Real-world experience with anti-PD-1/PD-L1 monotherapy in patients with non-small cell lung cancer : A retrospective Austrian multicenter study.

OBJECTIVE: As real-world data regarding immunotherapy for non-small cell lung cancer are lacking for Austria, we conducted a retrospective study in six hospitals to present data from real-world practice. METHODS: Patients with metastatic non-small cell lung cancer were stratified into two groups, either patients with first-line pembrolizumab monotherapy (cohort 1) or patients with second-line nivolumab, pembrolizumab or atezolizumab monotherapy (cohort 2). Primary outcome measures were objective response rate and overall survival. A matched-pair analysis was performed to compare overall survival to patients from the Tyrolean Lung Cancer Project as a historical control group. RESULTS: In total, 89 patients were identified, 42 patients in cohort 1 and 47 patients in cohort 2. The objective response rates were 43.3% and 31.4%, respectively. The median overall survival was 17.0 months (95% CI 11.7-21.5 months) in cohort 1 and 18.7 months (95% CI 9.5-23.4 months) in cohort 2. Treatment-related adverse events grades 3 and 4 were reported in 11.2% of patients. The matched-pair analysis showed a median overall survival of 15.2 months (95% CI 7.6-20.4 months) for first-line pembrolizumab monotherapy compared to 9.8 months (95% CI 7.8-11.6 months) for the historical control (p = 0.43). In cohort 2, a median overall survival of 20.3 months (95% CI 6.9-26.2 months) for second-line immunotherapy compared to 5.4 months (95% CI 3.2-11.7 months) for the historical control (p = 0.18) was shown. CONCLUSION: The results are comparable with other real-world studies and, when matched to historical controls, support the improvement in outcomes made possible by these agents.

Response of nematode communities to metals and PAHs in freshwater microcosms.

Freshwater microcosms were used to investigate the effects of various metals and polycyclic aromatic hydrocarbons (PAHs), applied as single substances and in mixtures, on sediment-inhabiting nematode assemblages. Several community measures, including nematode abundance, biomass, species richness, species composition and the NemaSPEAR[%]-index, were assessed and their changes in response to the chemical treatments were compared with the results of single-species toxicity testing using the nematode Caenorhabditis elegans. The diagnostic power of endpoints revealing toxic effects was then evaluated to assess the general suitability of nematodes as bioindicators of sediment contamination by a set of relevant chemicals. Overall, community measures based on species-level (detected with principle response curves) responded most sensitively to chemical stress in the microcosms, especially in terms of biomass, with the C. elegans toxicity test was for the most part as sensitive to the chemicals as the nematode species composition. Generally, this study justified the suitability of nematodes in assessments of the risk of chemicals frequently associated with freshwater sediments and underlined the value of C. elegans in the prospective risk assessment of chemicals, as this species was able to indicate the negative effects of comparatively low sediment concentrations of the contaminants.

Is Caenorhabditis elegans representative of freshwater nematode species in toxicity testing?

Multi-species toxicity tests were conducted using a broad range of freshwater nematode species to assess interspecific differences in sensitivity to chemical stress and to compare the toxicity to that on the standard test organism Caenorhabditis elegans. The lethal effects of nine different chemical treatments, including metals and polycyclic aromatic hydrocarbons (PAHs) in single and mixture application, were determined for nematodes exposed for 48 h to spiked aqueous solutions. The investigated freshwater nematodes exhibited distinct differences in their sensitivity. Ranking of the susceptibility of 27 species to chemical stress showed that the effects were largely independent of the tested chemical compounds. Overall, the responses of C. elegans were well within the range of those of freshwater nematode species, being slightly less tolerant to metals, but more tolerant to PAHs than the average freshwater species response. Therefore, this study justified the use of C. elegans as representative model for freshwater nematode species in toxicity testing.

Nontarget Analysis via LC-QTOF-MS to Assess the Release of Organic Substances from Polyurethane Coating.

A novel approach was investigated for the assessment of leaching from a one-component polyurethane (1C-PU) coating used for hydraulic structures using nontarget analysis via LC-QTOF-MS. Leaching behavior of the 1C-PU coating was studied using experiments in which the coating was exposed to water for defined periods (6 h, 24 h, 3 d, 14 d). Three hardening durations for the 1C-PU coating were tested (0 h, 24 h, 14 d) as well as two water matrices (ultrapure water and river water), including a successive water renewal. Dissolved organic carbon, total nitrogen bound and nontarget measurements via LC-QTOF-MS showed that under all tested conditions organic substances were leached out of the 1C-PU coating. The shorter the hardening duration and the longer the leaching duration, the higher were the number and quantities of the eluted substances, while the influence of the water matrix was minor with respect to substance elution. Based on the MS2 spectra from the LC-QTOF-MS measurements, 30 substances released from the 1C-PU coating were tentatively identified. These substances belong to five chemical groups: derivatives of (i) N-(tosyl)carbamate, (ii) p-toluenesulfonamide (PTSA), (iii) 4,4'-methylenediphenyl diisocyanate (4,4'-MDI), (iv) toluenediisocyanate (TDI), and (v) oligo(ethylene) ([C2H4O]n) as well as p-toluenesulfonic acid. The identity of seven substances was confirmed by authentic reference standards, all of which exhibited an elevated bacterial toxicity to Aliivibrio fischeri. p-Toluenesulfonic acid was present in a German canal (Teltowkanal) with concentrations of up to 11 µg L-1.

A Framework to Evaluate the Impact of Armourstones on the Chemical Quality of Surface Water.

Today, basic requirements for construction works include the protection of human health and of the environment. In the tension area between economic demands, circular flow economy and environmental safety, a link between the results from standardized leaching tests and the respective environmental quality standards must be created. To derive maximum release limits of metals and metalloids for armourstones in hydraulic engineering, this link is accomplished via a simple model approach. By treating natural materials and industrial by-products the same way, the article delivers an overview on the recent regulative situation in Europe as well as describes and discusses an innovative approach to derive maximum release limits for monolithic construction products in hydraulic engineering on a conceptual level. On a practical level, a list of test parameters is derived by connecting an extensive dataset (seven armourstone materials with five repetitions and 31 elements tested with the worldwide applied dynamic surface leaching test) with surface water quality standards and predicted no effect concentrations. Finally, the leaching tests results are compared with the envisaged maximum release limits, offering a direct comparison between natural materials and industrial by-products.

A comparative approach using ecotoxicological methods from single-species bioassays to model ecosystems.

Soft sediments are often hotspots of chemical contamination, and a thorough ecotoxicological assessment of this habitat can help to identify the causes of stress and to improve the health of the respective ecosystems. As an important component of the ecologically relevant meiobenthic fauna, nematodes can be used for sediment assessments, with various assay tools ranging from single-species toxicity tests to field studies. In the present study, microcosms containing sediment were used to investigate direct and indirect effects of zinc on natural nematode assemblages, and acute community toxicity tests considering only direct toxicity were conducted. The responses of the various freshwater nematode species in both approaches were compared with those of Caenorhabditis elegans, determined in standardized tests (ISO 10872). At a median lethal concentration (LC50) of 20 mg Zn/L, C. elegans represented the median susceptibility of 15 examined nematode species examined in the acute community toxicity tests. In the microcosms, Zn affected the nematodes dose-dependently, with changes in species composition first detected at 13 mg Zn/kg to 19 mg Zn/kg sediment dry weight. The observed species sensitivities in the microcosms corresponded better to field observations than to the results of the acute community toxicity tests. Environ Toxicol Chem 2016;35:2987-2997. © 2016 SETAC.

[Non-invasive and invasive out of hospital ventilation in chronic respiratory failure : Consensus report of the working group on ventilation and intensive care medicine of the Austrian Society of Pneumology]. / Nichtinvasive und invasive außerklinische Beatmung beim chronisch respiratorischen Versagen : Konsensus-Report des Arbeitskreises für Beatmung & Intensivmedizin der Österreichischen Gesellschaft für Pneumologie.

The current consensus report was compiled under the patronage of the Austrian Society of Pneumology (Österreichischen Gesellschaft für Pneumologie, ÖGP) with the intention of providing practical guidelines for out-of-hospital ventilation that are in accordance with specific Austrian framework parameters and legal foundations. The guidelines are oriented toward a 2004 consensus ÖGP recommendation concerning the setup of long-term ventilated patients and the 2010 German Respiratory Society S2 guidelines on noninvasive and invasive ventilation of chronic respiratory insufficiency, adapted to national experiences and updated according to recent literature. In 11 chapters, the initiation, adjustment, and monitoring of out-of-hospital ventilation is described, as is the technical equipment and airway access. Additionally, the different indications-such as chronic obstructive pulmonary diseases, thoracic restrictive and neuromuscular diseases, obesity hypoventilation syndrome, and pediatric diseases-are discussed. Furthermore, the respiratory physiotherapy of adults and children on invasive and noninvasive long-term ventilation is addressed in detail.

Benzotriazole UV stabilizers in sediments, suspended particulate matter and fish of German rivers: New insights into occurrence, time trends and persistency.

Benzotriazole UV stabilizers (BUVSs) are widely applied in plastics to prevent discoloration and to enhance product stability. This study describes for the first time the occurrence of nine different lipophilic BUVSs (UV-326, UV-320, UV-329, UV-350, UV-328, UV-327, UV-928, UV-234 and UV-360) in sediment, suspended particulate matter (SPM) and bream liver samples of German rivers. All investigated BUVSs were detected in sediments and SPM at concentrations in the low ng/g dry weight (dw) range. The so far rarely analyzed compound UV-360 as well as UV-326 were the predominant BUVSs in sediments and SPM from the river Rhine reaching maximum concentrations of 62 and 44 ng/g dw, respectively. Five BUVSs were also confirmed to bioaccumulate in bream liver, but neither UV-360 nor UV-326 was detected above the limit of quantification (LOQ). In contrast, highest concentrations in bream liver were determined for UV-327 (65 ng/g dw) and UV-328 (40 ng/g dw). A retrospective time trend analysis of BUVSs in SPM from two sites (river Rhine, 2005 to 2013; river Saar, 2006 to 2013) revealed increasing contamination levels of UV-329 and decreasing levels of UV-320 and UV-350. At one site (river Rhine) time trends of BUVS concentrations were also investigated in bream liver (1995-2013) and supported a considerably reduced exposure to UV-350. A first assessment of the environmental fate of BUVSs by sediment-water batch systems revealed a rapid partitioning into the sediment and no considerable degradation within 100 d.

Equilibrium sampling of polychlorinated biphenyls in River Elbe sediments--Linking bioaccumulation in fish to sediment contamination.

Equilibrium sampling can be applied to measure freely dissolved concentrations (cfree) of hydrophobic organic chemicals (HOCs) that are considered effective concentrations for diffusive uptake and partitioning. It can also yield concentrations in lipids at thermodynamic equilibrium with the sediment (clip⇌sed) by multiplying concentrations in the equilibrium sampling polymer with lipid to polymer partition coefficients. We have applied silicone coated glass jars for equilibrium sampling of seven 'indicator' polychlorinated biphenyls (PCBs) in sediment samples from ten locations along the River Elbe to measure cfree of PCBs and their clip⇌sed. For three sites, we then related clip⇌sed to lipid-normalized PCB concentrations (cbio,lip) that were determined independently by the German Environmental Specimen Bank in common bream, a fish species living in close contact with the sediment: (1) In all cases, cbio,lip were below clip⇌sed, (2) there was proportionality between the two parameters with high R(2) values (0.92-1.00) and (3) the slopes of the linear regressions were very similar between the three stations (0.297; 0.327; 0.390). These results confirm the close link between PCB bioaccumulation and the thermodynamic potential of sediment-associated HOCs for partitioning into lipids. This novel approach gives clearer and more consistent results compared to conventional approaches that are based on total concentrations in sediment and biota-sediment accumulation factors. We propose to apply equilibrium sampling for determining bioavailability and bioaccumulation potential of HOCs, since this technique can provide a thermodynamic basis for the risk assessment and management of contaminated sediments.

Experimental studies with nematodes in ecotoxicology: an overview.

With respect to their high abundances, their role as intermediaries between microorganisms and higher trophic levels, and their ubiquitous occurrence in all habitats, nematodes are of strong potential interest as environmental indicators. Ecotoxicological methods to evaluate the risk of anthropogenic pollutants on ecosystems require both in vitro and in vivo toxicity tests to investigate either mechanisms or pathways of toxicity and to set accurate toxicity thresholds. For this, the interest in nematodes as model organisms in ecotoxicology increased over the past few decades and existing appropriate experimental methods are reviewed in this manuscript. An overview of the various existing ecotoxicological tools for nematodes, ranging from molecular laboratory methods to experimental model ecosystem approaches, and their role as indicator organisms is given. The reviewed studies, approaches that range from species-based to community-based methods, reveal exciting possibilities for the future use of nematodes in ecotoxicological studies. Suitable ecotoxicological tools and ecological indices for nematodes should be integrated in weight-of-evidence approaches for assessing the ecological risk of contamination.

Bioaccumulation in aquatic systems: methodological approaches, monitoring and assessment.

Bioaccumulation, the accumulation of a chemical in an organism relative to its level in the ambient medium, is of major environmental concern. Thus, monitoring chemical concentrations in biota are widely and increasingly used for assessing the chemical status of aquatic ecosystems. In this paper, various scientific and regulatory aspects of bioaccumulation in aquatic systems and the relevant critical issues are discussed. Monitoring chemical concentrations in biota can be used for compliance checking with regulatory directives, for identification of chemical sources or event-related environmental risk assessment. Assessing bioaccumulation in the field is challenging since many factors have to be considered that can affect the accumulation of a chemical in an organism. Passive sampling can complement biota monitoring since samplers with standardised partition properties can be used over a wide temporal and geographical range. Bioaccumulation is also assessed for regulation of chemicals of environmental concern whereby mainly data from laboratory studies on fish bioaccumulation are used. Field data can, however, provide additional important information for regulators. Strategies for bioaccumulation assessment still need to be harmonised for different regulations and groups of chemicals. To create awareness for critical issues and to mutually benefit from technical expertise and scientific findings, communication between risk assessment and monitoring communities needs to be improved. Scientists can support the establishment of new monitoring programs for bioaccumulation, e.g. in the frame of the amended European Environmental Quality Standard Directive.

Estrogenic effects along the river Saale.

Sediments along the river Saale, one of the main tributaries of the river Elbe, were characterized with the yeast estrogen screen to elucidate possible sources of endocrine-disrupting compounds that might contribute to the downstream contamination of the river Elbe. At two sampling sites, elevated levels of estrogenic activity up to 55,000 ng ethinylestradiol equivalents per kilogram sediment dry weight were detected in the respective sediment extracts. Aliquots of the sediment extracts were analyzed for 4-nonylphenols and natural steroidal estrogens as possible candidates with an estrogenic potential. The maximal concentrations of 4-iso-nonylphenol and estrone were 115 mg/kg dry weight and 20 µg/kg dry weight at the sampling site Luppe, which showed in accordance the highest biological activity. Under consideration of compound concentration and compound specific estrogenic activity the 4-iso-nonylphenols contributed most to the observed estrogenic effect. A strong correlation between the measured estrogenic activity and the concentration of the sediment-associated 4-iso-nonylphenol underlines the relevance of this compound class as a xenoestrogen in the catchment area of the river Saale.

Effects of a bioassay-derived ivermectin lowest observed effect concentration on life-cycle traits of the nematode Caenorhabditis elegans.

The pharmaceutical ivermectin is used to treat parasitic infections, such as those caused by nematodes. While several studies have demonstrated the severe effects of ivermectin on non-target organisms, little is known about the drug's impact on free-living nematodes. In the present work, a full life-cycle experiment was conducted to estimate how an ivermectin lowest observed effect concentration derived from a Caenorhabditis elegans bioassay (endpoint reproduction) might translate into effects at the population level of this free-living nematode. The results showed that fecundity decreased to levels similar to those determined in the bioassay after a time of corresponding duration (18.6 % inhibition compared to the control), but the impact then rather weakened until the end of the experiment, at which point the net reproductive rate (R(0)) was still, but not significantly, reduced by 12.4 %. Moreover, the average lifespan, length of the reproductive period, maximum daily reproduction rate, and intrinsic rate of increase (r(m)) were significantly reduced by 30.0, 25.9, 11.2, and 3.5 %, respectively. The experiment revealed that a 4-day bioassay is protective enough for C. elegans with respect to ivermectin's effects on fecundity. However, the pronounced effects of a low drug concentration on survival, a highly elastic trait, may better account for the observed population-level response, i.e., a decrease of r(m), than the effects on fecundity. These results emphasize that full life-cycle experiments are valuable for assessment of pollutants, because the effects on several life-cycle traits can be simultaneously measured and integrated into an ecologically relevant parameter, the population growth rate, that reflects a population's response to a specific pollutant.

Sediment contact tests as a tool for the assessment of sediment quality in German waters.

A sediment contact test (SCT) battery consisting of five ecotoxicological test systems was applied to 21 native freshwater sediments characterized by a broad variety of geochemical properties and anthropogenic contamination. Higher plants (Myriophyllum aquaticum), nematodes (Caenorhabditis elegans), oligochaetes (Lumbriculus variegatus), zebrafish embryos (Danio rerio), and bacteria (Arthrobacter globiformis), representing various trophic levels and exposure pathways, were used as test organisms. The test battery detected sediment toxicity caused by anthropogenic pollution, whereas the various tests provided site-specific, nonredundant information to the overall toxicity assessment. Based on the toxicity pattern derived from the test battery, the sediments were classified according to a newly proposed classification system for sediment toxicity assessment. The SCT-derived classification generally agreed well with the application of consensus-based sediment quality guidelines (SQGs), especially with regard to sediments with high toxic potential. For sediments with low to medium toxic potential, the SQGs often underestimated the toxicity that was detected by the SCTs, underpinning the need for toxicity tests in sediment quality assessment.

A semi-fluid gellan gum medium improves nematode toxicity testing.

This study examined an alternative test medium for nematodes that use gellan gum as the gelling agent instead of agar. The semi-fluid consistency of the gel-like component nematode growth gellan gum (CNGG) supports three-dimensional distribution of the nematodes and food bacteria, but still allows free movement of the former. Moreover, flexible preparation of the medium and easy recovery of the test organisms are possible. Here, the effects of the nematicides ivermectin (pharmaceutical) and aldicarb (pesticide) and of the metal cadmium on the growth and reproduction of the free-living nematodes Caenorhabditis elegans and Panagrolaimus cf. thienemanni were studied in CNGG media. Results were compared to those obtained with the standard liquid test media in order to evaluate the applicability of CNGG for nematode toxicity testing. The sensitivity of P. cf. thienemanni to all three substances was found to be higher than that of C. elegans, but both nematodes showed the highest sensitivity to ivermectin exposure. This raises concerns about the risk posed by the pharmaceutical to non-target nematodes. In contrast to ivermectin bioassays carried out in CNGG medium, those conducted in liquid medium resulted in wide-ranging variability between and within replicates. Thus, CNGG seems to be particularly valuable for testing hydrophobic substances with a high sorption affinity as it favors their sorption to food bacteria and minimizes contact with the surfaces of the test vessels. However, the medium was less suitable for deriving toxicity thresholds for cadmium and may likewise not be an appropriate choice for testing other metals. The medium introduced herein was shown to be appropriate for sublethal nematode toxicity testing and likely provides a convenient environment for testing other nematode species. Besides improved testing of hydrophobic substances, CNGG also offers advantages for long-term studies, such as full life-cycle experiments, in which fresh medium is regularly needed. Moreover it may be beneficial for testing other poorly soluble or insoluble substances, such as nanoparticles.

Using meiofauna to assess pollutants in freshwater sediments: a microcosm study with cadmium.

The direct and indirect effects of Cd on benthic communities were assessed in a freshwater microcosm study over a period of seven months (218 d). Cadmium was regarded as a model substance to evaluate the usefulness of small-scale laboratory microcosm with microscopic fauna. In particular, effects on the meiofauna community, an ecologically important but rather neglected benthic component, were investigated. In addition, some microfaunal parameters (protozoan abundance and microbial activity) were determined. The sediment was spiked with nominal Cd concentrations of 10, 100, and 1,000 mg/kg dry weight. Because of the strong binding of Cd to sediment particles, measured Cd pore-water concentrations never exceeded 129.5 ± 40.7 µg/L. At 1,000 mg/kg dry weight, the abundances of the two dominant meiofauna taxa, nematodes and oligochaetes, were significantly reduced throughout the present study. Regarding nematodes, species of bacterivorous taxa (Daptonema, Eumonhystera) decreased, whereas species of predacious and omnivorous taxa (Mononchus, Dorylaimus, and Ironus) increased in dominance in microcosms of the highest Cd concentration. Transient effects on microfauna were observed, especially in the first half of the present study, with a reduction in microbial activity and protozoan abundance. However, in microcosms receiving the highest Cd concentration, the abundance of the flagellate Euglena mutabilis increased significantly toward the end of the present study. The results of the present study support the use of small-scale microcosms with natural meiofauna communities as a suitable tool to assess the impact of pollutants in freshwater sediments.

Assessing effects of the pharmaceutical ivermectin on meiobenthic communities using freshwater microcosms.

Ivermectin is a widely applied veterinary pharmaceutical that is highly toxic to several non-target organisms. So far, little is known about its impact on benthic freshwater species, although its rapid sorption to sediment particles and high persistence in aquatic sediments have raised concerns about the risk for benthic organisms. In the present study, indoor microcosms were used to assess the impact of ivermectin on freshwater meiobenthic communities over a period of 224 days. Microcosm sediments were directly spiked with ivermectin to achieve nominal concentrations of 0.9, 9, and 45 microg kg(-1) dw. Initially measured ivermectin concentrations (day 0) were 0.6, 6.2, and 31 microg kg(-1) dw. In addition to abundance of major meiobenthic organism groups, the nematode community was assessed on the species level, assuming a high risk for free-living nematodes due to their close phylogenetic relationship to the main target organisms of ivermectin, parasitic nematodes. Benthic microcrustaceans (cladocerans, ostracods) and nematodes showed the most sensitive response to ivermectin, while tardigrades profited from the presence of the pharmaceutical. The most pronounced effects on the meiofauna community composition occurred at the highest treatment level (31 microg kg(-1) dw), leading to a no observed effect concentration (NOEC(Community)) of 6.2 microg kg(-1) dw. However, the nematode community was already seriously affected at a concentration of 6.2 microg kg(-1) dw with two bacterivorous genera, Monhystera and Eumonhystera, being the most sensitive, whereas species of omnivorous genera (Tripyla, Tobrilus) increased in abundance after the application of ivermectin. Thus, a NOEC(Community) of 0.6 microg kg(-1) dw was derived for nematodes. Direct and indirect effects of ivermectin on meiobenthic communities could be demonstrated. The pharmaceutical is likely to pose a high risk, because its NOECs are close to predicted environmental concentrations (PECs) in sediments (0.45-2.17 microg kg(-1) dw), resulting in worst case risk quotients (RQs) of 1.05-36.2. This observation lends support to efforts aimed at preventing the repeated entry of ivermectin in aquatic environments and thus its accumulation in sediments. Moreover, this study points out that model ecosystem studies should be part of environmental risk assessments (ERAs) of veterinary medicinal products (VMPs).

Integral assessment of estrogenic potentials in sediment-associated samples: Part 2: Study of estrogen and anti-estrogen receptor-binding potentials of sediment-associated chemicals under different salinity conditions using the salinity-adapted enzyme-linked receptor assay.

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-beta-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 alpha (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-beta-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-beta-estradiol, the ELRA showed sigmoid concentration-effect relations over a broad range from 0.05 mug/l to 100 mug/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 per thousand. 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-beta-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 per thousand. 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.

Integral assessment of estrogenic potentials of sediment-associated samples. Part 1: The influence of salinity on the in vitro tests ELRA, E-Screen and YES.

GOAL, SCOPE AND BACKGROUND: Exogenic endocrine-active substances are also called 'Endocrine Disrupting Chemicals' (EDC). They imitate or hinder the function of natural endogenic hormones or disturb the synthesis or the metabolism of hormones or of hormone receptors. The Enzyme-Linked Receptor Assay (ELRA) can detect estrogenic and anti-estrogenic effects at the level of receptor binding and is a useful tool for the integrative detection of contaminant effects. Although the test system has been used repeatedly in sediment assessments, the questions have remained concerning how it responds to variations in the physico-chemical matrix. For some bioassays, the salinity of the sample is a critical factor. This is especially relevant when testing wastewater samples or when sediment-associated samples in the tidal reaches of rivers are tested. Sediments in the tidal reaches of rivers change their salinity several times a day. Against this background, it would be beneficial to have a test procedure of known salinity tolerance. On account of this, the salinity tolerance of the ELRA was tested, assessed with reference substances at several salinity levels, and compared with the E-Screen method and a Yeast Estrogen Screen (YES), which are also frequently applied in environmental testing. The aim of this paper was to explore when the salinity limits within these test procedures are applicable. The trials should reveal the working range to be expected, characterize the salinity-dependent variations in sensitivity of the test, and provide options for methodological adjustments to improve the stability against increased salinity. METHODS: The ELRA was carried out with the human Estrogen Receptor alpha. (ER) using the same principle like a competitive immunoassay based on ligand-protein interaction. However, an essential difference is the use of a physiologically relevant receptor instead of an antibody as a linking protein. The ELRA measures the competition of sample estrogens and anti-estrogens against estradiol supplied as a BSA-coating conjugate for the binding site of dissolved ER. Estradiol or xeno-estrogen binding is quantified by a biotynilated anti-ER antibody and the subsequent measurement of peroxidase activity by a streptavidin-POD-biotin complex. The E-Screen was performed with the human breast cancer cell line MCF-7, which expresses the estrogen receptor constitutively. Cell proliferation depends on binding of estrogens or xeno-estrogens with the receptor. After incubation, estrogen-dependent cell growth was measured by sulforhodamin B staining. The YES was performed with a recombinant yeast strain, transfected with a receptor and a reporter plasmid bearing the estrogen receptor and a vitellogenin gene fused with the reporter gene lacZ. Estrogen or xeno-estrogen-dependent gene induction was measured indirectly by LacZ activity. The salinity levels were simulated in varying concentrations with NaCl from 0 to 40 per thousand or Artificial Sea Water (ASW) from 0 to 32 per thousand. RESULTS: The study characterized the factor 'salinity' for the prospective application fields of the ELRA. With reference substances such as 17-beta-estradiol, the ELRA showed classical sigmoidal concentration-effect relations in a range from 0.05 to 100 microg/l under physiological conditions. After a methodological adjustment to compensate decreasing receptor-binding affinity of estrogens and xeno-estrogens at higher salinity levels, the ELRA became applicable under salinity conditions up to concentrations of 20.5 per thousand. In tests, the ELRA reached under the influence of salinity a mean limit of detection of 0.062 microg/l 17-beta-estradiol. The mean relative inter-test error was around 11%. Above concentrations of 20.5 per thousand there is a risk of false negative assessment. Compared with the E-Screen method using the MCF7 cell line and the yeast estrogen test system (YES), the ELRA shows a lower sensitivity to 17-beta-estradiol. In the E-Screen, the cell proliferation was strongly reduced by sodium chloride induced cytotoxicity. In comparison with the E-Screen, the salinity tolerance of the YES and YAS methods is significantly higher. DISCUSSION: Despite adaption, total salinity tolerance could not be achieved with the ELRA. Freshwater samples were generally appraisable. Higher salinity levels above 20.5 per thousand would tend towards false negative results. The low inter-test error of 11% makes the ELRA suitable for the detection of estrogenic and anti-estrogenic potentials of single substances, substance mixtures, and of environmental samples. CONCLUSIONS: The ELRA is very fast and reproducible, it can be used for high-throughput screening in a microplate format at low cost, it is robust to microbial contamination, and is less susceptible to cytotoxic interferences than cell culture methods. RECOMMENDATIONS AND PERSPECTIVES: In their established form, the YES and the E-Screen methods are not applicable for liquid phase testing at higher salinity conditions. The salinity-adapted test version of the ELRA described here shows a broader working range for samples. Native water samples of more or less brackish origin or high-salinity effluent samples are testable. Results of tests with sediment associated samples of different salinity will be subject of a forthcoming publication.