Comparison between intrauterine application of an antibiotic and an herbal product to treat clinical endometritis in dairy cattle - A randomized multicentre field study.

Uterine diseases are main indications for antibiotic use in dairy cows. To test a non-antibiotic treatment option, we compared the effect of an intrauterine cephapirin (Metricure®; cefapirin benzathin 500 mg per dosis; CEPH) with an intrauterine applied herbal product (25 ml of EucaComp® PlantaVet containing alcoholic extracts of Calendula officinalis L., Mellissa officinalis L., Origanum majorana L. and Eucalyptus globulus Labill. (EUC)) on the clinical cure of endometritis. Examinations of 816 cows between 21 and 35 days after calving were performed and cases of clinical endometritis (n = 169) were included. Diagnosis based on a scoring system for vaginal discharge. Study animals were randomly assigned to one of two treatment groups and treated immediately. After excluding animals with incomplete datasets, 136 cows (EUC: n = 61; CEPH: n = 75) remained for the final analysis. In total, 64% (EUC: 61%, CEPH: 67%) of analysed endometritis cases were considered as clinically cured 14 ± 2 days after the first treatment, 15% stayed uncured after the application of a maximum of two consecutive treatments, leading to an overall clinical cure rate of 85% (EUC: 82%, CEPH: 88%). No statistically difference in clinical cure rates could be observed between both treatment groups nor 14 ± 2 days after the first treatment (p = 0.956) neither regarding the overall cure rate (p = 0.923). In conclusion, the clinical cure of dairy cows' endometritis after the intrauterine application of the herbal product was non-inferior to the intrauterine application of the antibiotic cephapirin. These results could contribute to reduce the antimicrobial use in the daily veterinary routine treatment of endometritis.

Alkyl-phenanthrenes in early life stage fish: differential toxicity in Atlantic haddock (Melanogrammus aeglefinus) embryos.

Tricyclic polycyclic aromatic hydrocarbons (PAHs) are believed to be the primary toxic components of crude oil. Such compounds including phenanthrene are known to have direct effects on cardiac tissue, which lead to malformations during organogenesis in early life stage fish. We tested a suite of 13 alkyl-phenanthrenes to compare uptake and developmental toxicity in early life stage haddock (Melanogrammus aeglefinus) embryos during gastrulation/organogenesis beginning at 2 days post fertilization via passive dosing. The alkyl-phenanthrenes were tested at their solubility limits, and three of them also at lower concentrations. Measured body burdens were linearly related to measured water concentrations. All compounds elicited one or more significant morphological defects or functional impairment, such as decreased length, smaller eye area, shorter jaw length, and increased incidence of body axis deformities and eye deformities. The profile of developmental toxicities appeared unrelated to the position of alkyl substitution, and gene expression of cytochrome 1 a (cyp1a) was low regardless of alkylation. Mortality and sublethal effects were observed below the expected range for baseline toxicity, thus indicating excess toxicity. Additionally, PAH concentrations that resulted in toxic effects here were far greater than when measured in whole crude oil exposures that cause toxicity. This work demonstrates that, while these phenanthrenes are toxic to early life stage fish, they cannot individually account for most of the developmental toxicity of crude oil, and that other compounds and/or mixture effects should be given more consideration.

Fate-directed risk assessment of chemical mixtures: a case study for cedarwood essential oil.

The environmental risk assessment of UVCBs (i.e., substances of unknown or variable composition, complex reaction products, or biological materials) is challenging due to their inherent complexity. A particular problem is that UVCBs can contain constituents with unidentified chemical structures and/or have variable composition of constituents from batch to batch. Moreover, the composition of a UVCB in the environment is not the same as that of the UVCB in a product, meaning that a risk assessment based on environmental exposure to the UVCB in a product does not represent the actual environmental risk. Here we propose an in silico fate-directed risk assessment framework for UVCBs using cedarwood oil as a case study. The framework uses Monte Carlo simulations and the mass-balance models SimpleTreat and RAIDAR to provide quantitative information on whether unidentified constituents within the physical-chemical property space of a UVCB can be the decisive factor for the environmental risk of the entire UVCB. Thereby the framework provides a robust decision tool to evaluate if a UVCB risk assessment requires additional tests or if the data on known constituents is representative for the risk of the entire UVCB. In the case of cedarwood oil, it could be shown that a risk assessment based on the known constituents (representing around 70% of the overall UVCB by weight) is representative for the environmental risk of the entire UVCB - reducing the need for additional testing and test animals.

Screening of Antimicrobial Activity of Essential Oils against Bovine Respiratory Pathogens - Focusing on Pasteurella multocida.

Administration of essential oils as natural plant products with antimicrobial activity might be an alternative to antibiotic treatment of bovine respiratory disease. The aim of this study was to analyse the in vitro antimicrobial activity of 11 essential oils against Pasteurella multocida isolated from the respiratory tract of calves using microdilution with determination of minimum inhibitory and bactericidal concentration as well as agar disc diffusion. Additionally, antimicrobial activity against Mannheimia haemolytica and bacteria in the Mannheimia clade was assessed by agar disc diffusion. Seven essential oil mixtures were also tested against all bacterial isolates. P. multocida was strongly inhibited by cinnamon cassia and lemongrass oil followed by coriander, winter savory, thyme, clove, and peppermint oil in the microdilution assays. Eucalyptus, wintergreen, spruce, and star anise oil showed lower activity. Comparison of both methods revealed an underestimation of cinnamon cassia oil activity by agar disc diffusion and conflicting results for wintergreen oil in microdilution, which precipitated in broth. Cinnamon cassia, thyme, wintergreen, lemongrass, and winter savory oil all showed strong antimicrobial activity against M. haemolytica. Bacteria in the Mannheimia clade were mostly inhibited by cinnamon cassia and thyme oil. Pasteurella isolates were more susceptible to inhibition by essential oils than Mannheimia isolates. Essential oil mixtures did not show stronger antibacterial activity than single essential oils. In conclusion, cinnamon cassia and lemongrass as well as coriander, winter savory, and thyme oil are promising candidates for treatment of P. multocida-associated bovine respiratory infections.

Linking biodegradation kinetics, microbial composition and test temperature - Testing 40 petroleum hydrocarbons using inocula collected in winter and summer.

Many factors affect the biodegradation kinetics of chemicals in test systems and the environment. Empirical knowledge is needed on how much test temperature, inoculum, test substances and co-substrates influence the biodegradation kinetics and microbial composition in the test. Water was sampled from the Gudenaa river in winter (2.7 °C) and summer (17 °C) (microbial inoculum) and combined with an aqueous stock solution of >40 petroleum hydrocarbons prepared by passive dosing. This resulted in low-concentration test systems that were incubated for 30 days at 2.7, 12 and 20 °C. Primary biodegradation kinetics, based on substrate depletion relative to abiotic controls, were determined with automated Solid Phase Microextraction coupled to GC/MS. Biodegradation kinetics were remarkably similar for summer and winter inocula when tested at the same temperature, except when cooling summer inoculum to 2.7 °C which delayed degradation relative to winter inoculum. Amplicon sequencing was applied to determine shifts in the microbial composition between season and during incubations: (1) the microbial composition of summer and winter inocula were remarkably similar, (2) the incubation and the incubation temperature had both a clear impact on the microbial composition and (3) the effect of adding >40 petroleum hydrocarbons at low test concentrations was limited but resulted in some proliferation of the known petroleum hydrocarbon degraders Nevskia and Sulfuritalea. Overall, biodegradation kinetics and its temperature dependency were very similar for winter and summer inoculum, whereas the microbial composition was more affected by incubation and test temperature compared to the addition of test chemicals at low concentrations.

Passive Dosing of Petroleum and Essential Oil UVCBs-Whole Mixture Toxicity Testing at Controlled Exposure.

Petroleum products and essential oils are produced and used in large amounts and are categorized as "Substances of Unknown or Variable composition, Complex reaction products or Biological materials (UVCBs)." These UVCBs are notorious difficult-to-test substances, since they are complex mixtures of hydrophobic and volatile compounds. This study introduces two passive dosing (PD) approaches for whole UVCB toxicity testing: (1) headspace PD applies the UVCB and purified lipid oil as a donor to control exposure via the headspace and (2) silicone rod PD applies UVCB-loaded silicone rods to control exposure via an aqueous test medium and headspace. Headspace gas chromatography-mass spectrometry measurements were used to cross-validate the approaches at the saturation level and to confirm exposure and maintain mixture composition at varying donor concentration levels. Both approaches were applied to whole-mixture toxicity tests of petroleum and essential oil UVCBs with daphnia and algae. Finally, the observed toxicity was linked to concentrations in the donor and in lipid membranes at equilibrium with the donors. Dose-response curves were similar across the dosing approaches and tested species for petroleum products but differed by an order of magnitude between essential oils and PD systems. All observed toxic effects were consistent with baseline toxicity, and no excess mixture toxicity was observed.

Assessing toxicity of hydrophobic aliphatic and monoaromatic hydrocarbons at the solubility limit using novel dosing methods.

Reliable delineation of aquatic toxicity cut-offs for poorly soluble hydrocarbons is lacking. In this study, vapor and passive dosing methods were applied in limit tests with algae and daphnids to evaluate the presence or absence of chronic effects at exposures corresponding to the water solubility for representative hydrocarbons from five structural classes: branched alkanes, mono, di, and polynaphthenic (cyclic) alkanes and monoaromatic naphthenic hydrocarbons (MANHs). Algal growth rate and daphnid immobilization, growth and reproduction served as the chronic endpoints investigated. Results indicated that the dosing methods applied were effective for maintaining mean measured exposure concentrations within a factor of two or higher of the measured water solubility of the substances investigated. Chronic effects were not observed for hydrocarbons with an aqueous solubility below approximately 5 µg/L. This solubility cut-off corresponds to structures consisting of 13-14 carbons for branched and cyclic alkanes and 16-18 carbons for MANHs. These data support reliable hazard and risk evaluation of hydrocarbon classes that comprise petroleum substances and the methods described have broad applicability for establishing empirical solubility cut-offs for other classes of hydrophobic substances. Future work is needed to understand the role of biotransformation on the observed presence or absence of toxicity in chronic tests.

Effects of a Multicomponent Herbal Extract on the Course of Subclinical Ketosis in Dairy Cows - a Blinded Placebo-controlled Field-study.

A blinded placebo-controlled multi-center on-farm trial was conducted in dairy cows with subclinical ketosis to investigate effects of a multicomponent herbal extract. Blood ketone levels were measured weekly in early lactating cows from 16 Swiss herds. Cows were subclassified based on their initial blood-ß-hydroxybutyrate levels (≥ 1.0 [KET-low, 84 cows] and > 1.2 mmol/L [KET-high, 39 cows]) and randomly distributed to 3 groups treated orally with herbal extract containing Camellia sinensis, Cichcorium intybus, Gentiana lutea, Glycyrrhiza glabra, Taraxacum officinale, Trigonella foenum-graecum, and Zingiber officinale, sodium propionate, or placebo twice a day for 5 days. Milk yield, milk acetone, blood-ß-hydroxybutyrate, glucose, nonesterified fatty acids, gamma-glutamyl transferase, and glutamate dehydrogenase were analyzed over 2 wk. Linear mixed effect models were used for data analysis. No effects were found for nonesterifed fatty acids, gamma-glutamyl transferase, and glucose. Significantly higher glutamate dehydrogenase (29.71 U/L) values were found in herbal extract-treated animals compared to sodium propionate on day 7 (22.33 U/L). By trend, higher blood-ß-hydroxybutyrate levels (1.36 mmol/L) were found in the placebo group of KET-high-cows on day 14 compared to the sodium propionate group (0.91 mmol/L). Milk yields of all treatment groups increased. Milking time and treatment showed a significant interaction for milk acetone: sodium propionate led to an immediate decrease, whereas herbal extracts resulted in a milk acetone decrease from day 7 on, reaching significantly lower milk acetone on day 14 (3.17 mg/L) when compared to placebo (4.89 mg/L). In conclusion, herbal extracts and sodium propionate are both likely to improve subclinical ketosis in dairy cows, however, by different modes of action.

Biodegradation kinetics testing of two hydrophobic UVCBs - potential for substrate toxicity supports testing at low concentrations.

The biodegradation kinetics of UVCB substances (unknown or variable composition, complex reaction products or biological materials) should be determined below the solubility limit to avoid experimental artefacts by the non-dissolved mixture. Recently, we reported delayed biodegradation kinetics of single petroleum hydrocarbons even at concentrations just below the solubility limit and attributed this to toxicity. The present study aimed to determine the concentration effect on biodegradation kinetics for constituents in two UVCBs, using surface water from a rural stream as the inoculum. Parallel biodegradation tests of diesel and lavender oil were conducted at concentrations just below the solubility limit and two orders of magnitude lower. The biodegradation kinetics of diesel oil constituents were generally similar at the two concentrations, which coincided with the stimulation of bacterial productivity (growth) at both concentrations, determined by [3H]leucine incorporation. By contrast, the biodegradation of lavender oil constituents was significantly delayed or even halted at the high test concentration. This was consistent with lavender oil stimulating bacterial growth at low concentration but inhibiting it at high concentration. The delayed biodegradation kinetics of lavender oil constituents at high concentration was best explained by mixture toxicity near the solubility limit. Consequently, biodegradation testing of hydrophobic UVCBs should be conducted at low, environmentally relevant concentrations ensuring that mixture toxicity does not affect the biodegradation kinetics.

Accelerated Passive Dosing of Hydrophobic Complex Mixtures-Controlling the Level and Composition in Aquatic Tests.

Petroleum products and essential oils are complex mixtures of hydrophobic and volatile chemicals and are categorized as substances of unknown or variable composition, complex reaction products, or biological materials (UVCBs). In aquatic testing and research of such mixtures, it is challenging to establish initial concentrations without the addition of cosolvents, to maintain constant concentrations during the test, and to keep a constant mixture composition in dilution series and throughout test duration. Passive dosing was here designed to meet these challenges by maximizing the surface area (Adonor/Vmedium = 3.8 cm2/mL) and volume (Vdonor/Vmedium > 0.1 L/L) of the passive dosing donor in order to ensure rapid mass transfer and avoid donor depletion for all mixture constituents. Cracked gas oil, cedarwood Virginia oil, and lavender oil served as model mixtures. This study advances the field by (i) showing accelerated passive dosing kinetics for 68 cracked gas oil constituents with typical equilibration times of 5-10 min and for 21 cederwood Virginia oil constituents with typical equilibration times < 1 h, (ii) demonstrating how to control mixture concentration and composition in aquatic tests, and (iii) discussing the fundamental differences between solvent spiking, water-accommodated fractions, and passive dosing.

A multicomponent herbal feed additive improves somatic cell counts in dairy cows - a two stage, multicentre, placebo-controlled long-term on-farm trial.

In contrast to natural and historical diets of wild and domesticated ruminants, the diversity of plant species is limited in diets of modern dairy cows. Are "production diseases" linked to this? We conducted a trial to test the effects of a multicomponent herbal feed additive (HFA) on health, performance and fertility traits. A dose-finding study (DF) with 62 cows on 11 commercial farms compared a low (50 g) and a high (100 g) dose of HFA (HFA-50, HFA-100) with a placebo (PL). In a subsequent field trial (FT) with 280 cows on 30 commercial farms, HFA-100 was compared to PL. Cows were randomly assigned to HFA and PL groups and received HFA or PL individually daily from 14 days pre- to 300 days post-calving. Data were analysed with mixed effects models. No differences between HFA and PL were found regarding performance, body condition score and overall culling rates. A tendency towards lower milk urea for HFA-100 compared to PL (p = .06) was found in DF. HFA significantly reduced elevated milk acetone observations (≥10 mg/L) in the first 10 lactation weeks (HFA-100: 4%; HFA-50: 4%; PL: 12%) in DF. HFA-50 significantly reduced lameness incidence (HFA-100: 11%; HFA-50: 2%; PL: 14%) in DF. Calving intervals were 15 days shorter in HFA compared to PL in both trials, which could be confirmed by tendency (p = .07) in FT. In both trials, the proportion of test days with elevated somatic cell score (≥3.0) was significantly lower in HFA compared to PL (DF: HFA-100: 40%, HFA-50: 45% and PL: 55%; FT: HFA-100: 38% and PL: 55%) which is also reflected by tendency (p = .08) in lower culling rates due to udder diseases in FT. HFA showed no negative impact on any of the measured parameters. The effects of HFA indicate a potential of phytochemically rich and diverse feed additives for dairy cows' nutrition and physiology.

Biodegradation of hydrocarbon mixtures in surface waters at environmentally relevant levels - Effect of inoculum origin on kinetics and sequence of degradation.

Biodegradation is a dominant removal process for many organic pollutants, and biodegradation tests serve as tools for assessing their environmental fate within regulatory risk assessment. In simulation tests, the inoculum is not standardized, varying in microbial quantity and quality, thereby potentially impacting the observed biodegradation kinetics. In this study we investigated the effect of inoculum origin on the biodegradation kinetics of hydrocarbons for five inocula from surface waters varying in urbanization and thus expected pre-exposure to petroleum hydrocarbons. A new biodegradation method for testing mixtures of hydrophobic chemicals at trace concentrations was demonstrated: Aqueous solutions containing 9 hydrocarbons were generated by passive dosing and diluted with surface water resulting in test systems containing native microorganisms exposed to test substances at ng-µg/L levels. Automated Headspace Solid Phase Microextraction coupled to GC-MS was applied directly to these test systems to determine substrate depletion relative to abiotic controls. Lag phases were generally less than 8 days. First order rate constants were within one order of magnitude for each hydrocarbon in four of the five waters but lower in water from a rural lake. The sequence of degradation between the 9 hydrocarbons showed similar patterns in the five waters indicating the potential for using selected hydrocarbons for benchmarking between biodegradation tests. Degradation half-times were shorter than or within one order of magnitude of BioHCwin predictions for 8 of 9 hydrocarbons. These results showed that location choice is important for biodegradation kinetics and can provide a relevant input to aquatic exposure and fate models.

Equilibrium sampling of persistent and bioaccumulative compounds in soil and sediment: comparison of two approaches to determine equilibrium partitioning concentrations in lipids.

The equilibrium sampling in silicone is increasingly applied to measure freely dissolved concentrations and chemical activities within bioaccumulation research of hydrophobic organic chemicals. Two equilibrium methods were applied to PCB-contaminated soil and sediment, and directly calibrated with respect to equilibrium partitioning concentrations in lipids (C(lipid,partitioning)): (i) Solid phase microextraction in the headspace above the sample (HS-SPME) required optimization for its application to PCBs, and it was calibrated above external partitioning standards in olive oil. (ii) Equilibrium sampling with internally coated glass jars with varying thicknesses of silicone (PDMS) resulted in proportionality between coating and analyte mass, which confirmed several validity criteria. C(lipid,partitioning) was here determined as product of PDMS concentration and PDMS to lipid partition ratio. The results of the two methods were in good agreement and thus validated each other. Finally, the coated glass jar method was applied to field sediment containing invertebrates, which lead to C(lipid,partitioning) that were about two times higher than measured lipid-normalized concentrations in the organisms. Temperature differences and animal lipid structure were discussed as possible reasons for this discrepancy. Both methods combine high analytical performance, reduced equilibration times and new calibration possibilities, which makes them suited for bioaccumulation research and environmental monitoring.

Equilibrium sampling: partitioning of organochlorine compounds from lipids into polydimethylsiloxane.

Polydimethylsiloxane (PDMS) can be used for equilibrium sampling of environmental pollutants in a large variety of matrices including biota. For comparison with lipid-normalized concentrations e.g. from biota monitoring programmes, reliable lipid to PDMS partition ratios (K(Lipid,PDMS)) are required. Additionally, K(Lipid,PDMS) facilitate comparison of equilibrium sampling data obtained in various environmental media and can be helpful to convert equilibrium sampling data into a more informative form. This work investigated the equilibrium partitioning of polychlorinated biphenyls (PCBs) and selected organochlorine pesticides (OCPs) between lipids from biota of different trophic levels and PDMS. One vegetable oil, a fish oil and seal oil were investigated. The lipid to PDMS partition ratios were compound-specific and ranged from 14.5 to 62.9 g/g with correction for lipid uptake into the PDMS and from 13.0 to 54.8 g/g without correction. Additionally, PDMS served as a reference partitioning phase for the accurate determination of lipid to lipid partition ratios, which for all analytes were close to unity. Evaluating the results in a bioaccumulation context, they indicate that the equilibrium partitioning of neutral lipophilic environmental contaminants into the lipids of the three investigated species will be very similar, although they represent three distinct trophic levels.

Diffusion of PAH in potato and carrot slices and application for a potato model.

A method for quantifying the effect of medium composition on the diffusive mass transfer of hydrophobic organic chemicals through thin layers was applied to plant tissue. The method employs two silicone disks, one serving as source and one as sink for a series of PAHs diffusing through thin layers of water, potato tissue, and carrot tissue. Naphthalene, phenanthrene, anthracene, and fluoranthene served as model substances. Their transfer from source to sink disk was measured by HPLC to determine a velocity rate constant proportional to the diffusive conductivity. The diffusive flux through the plant tissue was modeled using Fick's first law of diffusion. Both the experimental results and the model suggest that mass transfer through plant tissue occurs predominantly through pore water and that, therefore, the mass transfer ratio between plant tissue and water is independent of the hydrophobicity of the chemical. The findings of this study provide a convenient method to estimate the diffusion of nonvolatile organic chemicals through various plant materials. The application to a radial diffusion model suggests that "growth dilution" rendersthe concentration of highly hydrophobic chemicals in potatoes below their equilibrium partitioning level. This is in agreement with field results for the bioconcentration of PAHs in potatoes.