Integral assessment of estrogenic potentials in sediment-associated samples

Background, aim, and scope  The enzyme-linked receptor assay (ELRA) detects estrogenic and anti-estrogenic effects at the molecular level of receptor binding and is a useful tool for the integrative assessment of ecotoxicological potentials caused by hormonally active agents (HAA) and endocrine disrupting compounds (EDC). The main advantage of the ELRA is its high sample throughput and its robustness against cytotoxicity and microbial contamination. After a methodological adaptation to salinity of the ELRA, according to the first part of this study, which increased its salinity tolerance and sensitivity for 17-β-estradiol, the optimised ELRA was used to investigate 13 native sediments characterised by different levels of salinity and chemical contamination. The applicability of the ELRA for routine analysis in environmental assessment was evaluated. Salinity is often a critical factor for bioassays in ecotoxicological sediment assessment. Therefore, salinity of the samples was additionally adjusted to different levels to characterise its influence on elution and binding processes of receptor-binding substances. Materials and methods  The ELRA was carried out with the human estrogen receptor α (ER) in a 96-well microplate format using the experimental setup known from the competitive immunoassay based on ligand–protein interaction. It is an important improvement that a physiologically relevant receptor was used as a linking protein instead of an antibody. The microplates were coated with a 17-β-estradiol-BSA conjugate, and dilution series of estradiol and of native sediment samples were added and incubated with the ER. After a washing step, a biotinylated mouse anti-ER antibody was added to each well. Receptor binding to estradiol, agonistic and antagonistic receptor binding, were determined by a streptavidin-POD-biotin complex with subsequent measurement of the peroxidase activity at the wavelength of 450 nm using a commercial ELISA multiplate reader. The sediment elutriates and pore water samples of sediments were tested in a dilution series to evaluate at which dilution step the receptor-binding potential ends. In the elution process (see Section 2.1 to 2.2), a method was developed to adjust the salinity to the levels of the reference testings, which offers an appropriate option to adjust the salinity in both directions. Statistical evaluation was made with a combination of the Mann–Whitney U test and the pT-method. Results  This part of the study characterised the environmental factor ‘salinity’ for prospective applications of the ELRA. Using reference substances such as 17-β-estradiol, the ELRA showed sigmoid concentration-effect relations over a broad range from 0.05 μg/l to 100 μg/l under physiological conditions. After methodological optimisation, both sensitivity and tolerance of the assay against salinity could be significantly raised, and the ELRA became applicable under salinity conditions up to concentrations of 20.5‰. The mean relative inter-test error (n = 3) was around 11% with reference substances and below 5% for single sediments elutriates in three replicates each. For sediment testings, the pore water and different salinity-adjusted elutriates of 13 sediments were used. A clear differentiation of the receptor-binding potential could be reached by application of the pT-method. Thereby, pT-values from one to six could be assigned to the sediments, and the deviation caused by the different salinity conditions was one pT-value. The mean standard deviation in the salinity adaptation procedure of the elutriates was below 5%. Discussion  Although the ELRA has already been used for assessments of wastewater, sludge and soil, its applicability for samples to different salinity levels has not been investigated so far. Even if the ELRA is not as sensitive as the E-screen or the YES-assay, with regard to reference substances like 17-β-estradiol, it is a very useful tool for pre-screening, because it is able to integrate both estrogenic as well as anti-estrogenic receptor-binding effects. According to the results of sediment testing, and given the integrative power to detect different directions of effects, the ELRA shows sufficient sensitivity and salinity tolerance to discriminate receptor-binding potentials in environmental samples. Conclusions  The optimised ELRA assay is a fast, cost-effective, reliable and highly reproducible tool that can be used for high-throughput screening in a microplate format in detecting both estrogenic and anti-estrogenic effects. Additionally, the ELRA is robust against microbial contaminations, and is not susceptible towards cytotoxic interferences like the common cell-culture methods. The general applicability and sufficient sensitivity of the ELRA was shown in freshwater environments. Marine and brackish samples can be measured up to salinity levels of 20.5‰. Recommendations and perspectives  In view of the proven sensitivity, functionality and the fastness of the ELRA, it is recommendable to standardise the test method. At the moment, no adequate in vitro test procedure exists which is standardised to DIN or ISO levels. The E-screen and the yeast estrogen/androgen screens (YES/YAS) sometimes underlie strong cytotoxic effects, as reported in the first part of this study. Further development of an ELRA assay using human androgen receptors appears to be very promising to gain information about androgenic and anti-androgenic effects, too. This would offer a possibility to use the ELRA as a fast and reliable pre-screening tool for the detection of endocrine potentials, thus minimising time and cost-expensive animal experiments.     

Combined use of molecular biology taxonomy, Raman spectrometry, and ESEM imaging to study natural biofilms grown on filter materials at waterworks

DNA-based population analysis was applied in combination with Raman spectrometry and Environmental Scanning Electron Microscopy for the characterisation of natural biofilms from sand and activated carbon filters operated for a long term at a municipal waterworks. Whereas the molecular biology polymerase chain reaction combined with denaturing gradient gel electrophoresis approach provides a deeper insight into the bacterial biofilm diversities, Raman spectrometry analyses the chemical composition of the extracellular polymer substances (EPS), microorganisms embedded in EPS as well as other substances inside biofilm (inorganic compounds and humic substances). Microscopy images the spatial distribution of biofilms on the two different filter materials. In addition, bacterial bulk water populations were compared with biofilm consortia using the molecular fingerprint technique mentioned.Population analysis demonstrated the presence of more diverse bacterial species embedded in a matrix of EPS (polysaccharides, peptides, and nucleic acids) on the sand filter materials. In contrast to this, activated carbon granules were colonised by reduced numbers of bacterial species in biofilms. Besides α-, β-, and γ-Proteobacteria, a noticeable specific colonisation with Actinobacteria was found on activated carbon particles. Here, the reduced biofilm formation came along with a decreased EPS synthesis. The taxonomy profiles of the different biofilms revealed up to 60% similarity on the same filter materials and 32% similarity of different materials. Similarity of adherent communities from filter materials and bulk water populations from the filter effluent varied between 36% and 58% in sand filters and 6–40% in granular activated carbon filters.The biofilm investigation protocols are most crucial to subsequent acquisition of knowledge on biofilm dynamics and bacterial contributions to transformation or adsorption processes in waterworks facilities.     

Effects of pen bedding and feeding high crude protein diets on manure composition and greenhouse gas emissions from a feedlot pen surface

Greenhouse gas (GHG) emissions from concentrated animal feeding operations vary by stage of production and management practices. The objective of this research was to study the effect of two dietary crude protein levels (12 and 16%) fed to beef steers in pens with or without corn stover bedding. Manure characteristics and GHG emissions were measured from feedlot pen surfaces. Sixteen equal-sized feedlot pens (19?×?23 m) were used. Eight were bedded approximately twice a week with corn stover and the remaining eight feedlot pens were not bedded. Angus steers (n = 138) were blocked by live weights (lighter and heavier) with 7 to 10 animals per pen. The trial was a 2?×?2 factorial design with factors of two protein levels and two bedding types (bedding vs. non bedding), with four replicates. The study was conducted from June through September and consisted of four ?28-day periods. Manure from each pen was scrapped once every 28 days and composite manure samples from each pen were collected. Air samples from pen surfaces were sampled in Tedlar bags using a Vac-U-Chamber coupled with a portable wind tunnel and analyzed with a greenhouse gas gas chromatograph within 24 hr of sampling. The manure samples were analyzed for crude protein (CP), total nitrogen (TN), ammonia (NH₃), total volatile fatty acid (TVFA), total carbon (TC), total phosphorus (TP), and potassium (K). The air samples were analyzed for methane (CH₄), carbon dioxide (CO₂), and nitrous oxide (N₂O) concentrations. The concentration of TN was significantly higher (p < 0.05) in manure from pens with cattle fed the high protein diets. The volatile fatty acids (VFAs) such as acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids concentrations were similar across both treatments. There were no significant differences in pen surface GHG emissions across manure management and dietary crude protein levels.

Implications: Livestock manure produces odor and emits GHGs (CO₂, CH₄, and N₂O) at different stages of production and management practices that have significant environmental concerns. Thus, it is important to measure GHG contributions from different sources and develop appropriate mitigation strategies for minimizing GHG contribution from livestock production facilities. Two dietary protein levels (12 and 16%) fed to beef steers in pens with or without corn stover bedding were studied. The results indicated that dietary protein levels and bedding vs. no bedding had very little effect on GHG emissions and manure composition under open feedlot conditions in North Dakota climatic conditions and management practices.     

Activated carbon amendment to sequester PAHs in contaminated soil: a lysimeter field trial

Activated carbon (AC) amendment is an innovative method for the in situ remediation of contaminated soils. A field-scale AC amendment of either 2% powder or granular AC (PAC and GAC) to a PAH contaminated soil was carried out in Norway. The PAH concentration in drainage water from the field plot was measured with a direct solvent extraction and by deploying polyoxymethylene (POM) passive samplers. In addition, POM samplers were dug directly in the AC amended and unamended soil in order to monitor the reduction in free aqueous PAH concentrations in the soil pore water. The total PAH concentration in the drainage water, measured by direct solvent extraction of the water, was reduced by 14% for the PAC amendment and by 59% for GAC, 12 months after amendment. Measurements carried out with POM showed a reduction of 93% for PAC and 56% for GAC. The free aqueous PAH concentration in soil pore water was reduced 93% and 76%, 17 and 28 months after PAC amendment, compared to 84% and 69% for GAC. PAC, in contrast to GAC, was more effective for reducing freely dissolved concentrations than total dissolved ones. This could tentatively be explained by leaching of microscopic AC particles from PAC. Secondary chemical effects of the AC amendment were monitored by considering concentration changes in dissolved organic carbon (DOC) and nutrients. DOC was bound by AC, while the concentrations of nutrients (NO(3), NO(2), NH(4), PO(4), P-total, K, Ca and Mg) were variable and likely affected by external environmental factors.     

Kinetic study of γ-hexabromocyclododecane orally given to laying hens (Gallus domesticus)

Introduction  

High concentrations of hexabromocyclododecane (HBCD) sometimes recorded in free-range hens' eggs are thought to be due to soil ingestion. Of the three stereoisomers of HBCD (α-, β-, and γ-HBCD), γ-HBCD is the main component in the commercial mixture, as well as in environmental matrices, whereas the isomer profile is α-dominated in biota. In fish and in mammals, this shift is thought to be due to a rapid elimination of γ-HBCD and to its bioisomerization to the more persistent α-HBCD. The aim of the current controlled study was to better understand the fate of ingested HBCD in laying hens. The isomer profile in soil being γ-dominated, excretion kinetics of γ-HBCD into egg yolk, and accumulation in liver and in abdominal fat were investigated.     

Contributions to ecological chemistry CXII balance of conversion of buturon‐14C in wheat under outdoor conditions 1

Abstract

The urea herbicide buturon (N‐[p‐chlorophenyl] ‐N’ ‐methyl‐N’ ‐isobutinyl‐urea), ¹⁴C‐labeled, was sprayed on winter wheat as an aqueous formulation (2.98 kg/ha) under outdoor conditions. Upon harvest (three months after application), a total of 49. 2% of the applied radiocarbon was recovered: 2.0% in the plants, 46.9% in the soil, and 0.3% in the leaching water (depth > 50 cm); less than 0.1% was in the grains (0.464 ppm). Only about half of the radioactivity present in plants could be recovered under mild extraction conditions; about half of this was unchanged buturon. In straw and husk extracts, the following metabolites were identified by gaschromatography/mass spectrometry: N‐(p‐chlorophenyl)‐N‐methyl‐O‐methyl‐carbamate (metabolite I), N‐phenyl‐N’ ‐formyl‐urea (metabolite II), two unstable metabolites giving (p‐chlorophenyl)‐isocyanate upon purification (metabolites III and IV), N‐(p‐chlorophenyl)‐N’ ‐methyl‐N’ ‐isobutenylol‐urea (metabolite V), p‐chloroformanilide (metabolite VI) and biologically bound p‐chloroaniline (metabolite VII). In the root and basal stem extract, the following metabolites were identified by gas chromatography/mass spectrometry: N‐(p‐chlorophenyl)‐O‐methyl‐carbamate (metabolite VIII) and N‐(p‐chlorophenyl)‐N’ ‐methyl‐urea (metabolite IX).     

Science with Society in the Anthropocene

Interdisciplinary scientific knowledge is necessary but not sufficient when it comes to addressing sustainable transformations, as science increasingly has to deal with normative and value-related issues. A systems perspective on coupled human–environmental systems (HES) helps to address the inherent complexities. Additionally, a thorough interaction between science and society (i.e., transdisciplinarity = TD) is necessary, as sustainable transitions are sometimes contested and can cause conflicts. In order to navigate complexities regarding the delicate interaction of scientific research with societal decisions these processes must proceed in a structured and functional way. We thus propose HES-based TD processes to provide a basis for reorganizing science in coming decades.     

Determination of 10 particle-associated multiclass polar and semi-polar pesticides from small streams using accelerated solvent extraction

A new analytical method using accelerated solvent extraction was developed for the determination of 10 particle-associated polar and semipolar pesticides. In addition, six deuterated analogues of the target compounds were evaluated as internal standards. The method yielded acceptable accuracy (73–103% recovery) and precision (<25% relative standard deviation) for eight compounds. Using size exclusion chromatography (SEC) as cleanup step resulted in higher recoveries compared to solid phase extraction (SPE) cleanup.

Deuterated standards with 10 or more deuterium atoms performed well as internal standards concerning similar recovery and correlation with the target analytes.

The method was employed to extract particle-associated pesticides from 16 streams located in an area with intense agriculture in France. Acetochlor, pirimicarb, tebuconazole, fenpropidin, -endosulfan and chlorfenvinphos were detected at concentrations up to 1 mg kg⁻¹ dry weight. A comparison with aquatic toxicity data indicated potential risk to the benthic fauna exposed to these concentrations of pirimicarb, -endosulfan and chlorfenvinphos.

We suggest that the method presented here be used for the extraction and quantitation of particle-associated polar pesticides.