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.     

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.     

Analysis of perfluorooctanoate (PFOA) and other perfluorinated compounds (PFCs) in the River Po watershed in N-Italy

C7-C11 perfluorinated carboxylates (PFACs) and perfluorooctansulfonate (PFOS) were analysed in selected stretches of the River Po and its major tributaries. Analyses were performed by solid-phase extraction (SPE) with Oasis HLB cartridges and methanol elution followed by LC-MS-MS detection using 13C-labelled internal standards. High concentration levels ( approximately 1.3 microg l(-1)) of perfluorooctanoate (PFOA) were detected in the Tánaro River close to the city Alessandria. After this tributary, levels between 60 and 337 ng l(-1) were measured in the Po River on several occasions. The PFOA concentration close to the river mouth in Ferrara was between 60 and 174 ng l(-1). Using the river discharge flow data in m3 s(-1) at this point (average approximately 920 m3 s(-1) for the year 2006), a mass load of approximately 0.3 kg PFOA per hour or approximately 2.6 tons per year discharged in the Adriatic Sea has been calculated. PFOS concentration levels in the Po River at Ferrara were approximately 10 ng l(-1).     

Screening zur Mobilisierbarkeit von Kupfer aus Böden und Schlämmen

In the presented paper, the pHstat-procedure is compared with four simple extraction procedures. These procedures make use of the following extraction solutions: distilled water (based on DIN 38414 part 4, DIN-S4), 1 mol/L ammonium nitrate, 1 mol/L ammonium acetate at pH 7.0 and 1 mol/L sodium acetate at pH 5.0, respectively. The extraction procedures were applied to 10 different soil samples and 2 sludges. The DIN-S4-procedure shows the lowest amounts of mobilized copper. The highest parts are extracted by sodium acetate solution. For the procedures investigated, the mobilized amounts extracted by ammonium acetate solution are most comparable with those of the pHstat-procedure. The simple sodium acetate procedure is useful for screening to select significant samples from a large number of soils. Thus, the high effort caused by the pHstat-procedure can be reduced to a minimum through the application of this screening procedure. The application of this screening, however, is difficult for samples containing large parts of organic compounds, e.g. sewage sludge. These samples show a different extracting behaviour.     

The BIOTA Biodiversity Observatories in Africa—a standardized framework for large-scale environmental monitoring

The international, interdisciplinary biodiversity research project BIOTA AFRICA initiated a standardized biodiversity monitoring network along climatic gradients across the African continent. Due to an identified lack of adequate monitoring designs, BIOTA AFRICA developed and implemented the standardized BIOTA Biodiversity Observatories, that meet the following criteria (a) enable long-term monitoring of biodiversity, potential driving factors, and relevant indicators with adequate spatial and temporal resolution, (b) facilitate comparability of data generated within different ecosystems, (c) allow integration of many disciplines, (d) allow spatial up-scaling, and (e) be applicable within a network approach. A BIOTA Observatory encompasses an area of 1?km² and is subdivided into 100 1-ha plots. For meeting the needs of sampling of different organism groups, the hectare plot is again subdivided into standardized subplots, whose sizes follow a geometric series. To allow for different sampling intensities but at the same time to characterize the whole square kilometer, the number of hectare plots to be sampled depends on the requirements of the respective discipline. A hierarchical ranking of the hectare plots ensures that all disciplines monitor as many hectare plots jointly as possible. The BIOTA Observatory design assures repeated, multidisciplinary standardized inventories of biodiversity and its environmental drivers, including options for spatial up- and downscaling and different sampling intensities. BIOTA Observatories have been installed along climatic and landscape gradients in Morocco, West Africa, and southern Africa. In regions with varying land use, several BIOTA Observatories are situated close to each other to analyze management effects.     

Assessing the ecological effects of human impacts on coral reefs in Bocas del Toro,Panama

Environmental and biological reef monitoring was conducted in Almirante Bay (Bahía Almirante) in Bocas del Toro, Panama, to assess impacts from anthropogenic developments. An integrated monitoring investigated how seasonal temperature stress, turbidity, eutrophication and physical impacts threatened reef health and biodiversity throughout the region. Environmental parameters such as total suspended solids [TSS], carbon isotopes (δ¹³C), C/N ratios, chlorophyll a, irradiance, secchi depth, size fractions of the sediments and isotope composition of dissolved inorganic carbon [DIC] of the water were measured throughout the years 2010 and 2011 and were analysed in order to identify different impact sources. Compared to data from Collin et al. (Smithsonian Contributions to the Marine Sciences 38:324–334, 2009) chlorophyll a has doubled at sites close to the city and the port Almirante (from 0.46–0.49 to 0.78–0.97 μg l^?1) and suspension load increased, visible by a decrease in secchi depth values. Visibility decreased from 9-13 m down to 4 m at the bay inlet Boca del Drago, which is strongly exposed to river run off and dredging for the shipping traffic. Eutrophication and turbidity levels seemed to be the determining factor for the loss of hard coral diversity, most significant at chlorophyll a levels higher than 0.5 μg l^?1 and TSS levels higher than 4.7 mg l^?1. Hard coral cover within the bay has also declined, at some sites down to <10 % with extremely low diversities (7 hard coral species). The hard coral species Porites furcata dominated the reefs in highly impacted areas and showed a strong recovery after bleaching and a higher tolerance to turbidity and eutrophication compared to other hard coral species in the bay. Serious overfishing was detected in the region by a lack of adult and carnivorous fish species, such as grunts, snappers and groupers. Study sites less impacted by anthropogenic activities and/or those with local protection showed a higher hard coral cover and fish abundance; however, an overall loss of hard coral diversity was observed.     

Comment on "Analysis of groundwater contamination using concentration-time series recorded during an integral pumping test: Bias introduced by strong concentration gradients within the plume" by Allelign Zeru and Gerhard Schäfer

We consider the results of a recent paper in this journal [Zeru, A. and Sch?fer, G., 2005. Analysis of groundwater contamination using concentration-time series recorded during an integral pumping test: Bias introduced by strong concentration gradients within the plume. Journal of Contaminant Hydrology 81 (2005) 106-124], which addresses the field-scale characterisation of contaminant plumes in groundwater. There, it is concluded that contaminant concentration gradients can bias Integral Pumping Test (IPT) interpretations considerably, in particular if IPTs are conducted in advective fronts of contaminant plumes. We discuss implications of this setting and also argue that the longitudinal and transverse dispersivities used in the examples of Zeru and Sch?fer (2005) of up to 30 m and 3 m, respectively, are generally very high for the here relevant capture zone scale (<20 m). However, regardless of both longitudinal and transverse concentration gradients, we further show through a counter-example that IPT results are unbiased as long as the concentration attenuation along the flow direction is linear over the capture zone extent.     

LC-MS-MS analysis and occurrence of octyl- and nonylphenol, their ethoxylates and their carboxylates in Belgian and Italian textile industry, waste water treatment plant effluents and surface waters

Alkylphenols (APs), alkylphenol ethoxylates (APEOs), ethoxycarboxylate metabolites (APECs) and bisphenol A were determined in surface water using solid-phase extraction (SPE) followed by triple-quadrupole LC-MS-MS. APs were separated by LC from APECs using an acetonitrile-water-gradient without the addition of any buffer. Nonylphenol ethoxycarboxylates (NPECs) interfere in the detection of nonylphenols (NPs) when using an acidic mobile phase, because they produce the same MS-MS fragment ions (219>133 and 147). 4n-NP shows the characteristic transition 219>106; it is well suited as internal standard. Nonylphenol ethoxylates NPE(n)Os (n=1-17) were analysed separately in a second run by positive ionization using an ammonium acetate mobile phase. Textile industry discharges, the corresponding wastewater treatment plant (WWTP) effluents and the receiving rivers in Belgium and Italy were analysed. Among the substances investigated, NPE1C and NPE2O exhibited the highest concentrations in the water samples, up to 4.5 microg l(-1) NPE1C in a WWTP effluent and 3.6 microg l(-1) NPE2O in a river. The highest NP levels were found in the receiving rivers (max. 2.5 microg l(-1)). The predicted no-effect concentration (PNEC) for NP of 0.33 microg l(-1) for water species was frequently exceeded in the surface waters investigated, suggesting potential adverse effects to the aquatic environment.     

Conservation of the Toromiro Tree: Case Study in the Management of a Plant Extinct in the Wild

Abstract: We reviewed the history and conservation of Sophora toromiro , a species that has been extinct in the wild since 1960 but has survived as scattered individuals in botanic gardens and private collections. The short-term conservation of S. toromiro is dependent on the management of surviving ex situ stocks. This is being achieved through international collaboration by a working group established to coordinate the conservation management of the species. Molecular evidence indicates that the species retains greater genetic variability than expected. The greatest amount of genetic variability was located in specimens outside botanic garden collections. No unmodified natural habitat survives on Rapa Nui ( Easter Island); so opportunities to establish a viable wild population are limited. Evidence from past reintroductions indicates that the best short-term opportunity for the species is through conventional horticultural management in botanic gardens and traditional farm plots on Rapa Nui. Some extinct-in-the-wild taxa (sensu World Conservation Union 1994), such as the Toromiro, retain genetic variability, and appropriate reintroduction sites exist. These taxa represent valid priorities for conservation management.