The spread of veterinary medicinal products (VMPs) and biocides via manure onto agriculturally used areas represents a very important emission into the environment for these product groups. Within this literature study, publicly available transformation studies with liquid manure are summarized. Transformation studies were evaluated regarding the transformation fate of tested substances, the origin and characteristics of used manure, the experimental setup, and the measured parameters. As main topics within the 42 evaluated transformation studies, the high dependency of transformation on temperature, redox potential, dry matter content, and other parameters is reported. Test duration throughout the studies ranged from 2 to 374 days and study temperature ranged from 5 to 55 °C. Only seven publications gave information on the redox potential of the manure. Further, the characterization of the matrix in many cases was inadequate due to missing parameters such as dry matter content or pH. Only three publications studied transformation of biocides. To allow for a consistent assessment of studies within the registration process, a harmonized internationally accepted and validated test method is needed. Additionally, monitoring data of VMPs in manure were collected from literature and evaluated regarding the origin and characteristics of the manure, the minimum/maximum found concentrations, and the percentage of identified compounds. Within the 27 evaluated publications, 1568 manure samples were analyzed and 39 different active substances for VMPs and 11 metabolites and transformation products of VMPs could be found in manure. Most often, the samples were analyzed for sulfonamides, tetracyclines, and fluoroquinolones. Not one study searched for biocides or worked with a non-target approach. For sulfadiazine and chlortetracycline, concentrations exceeding the predicted environmental concentrations were found.
Antibiotics and their metabolized products in human and animal waste are the major cause of antibiotic resistance in bacteria. Understanding what happens to drugs after their use could improve methods to remove antibiotics from rivers and soils and reduce the prevalence of resistance in bacterial populations in the environment.
The Fish Sexual Development Test (FSDT) is a non-reproductive test to assess adverse effects of endocrine disrupting chemicals. With the present study it was intended to evaluate whether gene expression endpoints would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDT according to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (test concentrations: 10μg/L, 32μg/L, 100μg/L) using zebrafish (Danio rerio). Gene expression analyses using quantitative RT-PCR were included at 48h, 96h, 28days and 63days post fertilization (hpf, dpf). The selection of genes aimed at finding molecular endpoints which could be directly linked to the adverse apical effects of aromatase inhibition. The most prominent effects of fadrozole exposure on the sexual development of zebrafish were a complete sex ratio shift towards males and an acceleration of gonad maturation already at low fadrozole concentrations (10μg/L). Due to the specific inhibition of the aromatase enzyme (Cyp19) by fadrozole and thus, the conversion of C19-androgens to C18-estrogens, the steroid hormone balance controlling the sex ratio of zebrafish was altered. The resulting key event is the regulation of directly estrogen-responsive genes. Subsequently, gene expression of vitellogenin 1 (vtg1) and of the aromatase cyp19a1b isoform (cyp19a1b), were down-regulated upon fadrozole treatment compared to controls. For example, mRNA levels of vtg1 were down-regulated compared to the controls as early as 48 hpf and 96 hpf. Further regulated genes cumulated in pathways suggested to be controlled by endocrine mechanisms, like the steroid and terpenoid synthesis pathway (e.g. mevalonate (diphospho) decarboxylase (mvd), lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase; lss), methylsterol monooxygenase 1 (sc4mol)) and in lipid transport/metabolic processes (steroidogenic acute regulatory protein (star), apolipoprotein Eb (apoEb)). Taken together, this study demonstrated that the existing Adverse Outcome Pathway (AOP) for aromatase inhibition in fish can be translated to the life-stage of sexual differentiation. We were further able to identify MoA-specific marker gene expression which can be instrumental in defining new measurable key events (KE) of existing or new AOPs related to endocrine disruption.
Antimicrobial resistance (AMR) is a threat to public and animal health on the global scale. The origin of the genes associated with resistance has long been unknown. Recently, there is a growing body of evidence demonstrating that environmental bacteria are resistant to a multitude of antibiotic substances and that this environmental reservoir of AMR is still growing. The analysis of the genomes of bacterial pathogens indicates that they have acquired their resistance profiles by incorporating different genetic elements through horizontal gene transfer. The ancestors of pathogenic bacteria, as well as the origin of resistance determinants, lay most likely in the environmental microbiota. Indeed, there is some evidence that at least some clinically relevant resistance genes have originated in environmental bacterial species. Thus, feasible measures are required to reduce the risks posed by AMR genes and resistant bacteria that occur in the environment. It has been shown that a concurrence of factors, such as high concentrations of antibiotics or heavy metals used as biocides and high bacterial densities, promote development and spread of antimicrobial resistance. For this purpose, it is essential to restrict the use of antibiotics for the treatment of livestock and humans to medical necessity, as well as to reduce the application of biocides and heavy metals in animal husbandry. Moreover, it is important to further develop sanitary measures at the interface between the environment and clinical settings or livestock farming.
Background: Spreading of manure on agricultural soils represents an important pathway by which veterinary medicinal and biocidal products enter the environment. To assess their environmental impact in the context of authorization processes, experimental testing of transformation of these compounds in manure is considered in regulatory guidance documents. However, there is no standardized experimental test method available so far. To fill this gap, an experimental test method was developed to examine transformation in liquid cattle and pig manure. Results: To account for manure specifics and for a possible influence of manure parameters on transformation rates, a comprehensive data acquisition and statistical analysis were performed. To address the variability of liquid manure of different type and origin, 30 manures were sampled and characterized. Thereof, three cattle and three pig manures were selected to perform anaerobic transformation studies with two radiolabeled veterinary medicinal active substances (14 C-salicylic acid and 14 C-paracetamol) and a confidential radiolabeled " 14 C-biocide B" serving as test compounds. Spatial and seasonal variability of manure parameters was quantified, and a sampling technique to obtain homogenous manure samples was developed. Transformation studies with six replicates per sampling point were conducted to examine the influence of test setup, of manure storage conditions, and of parameter variations between manure of the same species and of different species on half-lives, formation of extractable and non-extractable residues and mineralization rates. Finally, a test design was established that yields reproducible results for transformation studies in liquid cattle and pig manure under anaerobic conditions. Conclusions: Results give a reliable basis for an experimental test method to perform anaerobic transformation studies in liquid cattle and pig manure. The developed test method comprises detailed guidance on selection of sampling date and site, collection from manure tank, storage duration and temperature in the laboratory, duration of manure acclimation period, and the incubation system design. It proved to give reproducible results in a validation ring test performed in a follow-up project and is planned to be submitted as draft test guideline for approval by the OECD.
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