Implications of the use of organic fertilizers for antibiotic resistance gene distribution in agricultural soils and fresh food products. A plot-scale study

The spread of antibiotic resistance genes (ARG) into agricultural soils, products, and foods severely limits the use of organic fertilizers in agriculture. In order to help designing agricultural practices that minimize the spread of ARG, we fertilized, sown, and harvested lettuces and radish plants in experimental land plots for two consecutive agricultural cycles using four types of fertilizers: mineral fertilization, sewage sludge, pig slurry, or composted organic fraction of municipal solid waste. The analysis of the relative abundances of more than 200,000 ASV (Amplicon Sequence Variants) identified a small, but significant overlap (<10%) between soil's and fertilizer microbiomes. Clinically relevant ARG were found in higher loads (up to 100 fold) in fertilized soils than in the initial soil, particularly in those treated with organic fertilizers, and their loads grossly correlated to the amount of antibiotic residues found in the corresponding fertilizer. Similarly, low, but measurable ARG loads were found in lettuce (tetM, sul1) and radish (sul1), corresponding the lowest values to samples collected from minerally fertilized fields. Comparison of soil samples collected along the total period of the experiment indicated a relatively year-round stability of soil microbiomes in amended soils, whereas ARG loads appeared as unstable and transient. The results indicate that ARG loads in soils and foodstuffs were likely linked to the contribution of bacteria from organic fertilizer to the soil microbiomes, suggesting that an adequate waste management and good pharmacological and veterinarian practicesmay significantly reduce the presence of these ARGs in agricultural soils and plant products.Postprint (published version

Development of quantitative RNA biomarkers for detecting dioxin-like and estrogenic pollutants in Costa Rican native fish species

IRETMonitoring the environmental impact on native species is crucial for the correct management of tropical ecosystems. The Costa Rican fish Parachromis dovii (Cichlidae) and Poecilia gillii (Poecillidae) were used as sentinel species for freshwater bodies under considerable pressure by intensive agriculture Cichlidae development. Suitable qRT-PCR probes for the quantification of hepatic mRNAlevels of two stress-related genes -vitellogen in (estrogenic effects) and cytochrome P4501A (CYP1A, dioxin-like compounds) - for both species were designed and validated in experimental treatments with model effectors (17β-estradiol and β-naphtoflavone respectively), demonstrating their usefulness as markers of exposure to these two kinds of Pollutants. Analysis of fish transplanted across pesticide contaminated sites near Palo Verde National Park, Pacific Coast of Costa Rica did show significant changes on hepatic Cyp1A in both species. In P. dovii, Cyp1A levels were enhanced in Barbubal and in the impacted Cabuyo sites in the rainy season whereas in P. gillii fish Cyp1A transcripts were down-regulated differently across rainy and dry seasons. Vitellogen in mRNA levels in P. gillii varied between males and females with males showing always low values which indicated no estrogenic effects. Within females, vitellogenin levels varied over 100, 000 fold depending on their maturation stage, further demonstrating the ability of the method to monitor changes (natural or induced) in the reproductive system of the fish.El monitoreo del impacto ambiental sobre las especies nativas es fundamental para el correcto manejo de los ecosistemas tropicales. Los peces costarricenses Parachromis dovii (Cichlidae) y Poecilia gillii (Poecillidae) fueron utilizados como especies centinelas para cuerpos de agua dulce bajo una presión considerable por el desarrollo de Cichlidae de agricultura intensiva. Sondas qRT-PCR adecuadas para la cuantificación de los niveles de ARNm hepático de dos genes relacionados con el estrés -vitelógeno in (efectos estrogénicos) y citocromo P4501A (CYP1A, compuestos similares a las dioxinas) - para ambas especies se diseñaron y validaron en tratamientos experimentales con efectores modelo ( 17β-estradiol y β-naftoflavona respectivamente), demostrando su utilidad como marcadores de exposición a estos dos tipos de contaminantes. El análisis de peces trasplantados a través de sitios contaminados con pesticidas cerca del Parque Nacional Palo Verde, Costa del Pacífico de Costa Rica, mostró cambios significativos en la Cyp1A hepática en ambas especies. En P. dovii, los niveles de Cyp1A aumentaron en Barbubal y en los sitios impactados de Cabuyo durante la temporada de lluvias, mientras que en los peces de P. gillii las transcripciones de Cyp1A se regularon negativamente de manera diferente en las estaciones lluviosa y seca. Los niveles de vitelógeno en ARNm en P. gillii variaron entre machos y hembras, mostrando los machos siempre valores bajos, lo que indica que no hay efectos estrogénicos. Dentro de las hembras, los niveles de vitelogenina variaron más de 100.000 veces dependiendo de su etapa de maduración, lo que demuestra aún más la capacidad del método para controlar los cambios (naturales o inducidos) en el sistema reproductivo de los peces.O monitoramento do impacto ambiental sobre as espécies nativas é fundamental para o correto manejo dos ecossistemas tropicais. Os peixes costarriquenhos Parachromis dovii (Cichlidae) e Poecilia gillii (Poecillidae) foram usados ​​como espécies sentinela para corpos de água doce sob considerável pressão pelo desenvolvimento de agricultura intensiva de Cichlidae. As sondas de qRT-PCR adequadas para a quantificação de níveis de mRNA hepático de dois genes relacionados ao estresse -vitelogênio em (efeitos estrogênicos) e citocromo P4501A (CYP1A, compostos semelhantes à dioxina) - para ambas as espécies foram projetadas e validadas em tratamentos experimentais com efetores modelo ( 17β-estradiol e β-naftoflavona respectivamente), demonstrando sua utilidade como marcadores de exposição a esses dois tipos de Poluentes. A análise de peixes transplantados em locais contaminados com pesticidas próximos ao Parque Nacional Palo Verde, na costa do Pacífico da Costa Rica, mostrou mudanças significativas no Cyp1A hepático em ambas as espécies. Em P. dovii, os níveis de Cyp1A foram aumentados em Barbubal e nos locais impactados de Cabuyo na estação chuvosa, enquanto em peixes P. gillii os transcritos de Cyp1A foram regulados negativamente de forma diferente nas estações chuvosa e seca. O vitelogênio nos níveis de mRNA em P. gillii variou entre machos e fêmeas, com os machos sempre apresentando valores baixos que não indicaram efeitos estrogênicos. Nas fêmeas, os níveis de vitelogenina variaram mais de 100.000 vezes dependendo do seu estágio de maturação, demonstrando ainda mais a capacidade do método de monitorar mudanças (naturais ou induzidas) no sistema reprodutivo dos peixes.Department of Environmental Chemistry, SpainUniversidad Nacional, Costa RicaInstituto Regional de Estudios en Sustancias Tóxica

Effects of ifosfamide and cisplatin exposure combined with a climate change scenario on the transcriptome responses of the mussel Mytilus galloprovincialis

Coastal ecosystems are currently exposed to pollutants and climate change. Namely, the increasing consumption of antineoplastic drugs and their potential release to aquatic ecosystems are raising concerns. Nevertheless, information regarding the toxicity of these drugs towards non-target species is scarce, especially considering climate change scenarios. Ifosfamide (IF) and cisplatin (CDDP) are among the antineoplastics already detected in aquatic compartments and due to their mode of action (MoA) can negatively affect aquatic organisms. This study evaluates the transcription of 17 selected target genes related to the MoA of IF and CDDP in Mytilus galloprovincialis gills exposed to environmentally relevant and toxicological meaningful concentrations (IF - 10, 100, 500 ng/L; CDDP - 10, 100, 1000 ng/L), under an actual (17 °C) and predicted warming scenario (21 °C). Results showed an upregulation of the cyp4y1 gene when exposed to the highest concentrations of IF, regardless of the temperature. Both drugs upregulated genes related to DNA damage and apoptosis (p53, caspase 8 and gadd45), especially under warmer conditions. Increased temperature also downregulated genes related to stress and immune responses (krs and mydd88). Therefore, the present results showed a gene transcriptional response of mussels to increasing concentrations of antineoplastics and that warmer temperatures modulated those effects.Vanessa Queirós benefited from a PhD grant (PD/BD/150590/2020) given by the National Funds through the Portuguese Science Foundation (Fundação para a Ciência e a Tecnologia, FCT). Thanks are due for the financial support to CESAM (UIDP/50017/2020+UIDB/50017/2020+LA/P/0094/2020) and to the Spanish Ministry of Science and Innovation (PID2020-113371RB-C21).Peer reviewe