganisms can transform these toxic dyes into colorless, nontoxic compounds or even achieve complete mineralization. This study investigates the biodegradation efficiency of a newly isolated Paramecium species in azo dye-contaminated industrial wastewater.

• Methods:

A Paramecium strain was isolated from industrial wastewater and identified as P. jenningsi via 18S rRNA sequencing (NCBI Accession No. MZ540265). Growth optimization was conducted at varying pH (6, 6.5, 7, 7.5, 8, and 8.5) and temperatures (20 °C, 25 °C, 30 °C, and 35 °C). Under optimum conditions, P. jenningsi was exposed to 20 ppm Dianix Yellow Brown (DYB) azo dye for 30 days. Growth kinetics, morphological changes, and vacuole activity were monitored to assess physiological responses. DYB decolorization was measured using a UV spectrophotometer at 260 nm over 0, 48, 96, and 144 h. FTIR was used to assess functional group changes, and GC-MS was used to identify dye-degradation metabolites.

• Results:

Under the optimum conditions of pH 7.0 and 25 °C, P. jenningsi exhibited distinct growth phases, with a 26-fold increase in growth by day 14 in 20 ppm DYB compared to the control. However, reductions in mitosis, motility, and vacuole activity indicated mild DYB toxicity. By 144 h, 85.43% DYB decolorization was observed. FTIR revealed the loss of azo (-N=N) and amine (C-N) groups, alongside the formation of hydroxyl (-OH) and carbonyl (-C=O) groups, indicating dye transformation. GC-MS profiling suggested a two-step degradation: by day 4, formation of 4-tert-butylaniline, N-trimethylsilyl, 3-hydroxyanthranilic acid, and bis(2-ethylhexyl) phthalate indicated azo bond cleavage; by day 8, pentacosane, octacosane, and eicosane suggested further DYB breakdown into less toxic compounds.

• Conclusions:

This study highlights P. jenningsi as a promising tool for the bioremediation of azo dye-contaminated wastewater.

5.3. Comparative Proteomic Analysis of Cadmium Stress Response in a Newly Identified Paramecium Species Isolated from Industrial Wastewater

• Itrat Zahra ¹, Tuba Arooj ¹, Michael Betenbaugh ² and Farah Rauf Shakoori ¹

¹ 

Institute of Zoology, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan

² 

Chemical and Biomolecular Engineering Department, Johns Hopkins University, Baltimore, MD 21218, USA

• Introduction:

Microbial-mediated bioremediation offers a sustainable solution for detoxifying polluted environments, but its molecular mechanisms remain poorly understood. Recent advances in multi-omics approaches, particularly transcriptomics, proteomics, and metabolomics, have enabled detailed insight into cellular responses to environmental stressors. In this study, a mass spectrometry-based quantitative proteomic profiling approach was applied to investigate alterations in protein expression in Paramecium multimicronucleatum exposed to cadmium (Cd²⁺), a toxic heavy metal known to cause cellular stress.

• Methods:

A mass spectrometry-based quantitative proteomic approach was applied to analyze protein abundance changes in P. multimicronucleatum after Cd²⁺ exposure as compared to a control. Samples were processed using the Proteome Discoverer software to identify and quantify peptide spectral matches, total peptides, and proteins. Differential abundance was determined using statistical analysis (p ≤ 0.05).

• Results:

The proteomic analysis identified 7416 peptide spectral matches, corresponding to 2824 unique peptides and 989 proteins. Among these, 29 proteins showed statistically significant differential abundance in cadmium-treated samples. Six proteins were upregulated, while twenty-three were downregulated. These proteins were functionally linked to stress responses, energy metabolism, protein degradation, cell growth, and hormone processing.

• Conclusions:

This comprehensive comparative proteomic analysis provides valuable insights into the molecular adaptations of Paramecium under cadmium stress. The findings contribute to our understanding of metal detoxification pathways, supporting future studies on bioremediation strategies for environmental sustainability.

5.4. Optimization of Three-Dimensional Cultures of the RTL-W1 Liver Cell Line to Model Polycyclic Aromatic Hydrocarbon Effects on Liver Detoxification

• Telma Esteves, Margarida Vilaça, Rosária Seabra, Fernanda Malhão, Eduardo Rocha and Célia Lopes

¹ 

Team of Animal Morphology and Toxicology, CIIMAR/CIMAR—Interdisciplinary Centre of Marine and Environmental Research, University of Porto, 4050-123 Porto, Portugal

² 

Laboratory of Histology and Embryology, Department of Microscopy, ICBAS—School of Medicine and Biomedical Sciences, University of Porto, 4050-313 Porto, Portugal

Climate change-induced stress and increased aquatic pollution, including persistent pollutants such as polycyclic aromatic hydrocarbons (PAHs), disrupt biological functions. Although 3D cell culture systems can be used to better understand these effects, their use in fish studies is currently limited. In this vein, developing and characterizing new models is of extreme importance, as these may help to disclose pollutant effects on temperature increase scenarios.

To generate spheroids from the rainbow trout liver-derived RTL-W1 cell line, four cell densities—10,000, 20,000, 40,000 and 60,000 cells per well—were cultured in 96-well ultra-low attachment (ULA) plates at 18 °C, with or without centrifugation after seeding. Over 18 days in culture, the spheroids were assessed for viability (using alamar blue and lactate dehydrogenase (LDH) assays), biometry (diameter, area and sphericity), and morphology (optical and electron microscopy). Protein expression of cytochrome P450 (CYP) 1A was assessed by immunocytochemistry (ICC) at 10, 14, and 18 days in culture. To determine its suitability for ecotoxicology research, spheroids (60,000 cells) were exposed from the 10th to the 14th day in culture to two concentrations of the model PAH benzo(k)fluoranthene (BkF), at 18 and 23 °C. CYP1A immunolabeling and mRNA expression were evaluated after the exposures.

In all culture densities, spheroids’ diameter and area decreased over time, while sphericity and viability increased. The biometric parameters remained stable from the 10th day onward. Centrifugation did not impact the spheroids’ formation dynamics. Morphological analysis showed unscathed cells and organelle content compatible with hepatocytic differentiation. Spheroids expressed CYP1A in ICC, denoting their functionality. BkF-treated spheroids exhibited increases in both ICC and mRNA expression, but there were no variations in gene expression across temperatures. The generated spheroids appear to be a promising alternative model for studying PAHs and warming effects on fish liver detoxification.