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Groundwater quality assessment in areas with intensive livestock: is manure recycling a major source of pollution and dissemination of antibiotic resistance genes?”

Periodic Reporting for period 1 - RESOURCE (Groundwater quality assessment in areas with intensive livestock: is manure recycling a major source of pollution and dissemination of antibiotic resistance genes?”)

Reporting period: 2017-10-02 to 2019-10-01

Groundwater is an extremely important resource, especially in areas where surface water is of limited supply or poor quality. About 75% of the European Union inhabitants depend on groundwater as a drinking water supply, being an important resource for industry and agriculture as well. In agricultural areas with intensive livestock activities, groundwater chemical and microbiological quality might be seriously compromised by the excessive use of animal manure as organic fertilizer. From a chemical perspective, animal manure is rich in nutrients that are beneficial for crop growth and soil conditioning and offers the possibility to close the nutrient cycle. However, it can also contain high concentrations of antibiotics and antibiotic resistance genes (ARGs) and its reuse as fertilizer can be an important pathway for groundwater contamination. The spread of antibiotic resistance has become a major global public health concern because several organisms that cause infections have become resistant to the most commonly prescribed antibiotics, reducing or eliminating their therapeutic effect. An alarming fact is that, approximately every year, 25000 European citizens (5.1 per 100000 inhabitants) die from infections caused by bacteria that have developed resistance towards antimicrobials.

The main objective of RESOURCE was to broaden the knowledge about the role of animal manure reuse as a major source of groundwater pollution in agricultural areas with high livestock production by: (i) the analysis of selected multiple-class antibiotics and ARGs and (ii) the use of an innovative approach, based on the bulk characterization of the dissolved organic matter (DOM), by high resolution mass spectrometry (HRMS), to uncover the chemical fingerprint of selected manure-impacted groundwater bodies, used as sources for drinking water production. DOM is a combination of numerous substances, such as humic and fulvic acids, polysaccharides, proteins, lipids, among others. Its characterization is of interest since it can react with disinfectants during drinking water treatment and generate disinfection by-products that are potentially toxic to human health.
Several tasks were performed within RESOURCE to accomplish the project objectives. Target analytical methodologies and protocols were developed to analyze selected antibiotics and ARGs in both manure (mostly pig slurry) and groundwater. A streamlined protocol for DOM characterization in groundwater, and subsequent statistical analysis, was set-up. Comprehensive field monitoring studies were performed, collecting both groundwater and manure, in areas with intensive livestock production and where groundwater is also used as a drinking water source. Besides analyzing antibiotics and ARGs in manure, their reduction under different treatment technologies was also evaluated as a strategy to reduce potential environmental and human health risks.

Results showed a widespread occurrence of antibiotics widely used in animal husbandry in animal manure (i.e. pig slurry) at high concentrations (from high µg L-1 to even mg L-1). Some of these antibiotics were also present in groundwater samples, mostly at low concentrations (ng L-1). Nevertheless, in some cases, they even reached remarkable levels (µg L-1). Genes conferring resistance to the antibiotic classes detected were also found in both manure and groundwater, with values from 4×102 to 5×106 copy numbers/L water in groundwater. The reduction of antibiotics during manure treatment was moderate (from 40% up to even complete elimination for some compounds), suggesting that its treatment prior to field application is a suitable alternative to reduce environmental risks. DOM analysis revealed that most molecular formulas were CHO compounds, while a remarkable percentage contained nitrogen and sulphur (~15-20%, respectively) as well. The use of van Krevelen diagrams (plots of H/C versus O/C atomic ratios) showed that all our samples were dominated by molecules falling in the lignin-like region. Finally, we could also establish relationships between antibiotics and DOM composition. The analysis of groundwater samples along different seasons showed large variability in the compounds detected and their concentrations. This hinders the assessment of groundwater quality for public uses as well as the implementation of efficient monitoring strategies.
Project results were disseminated through different strategies, such as: (i) the publication of open-access journal articles (two papers are under preparation and will be submitted once completed); (ii) oral and poster contributions in national and international conferences; (iii) the organization of a workshop with stakeholders, farmers and end-users: (iv) the elaboration of an open-access technical guide about the reduction of antibiotics in manure treatment and to draft guidelines for a safe manure reuse; (v) disseminating the project activities and results at the institute’s annual newsletter, 10th anniversary conference “Water research in perspective: beyond 2020” and the EU Climate Change Initiative, all open to the general public and (vi) the presentation of a master thesis, among others.
RESOURCE contributed in filling in the existing knowledge gaps about the role of animal manure reuse as a major source of groundwater contamination by antibiotics and ARGs. Furthermore, it provided novel information about the DOM fingerprint, and its hydrogeological flow system, in manure-impacted groundwater bodies. The combination of chemical (target methods and DOM fingerprinting) and microbiological tools to assess groundwater quality was also a novel aspect of the project. The research conducted within RESOURCE had great social relevance at both national and international level. Spain is one of the major livestock producers in Europe. Due to this intensive livestock production, there is a large surplus of manure that is being applied in agricultural fields and this may lead to a degradation of groundwater resources. On the other hand, antibiotic resistance and its dissemination to the environment is a major global public health concern and a topic of broad interest for EU policy objectives. RESOURCE contributed to set light to these important environmental and human health issues. Results achieved allowed the identification of priority antibiotics to focus on in future monitoring studies and that should be included in future legislations as priority pollutants. They also highlighted the need to ensure safe manure management practices and stressed the need to reduce antibiotic usage in animal breeding to avoid any potential environmental and human health risks.
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