Residue analysis of tetracyclines and their metabolites in eggs and in the environment by HPLC coupled with a microbiological assay and tandem mass spectrometry

Tetracyclines are widely used in farm animals. This can cause drug residues in products of animal origin and, after excretion of these substances, in animal slurry and in soil fertilized with that slurry. In this paper, we present a method based on a microbiological assay coupled with HPLC for the detection of oxytetracycline, tetracycline, and chlortetracycline in eggs. After a simple liquid extraction of the samples and HPLC separation, fractions were collected on microtiter plates, and the tetracyclines were analyzed using the Staphylococcus aureus assay. This method was able to identify residues of tetracyclines in eggs at a level set by regulatory agencies (i.e., 200 microg/kg). In addition, it was shown that the described microbiological method can be used as a screening assay for the detection of tetracyclines and possible biologically active metabolites in animal slurry and soil samples. Employing the same extraction procedure, it was demonstrated that LC-MS-MS allowed the quantification of 20-400 microg/kg in eggs with recoveries ranging from 71 to 109% and RSDs of 3-15%.     

Metal accumulation and hydraulic performance of bioretention systems after long-term operation

Purpose

Stormwater bioretention systems are widely used to treat diffuse infiltration of runoff from paved surfaces and roofs. Substantial questions remain about the hydraulic performance and the accumulation of pollutants in systems over the long term. Data of metal accumulation of systems with operational times >10 years currently is limited. This study deals with the accumulation of metals in a variety of long-term operational bioretention systems (11–22 years) to derive further operation recommendations for the water authorities.

Materials and methods

The hydraulic conductivity of the bioretention systems in field was measured using a double ring infiltrometer. Media soil samples from 22 diverse designed systems were collected across the surface and at intervals up to a depth of 65 cm to determine the spatial accumulation of Zn, Cu, Pb and Cd. Leaching experiments of selected bioretention media soils were derived to assess the metal leachability by water.

Results and discussion

The hydraulic performance of most bioretention systems still met the technical guidelines of Germany even after long-term operation. Considerable metal accumulation occurred in the topsoil (0–20 cm). Median concentrations of all metals are highest at the soil surface (0–10 cm), decreasing with increasing depth. High concentrations were determined at the inflow points of the runoff waters, whereas concentrations at more than 1.5 m distance from the inflow were only slightly increased compared to the initial soil concentrations. Leachability tests have shown that most of the metals deposited in bioretention soils are only slightly water soluble. No concentrations exceeding the threshold values of the German Soil Contamination Ordinance for the pathway soil to groundwater could be determined.

Conclusions

The hydraulic conductivity of the bioretention systems is given even well after long-term operation. Most of the metal accumulation is concentrated in the top 20 cm; concentrations decrease rapidly and mostly reach background/initial concentrations after depths of 30 cm. The water-soluble metals are all below the trigger values of the German Soil Act. This underlines the strong retention capacity of long-term bioretention systems after long-term operational times.

     

The impact of environmental change on the intensity and spatial pattern of water erosion in a semi-arid mountainous Andean environment

Aerial photographs taken in 1976 and 1989 and a field survey in 1999 showed that land use in a 900-ha catchment in the southern part of the Ecuadorian Andes is highly dynamic. Over 23 years, ca. 83 ha of arable land was abandoned and ca. 70 ha was taken into agricultural production. Changes in land use were not spatially homogeneous. Parcels on unstable geologic formations and close to village centres were preferably set-aside. Land taken into cultivation was preferably located on gently sloping areas close to newly built sites and arterial roads. The area with bush vegetation increased by regeneration of natural vegetation on fields set-aside in the late 1970s and early 1980s. There was a complex interaction between water erosion and environmental change in the study area. Land taken into production was levelled for furrow irrigation: this led to a net reduction in the area susceptible to water erosion. However, one quarter of the area affected only by sheet and rill erosion 23 years ago has since become incised by deep gullies. This increase in gully density was related to inadequate construction and management of irrigation infrastructure, rather than to change in vegetation cover and/or soil erodibility caused by agricultural practices. This factor is often overlooked in studies of the effects of environmental change on geomorphologic processes.     

Studies of Coxiella burnetii infections in dairy herds with special regard to infections in men

Investigations of 1167 dairy cows out of 105 herds with fertility disorders on secretion of Coxiella burnetii (C. burnetii) by the genitals as well as serological studies of these animals using ELISA demonstrated that C. burnetii infections are significantly associated with abortions but not with repeated inseminations without success or vaginal excretions. The demonstration of an increased risk of infections for persons caring for those dairy herds could be shown by a total of 253 serological studies. A comparison of these studies of farmers caring for dairy herds suffering from abortions a seroprevalence of > or = 20% showed that these persons revealed significantly more frequent antibodies against C. burnetii than farmers of the group compared with. Further studies showed that in herds suffering from abortions a seroprevalence of > or = 20% means an additional risk of infections of farmers. Vice versa abortions of the cows in herds with a seroprevalence of > or = 20% imply an additional infectious risk.     

Repeated administrations of adrenocorticotropic hormone during gestation in gilts: Effects on growth, behaviour and immune responses of their piglets

This study investigated whether repeated administrations of adrenocorticotropic hormone (ACTH) during mid or late gestation, a treatment which induces endogenous cortisol release, affect growth performance, early vitality, open-field behaviour and immune responses of neonatal pigs. Administrations of ACTH (100 IU per animal, Synacthen^® Depot) were given intramuscularly to gilts every second day either during mid (Day 49 until 75, Experiment 1) or late gestation (Day 85 until 107, Experiment 2). Control gilts received repeated injections of saline. The repeated ACTH stimulation of gilts during late gestation significantly reduced their daily weight gain during this period, but not when applied during mid gestation. Gestation length, number of born piglets and vitality measures of the newborn piglets, such as the rectal temperatures after birth and times elapsed between birth and first udder contact or milk uptake were not affected by the prenatal treatments. Administration of ACTH during late but not during mid gestation significantly increased the birth weights of piglets, and this difference in postnatal body weight was detectable until an age of 21 days. In addition, only the stimulation with ACTH during late gestation had an immunosuppressive effect on the lymphocyte proliferation of piglets 1 day after birth in response to the T-cell mitogen ConA and, in tendency, on the proliferation in response to the B-cell mitogen LPS. Twenty-four day old piglets from gilts treated during late gestation showed significantly more escape behaviour in an open-field than piglets from control litters. In conclusion, elevated maternal glucocorticoid levels during critical periods of prenatal development in pigs may affect prenatal growth, cell-mediated immunity and emotional reactivity in the neonatal piglets. The occurrence of these effects depends on the timing of increased maternal cortisol levels during gestation.     

IGF-I- and EGF-dependent DNA synthesis of porcine myoblasts is influenced by the dietary isoflavones genistein and daidzein

Soy-derived isoflavones have been reported to be specific inhibitors of protein tyrosine kinases like the type 1 insulin-like growth factor receptor (IGF-1R) and the epidermal growth factor receptor (EGFR). This study was conducted to investigate, whether IGF-I and EGF stimulate porcine myoblast growth and whether the responses are influenced by isoflavones. Satellite cell-born myoblasts derived from the semimembranosus muscle of newborn piglets were treated for 26h with IGF-I or EGF alone and in combination with genistein or daidzein. The DNA amount was measured and DNA synthesis was recorded as 6 h-[(3)H]thymidine incorporation during exponential growth in serum-free basal medium. IGF-I and EGF synergistically stimulated DNA synthesis of porcine myoblast with EGF causing a greater response. Genistein (100mumol/l) effectively reduced the growth factor-mediated DNA synthesis, which was associated with an inhibition of growth factor receptor protein expression. In response to daidzein no reduction in growth factor-mediated DNA synthesis was found. Daidzein (1; 10mumol/l) combined with IGF-I caused even a slight increase in DNA amount compared with the untreated control. The expression of the IGF-1R precursor protein was reduced with 10 and 100mumol/l daidzein, whereas the EGFR expression remained unchanged with daidzein. The results suggest that dietary isoflavones may interact with growth factor-induced stimulation of pig skeletal muscle growth.     

Overstory-specific effects of litter fall on the microbial carbon turnover in a mature deciduous forest

Mature deciduous forests can serve as important carbon (C) sinks, but the C storage differs significantly in dependency on the tree species. To specify the significance of overstory-specific effects of litter fall on the soil microbial C turnover, we have investigated the ¹³C isotopic signature of microbial biomarker phospholipid fatty acids (PLFAs). Samples were taken under pure Fagus sylvatica and mixed overstory (F. sylvatica and Fraxinus excelsior or F. excelsior, Acer spp. and F. sylvatica) in a mature temperate deciduous forest in Central Germany 4 weeks prior to and 3 weeks after litter fall. Accordingly, the CO₂ emission from soil was measured before, during and after the litter fall to investigate the response of decomposition. At all sites and at both sampling dates the fungal biomarker PLFA 18:2ω6,9 had predominantly lower δ¹³C values (from −32 to −43‰) than the bacterial biomarker PLFAs (δ¹³C values from −23 to −39‰). This difference indicated that fungi generally used preferentially plant derived C, whereas the bacterial populations include groups which used SOM derived C, independent on the overstory trees. Under pure F. sylvatica overstory the δ¹³C values of microbial biomarker PLFAs were slightly decreased (up to 2‰ for 17:0br) or unchanged after litter fall. By contrast, under both variants of mixed overstory the δ¹³C values of biomarker PLFAs of fungi (18:2ω6,9) were increased after litter fall (+3.5 and +3.8‰). This might be explained partly by a faster initial decomposition of foliar litter from mixed overstory already during litter fall as confirmed by higher CO₂ emission under mixed F. excelsior, Acer spp. and F. sylvatica than under pure F. sylvatica in this period. However, the involved microbial populations differed overstory-specific. Bacterial biomarker PLFAs with strongest overstory-specific differences in the response on litter fall were 17:0br (Gram-positive bacteria), 18:1 and 19:0cy (Gram-negative bacteria). The present results indicate that a tree species conversion even exclusively between deciduous tree species might alter the soil microbial C turnover during litter decomposition and suggest that it would in the long-term change the SOM stability and C storage.     

Speciation in the parthenogenetic oribatid mite genus Tectocepheus (Acari, Oribatida) as indicated by molecular phylogeny

Despite the lack of universal concepts for species and speciation, both sexual and asexual organisms are expected to diversify into discrete genotypic and morphological clusters. Species-rich clusters of parthenogenetic oribatid mites likely evolved in the absence of sexual reproduction. We used nucleotide sequences of the large and small rDNA genes (18S and 28S) and the coding genes for heat shock protein 82 (hsp82) and elongation factor-1 alpha (ef-1α) for phylogenetic analyses of three morphotypes of the parthenogenetic oribatid mite genus Tectocepheus. DNA sequence divergences of the different morphotypes were similar to those of sexual species in other organisms. Maximum likelihood analyses of single genes and combined data sets were largely congruent in reconstructing the phylogeny of the Tectocepheus species. The results suggest that the different morphotypes of Tectocepheus indeed evolved in the absence of sexual reproduction, and that Tectocepheus minor, Tectocepheus velatus and Tectocepheus sarekensis are best considered distinct species. Further, the results suggest that 18S rDNA, hsp82 and ef-1α are powerful markers for resolving phylogenetic relationships of oribatid mites. Saturation plots indicated that the D3-region of 28S is much more saturated than all other examined genes. This indicates that the D3-region is unsuitable for resolving ancient splits in oribatid mites.