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

Assessment of the biological risks associated to the use of biosolids and wastewater

Tesi en modalitat de compendi de publicacions(English) The circular economy is an emerging concept presented as a solution to counteract the damage caused by the linear economic scheme followed in the past decades. It is based on the principle of "closing the life cycle" and seeks, through reuse and revaluation, to extend the value of products, materials, and resources, such as water and energy, minimizing the generation of waste. In this context, the European Union (EU) aims to build a food system that could work under all circumstances, ensuring a safe and sustainable food and water supply for all citizens by applying circularity. Ensuring the availability of water and fertilizers for agriculture is probably the major challenge for achieving this goal. In this respect, the EU contemplates the use of effluents collected from water treatment plants (WWTP) for irrigation or aquifer recovery. Similarly, the use of solid waste such as sludge from WWTPs, the organic fraction of urban solid waste (OFMSW), or livestock waste, at different processing levels, is a promising alternative to mineral/chemical fertilization, promoting a self-sustainable, circular food system. However, the use of these recycled materials is not free from risks. They contain hazards of emerging concern including Contaminants of Emerging Concern (CECs) (like prescription drugs, endocrine disruptors and antibiotics) as well as different microbial hazards (not only pathogens, which are legally regulated and tested, but also antibiotic resistant bacteria known to spread the resistance to different antibiotics to the microbiomes from waterbodies, soils, and crops). Upcoming legislations that aim to promote and regulate their use agree on the fact that the main challenge relies on correctly assessing their associated risks for environmental and human health. This thesis aims to evaluate the biological activities associated to livestock wastes, OFMSW and WWTP effluents and associated biosolids in order to determine their potential hazards and the level of exposure for the environment and for the general population in the context of their use. For this purpose, we specifically evaluated the impact of organic fertilization on agroecosystems through ABs, ARGs and ARBs transmission and microbiome modulation monitoring. Moreover, we assessed the impact linked to WWTP effluents through the screening of ARGs and ARBs and associated toxic activities. Consequently, we developed an environmental risk analysis framework associated to biosolids and wastewater reuse considering the occurrence of the monitored hazards and their potential impacts on exposed environments. In the studies included in this thesis we observed that the selective pressure posed by the analyzed organic fertilizers was enough to favor the soil colonization by antibiotic-resistant bacteria and to increase horizontal gene transfer (HGT) in both target soils and crops. The fact that this impact appeared transient highlights the importance of respecting fertilization rates and the time elapsing from fertilization to harvest. Moreover, the processing of wastes favoring bacterial succession appeared as a valuable tool to reduce this risk. On the other hand, we detected high loads of both chemical and microbial hazards in WWTP effluents, underscoring the need of tertiary treatments able to reduce them in order to ensure a safe reuse of wastewater. In this regard, Soil Aquifer Treatment (SAT) technologies appeared in our studies as cost-effective alternatives to other tertiary treatments given their efficiency on removing biological (particularly ARG loads) and chemical hazards and their potential ability to promote the displacement of gut-related, copiotrophic bacteria by groundwater-like microbiomes, enhancing in this way the removal of ARGs, CECs and toxic activities.(Català) L'economia circular és un concepte emergent que es presenta com una solució per contrarestar els danys causats per l'esquema econòmic lineal en les últimes dècades. Es basa en el principi de "tancar el cicle de vida" i busca, a través de la reutilització i la revaloració, allargar la vida i el valor de productes, materials i recursos, com l'aigua i l'energia, minimitzant la generació de residus. En aquest context, la Unió Europea (UE) té com a objectiu construir un sistema alimentari resilient, garantint un subministrament d'aliments i aigua segur i sostenible per a tots els ciutadans aplicant els principis de la circularitat. Assegurar la disponibilitat d'aigua i fertilitzants per a l'agricultura és probablement el gran repte per assolir aquest objectiu. En aquest sentit, la UE contempla l'ús d'efluents de les estacions de depuració d'aigües residuals (EDAR) per al reg o la recuperació d'aqüífers. De la mateixa manera, l'ús de residus sòlids com els fangs de les EDAR, la fracció orgànica dels residus sòlids urbans (FORM) o els residus ramaders, a diferents nivells de processament, és una alternativa prometedora a la fertilització mineral/química, promovent un sistema alimentari autosostenible i circular. No obstant això, l'ús d'aquests subproductes no està exempt de riscos químics i biològics. Això és degut a què en la composició d'aquests efluents i residus es poden detectar concentracions (rellevants) de contaminants emergents (CE) (com ho són els medicaments, els disruptors endocrins i els antibiòtics), així com la presència d’altres vectors de contaminació d'origen microbià (no només patògens regulats, sinó també bacteris resistents als antibiòtics coneguts per propagar la resistència a diferents antibiòtics a través dels microbiomes aquàtics i dels sòls i cultius). L'elaboració de polítiques que tenen com a objectiu promoure i regular el seu ús identifiquen la l’avaluació de riscos associats a la salut ambiental i humana com el principal repte. Aquesta tesi té com a objectiu avaluar les activitats biològiques associades als residus ramaders, FORM i efluents d’EDAR i biosòlids associats per tal de determinar els seus riscos potencials i el nivell d'exposició per al medi ambient i per a la població en general en el context del seu ús. Amb aquesta finalitat, hem avaluat específicament l'impacte de la fertilització orgànica en els agroecosistemes mitjançant la transmissió d'ABs, ARGs i ARBs i el monitoratge de la modulació del microbioma. A més, hem avaluat l'impacte vinculat als efluents de les EDAR mitjançant el cribratge d'ARGs i ARBs i activitats tòxiques associades. En conseqüència, aquset treball ha desenvolupat un marc d'anàlisi de riscos ambientals associat als biosòlids i a la reutilització d'aigües residuals tenint en compte l'ocurrència dels perills monitoritzats i els seus impactes potencials en els ambients exposats. En els estudis inclosos en aquesta tesi hem observat que la pressió selectiva que exerceixen els fertilitzants orgànics analitzats és suficient per afavorir la colonització del sòl per bacteris resistents als antibiòtics i per augmentar la transferència horitzontal de gens (THG) tant en sòls diana com en cultius. El fet que aquest impacte sigui transitori posa de manifest la importància de respectar les freqüències d'aplicació de fertilitzant als sòls i el temps transcorregut des de la fertilització fins a la collita. A més, s'ha identificat que el processament de residus afavorint la successió bacteriana és una eina molt valuosa/efectiva/útil per reduir aquest risc. D'altra banda, hem detectat altes càrregues de riscos tant químics com microbians als efluents de les EDAR, fet que evidencia la necessitat de tractaments terciaris capaços de reduir-los per garantir una reutilització segura de les aigües residuals. En aquest sentit, les tecnologies basades en la recàrrega artificial d'aqüífers (SAT) apareixen als nostres estudis com a alternatives altament rendibles.DOCTORAT EN CIÈNCIES DEL MAR (Pla 2013

Efecto de nanoplásticos de poliestireno carboxílicos sobre algunos aspectos fisiológicos y bioquímicos en Artemia parthenogenetica Artemia parthenogenetica

La contaminación de los ecosistemas acuáticos con desechos plásticos y sus fragmentos se reconoce como uno de los mayores problemas de calidad medioambiental. Dentro de esta problemática, los nanoplásticos revisten gran interés por sus especiales características fisicoquímicas y porque los efectos que pueden llegar a originar han sido mucho menos estudiados que los provocados por los macro y microplásticos. Su entrada en los ecosistemas acuáticos puede producirse por su vertido directo o por acción de la degradación. El objetivo principal del presente estudio fue la caracterización de los efectos ocasionados por nanoplásticos de poliestireno funcionalizados con grupos aniónicos carboxílicos (NP(PS-COOH)) en crustáceos, utilizando como modelo Artemia parthenogenetica. Para ello se determinó su toxicidad crónica analizando los efectos sobre el crecimiento, la supervivencia y el comportamiento alimenticio y, además, se evaluaron algunas respuestas subcelulares enzimáticas relacionadas con la biotransformación de xenobióticos (carboxilesterasa, CbE y glutatión-S-transferasa, GST), la actividad nerviosa (colinesterasa, ChE), la defensa antioxidante (catalasa, CAT), y de protección frente al estrés general (proteínas de estrés HSP70). Los resultados obtenidos demostraron que la exposición crónica de A. parthenogenetica a NP(PS-COOH) no alteró la supervivencia, la actividad CAT, ni la expresión de las HSP70, pero sí redujo el crecimiento y la tasa de filtración. Además, la actividad CbE y ChE disminuyeron y la actividad GST aumentó. Estos resultados ponen de manifiesto que los nanoplásticos funcionalizados con grupos carboxílicos (PS-COOH) son capaces de producir efectos tóxicos en A. parthenogenetica, aunque en menor medida que los descritos para nanoplásticos similares funcionalizados con grupos amínicos (PS-NH2).Peer reviewe

Uptake and transcriptional effects of polystyrene microplastics in larval stages of the Mediterranean mussel Mytilus galloprovincialis

The widespread occurrence of microplastics (MP) in the marine environment is cause of increasing concerns about the safety of the exposed ecosystems. Although the effects associated to the MP uptake have been studied in most marine taxa, the knowledge about their sub-lethal impacts on early life stages of marine species is still limited. Here, we investigated the uptake/retention of 3-\u3bcm polystyrene MP by early stages of the Mediterranean mussel Mytilus galloprovincialis, and the related effects on gut clearance, feeding efficiency, morphological and transcriptional parameters involved in embryo-larval development. Uptake measurements were performed on larvae at 48 h, 3, 6 and 9 days post fertilization (pf) after exposure to a range of 50\u201310,000 particles mL 121. At all tested pf periods, treatments resulted in a significant and linear increase of MP uptake with increasing concentrations, though levels measured at 48 h pf were significantly lower compared to 3\u20139 d pf. Ingested MP were retained up to 192 h in larvae's gut, suggesting a physical impact on digestive functions. No change was noted between the consumption of microalgae Nannochloropsis oculata by larvae when administered alone or in the presence of an identical concentration (2000 items mL 121) of MP. The exposure to 50\u201310,000 MP mL 121did not alter the morphological development of mussel embryos; however, transcriptional alterations were observed at 50 and 500 MP mL 121, including the up-regulation of genes involved in shell biogenesis (extrapallial protein; carbonic anhydrase; chitin synthase) and immunomodulation (myticin C; mytilin B), and the inhibition of those coding for lysosomal enzymes (hexosaminidase; \u3b2-glucorinidase; catepsin-L). In conclusion, though not highlighting morphological or feeding abnormalities, data from this study revealed the onset of physical and transcriptional impairments induced by MP in mussel larvae, indicating sub-lethal impacts which could increase their vulnerability toward further environmental stressors

Dose effect of Zn and Cu in sludge-amended soils on vegetable uptake of trace elements, antibiotics, and antibiotic resistance genes: Human health implications

The application of sewage sludge to agricultural fields reduces the need for mineral fertilizers by increasing soil organic matter, but may also increase soil pollution. Previous studies indicate that zinc and copper, as the most abundant elements in sewage sludge, affect plant uptake of other contaminants. This paper aims to investigate and compare the effect of increasing amounts of Zn and Cu in sludge-amended soils on the accumulation of trace elements (TEs), antibiotics (ABs), and antibiotic resistance genes (ARGs) in lettuce and radish. The vegetables were grown under controlled conditions, and the influence on plant physiology and human health were also evaluated. The results show that the addition of Zn and Cu significantly increased the concentration of TEs in the edible tissue of both vegetables. According to the hazard quotient (HQ) of the TEs, the human health risk increased 2 to 3 times and was 3 to 4 times greater in lettuce than in radish. In contrast to the TEs, the occurrence of ABs and most of the ARGs was higher in radish roots than lettuce leaves. ABs were not detected in lettuce leaves, and the amount of all ARGs except blaTEM was 10 times lower than in radish roots. On the other hand, the addition of Zn and Cu had no significant effect on the occurrence of ABs and ARGs in the edible part of the vegetables, and no damage was found to plant productivity or physiology. The results show that the consumption of lettuce and radish grown in sewage-sludge-amended soils under our dosage does not pose an adverse human health effect, as the total HQ value was always less than 1, and the presence of ABs and ARGs was not found to have any potential impact. Nevertheless, further studies are needed to estimate the long-term effect on human health of crops grown under frequent application of biosolids in arable soil.The authors gratefully acknowledge the financial support of the Spanish Ministry of Economy, Industry, and Competitiveness (MEIC) through Project AGL2017-89518-R. Rui You gratefully acknowledges a fellowship from the China Scholarship Council. The authors would further like to thank Mr. M. Massip for his kind assistance at the greenhouse facility.Peer reviewe