Toxicity assessment of silver nanoparticles in soils through standard tests, an integrated battery of biomarkers at different levels of biological complexity and in vitro approaches with coelomocytes

222 p.Existe un riesgo potencial de aumentar la entrada de nanopartículas de plata (Ag NPs) en los ecosistemas. Así se ha estimado un incremento anual de 0.007 mg Ag NPs/kg en suelos que reciben descargas de lodos de depuradora o residuos de su incineración. De forma simultánea crece la preocupación respecto a posibles daños que estas Ag NPs puedan provocar en el ecosistema terrestre y en los invertebrados que lo habitan. En la presente tesis, se han estudiado los efectos de las Ag NPs (recubiertas de PVP-PEI) en el suelo, mediante test estandarizados (OECD), biomarcadores a diferentes niveles de complejidad biológica y ensayos in vitro con celomocitos de la lombriz Eisenia fetida. Previamente se establecieron la principal ruta de toma de las Ag NPs, el suelo estándar más apropiado para el mantenimiento de lombrices y las condiciones para los ensayos in vitro (medio de cultivo, test en microplaca). Tras realizar un primer screening, las lombrices se expusieron durante diferentes tiempos (3 y 14d) a una concentración alta pero subletal y a otra concentración próxima a la estimada para el medio ambiente. Aparte de efectos a nivel de organismo (pérdida de peso, reproducción), concentraciones altas pero subletales de Ag NPs causaron citotoxicidad en celomocitos, genotoxicidad y alteraciones en los sistemas de detoxificación de metales y respuesta antioxidante. En conclusión, la combinación de test estándares, la integración de los biomarcadores medidos a diferentes niveles de complejidad biológica y los parámetros medidos en celomocitos extruidos de lombrices expuestas (citotoxicidad y número relativo de las subpoblaciones), han permitido hacer un riguroso diagnóstico de la toxicidad de las Ag NPs en el medio terrestre.PIE : Plentziako Itsas Estazio

Toxicity assessment of silver nanoparticles in soils through standard tests, an integrated battery of biomarkers at different levels of biological complexity and in vitro approaches with coelomocytes

222 p.Existe un riesgo potencial de aumentar la entrada de nanopartículas de plata (Ag NPs) en los ecosistemas. Así se ha estimado un incremento anual de 0.007 mg Ag NPs/kg en suelos que reciben descargas de lodos de depuradora o residuos de su incineración. De forma simultánea crece la preocupación respecto a posibles daños que estas Ag NPs puedan provocar en el ecosistema terrestre y en los invertebrados que lo habitan. En la presente tesis, se han estudiado los efectos de las Ag NPs (recubiertas de PVP-PEI) en el suelo, mediante test estandarizados (OECD), biomarcadores a diferentes niveles de complejidad biológica y ensayos in vitro con celomocitos de la lombriz Eisenia fetida. Previamente se establecieron la principal ruta de toma de las Ag NPs, el suelo estándar más apropiado para el mantenimiento de lombrices y las condiciones para los ensayos in vitro (medio de cultivo, test en microplaca). Tras realizar un primer screening, las lombrices se expusieron durante diferentes tiempos (3 y 14d) a una concentración alta pero subletal y a otra concentración próxima a la estimada para el medio ambiente. Aparte de efectos a nivel de organismo (pérdida de peso, reproducción), concentraciones altas pero subletales de Ag NPs causaron citotoxicidad en celomocitos, genotoxicidad y alteraciones en los sistemas de detoxificación de metales y respuesta antioxidante. En conclusión, la combinación de test estándares, la integración de los biomarcadores medidos a diferentes niveles de complejidad biológica y los parámetros medidos en celomocitos extruidos de lombrices expuestas (citotoxicidad y número relativo de las subpoblaciones), han permitido hacer un riguroso diagnóstico de la toxicidad de las Ag NPs en el medio terrestre.PIE : Plentziako Itsas Estazio

Impacts of sewage sludges deposition on agricultural soils: Effects upon model soil organisms

During years sewage sludges have been worldwide poured in agricultural soils to enhance vegetal production. The "Landfill 17" located in Gernika-Lumo town (43°19'28.9"N 2°40'30.9"W) received for decades sewage sludges from the local Waste Water Treatment Plant (WWTP) with agricultural purposes. To this WWTP, several pollutants as heavy metals (Cd, Cr, Ni, Pb), PAHs (benzo(a)pyrene among many others) and pesticides (i.e. dieldrin) could have arrived from local industry and be widespread all over the landfill. Soil invertebrates like earthworms and plants are of special interest due to their close contact with the polluted matrix and their potential effects by the presence of pollutants. In this context, the aim of the present work was to determine the health status of landfill soils by evaluating the effects on model soil organisms exerted by long-lasted pollutants after on site deposition of WWTP active sludges. With such a purpose, different standard toxicity tests and cellular level endpoints were performed on lettuce and earthworms. Indeed, germination (EPA 850.4100) and root elongation (EPA 850.4230) tests were carried out in Lactuca sativa, while OECD acute toxicity test (OECD-204), reproduction test (OECD-222) and Calcein-AM viability test with coelomocytes were applied in Eisenia fetida worms. For the exposure, soils collected in the landfield containing low, medium and high concentrations of pollutants were selected, and as reference LUFA 2.3 natural standard soil was chosen. While no differences were shown in the assays with L. sativa, significant differences between sludge exposed groups and control group were recorded with E. fetida, with lower coelomocyte number and viability and higher tissue metal accumulation after 28days of exposure to polluted soils. These results confirmed the impact of contaminants to soil biota even after long periods of time.Basque Government (IT810-13), CTM2017-87766-R from MINECO, PhytoSUDOE-SOE1/P5/E0189, and Gernika City Hall

Predicting environmental concentrations and the potential risk of Plant Protection Products (PPP) on non-target soil organisms accounting for regional and landscape ecological variability in european soils

[EN] Plant Protection Products (PPP) raise concerns as their application may cause effects on some soil organisms considered non-target species which could be highly sensitive to some pesticides. The European Food and Safety Authority (EFSA), in collaboration with the Joint Research Centre (JRC) of the European Commission, has developed guidance and a software tool, Persistence in Soil Analytical Model (PERSAM), for conducting soil exposure assessments. EFSA PPR Panel has published recommendations for the risk assessment of non-target soil organisms. We have used PERSAM for calculating PPPs predicted environmental concentrations (PECs); and used the estimated PEC for assessing potential risks using Toxicity Exposure Ratios (TER) for selected soil organisms and good agricultural practices. Soil characteristics and environmental variables change along a latitudinal axis through the European continent, influencing the availability of PPP, their toxicity upon soil biota, and hence, impacting on the risk characterization. Although PERSAM includes as input geographical information, the in-formation is aggregated and not further detailed in the model outputs. Therefore, there is a need to develop landscape based environmental risk assessment methods addressing regional variability. The objective was to integrate spatially explicit exposure (PECs) and effect data (biological endpoints i.e. LC50, NOEC, etc.) to estimate the risk quotient (TER) of four PPP active substances (esfenvalerate, cyclaniliprole, picoxystrobin, fenamidone) on non-target species accounting European landscape and agricultural variability. The study was focused on the effects produced by the above-mentioned pesticides on two soil organisms: E. fetida earthworms and Folsomia sp. collembolans. After running PERSAM assuming a worst case application of PPPs, PECs in total soil and pore water were obtained for different depths in northern, central and southern European soils. With this data, soil variability and climatic differences among soils divided in three large Euroregions along a latitudinal transect (Northern, Central, Southern Europe) were analysed. Summarising, a trend to accumulate higher PECs and TERs in total soil was observed in the north decreasing towards the south. Higher PECs and TERs could be expected in pore water in southern soils, decreasing towards the north. The risk disparity between pollutant concentrations at different soils compartments should be taken into account for regulatory purposes, as well as the potential landscape variabilities among different Euroregions.This work has been partially funded by European Food Safety Authority (EFSA) through the grant NP/EFSA/AFSCO/2016/03. The authors Miguel Santos and Jose V Tarazona are employed with the EFSA in the Pesticides Peer Review and the Scientific Committee and Emerging Risks Units. However, the present article is published under the sole responsibility of the authors and may not be considered as an EFSA scientific output. The positions and opinions presented in this article are those of the authors alone and do not represent the views of EFSA

Misinterpretation in microplastic detection in biological tissues: When 2D imaging is not enough

The presence of microplastics in the food chain is a public concern worldwide, and its analysis is an analytical challenge. In our research, we apply Raman imaging to study the presence of 1mum polystyrene microplastics in cryosections of Mytilus galloprovincialis due to its wide geographic distribution, widespread occurrence in the food web, and general high presence in the environment. Ingested microplastics are accumulated in the digestive tract, but a large number can also be rapidly eliminated. Some authors state that the translocation of microplastics to the epithelial cells is possible, increasing the risk of microplastics transmission along the food chain. However, as seen in our study, a surface imaging approach (2D) is probably not enough to confirm the internalization of particles and avoid misinterpretation. In fact, while some microplastic particles were detected in the epithelium by 2D Raman imaging, further 3D Raman imaging analysis demonstrated that those particles were dragged from the lumens to the epithelium during sample preparation due to the blade drag effect of the cryotome, and subsequently located on the surface of the analyzed cryosection, discarding the translocation to the epithelial cells. This effect can also happen when the samples are fortuitously contaminated during sample preparation. Several research articles that use similar analytical techniques have shown the presence of microplastics in different types of tissue. It is not our intention to put such results in doubt, but the present work points out the necessity of appropriate three-dimensional analytical methods including data interpretation and the need to go a step further than just surface imaging analysis.This work was funded by Basque Government (KK 2021/00001 ELKARTEK 2021/2022, IT1743-22); MINECO (PID 2020-118685RB-I00, PLASTeMER); further financial support by grant CEX2020-001038-M funded by MCIN/AEI/ 10.13039/50110001103

Innovative in vivo and in vitro bioassays for the establishment of toxicity thresholds of pollutants in sediment quality assessment using polychaetes and their immune cells.

Sediment toxicity testing has become a crucial component for assessing the risks posed by contaminated sediments and for the development of sediment quality assessment strategies. Commonly used organisms for bioassays with estuarine sediments include amphipods, Arenicola marina polychaetes and echinoids. Among the latter, the Sea Urchin Embryo test (SET) is the most widely used. However, one relevant limitation of this bioassay is the unavailability of gametes all year-round, particularly outside the natural spawning seasons. Consequently, the establishment of an appropriate and complementary model organism for a continuous assessment of sediment quality is recommended. A reliable assessment of the hazards resulting from pollutants in sediments or pore water, can be achieved with ecologically relevant species of sediment such as the polychaete Hediste diversicolor, which is widespread in estuaries and has the capacity to accumulate pollutants. The aim of this work was to develop reliable in vivo and in vitro bioassays with H. diversicolor and its coelomocytes (immune cells) to determine the toxicity thresholds of different contaminants bounded to sediments or resuspended into water. Polychaetes were exposed to sublethal concentrations of CuCl2 (in vivo) and a non-invasive method for collection of polychaetes coelomocytes was applied for the in vitro bioassay, exposing cells to a series of CuCl2 and AgNPs concentrations. Same reference toxicants were used to expose Paracentrotus lividus following the SET (ICES N 51; Beiras et al., 2012) and obtained toxicity thresholds were compared between the two species. In vivo exposure of polychaetes to high concentrations of Cu produced weight loss and histopathological alterations. After in vitro approaches, a significant decrease in coelomocytes viability was recorded for both toxicants, in a monotonic dose-response curve, at very short-exposure times (2h). The toxicity thresholds obtained with polychaetes were in line with the ones obtained with the SET, concluding that their sensitivity is similar. In conclusion, in vivo and in vitro bioassays developed with H. diversicolor are accurate toxicity screenings of pollutants that could be bounded to sediments or dissolved in the pore water, and may complement the SET outside the spawning period of the echinoderms. The bioassays herein developed could be applied not only to establish the toxicity thresholds of individual compounds or mixtures, but also to assess the toxicity of field collected sediments.The authors thank Basque Government (Grant to Consolidated Research Groups; IT1302-19, IT1213-19) and Spanish Ministry of Economy and Competitiveness (SEAdimenTOX project, CTM 2017-87766-R)

Lurzoru kutsatuen karakterizazio intentsiboa in vivo eta in silico fokatzeak erabiliz

The anthropic activities during the last decade are causing damages on ecosystems. In fact, animal husbandry, agriculture, and industrial activities have increased the proportion of soils contaminated with pesticides and metals. The presence of pollutants could affect soil organisms and the Ecosystem Services provided; thus, a proper risk evaluation is required in order to assess the final environmental impacts of pollutants. In silico models are tools designed to predict environmental concentrations of released pollutants that could assist on substances evaluation and regulation. Meanwhile, the health paradigm, understanding the natural environment as an inter-related compartment, has been spread within the scientific community. Hence, the measurement of toxicological effects produced by contaminants on soil organisms (using in vivo tests and biomarkers) gained a high reputation for evaluating environmental health. This work presents different approaches, techniques, software, guidelines and tests for assessing contaminated soils; crucial for an integrative soil assessment; Azken hamarkadatako jarduera antropikoak ekosistemetan desorekak sortzen hasiak dira. Horrela, abeltzaintzak, nekazaritzak eta aktibitate industrialek pestizida eta metalekin kutsaturiko lurzoruen proportzioa emendarazi dute. Kutsatzaileen presentziak, lurzoruetan bizi diren organismoak eta beraz, lurzoruak eskaintzen dituen Zerbitzu Ekosistemikoak erasatea ekarri dezake, arrisku ebaluazio egokien beharra eskatuz. In silico modeloak, kutsatzaile baten aplikazio osteko ingurumen kontzentrazioak aurreikusteko tresna egokiak dira, substantzien arrisku ebaluazioan eta erregulazioan lagun dezaketenak. Bestetik, kutsatzaileak lurzoruan duten inpaktua ebaluatze aldera, osasunaren paradigma ikuspuntua; hots, medio naturalak inter-erlazionatutako konpartimentu gisa ulertzen dituena, hedatzen hasi da komunitate zientifikoaren baitan. Era honetan, kutsatzaileek lurzoru organimoengan eragindako efektu toxikologikoen neurketak (in vivo test-ak eta biomarkatzaileak erabiltzen dituztenak) omen handia irabazi dute ingurumenaren osasuna ebaluatzerako orduan. Lan honetan, kutsatutako lurzoruak ebaluatzeko ikuspuntu, teknika, software, gidalerro eta test ezberdinak aurkezten dira; lurzoru ebaluazio integral bat burutzeko beharrezkoak direnak

Time-course distribution of fluorescent microplastics in target tissues of mussels and polychaetes.

The majority of the plastic produced in the last century is accumulated in the environment, leading to an exacerbated contamination of marine environments due to transport from land to the ocean. In the ocean, mechanical abrasion, oxidation, and photodegradation degrade large plastics into microplastics (MPs) - 0.1mum to 5mm (EFSA, 2016) which are transported through water currents reaching the water surface, water column, and sediments. Further, they can be accumulated by aquatic and benthic species, entering the trophic chain and becoming a potential threat to humans. In the present research, we aimed to decipher the accumulation and distribution time-courses between different organs or target tissues of organisms inhabiting coastal areas such as mussels Mytilus galloprovincialis and polychaetes Hediste diversicolor. Both were exposed in microcosm experiments to fluorescent polystyrene MPs (1mum) which were spiked at two doses (103 and 105 particles/mL) for 1, 4, 24, and 72h. Mussels and polychaetes were digested with 10% KOH and filtered to quantify the number of MPs incorporated. Different anatomical parts of the body were selected and processed for cryosectioning and posterior microscopic localisation of MPs. Both species accumulate MPs spiked in water column, mainly after exposure to the highest dose. In mussels, particles were found in distinct parts of the digestive tract (stomach, digestive diverticula, ducts) and gills. Even if the majority of MPs were localised in the lumen of the digestive tract, in some cases, were inside the digestive epithelium. The identification of MPs and their internalization in the digestive system was studied using Raman spectroscopy. A decreasing trend with time regarding MPs number in the digestive tract (stomach) of mussels was observed while the opposite was recorded for polychaetes and sediments. The combination of microscopical observations of frozen sections and Raman, appeared to be accurate methodologies to address MPs abundances and to reveal their localisation in different organs. This work has enabled to understand the distribution and fate of MPs in different environmental compartments and it could contribute to gain knowledge about their impact after ingestion by coastal organisms.This work was funded by MINECO (PID 2020-118685RB-I00, PLASTeMER); Basque Government (KK 2021/00001 ELKARTEK 2021 2022) and Basque Government (IT1743-22)

Araztegi-lokatzak jasotako lurzoruaren analisi toxikologikoa zizare eta landareak erabiliz

Urtetik urtera areagotzen diren giza populazioak eta jarduera industrialak zabortegi, isuri-puntu eta betetze guneen emendioa eragin du. Zabortegi horien artean, 48046-00181 kodearekin, "17-zabortegia" dago, Gernika-Lumon kokatua (43°19’28,9”N 2°40’30,9”W.). Zabortegi horretan, Gernikako araztegiko arazketa-lokatzak isuri izan ziren ongarri gisa urteetan zehar; eta, horrela, Gernikako industrian sortutako hainbat kutsatzaile (metal astunak, PAHak, pestizidak, etab.) bertan lurperatu ziren. Isurketaren eraginez gehien kaltetutako espezieen artean, azpimarratzekoak dira landareak eta zizareak edo antzeko lurzoruko ornogabeak; batez ere, berorien eta lurzoru-matrizearen arteko kontaktu estuagatik. Testuinguru horretan, lan honen helburua da lokatz horien isurketak eragindako efektuen ebaluazio bat gauzatzea, lurzoruari beste erabilera bat emate aldera; eta, horretarako, zabalki ezagunak diren test estandarizatu eta biomarkatzaileak erabiliko dira: organismo (uraza eta zizare), denbora (3, 7, 28 eta 56 egunera) eta konplexutasun biologiko desberdinetan (zelula-mailatik populazio-mailara arte). Besteak beste, OECD-204 (toxikotasun akutuaren testa), OECD-222 (ugalketa-testa) eta kaltzeina AM bideragarritasun-testak aplikatu ziren Eisenia fetida zizarean, paraleloki, Lactuca sativa landareetan ernetze- eta elongazio-testak gauzatu ziren heinean. Azterketa toxikologikoen ostean, kontrolarekiko desberdintasun esanguratsuak ikusi ziren: erauzitako zelomozito kantitatean, bideragarritasun zelularrean, ehunetan metatutako metal-kontzentrazioetan, hazkuntza-parametroetan (pisu-galeran) eta ugalketa-parametroetan, kutsatzaileek lurzoru biotan eragindako afekzioa berretsiz. L. sativa espeziarekin egindako frogetan, aldiz, ez zen desberdintasun nabarmenik preziatu talde esperimentalen artean.; Increasing global population, along with a rising industrial activity and soil scarceness led to landfill, spill and filling point extension. Among this landfills, “Landfill 17” can be found with 48046-00181 code; located in Gernika-Lumo (43°19'28,9"N 2°40'30,9"W, Basque Country). There, sewage sludges coming from Gernika waste water treatment plant were poured with a hose with agricultural purposes; thus, many pollutants with industrial origin (heavy metals, PAHs and pesticides among others) ended up in the mentioned soils. Among the species mostly affected by the contamination, plants and earthworms must be highlighted due to their close relation with soil matrix; and therefore, soil pollution. In this context, the aim of this work is to evaluate the effects exerted by pollutants in the spilling in order to assess potential Landfill future uses; using for that widely known biomarkers and standardized tests in different organisms (lettuce and earthworms), exposure times (3, 7, 28 and 56 days) and complexity levels (from cellular level to populational level) for that. Indeed, OECD-204 (acute toxicity test), OECD-222 (reproduction test) and calcein AM tests were applied in Eisenia fetida earthworms; while, germination and elongation tests were applied on Lactuca sativa plants. After carrying out ecotoxicological assays, significant differences respect to the control were observed in: extruded coelomocyte quantity, cell viability, tissue metal accumulations, growth (weight loss) parameters or reproductive parameters; proving pollutants affection upon soil biota. However, no significant differences were observed between experimental groups in those tests carried out with L. sativa

Effects of sulfamethazine and tetracycline at molecular, cellular and tissue levels in Eisenia fetida earthworms

Soil contamination by antibiotics is a global issue of great concern that contributes to the rise of bacterial antibiotic resistance and can have toxic effects on non-target organisms. This study evaluated the variations of molecular, cellular, and histological parameters in Eisenia fetida earthworms exposed to sulfamethazine (SMZ) and tetracycline (TC), two antibiotics commonly found in agricultural soils. The earthworms were exposed for 14 days to a series of concentrations (0, 10, 100, and 1000 mg/kg) of both antibiotics. SMZ and TC did not affect the survival of E. fetida, however, other effects at different levels of biological complexity were detected. The two highest concentrations of SMZ reduced the viability of coelomocytes. At the highest TC concentration, there was a noticeable decline in cell viability, acetylcholinesterase activity (neurotoxicity), and the relative presence of mucopolysaccharides in the epidermis (mucous production). Glutathione S-transferase activity decreased in all TC treatments and at the highest SMZ concentration. However, levels of malondialdehyde and protein carbonyls did not change, suggesting an absence of oxidative stress. Tetracycline was neurotoxic to E. fetida and changed the integrity of the epidermis. Both antibiotics altered the intestinal microbiota of E. fetida, leading to a reduction in the relative abundance of bacteria from the phyla Proteobacteria and Bacteroidetes, while causing an increase in the phylum Actinobacteroidota. All observed changes indicate that both SMZ and TC can disrupt the earthworms' immune system and gut microbiome, while fostering the growth of bacteria that harbour antibiotic resistance genes. Finally, both antibiotics exerted additional metabolic and physiological effects that increased the vulnerability of E. fetida to pathogens.Partially funded by the Basque Government (KONTRAE, KK2020-00007; Consolidate groups, IT1446-22, IT1743-22), Joint Research Laboratory on Environmental Antibiotic Resistance (Euskampus), “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES” (001), and “Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq” (313503/2021-4). Also grateful to the University of the Basque Country (UPV/EHU) for the pre-doctoraI fellowship of I. Vergara-Luis. We extend our deepest gratitude to Ainhoa Ugarte and Ane Arrizabalaga for the micrographs