Prevalence of veterinary antibiotics in natural and organic fertilizers from animal food production and assessment of their potential ecological risk.

BACKGROUND: Veterinary antibiotics are emerging contaminants and enter into soil principally by agricultural application of organic fertilizers. This article presents the results of the research obtained for the analyzed 70 samples of fertilizers (pig and poultry manure and slurry and digestate) for various classes of antibiotics. RESULTS: Doxycycline, oxytetracycline, tetracycline, lincomycin, tiamulin and enrofloxacin were found in tested samples. Doxycycline was found as a dominant compound, and its highest concentration was 175 mg/kg in pig manure. This investigation indicated that fertilization with manure, especially animal feces, might be the primary source of antibiotics. Additionally, a risk assessment based on a risk quotient was carried out, which showed that the determined concentrations of antibiotics in fertilizers may pose a threat to soil microorganisms. CONCLUSIONS: Results suggested that the ecological risk effects of antibiotic contamination on soil bases and their potential adverse risk on human health needs special attention. © 2023 Society of Chemical Industry.

Effect of a recent parenteral dexamethasone and ketoprofen administration on the immunological diagnosis of tuberculosis in goats.

Caprine tuberculosis (TB) is a zoonosis caused by members of the Mycobacterium tuberculosis complex (MTBC). Caprine TB eradication programmes are based mainly on intradermal tuberculin tests and slaughterhouse surveillance. Different factors may affect the performance of the TB diagnostic tests used in caprine herds and, therefore, their ability to detect infected animals. The present study evaluates the effect of the fraudulent administration of two anti-inflammatory substances, dexamethasone and ketoprofen, on the performance of the TB diagnostic techniques used in goats, as well as the suitability of high performance liquid chromatography (HPLC) for their detection in hair samples. The animals (n = 90) were distributed in three groups: (1) a group treated with dexamethasone (n = 30); a second group treated with ketoprofen (n = 30); and a third non-treated control group (n = 30). Both dexamethasone and ketoprofen groups were subjected to intramuscular inoculation with the substances 48 h after the administration of bovine and avian purified protein derivatives (PPDs), that is, 24 h before the tests were interpreted. All the animals were subjected to the single and comparative intradermal tuberculin (SIT and CIT, respectively) tests, interferon-gamma release assay (IGRA) and P22 ELISA. The number of SIT test reactors was significantly lower in the dexamethasone (p = 0.001) and ketoprofen (p < 0.001) groups 72 h after the bovine PPD inoculation compared with the control group. A significantly higher number of positive reactors to IGRA was detected within the dexamethasone group (p = 0.016) 72 h after PPD administration compared to the control group. Dexamethasone and ketoprofen detection in either hair or serum samples was challenging when using HPLC since these substances were not detected in animals whose skin fold thickness (SFT) was reduced, what could be an issue if they are used for fraudulent purposes. In conclusion, the parenteral administration of dexamethasone or ketoprofen 48 h after the PPDs administration can significantly reduce the increase in SFT (mm) and subsequently the number of positive reactors to SIT test.

Development and Validation of Multi-Residue Method for Drugs Analysis in Human Feces by Liquid Chromatography-Tandem Mass Spectrometry.

The use of veterinary drugs in animal production is a common practice to secure animal and human health. However, residues of administrated drugs could be present in animal food products. Levels of drugs in food of animal origin are regulated within the European Union. In recent years, residues have been detected not only in food, but also in the environmental elements such as water or soil, meaning that humans are involuntarily exposed to these substances. This article presents a multiclass method for the analysis of various therapeutic groups of pharmaceuticals in human feces. Pharmaceuticals are extracted from feces with an acid extraction solvent, and after filtration the extract was analyzed by HPLC-MS/MS. A limit of detection of 10 ng/g was achieved for 9 pharmaceuticals, with linearity over 0.99 and repeatability and reproducibility lower than 20%. The method was satisfactorily applied in 25 feces samples of individuals that had declared not to be under medical treatment for the last two months. Results indicate the presence of six different compounds at concentration between 10 and 456 ng/g. This preliminary study showed the involuntary exposure of human gut microbiota to active substances such as pharmaceuticals.

Identification and Quantification of 29 Active Substances by HPLC-ESI-MS/MS in Lyophilized Swine Manure Samples.

Veterinary drugs are frequently employed to treat and prevent diseases in food-producing animals to improve animal health and to avoid the introduction of microorganisms into the food chain. The analysis of the presence of pharmaceutical residues in animal manure could help to evaluate the legal and illegal practices during food production without harming the animals and to correctly manage manure when it is going to be applied as a fertilizer. This article describes a method for the simultaneous analysis of 29 active substances, mostly antibiotics and antiparasitic agents. Substances were extracted from lyophilized manure with a methanol:McIlvaine solution and analyzed with HPLC-ESI-MS/MS and a C18 HPLC column. The method was validated following European guidelines, the achieved trueness was between 63 and 128% (depending on the analytes), and the linearity was between 100 and 1500 µg/kg. The applicability of the method was demonstrated in 40 manure samples collected from pig farms where tetracycline was quantified in 7.5% of the samples. These results show the viability of this non-invasive method for the control of the legal and illegal administration of pharmaceuticals in food-producing animals.

Determination of pharmaceuticals and heavy metals in groundwater for human and animal consumption and crop irrigation in Galicia.

Pharmaceuticals and heavy metals are contaminants present in groundwaters, which are the main source of drinking water in most parts of the world. In the northwest region of Spain, Galicia, groundwater harvesting is a common practice for drinking water supply, crop irrigation, cattle watering, as well as recreational use such as filling pools. In order to assess the quality of Galician groundwaters, the presence of 21 pharmaceuticals and 10 heavy metals was analysed by UPLC-MS/MS and ICP/MS methods, respectively, in a total of 118 groundwater samples from private wells. Seventeen of the 21 compounds studied were detected in 28% of the samples, with the highest presence of pharmaceuticals belonging to the antimicrobial group (52%), specifically the sulphonamides group in a range of concentration between 21 and 14.9 ng/L. In addition, 30% of the samples contained at least one heavy metal (Mn, As and Fe) above the legally permitted levels. Evaluation of the risk associated with the consumption of the analysed groundwater indicated no human risk for any of the detected pharmaceuticals but high cancer risk for children due to Cd, Cr and As concentrations was observe.

Quantification of Veterinary Antibiotics in Pig and Poultry Feces and Liquid Manure as a Non-Invasive Method to Monitor Antibiotic Usage in Livestock by Liquid Chromatography Mass-Spectrometry.

Antibiotics are active substances frequently used to treat and prevent diseases in animal husbandry, especially in swine and poultry farms. The use of manure as a fertilizer may lead to the dispersion of antibiotic residue into the environment and consequently the development of antibiotic-resistant bacteria. Most pharmaceutical active ingredients are excreted after administration, in some cases up to 90% of the consumed dose can be found in the feces and/or urine as parent compound. Therefore, due to antibiotic metabolism their residues can be easily detected in manure. This article describes a method for simultaneous analysis of ciprofloxacin, chlortetracycline, doxycycline, enrofloxacin, lincomycin, oxytetracycline, tetracycline, tiamulin, trimethoprim and tylosin in feces, liquid manure and digestate. Antibiotics were extracted from the different matrices with McIlvaine-Na2EDTA buffer solution and the extract was purified by the use two techniques: d-SPE and SPE (Strata-X-CW cartridges) and final eluent was analyzed by LC-MS and LC-MS/MS. The European Commission Decision 2002/657/EC was followed to conduct the validation of the method. Recoveries obtained from spiked pig and poultry feces and liquid manures samples ranged from 63% to 93% depending on analytes. The analysis of 70 samples (feces, liquid manure and digestate) revealed that 18 samples were positive for the presence of doxycycline, oxytetracycline, tetracycline, chlortetracycline, enrofloxacin, tiamulin and lincomycin. The results obtained in the presented study demonstrated that animal feces can be used as a non-invasive method detection antibiotic usage in animal production.

Influence of the Intestinal Microbiota on Diabetes Management.

In recent decades, there has been a very rapid increase in the prevalence of diabetes globally, with serious health and economic implications. Although today there are several therapeutic treatments for this disease, these do not address the causes of the disease and have serious side effects, so it is necessary to seek new treatments to replace or complement the existing ones. Among these complementary treatments, a strong link between the intestinal microbiota and diabetes has been demonstrated, which has focused attention on the use of biotherapy to regulate the function of the intestinal microbiota and, thus, treat diabetes. In this way, the main objective of this work is to provide a review of the latest scientific evidence on diabetes, gathering information about new trends in its management, and especially, the influence of the intestinal microbiota and microbiome on this pathology. It is possible to conclude that the relationship between the intestinal microbiota and diabetes is carried out through alterations in energy metabolism, the immune system, changes in intestinal permeability, and a state of low-intensity systemic inflammation. Although, currently, most of the experimental work, using probiotics for diabetes management, has been done on experimental animals, the results obtained are promising. Thus, the modification of the microbiota through biotherapy has shown to improve the symptoms and severity of diabetes through various mechanisms related to these alterations.

Determination of Florfenicol, Thiamfenicol and Chloramfenicol at Trace Levels in Animal Feed by HPLC⁻MS/MS.

Administration of florfenicol and thiamfenicol through medicated feed is permitted within the European Union, always following veterinary prescription and respecting the withdrawal periods. However, the presence of low levels of florfenicol, thiamfenicol, and chloramfenicol in non-target feed is prohibited. Since cross-contamination can occur during the production of medicated feed and according to Annex II of the European Regulation 2019/4/EC, the control of residue levels of florfenicol and thiamfenicol in non-target feed should be monitored and avoided. Based on all the above, a sensitive and reliable method using liquid chromatography tandem mass spectrometry was developed for the simultaneous detection of chloramfenicol, florfenicol, and thiamfenicol at trace levels in animal feed. Analytes were extracted from minced feed with ethyl acetate. Then, the ethyl acetate was evaporated, the residue was resuspended in Milli-Q water and the extract filtered. The method was in-house validated at carryover levels, with concentration ranging from 100 to 1000 µg/kg. The validation was conducted following the European Commission Decision 2002/657/EC and all performance characteristics were successfully satisfied. The capability of the method to detect amfenicols at lower levels than any prior perspective regulation literature guarantees its applicability in official control activities. The developed method has been applied to non-compliant feed samples with satisfactory results.

Simultaneous analysis of coccidiostats and sulphonamides in non-target feed by HPLC-MS/MS and validation following the Commission Decision 2002/657/EC.

Taking into consideration the maximum level (ML) for coccidiostats included in the European Regulation 574/2011 and the fact that the presence of residues of sulphonamides in non-target feed is forbidden, the aim of this article is to present an analytical method based on HPLC-MS/MS for the identification and quantification of sulphonamides and coccidiostats in non-target feeds. The method was validated following Commission Decision 2002/657/EC, and recovery, repeatability and reproducibility were within the limits established in the Decision. For coccidiostats, the decision limit and detection capability were calculated for the different species taking into account the ML allowed in Regulation 574/2011. The applicability of the method was investigated in 50 feed samples collected from dairy farms, 50 obtained from feed mills and 10 interlaboratory feed samples.

Development and validation of an LC-MS/MS method for the quantification of tiamulin, trimethoprim, tylosin, sulfadiazine and sulfamethazine in medicated feed.

A new multi-compound method for the analysis of veterinary drugs, namely tiamulin, trimethoprim, tylosin, sulfadiazine and sulfamethazine was developed and validated in medicated feeds. After extraction, the samples were centrifuged, diluted in Milli-Q water, filtered and analysed by high performance liquid chromatography coupled to tandem mass spectrometry. The separation of the analytes was performed on a biphenyl column with a gradient of 0.1% formic acid in acetonitrile and 0.1% formic acid in Milli-Q water. Quantitative validation was done in accordance with the guidelines laid down in European Commission Decision 2002/657/EC. Method performances were evaluated by the following parameters: linearity (R2 < 0.99), precision (repeatability <14% and within-laboratory reproducibility <24%), recovery (73.58-115.21%), sensitivity, limit of detection (LOD), limit of quantification (LOQ), selectivity and expanded measurement uncertainty (k. = 2). The validated method was successfully applied to the 2 medicated feeds obtained from the interlaboratory studies and feed manufactures from Spain in August 2017. In these samples, tiamulin, tylosin and sulfamethazine were detected at the concentration levels declared by the manufacturers. The developed method can therefore be successfully used to routinely control the content and homogeneity of these antibacterial substances in medicated feed. Abbreviations AAFCO - Association of American Feed Control Officials; TYL - tylosin; TIAM - tiamulin fumarate; TRIM - trimethoprim; SDZ - sulfadiazine; SMZ - sulfamethazine; UV - ultraviolet detector; FLD - fluorescence detector; HPLC - high performance liquid chromatography; MS/MS - tandem mass spectrometry; LOD - limit of detection; LOQ - limit of quantification; CV - coefficient of variation; SD - standard deviation; U - uncertainty.

Influence of food consumption patterns and Galician lifestyle on human gut microbiota.

The proportion of different microbial populations in the human gut is an important factor that in recent years has been linked to obesity and numerous metabolic diseases. Because there are many factors that can affect the composition of human gut microbiota, it is of interest to have information about what is the composition of the gut microbiota in different populations in order to better understand the possibilities for improving nutritional management. A group of 31 volunteers were selected according to established inclusion and exclusion criteria and were asked about their diet history, lifestyle patterns, and adherence to the Southern European Atlantic Diet. Fecal samples were taken and subsequently analyzed by real-time PCR. The results indicated different dietary patterns for subjects who consumed a higher amount of fruits, vegetables, legumes, and fish and a lower amount of bakery foods and precooked foods and snacks compared to Spanish consumption data. Most participants showed intermediate or high adherence to Southern European Atlantic Diet, and an analysis of gut microbiota showed high numbers of total bacteria and Actinobacteria, as well as high amounts of bacteria belonging to the genera Lactobacillus spp. and Bifidobacterium spp. A subsequent statistical comparison also revealed differences in gut microbiota depending on the subject's body weight, age, or degree of adherence to the Southern European Atlantic Diet.

Food additives, contaminants and other minor components: effects on human gut microbiota-a review.

Gut bacteria play an important role in several metabolic processes and human diseases, such as obesity and accompanying co-morbidities, such as fatty liver disease, insulin resistance/diabetes, and cardiovascular events. Among other factors, dietary patterns, probiotics, prebiotics, synbiotics, antibiotics, and non-dietary factors, such as stress, age, exercise, and climatic conditions, can dramatically impact the human gut microbiota equilibrium and diversity. However, the effect of minor food constituents, including food additives and trace contaminants, on human gut microbiota has received less attention. Consequently, the present review aimed to provide an objective perspective of the current knowledge regarding the impacts of minor food constituents on human gut microbiota and consequently, on human health.

Development and validation of multi-residue and multi-class method for antibacterial substances analysis in non-target feed by liquid chromatography - tandem mass spectrometry.

A confirmatory HPLC-MS/MS method for the determination of residues of 11 antibacterial substances from different therapeutic class (ß-lactams, lincosamides, fluoroquinolones, macrolides, pleuromutilins and sulfonamides) in animal feeds has been developed. The sample preparation is based on extraction with 0.1% formic acid in acetonitrile. Separation of the analytes was performed on biphenyl column with a gradient of 0.1% formic acid in acetonitrile and 0.1% formic acid in Milli-Q water. The developed method was validated following the guidelines included in the European Union Commission Decision 2002/657/EC. Limits of detection ranging from 79.22 to 193.60 µg/kg; instrumental and analytical linearity coefficients were above 0.99 for matrix-match calibration; and relative recoveries ranging from 76.04% to 117.39%. Repeatability of the method was in the range of 2.41-19.76% (CV, %), whereas reproducibility ranged from 6.52 to 28.40% (CV %). The method shown to be efficient and precise for quantification of the 11 antibacterial substances in animal feed. The results demonstrate the feasibility of the method for routine use to monitor these substances in feed. The validated method was successfully applied to eight suspect feed samples collected from the Association of American Feed Control Officials (AFFCO) and feed manufactures from Galicia (Spain) in June and July 2017. Of these 8 non-target feeds, 5 were positive for the presence of tiamulin, tylosin and sulfadiazine.

Identification and quantification of 12 pharmaceuticals in water collected from milking parlors: Food safety implications.

The introduction of drug residues into the food chain and their presence in drinking water has been recently investigated. The aim of this work was to monitor the presence of 19 active drugs in water samples collected from milking parlors of dairy farms located in Galicia (northwest Spain), one of the main Spanish milking areas. Overall, 65% of the samples tested positive for at least one of the compounds analyzed. A total of 12 drugs were measured, with concentrations ranging between 17 and 3,941 ng/L. Considering that a mixture of compounds may contribute to the overall effect of each compound and might increase or reduce its toxicity, it should be noted that 29% of the samples tested contained more than one pharmaceutical. To date, the effects of the continuous consumption of these mixtures of drugs in water or milk are unknown; however, antimicrobials may affect the human gut microbiota or have toxic effects in sensitive individuals.

A Confirmatory Method Based on HPLC-MS/MS for the Detection and Quantification of Residue of Tetracyclines in Nonmedicated Feed.

The Commission Regulation 574/2011/EC set up maximum levels of coccidiostats and histomonostats in nonmedicated feed as a consequence of carry-over during manufacturing. Carry-over takes place from medicated to nonmedicated feed during feed production. Similar contamination could also occur for other pharmaceuticals such as tetracyclines, a group of antibiotics commonly employed in food production animal. The objective of this work is to present a simple and fast method for the simultaneous detection of four tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline) in nontarget feed at a µg/kg level. Validation of the method was performed according to the guideline included in the Commission Decision 2002/657/EC for official method. The validated method was successfully applied to 50 feed samples collected from different milk farms and 25 samples obtained from feed manufacturers. While oxytetracycline was the tetracycline most frequently detected, chlortetracycline was the analyte measured at the highest concentration 15.14 mg/Kg. From 75 nonmedicated feed analysed 15% resulted to be positive for the presence of one tetracycline.

Environmental concentrations of prednisolone alter visually mediated responses during early life stages of zebrafish (Danio rerio).

The development of the eye in vertebrates is dependent upon glucocorticoid signalling, however, specific components of the eye are sensitive to synthetic glucocorticoids. The presence of synthetic glucocorticoids within the aquatic environment may therefore have important consequences for fish, which are heavily reliant upon vision for mediating several key behaviours. The potential ethological impact of synthetic glucocorticoid oculotoxicity however has yet to be studied. Physiological and behavioural responses which are dependent upon vision were selected to investigate the possible toxicity of prednisolone, a commonly occurring synthetic glucocorticoid within the environment, during early life stages of zebrafish. Although exposure to prednisolone did not alter the morphology of the external eye, aggregation of melanin within the skin in response to increasing light levels was impeded and embryos exposed to prednisolone (10 µg/l) maintained a darkened phenotype. Exposure to prednisolone also increased the preference of embryos for a dark environment within a light dark box test in a concentration dependent manner. However the ability of embryos to detect motion appeared unaffected by prednisolone. Therefore, while significant effects were detected in several processes mediated by vision, changes occurred in a manner which suggest that vision was in itself unaffected by prednisolone. Neurological and endocrinological changes during early ontogeny are considered as likely candidates for future investigation.

Physiological and Behavioral Effects of Exposure to Environmentally Relevant Concentrations of Prednisolone During Zebrafish (Danio rerio) Embryogenesis.

The presence of synthetic glucocorticoids within the aquatic environment has been highlighted as a potential environmental concern as they may mimic the role of endogenous glucocorticoids during vertebrate ontogeny. Prednisolone is a commonly prescribed synthetic glucocorticoid which has been repeatedly detected in the environment. This study investigated the impact of environmentally relevant concentrations of prednisolone (0.1, 1, and 10 µg/L) during zebrafish embryogenesis using physiological and behavioral end points which are known to be mediated by endogenous glucocorticoids. The frequency of spontaneous muscle contractions (24 hpf) was significantly reduced by prednisolone and 0.1 µg/L increased the distance embryos swam in response to a mechanosensory stimulus (48 hpf). The percentage of embryos hatched significantly increased following prednisolone treatment (1 and 10 µg/L), while growth and mortality were unaffected. The onset of heart contraction was differentially affected by prednisolone while heart rate and oxygen consumption both increased significantly throughout embryogenesis. No substantial effect on the axial musculature was observed. Morphological changes to the lower jaw were detected at 96 hpf in response to 1 µg/L of prednisolone. Several parameters of swim behavior were also significantly affected. Environmentally relevant concentrations of prednisolone therefore alter early zebrafish ontogeny and significantly affect embryo behavior.

A study on toxic and essential elements in wheat grain from the Republic of Kazakhstan.

Little information is currently available about the content of different elements in wheat samples from the Republic of Kazakhstan. The concentrations of toxic (As, Cd, Cr, Hg, Pb, and U) and essential (Co, Cu, Fe, Mn, Ni, Se, and Zn) elements in 117 sampled wheat grains from the Republic of Kazakhstan were measured. The results indicated that the mean and maximum concentrations of most investigated elements (As, Cd, Co, Cr, Mn, Se, Pb, and U) were higher in samples collected from southern Kazakhstan. The mean and maximum concentrations of toxic elements such as As, Cd, Hg, and Pb did not exceed levels specified by European, FAO, or Kazakh legislation, although the hazard quotient (HQ) values for Co, Cu, Mn, and Zn were higher than 1 and the hazard index (HI) was higher than 1 for samples collected from all areas of Kazakhstan. This indicates that there should be concern about the potential hazards of the combination of toxic elements in Kazakh wheat.

Introduction of human pharmaceuticals from wastewater treatment plants into the aquatic environment: a rural perspective.

Incomplete removal of pharmaceuticals during wastewater treatment can result in their discharge into the aquatic environment. The discharge of pharmaceuticals in wastewater treatment plant (WWTP) effluents into rivers, lakes and the oceans has led to detectable concentrations of pharmaceuticals in the aquatic environment in many countries. However, to date studies of WWTP discharges into the aquatic environment have largely been confined to areas of relatively high population density, industrial activity or systems impacted on by such areas. In this work, two sites in the far north of Scotland were used to assess whether, and which, pharmaceuticals were being introduced into natural waters in a rural environment with low population density. Samples from two WWTPs (with differing modes of operation), and one receiving water, the River Thurso, were analysed for the presence of 12 pharmaceuticals (diclofenac, clofibric acid, erythromycin, ibuprofen, mefenamic acid, paracetamol, propranolol, sulfamethoxazole, tamoxifen, trimethoprim and dextropropoxyphene). Ten of the 12 pharmaceuticals investigated were detected in at least one of the 40 WWTP effluent samples. Maximum concentrations ranged from 7 ng L(-1) (sulfamethoxazole) to 22.8 µg L(-1) (paracetamol) with diclofenac and mefenamic acid being present in all of samples analysed at concentrations between 24.2 and 927 ng L(-1) and 11.5 and 22.8 µg L(-1), respectively. Additionally, the presence of four pharmaceuticals at ng L(-1) levels in the River Thurso, into which one of the WWTPs discharges, shows that such discharges result in measurable levels of pharmaceuticals in the environment. This provides direct evidence that, even in rural areas with low population densities, effluents from WWTPs can produce quantifiable levels of human pharmaceutical in the natural aquatic environment. These observations indicate that human pharmaceuticals may be considered as contaminants, with potential to influence water quality, management and conservation not only in urban and industrial regions but also those more rural in nature.

Sulfathiazole: analytical methods for quantification in seawater and macroalgae.

The awareness of the interconnection between pharmaceutical residues, human health, and aquaculture has highlighted the concern with the potential harmful effects it can induce. Furthermore, to better understand the consequences more research is needed and to achieve that new methodologies on the detection and quantification of pharmaceuticals are necessary. Antibiotics are a major class of drugs included in the designation of emerging contaminants, representing a high risk to natural ecosystems. Among the most prescribed are sulfonamides, with sulfathiazole being the selected compound to be investigated in this study. In the environment, macroalgae are an important group of producers, continuously exposed to contaminants, with a significant role in the trophic web. Due to these characteristics are already under scope for the possibility of being used as bioindicators. The present study describes two new methodologies based on liquid chromatography for the determination of sulfathiazole in seawater and in the green macroalgae Ulva lactuca. Results show both methods were validated according to international standards, with MS/MS detection showing more sensitivity as expected with LODs of 2.79ng/g and 1.40ng/mL for algae and seawater, respectively. As for UV detection the values presented were respectively 2.83µg/g and 2.88µg/mL, making it more suitable for samples originated in more contaminated sites. The methods were also applied to experimental data with success with results showing macroalgae have potential use as indicators of contamination.