Contaminations of organic fertilizers with antibiotic residues, resistance genes, and mobile genetic elements mirroring antibiotic use in livestock?

Pig manures are frequently used as fertilizer or co-substrate in biogas plants (BGPs) and typically contain antibiotic residues (ARs), as well as bacteria carrying resistance genes (RGs) and mobile genetic elements (MGEs). A survey of manures from eight pig fattening and six pig breeding farms and digestates from eight BGPs in Lower Saxony, Germany was conducted to evaluate the link between antibiotic usage and ARs to RGs and MGEs present in organic fertilizers. In total, 11 different antibiotics belonging to six substance classes were applied in the farms investigated. Residue analysis revealed concentrations of tetracycline up to 300 mg kg-1 dry weight (DW) in manures and of doxycycline up to 10.1 mg kg-1 DW in digestates indicating incomplete removal during anaerobic digestion. RGs (sul1, sul2, tet(A), tet(M), tet(X), qacE∆1) were detected in total community DNA of all samples by PCR-Southern blot hybridization. Broad-host range plasmids (IncP-1, IncQ, IncN, and IncW) and integron integrase genes (intI1, intI2) were found in most manure samples with IncN and IncW plasmids being more abundant in manure from pig breeding compared to pig fattening farms. IntI1, IncQ, and IncW plasmids were also detected in all digestates, while IncP-1, IncN, and LowGC plasmids were detected only sporadically. Our findings strongly reinforce the need for further research to identify mitigation strategies to reduce the level of contamination of organic fertilizers with ARs and transferable RGs that are applied to soil and that might influence the mobile resistome of the plant microbiome.

From the application of antibiotics to antibiotic residues in liquid manures and digestates: A screening study in one European center of conventional pig husbandry.

In conventional pig husbandry, antibiotics are frequently applied. Together with excreta, antibiotic residues enter liquid manures finally used as organic soil fertilizers or input materials for biogas plants. Therefore, this first screening study was performed to survey the application patterns of antibiotics from fall 2011 until spring 2013. Manures and digestates were then analyzed for selected antibiotic residues from spring 2012 to 2013. The data analysis of veterinary drug application documents revealed the use of 34 different antibiotics belonging to 11 substance classes at 21 farms under study. Antibiotics, particularly tetracyclines, frequently administered to larger pig groups were detected in manure samples up to higher mg kg(-1) dry weight (DW) concentrations. Antibiotic residues in digestates, furthermore, show that a full removal capacity cannot be guaranteed through the anaerobic digestion process in biogas plants.

Acute Toxicity and Environmental Risks of Five Veterinary Pharmaceuticals for Aquatic Macroinvertebrates.

Due to the high use of antibiotics and antiparasitics for the treatment of livestock, there is concern about the potential impacts of the release of these compounds into freshwater ecosystems. In this context, the present study quantified the acute toxicity of two antibiotics (sulfadiazine and sulfadimidine), and three antiparasitic agents (flubendazole, fenbendazole, ivermectin) for nine freshwater invertebrate species. These experiments revealed a low degree of toxicity for the sulfonamide antibiotics, with limited implications in the survival of all test species at the highest test concentrations (50 and 100 mg/L). In contrast, all three antiparasitic agents indicated on the basis of their acute toxicity risks for the aquatic environment. Moreover, chronic toxicity data from the literature for antiparasitics, including effects on reproduction in daphnids, support the concern about the integrity of aquatic ecosystems posed by releases of these compounds. Thus, these pharmaceuticals warrant further careful consideration by environmental risk managers.

Comparison of different salt solutions for density separation of conventional and biodegradable microplastic from solid sample matrices.

Microplastics are the new emerging pollutants ubiquitously detectable in aquatic and terrestrial ecosystems. Fate and behavior, as well as ecotoxicity, are of increasing environmental concern, particularly in sediments and soils as natural sinks. For a global environmental risk assessment, reliable and easy to apply analytical methods are mandatory to obtain comparable data. This is based on the isolation of microplastics out of the solid sample matrices prior to instrumental detection. Thus, this study provides an easy to apply approach for density separation. The technique emerged from a comparative study using different salt solutions to isolate conventional, and for the first time biodegradable, microplastics from different solid sample matrices, i.e., sand, artificial soil, and compost. Four solutions (water, sodium chloride, sodium hexametaphosphate, and sodium bromide) of different densities were applied followed by oxidizing digestion. Finally, the impact of the procedures on size and surface properties of microplastics was tested. Dependent on the sample matrix, the highest recovery rates of 87.3-100.3% for conventional polymers, and 38.2-78.2% for biodegradable polymers, were determined with sodium bromide. It could be shown that the type of solid sample matrix influences the recovery rates and has to be considered when choosing a sample preparation technique.

Reclaimed water driven lettuce cultivation in a hydroponic system: the need of micropollutant removal by advanced wastewater treatment.

For a novel approach of resource-efficient water reuse, a municipal wastewater treatment plant was extended at pilot scale for advanced wastewater treatment, i.e., ozonation and biological activated carbon filtration, and a hydroponic system for reclaimed water driven lettuce cultivation. The treatment specific wastewater lines with the corresponding lettuce plants, differentiated into roots and shoots, were monitored for priority wastewater micropollutants, i.e., acesulfame (sweetener), caffeine (stimulant), carbamazepine, diclofenac, ibuprofen, sulfamethoxazole with acetyl-sulfamethoxazole (human pharmaceuticals), 1H-benzotriazole, and 4/5-methylbenzotriazole (industrial chemicals). As clearly demonstrated, conventional tertiary treatment could not efficiently clean up wastewater. Removal efficiencies ranged from 3% for carbamazepine to 100% for ibuprofen. The resulting pollution of the hydroponic water lines led to the accumulation of acesulfame, carbamazepine, and diclofenac in lettuce root systems at 32.0, 69.5, and 135 µg kg-1 and in the uptake of acesulfame and carbamazepine into lettuce shoots at 23.4 and 120 µg kg-1 dry weight, respectively. In contrast, both advanced treatment technologies when operating under optimized conditions achieved removal efficiencies of > 90% also for persistent micropollutants. Minimizing the pollution of reclaimed water thus met one relevant need for hydroponic lettuce cultivation.

Bulk soil and maize rhizosphere resistance genes, mobile genetic elements and microbial communities are differently impacted by organic and inorganic fertilization.

Organic soil fertilizers, such as livestock manure and biogas digestate, frequently contain bacteria carrying resistance genes (RGs) to antimicrobial substances and mobile genetic elements (MGEs). The effects of different fertilizers (inorganic, manure, digestate) on RG and MGE abundance and microbial community composition were investigated in a field plot experiment. The relative abundances of RGs [sul1, sul2, tet(A), tet(M), tet(Q), tet(W), qacEΔ1/qacE] and MGEs [intI1, intI2, IncP-1, IncP-1ε and LowGC plasmids] in total community (TC)-DNA from organic fertilizers, bulk soil and maize rhizosphere were quantified by qPCR before/after fertilization and prior to maize harvest. Microbial communities were analyzed via Illumina sequencing of 16S rRNA gene fragments amplified from TC-DNA. Compared to inorganic fertilization, manure treatments increased relative abundances of all RGs analyzed, integrons and few genera affiliated to Bacteroidetes and Firmicutes in bulk soil, while digestate increased sul2, tet(W) and intI2. At harvest, treatment effects vanished in bulk soil. However, organic fertilizer effects were still detectable in the rhizosphere for RGs [manure: intI1, sul1; digestate: tet(W)] and Clostridium related sequences (digestates) with increased relative abundance. Our data indicated transient organic fertilizer effects on RGs, MGEs and microbial community composition in bulk soil with long-term history of digestate or manure application.

Soil texture-depending effects of doxycycline and streptomycin applied with manure on the bacterial community composition and resistome.

Veterinary antibiotics, bacteria carrying antibiotic resistance determinants located on mobile genetic elements and nutrients are spread on agricultural soil using manure as fertilizer. However, systematic quantitative studies linking antibiotic concentrations and antimicrobial resistance genes (ARGs) in manure and the environment are scarce but needed to assess environmental risks. In this microcosm study, a sandy and a loamy soil were mixed with manure spiked with streptomycin or doxycycline at five concentrations. Total-community DNA was extracted on days 28 and 92, and the abundances of ARGs (aadA, strA, tet(A), tet(M), tet(W), tet(Q), sul1, qacE/qacEΔ1) and class 1 and 2 integron integrase genes (intI1 and intI2) were determined by qPCR relative to 16S rRNA genes. Effects on the bacterial community composition were evaluated by denaturing gradient gel electrophoresis of 16S rRNA gene amplicons. Manure application to the soils strongly increased the relative abundance of most tested genes. Antibiotics caused further enrichments which decreased over time and were mostly seen at high concentrations. Strikingly, the effects on relative gene abundances and soil bacterial community composition were more pronounced in sandy soil. The concept of defining antibiotic threshold concentrations for environmental risk assessments remains challenging due to the various influencing factors.

Chemical and biological characterization of non-extractable sulfonamide residues in soil.

For sulfonamides, the formation of non-extractable residues has been identified by laboratory testing as the most relevant concentration determining process in manured soil. Therefore, the present study has been focused on the chemical and biological characterization of non-extractable residues of (14)C-labeled sulfadiazine or sulfamethoxazole. In laboratory batch experiments, the test substances were spiked via standard solution or test slurry to microbially active soil samples. After incubation periods of up to 102d, a sequential extraction technique was applied. Despite the exhaustive extraction procedure, sulfadiazine residues mainly remained non-extractable, indicating the high affinity to the soil matrix. The remobilization of non-extractable (14)C-sulfadiazine residues was monitored in the activated sludge test and the Brassica rapa test. Only small amounts (<3%) were transferred into the extractable fractions and 0.1% was taken up by the plants. In the Lumbricus terrestris test A, the release of non-extractable (14)C-sulfamethoxazole residues by the burrowing activity of the earthworms was investigated. The residues mainly remained non-extractable (96%). The L. terrestris test B was designed to study the immobilization of (14)C-sulfamethoxazole in soil directly after the test slurry application. The mean uptake by earthworms was 1%. Extractable and non-extractable residues amounted to 5% and 93%, respectively. Consequently, the results of all tests confirmed the high affinity of the non-extractable sulfonamide residues to the soil matrix.

Full-scale mesophilic biogas plants using manure as C-source: bacterial community shifts along the process cause changes in the abundance of resistance genes and mobile genetic elements.

The application of manure, typically harboring bacteria carrying resistance genes (RGs) and mobile genetic elements (MGEs), as co-substrate in biogas plants (BGPs) might be critical when digestates are used as fertilizers. In the present study, the relative abundance of RGs and MGEs in total community (TC-) DNA from manure, fermenters and digestate samples taken at eight full-scale BGPs co-fermenting manure were determined by real-time PCR. In addition, the bacterial community composition of all digestates as well as manure and fermenter material from one BGP (BGP3) was characterized by 454-pyrosequencing of 16S rRNA amplicons from TC-DNA. Compared to respective input manures, relative abundances determined for sul1, sul2, tet(M), tet(Q), intI1, qacEΔ1, korB and traN were significantly lower in fermenters, whereas relative abundances of tet(W) were often higher in fermenters. The bacterial communities in all digestates were dominated by Firmicutes and Bacteroidetes while Proteobacteria were low in abundance and no Enterobacteriaceae were detected. High-throughput sequencing revealed shifts in bacterial communities during treatment for BGP3. Although in comparison to manure, digestate bacteria had lower relative abundances of RGs and MGEs except for tet(W), mesophilic BGPs seem not to be effective for prevention of the spread of RGs and MGEs via digestates into arable soils.

Laboratory testing on the removal of the veterinary antibiotic doxycycline during long-term liquid pig manure and digestate storage.

The veterinary antibiotic doxycycline (DOXY) is today frequently applied in conventional pig husbandry for the control of respiratory diseases. After the treatment, pigs excrete major amounts of DOXY as the unchanged active substance. Thus, DOXY residues were found in liquid manures and digestates of biogas plants at concentrations of mg kg(-1) dry weight. In order to assess the impact of field applications of contaminated manures and digestates on the entry of DOXY residues into arable and grassland soils, thorough information about the removal of DOXY during long-term storage of farm fertilizers is required. Since this aspect has been only less investigated for manures but not for digestates, first long-term storage simulation tests were performed at laboratory scale. Within the 170-d incubation periods under strictly anaerobic conditions, doxycycline was removed in liquid pig manure by 61% and in digestate by 76%. The calculated half-lives of 120 d and 91 d thus emphasized the persistence of doxycycline in both matrices. Due to the substance specific properties of DOXY, this removal was caused neither by mineralization, epimerization nor biotransformation. According to the high affinity of DOXY to manure and digestate solids, however, the formation of non-extractable residues has to be taken into account as the predominant concentration determining process. This was indicated by the sequential extraction procedure applied. Hence, these results confirmed that a full removal capacity for doxycycline cannot be reached through the long-term storage of farm fertilizers.

Investigations on the fate of sulfadiazine in manured soil: laboratory experiments and test plot studies.

The fate of 14C-labeled sulfadiazine (SDZ) in manured soil has been investigated in laboratory test systems. In the first approach, stability of 14C-SDZ in liquid bovine manure has been tested. Only 1% of the initially applied radiotracer was mineralized to 14C-carbon dioxide and 82% were transferred to nonextractable residues within a 102-d incubation period. Test slurries with defined aged residues were prepared and, supplementary to standard solutions, applied to silty-clay soil samples. These tests showed the high affinity of 14C-SDZ residues to the soil matrix. In the second approach, basic data on microbial, chemical, and photoinduced degradability in soil were gathered. The data indicated the formation of nonextractable residues as the predominant process in soil, which was accelerated by the test slurry application. In the third approach, laboratory lysimeter tests were conducted to investigate leaching and degradation as simultaneously occurring processes. The 14C-SDZ residues (64%) mainly were retained in the surface layer as nonextractable residues. Although a high mobility in soil was revealed by a soil/water distribution coefficient of 2 L kg(-1), percolate contamination amounted to only 3% of the initially applied 14C-SDZ. The tendencies of leaching and degradability in soil also were observed in test plot studies under field conditions.

Test-plot studies on runoff of sulfonamides from manured soils after sprinkler irrigation.

Three test-plot series have been performed to gather information on runoff of sulfonamides from manured arable and grassland after sprinkler irrigation. To prepare test slurries with defined aged residues, liquid bovine manure was fortified with sulfadiazine, sulfadimidine, and sulfamethoxazole and stored short-term. After test-slurry application, the arable land was treated by soil cultivation before irrigation, and the manured grassland was irrigated directly with 50 mm h(-1) for 2 h. The runoff suspensions were sampled at 5- to 10-min intervals, separated into aqueous phase and suspended matter and residue analyzed. Higher runoff emissions were found from manured grassland plots. The discharge volumes ranged from 106 to 252 L and the total runoff emissions ranged from 13 to 28% of sulfonamides applied initially. Within the first 20 min of the irrigation period that represented a rainfall of 17 mm, emissions, on average, were 4%. The loads of sulfonamides predominantly occurred in the runoff water. The only emissions via suspended matter, on average, were 0.02%. On arable land, however, the runoff was reduced by soil cultivation. Discharge volumes and sulfonamide emissions were 36 to 128 L and 0.1 to 2.5%, respectively. Despite the high-intensity sprinkler irrigation, major emissions did not occur until a 60-min delay.

Transferable antibiotic resistance plasmids from biogas plant digestates often belong to the IncP-1ε subgroup.

Manure is known to contain residues of antibiotics administered to farm animals as well as bacteria carrying antibiotic resistance genes (ARGs). These genes are often located on mobile genetic elements. In biogas plants (BGPs), organic substrates such as manure and plant material are mixed and fermented in order to provide energy, and resulting digestates are used for soil fertilization. The fate of plasmid carrying bacteria from manure during the fermentation process is unknown. The present study focused on transferable antibiotic resistance plasmids from digestates of seven BGPs, using manure as a co-substrate, and their phenotypic and genotypic characterization. Plasmids conferring resistance to either tetracycline or sulfadiazine were captured by means of exogenous plasmid isolation from digestates into Pseudomonas putida KT2442 and Escherichia coli CV601 recipients, at transfer frequencies ranging from 10(-5) to 10(-7). Transconjugants (n = 101) were screened by PCR-Southern blot hybridization and real-time PCR for the presence of IncP-1, IncP-1ε, IncW, IncN, IncP-7, IncP-9, LowGC, and IncQ plasmids. While 61 plasmids remained unassigned, 40 plasmids belonged to the IncP-1ε subgroup. All these IncP-1ε plasmids were shown to harbor the genes tet(A), sul1, qacEΔ1, intI1, and integron gene cassette amplicons of different size. Further analysis of 16 representative IncP-1ε plasmids showed that they conferred six different multiple antibiotic resistance patterns and their diversity seemed to be driven by the gene cassette arrays. IncP-1ε plasmids displaying similar restriction and antibiotic resistance patterns were captured from different BGPs, suggesting that they may be typical of this environment. Our study showed that BGP digestates are a potential source of transferable antibiotic resistance plasmids, and in particular the broad host range IncP-1ε plasmids might contribute to the spread of ARGs when digestates are used as fertilizer.

Laboratory tests on sorption and transformation of the insecticide flubendiamide in Japanese tea field soil.

Flubendiamide belongs to the modern insecticides applied in Japanese tea cultivation to control smaller tea tortrix and tea leaf roller. Since fate and behavior in soil have been only monitored sparsely and fragmentarily until today, laboratory tests were performed on sorption, leaching, biotransformation and photo-induced biotransformation of flubendiamide in two different soils. In batch equilibrium tests, K(d) and K(OC) values were 15 and 298 L kg(-1) for the Japanese tea field soil as well as 16 and 1610 L kg(-1) for the German arable field soil classifying flubendiamide to be moderately mobile and slightly mobile, respectively. The affinity to the tea field soil was additionally confirmed by soil column tests where flubendiamide was predominantly retarded in the topsoil layers resulting in a percolate contamination of only 0.002 mg L(-1). In the aerobic biotransformation tests, flubendiamide did not substantially disappear within the 122-d incubation period. Due to DT(50)>122 d, flubendiamide was assessed very persistent. Supplementary, photo-induced impacts on biotransformation were studied in a special laboratory irradiation system. Despite a 14-d irradiation period, photo-induced biotransformation in the tea field soil was not identifiable, neither by HPLC/DAD nor by LC/MS/MS. 3-d irradiation tests in photosensibilizing acetone, however, showed that the primary photo-transformation product desiodo-flubendiamide was formed. How far this photochemical reaction may also occur in soil of perennial tea plant stands, however, has to be checked in field studies.

Process dominance analysis for fate modeling of flubendazole and fenbendazole in liquid manure and manured soil.

Fate monitoring data on anaerobic transformation of the benzimidazole anthelmintics flubendazole (FLU) and fenbendazole (FEN) in liquid pig manure and aerobic transformation and sorption in soil and manured soil under laboratory conditions were used for corresponding fate modeling. Processes considered were reversible and irreversible sequestration, mineralization, and metabolization, from which a set of up to 50 different models, both nested and concurrent, was assembled. Five selection criteria served for model selection after parameter fitting: the coefficient of determination, modeling efficiency, a likelihood ratio test, an information criterion, and a determinability measure. From the set of models selected, processes were classified as essential or sufficient. This strategy to identify process dominance was corroborated through application to data from analogous experiments for sulfadiazine and a comparison with established fate models for this substance. For both, FLU and FEN, model selection performance was fine, including indication of weak data support where observed. For FLU reversible and irreversible sequestration in a nonextractable fraction was determined. In particular, both the extractable and the nonextractable fraction were equally sufficient sources for irreversible sequestration. For FEN generally reversible formation of the extractable sulfoxide metabolite and reversible sequestration of both the parent and the metabolite were dominant. Similar to FLU, irreversible sequestration in the nonextractable fraction was determined for which both the extractable or the nonextractable fraction were equally sufficient sources. Formation of the sulfone metabolite was determined as irreversible, originating from the first metabolite.

Development of a novel concept for fate monitoring of biocides in liquid manure and manured soil taking 14C-imazalil as an example.

Biocides are frequently applied in animal houses for veterinary hygiene or pest control. Thus, they may reach liquid manure tanks. Biocides that are not transformed during manure storage enter soil by the application of manure as organic fertilizer. Due to this environmentally relevant entry route, biocidal substances and products undergo a regulatory fate monitoring in liquid manure and soil. According to this, a novel concept was developed investigating the biocide imazalil as an example. For this purpose, excrements of test animals individually kept in experimental animal houses under standard nutrition were sampled. After matrix characterization, bovine and pig reference manures of defined dry substance contents were prepared. They were used for long-term transformation tests of (14)C-imazalil under strictly anaerobic conditions typical for manure storage in tanks. During the 177-d incubation period, however, imazalil was not substantially transformed. Furthermore, test manures with 7-d aged (14)C-imazalil residues were applied to study aerobic transformation and sorption in manured soil. Both concentration determining processes in soil were affected by the manure matrices. Comparing disappearance times (DT(50)) and sorption coefficients (K(OC)) after standard application (DT(50): 83 d; K(OC): 4059 L kg(-1)), (14)C-imazalil disappeared more rapidly after test manure application. DT(50) values were 29 or 48 d depending on whether bovine or pig test manure was applied. Mobility was slightly enhanced revealed by K(OC) of 1852 and 1385 L kg(-1), respectively.