Biotoxizit?tstests mit PAK Die Nodulation in der Leguminosen-Rhizobien-Symbiose als sensibler Wirkungsparameter

Im Zusammenhang mit der Umweltchemikalienprüfung und der Untersuchung und Kontrolle kontaminierter B?den besteht gro?er Bedarf an Testverfahren mit h?heren Pflanzen, die schnell, einfach und preisgünstig durchzuführen sind und bereits bestehende Testverfahren nach OECD- und BBA-Richtlinien sinnvoll erg?nzen k?nnen. Dieser Beitrag stellt einen Biotoxizit?tstest vor, der die Nodulation in der Leguminosen-Kn?llchen-Symbiose als sensiblen Wirkungsparameter für die Belastung mit polyzyklischen aromatischen Kohlenwasserstoffen (PAK) benutzt. Fremdstoff, Pflanze und Mikroorganismus werden dabei auf einer festen Matrix in Kontakt miteinander gebracht; das axenische System erlaubt die Untersuchung der Fremdstoffwirkung sowohl auf den Gesamtorganismus Pflanze, als auch auf die Interaktion von Pflanze und Mikroorganismus (Kn?llchenbildung). In Dose-Response-Versuchen zeigte sich der Wirkungsparameter Nodulation, gemessen als Anzahl Wurzelkn?llchen pro Pflanze, sensibler als andere pflanzliche Wachstumsparameter. There is great need for toxicity tests with higher plants for the investigation and monitoring of chemicals in the environment — especially for tests which are quick, easy, and inexpensive. Such tests should add to and improve current guidelines, e.g., OECD- and BBA-Guidelines. This article describes the nodulation of leguminous plants withRhizobium bacteria used in a bioassay with polycyclic aromatic hydrocarbons (PAH). Plants growing on a solid medium were exposed simultaneously to bacteria and PAH. This axenic culture system allows to observe the impact of PAH on plant growth and the interaction between plant and bacteria (nodulation). Dose-response-experiments indicated that nodulation measured as number of nodules per plant, was more sensitive to the inhibition by PAH than other plant growth parameters.     

Chapter three: methodology of exposure modeling

In this chapter, the concept of exposure assessment and its evolution is introduced, and evaluated by critically appraising the pertinent literature as it applies to exposures to Particulate Matter (PM). Exposure measurement or estimation methodologies and models are reviewed. Three exposure/measurement methodologies are assessed. Estimation methods focus on source evaluation and attribution, sources include those outdoors and indoors as well as in occupational and in-transit environments. Fate and transport models and their inputs are addressed to estimate concentrations outdoors and indoors; source attribution techniques help focus on the contributing sources. Activity pattern techniques are also reviewed and their use in exposure models to estimate inhalation exposure to PM is presented. Deterministic, regression and other stochastic models of exposure to PM are reviewed and evaluated. Strengths, limitations, assumptions and affirmations of the use of exposure assessment as an integral component of risk assessment and risk management are discussed in the conclusions and discussions section of this work.     

Endocrine disruption of water and sediment extracts in a non-radioactive dot blot/RNAse protection-assay using isolated hepatocytes of rainbow trout

Goal, Scope and Background In order to evaluate the estrogenic activity of sediments and XAD water extracts of selected sites of the catchment area of the River Neckar, a river system in Southern Germany, an integrative assessment approach was used to assess the ecological hazard potential of endocrine-disrupting compounds in sediment and water. Methods The approach is based on estrogen receptor-mediated vitellogenin synthesis induced in isolated hepatocytes of rainbow trout and quantified in a non-radioactive dot blot/RNAse protection-assay in parallel to comprehensive chemical analyses of estrogenic substances. Results and Discussion Numerous investigated extracts revealed an estrogen activity comparable to that of the positive control (1 nM 17?-estradiol corresponding to 270 ng/L in the test medium). Based on a concentration factor of 30 in the extracts and a recovery of XAD resins of approximately 80 %, 17?-estradiol equivalent concentrations between 20 and 26.7 ng/L could be calculated downstream of a sewage treatment plant (< 0.1 ng/L for a reference site). A comparison of the bioassay-derived Bio-TEQs (toxicity equivalents) and the Chem-TEQs revealed a high correlation with a Pearson coefficient of 0.85, indicating that the same ranking of the samples could be obtained with respect to the endocrine disrupting potential with both chemical and bioanalytical analysis. However, the TEQ concentrations computed from chemical analyses were significantly lower than the bioassay-derived TEQ concentrations. In fact, in none of the samples, more than 14 % of the vitellogenin-inducing potency could be attributed to the substances (steroids, alkylphenols, bisphenol A, diethylstilbestrol) analyzed. A comparison of the endocrine disrupting potential of sediments extracted by the solvents acetone and methanol revealed lower biological effects for acetone-extracted samples. Possible reasons may be a masking of endocrine effects in acetone extracts by cytotoxicity, a low extraction efficiency of the solvent acetone, or anti-estrogen potencies of some extracted sediment compounds. Using a mass balance approach, the contribution of the compounds analyzed chemically (Chem-TEQs) to the total endocrine activity (Bio-TEQs) was calculated. Based on the very low detection limits, particularly of the steroids with their high TEF factors, results revealed that a calculation of the Chem-TEQs is associated with considerable scale inaccuracy: Whereas only 7-15 % of the biological effectiveness (Bio-TEQs) could be explained by endocrine substances identified above the detection limits, the assumption of concentrations slightly below the given detection limits would result in a significant over estimation (137-197 %) of the Bio-TEQs. Even the interassay variation of the dot blot assay with different fish donors for primary hepatocyte (factor 2 - 2.5) is relatively low, when compared to the large range of the Chem-TEQ concentrations (factor 20) obtained when applying different modes of calculation. Conclusions and Outlook Overall, only a minor portion of the endocrine activity detected by bioassays could be linked to compounds identified by chemical analysis. In vitro assays for assessment of endocrine activities are useful as sensitive integrating methods that provide quantitative estimates of the total activity of particular receptor-mediated responses. Although discrepancies may also result from different bioanalytical approaches, it is overall likely that bioanalytical and not chemical analytical approaches give the correct estimate of endocrine disrupting potencies in environmental samples. As a conclusion, assessment of endocrine disruption based on chemical analysis alone does not appear sufficient and further research into the spectrum of substances with potential endocrine activity as well as into additive or even synergistic effects in complex environmental samples is urgently needed.     

Uptake and metabolism of 2,4,6-trinitrotoluene in higher plants

The fate of the explosive 2,4,6-TNT in plants is of major interest. Therefore, a method was developed to analyse TNT and derivatives in plant tissue. The method was utilized to investigate the uptake and metabolism of TNT inMedicago sativa andAllium schoenoprasum grown in hydroponic cultures containing TNT levels of 0.1 to 10 mg/1. Detectable concentrations of nitrotoluenes were significantly higher inAllium schoenoprasum than inMedicago sativa. The uptake of TNT in plants was directly related to the initial TNT level. The principal nitroaromatic components in roots and shoots of both plant species were identified as 4-ADNT and 2-ADNT in equal amounts, with substantially less TNT.     

Persistent organic pollutants in atmospheric deposition and biomonitoring with Tillandsia usneoides (L.) in an industrialized area in Rio de Janeiro state, southeast Brazil--Part II: PCB and PAH

Monitoring of immission of persistent organic pollutants in the industrialized area of Volta Redonda (V.R.) and in the National Park of Itatiaia (PNI) in southeast Brazil was performed using an endemic bromeliad species as biomonitor and measuring bulk deposition rates of polychlorinated biphenyls (PCB) and polycyclic aromatic hydrocarbons (PAH). For the sum of PCB, overall deposition rates were between 17 and 314 ng/(m2 day) in winter and between 43 and 81 ng/(m2 day) in summer, respectively. Deposition rates of dioxin-like PCBs ranged from 0.14 to 2.8 pg WHO-TEQ/(m2 day) in winter and from 0.90 to 4.3 pg WHO-TEQ/(m2 day) in summer. PCB deposition rates (total PCB and WHO-TEQ) were in the same range in winter in V.R. and PNI. In summer, contamination levels in V.R. were 6-10-folds higher than in PNI. PCB concentrations in biomonitor samples from V.R. and PNI were in the same range in summer and in winter. Concentrations of total PCB ranged from 14 to 95 microg/kg dry matter (d.m.) in winter and from 18 to 27 microg/kg d.m. in summer, respectively. The TEQ values were between 1.7 and 4.1 ng WHO-TEQ/kg d.m. in winter and between 1.9 and 2.9 ng WHO-TEQ/kg d.m. in summer. PCB concentrations of di-ortho PCB but not of non-ortho PCB were a factor of 2-4 lower in summer in both areas. PCB congener profiles resembled those from technical formulations. The profiles shifted to the higher chlorinated congeners in summer, probable due to revolatilisation of the lighter components at higher temperatures. PCB profiles in biomonitor resembled those from deposition samples and the shift to the heavier congeners in summer was even more pronounced. PAH deposition rates were in a similar range in both areas (131-2415 ng/(m2 day)). PAH levels in biomonitor samples from V.R. were about one order of magnitude higher than in samples from PNI indicating the impact of local sources. PAH profiles revealed stationary thermal processes as main source of contamination in V.R. whereas in PNI, biomass burning seems to be the main contamination source.     

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.     

Geochemistry and bioavailability of metals in sediments from northern San Francisco Bay

In this study, metals (Be, Cr, Mn, Fe, Ni, Cu, Zn, Ag, Cd, Pb and Hg) in the fine-grained fraction (<63 microm) from 12 sites at different locations in northern San Francisco Bay over a year period from March 2000 to March 2001 were analyzed after acid extraction. The results showed that metal concentrations in the sediments varied from site to site, whereas some of them were found elevated with respect to the sediment of Tomales Bay, CA, which has little contamination history, indicating an enrichment of the metals in the sediment samples analyzed. Sediment toxicity and bioaccumulation evaluation by a clam species, Macoma nasuta, exposed to the sediment samples collected from the six sampling sites was carried out. The results showed that the sediment samples tested significantly reduced clam survival. Toxicity of the sediments to the clam was, in part, related to elevated metal concentrations in the sediments. In order to examine geochemistry of the metals and to understand potential correlations between metal concentrations and geochemical matrix elements of the sediments, bioavailability and toxicity of the metals, detailed analysis of metal concentrations associated with total organic carbon and the Fe-oxy-hydroxides in the sediment samples was performed. The analysis showed that sediment geochemistry appeared to influence metal bioavailability and may have important impacts on the toxicity of these metals to the clam.     

Methane and nitrous oxide emissions from a subtropical coastal embayment (Moreton Bay, Australia)

Surface water methane (CH₄) and nitrous oxide (N₂O) concentrations and fluxes were investigated in two subtropical coastal embayments (Bramble Bay and Deception Bay, which are part of the greater Moreton Bay, Australia). Measurements were done at 23 stations in seven campaigns covering different seasons during 2010-2012. Water-air fluxes were estimated using the Thin Boundary Layer approach with a combination of wind and currents-based models for the estimation of the gas transfer velocities. The two bays were strong sources of both CH₄ and N₂O with no significant differences in the degree of saturation of both gases between them during all measurement campaigns. Both CH₄ and N₂O concentrations had strong temporal but minimal spatial variability in both bays. During the seven seasons, CH₄ varied between 500% and 4000% saturation while N₂O varied between 128 and 255% in the two bays. Average seasonal CH₄ fluxes for the two bays varied between 0.5 ± 0.2 and 6.0 ± 1.5 mg CH₄/(m²·day) while N₂O varied between 0.4 ± 0.1 and 1.6 ± 0.6 mg N₂O/(m²·day). Weighted emissions (t CO₂-e) were 63%-90% N₂O dominated implying that a reduction in N₂O inputs and/or nitrogen availability in the bays may significantly reduce the bays' greenhouse gas (GHG) budget. Emissions data for tropical and subtropical systems is still scarce. This work found subtropical bays to be significant aquatic sources of both CH₄ and N₂O and puts the estimated fluxes into the global context with measurements done from other climatic regions.     

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.     

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.