Target-site sensitivity in a specialized herbivore towards major toxic compounds of its host plant: the Na+K+-ATPase of the oleander hawk moth (Daphnis nerii) is highly susceptible to cardenolides

The caterpillars of the oleander hawk moth, Daphnis nerii (Linnaeus, 1758) (Lepidoptera: Sphingidae) feed primarily on oleander (Nerium oleander). This plant is rich in cardenolides, which specifically inhibit the Na⁺K⁺-ATPase. Since some insects feeding on cardenolide plants possess cardenolide-resistant Na⁺K⁺-ATPases, we tested whether D. nerii also possesses this strategy for circumventing cardenolide toxicity. To do so, we established a physiological assay, which allowed direct measurement of Na⁺K⁺-ATPase cardenolide sensitivity. Using Schistocerca gregaria, as a cardenolide-sensitive reference species, we showed that D. nerii Na⁺K⁺-ATPase was extremely sensitive to the cardenolide ouabain. Surprisingly, its sensitivity is even higher than that of the cardenolide-sensitive generalist, S. gregaria. The presence or absence of cardenolides in the diet of D. nerii did not influence the enzyme’s cardenolide sensitivity, indicating that target-site insensitivity is not inducible in this species. However, despite the sensitivity of their Na⁺K⁺-ATPase, caterpillars of D. nerii quickly recovered from an injection of an excessive amount of ouabain into their haemocoel. We conclude that D. nerii possesses adaptations, which enable it to feed on a cardenolide-rich diet other than that previously described in cardenolide specialized insects, and discuss other potential resistance mechanisms.     

Main differences on European regulations in the frame of the Seveso Directive

Since the first Seveso Directive was issued 25 years ago, each Member State of the European Union developed own regulations to enforce its application. The lack of a common approach generated differences between Member States, or even between regions within a Member State, which in practical terms caused that establishments falling under the field of application of the Seveso Directive face a different level of requirements. This situation causes significantly different impact on social, technical and financial aspects.Factors like industrial background, accidental history, actual land use, social attitude towards risk, economical and political structures play an important role in how the decisions about hazardous activities are perceived and handled.Answering the question: “Is there a right methodology for the assessment of the risk of major accidents?” may be sterile or impossible. Many tools are appropriate and the main concern is to use them properly. Technically, the causes and consequences of, e.g., a fire in a port in Spain on the Mediterranean Sea or in the port of Basel (Switzerland) in the Rhine River could be assessed using the same methods and criteria, but this is not usually the case.This paper shows, with practical examples, the differences existing while applying different regulations in the field of prevention of major accidents and the potential effects that these differences may generate regarding the definition and management of emergency plans outside establishments.If the existing unequal situation is not faced, the comparative disagreements between Member States will prevail. Therefore, the application of the Seveso Directive will generate a different impact on the society, operators and regulators, and may become a factor which may cause a risk transfer between Member States. Efforts should be addressed to harmonize criteria in this subject.     

Determinants of brominated flame retardants in breast milk from a large scale Norwegian study

Brominated flame retardants (BFRs), particularly polybrominated diphenyl ethers (PBDEs), are widely present in human populations. In order to investigate human exposure pathways and associations with socioeconomic and lifestyle factors, 393 breast milk samples from mothers living in various regions throughout Norway were analyzed. Up to ten PBDE congeners were measured in all the samples, hexabromocyclododecane (HBCD) and BDE-209 in subsets of 310 and 46, respectively. The median concentrations of the sum of the seven most prominent PBDEs (BDE-28, 47, 99, 100, 153, 154 and 183), BDE-209 and HBCD were 2.1, 0.32 and 0.86 ng/g lipids, respectively. These concentrations are comparable to the levels generally observed in human populations in Europe. The frequency distributions were quite skewed with long tails towards higher concentrations. Maternal age, parity, education, having a cohabitant employed as electrician, and ventilation were factors significantly associated with some of the BFRs, although these factors only explained a small amount of the variability (R² 0.04–0.16). The mothers' diet was not found to influence the breast milk PBDE and HBCD levels. Our results show that sources other than the diet are important for the variability seen in breast milk BFR concentrations and that exposure from the indoor atmosphere should be emphasized in future studies.     

Origin of middle rare earth element enrichment in acid mine drainage-impacted areas

The commonly observed enrichment of middle rare earth elements (MREE) in water sampled in acid mine drainage (AMD)-impacted areas was found to be the result of preferential release from the widespread mineral pyrite (FeS₂). Three different mining-impacted sites in Europe were sampled for water, and various pyrite samples were used in batch experiments with diluted sulphuric acid simulating AMD-impacted water with high sulphate concentration and high acidity. All water samples independent on their origin from groundwater, creek water or lake water as well as on the surrounding rock types showed MREE enrichment. Also the pyrite samples showed MREE enrichment in the respective acidic leachate but not always in their total contents indicating a process-controlled release. It is discussed that most probably complexation to sulphite (SO₃ ^2?) or another intermediate S-species during pyrite oxidation is the reason for the MREE enrichment in the normalized REE patterns.     

Polycyclic aromatic hydrocarbon (PAH) contamination of surface sediments and oysters from the inter-tidal areas of Dar es Salaam, Tanzania

Surface sediment and oyster samples from the inter-tidal areas of Dar es Salaam were analyzed for 23 polycyclic aromatic hydrocarbons (PAHs) including the 16 compounds prioritized by US-EPA using GC/MS. The total concentration of PAHs in the sediment ranged from 78 to 25,000 ng/g dry weight, while oyster concentrations ranged from 170 to 650 ng/g dry weight. Hazards due to sediment contamination were assessed using Equilibrium Partitioning Sediment Benchmarks and Threshold Effect Levels. Diagnostic indices and principle component analysis were used to identify possible sources. Interestingly, no correlation between sediment and oyster concentrations at the same sites was found. This is supported by completely different contamination patterns, suggesting different sources for both matrices. Hazard assessment revealed possible effects at six out of eight sites on the benthic communities and oyster populations. The contribution of PAH intake via oyster consumption to carcinogenic risks in humans seems to be low.     

Reproduction and survival of tawny owls in relation to persistent organic pollutants

The potential effects of organochlorines (OCs) and brominated flame retardants on reproduction and survival were studied in tawny owls (Strix aluco) in Central Norway over a period of 19 years (1986-2004). Concentrations of 14 OCs and five polybrominated diphenyl ethers (PBDEs) were measured in eggs (n=104), and Principal Component Analysis was used to produce composite measurements of pollutants; i.e. PC1 and PC2, which accounted for 85% of the variation in contaminant concentrations. There was no evidence for adverse associations between pollutants (PC scores) and life-history traits such as clutch size, probability of producing fledglings and survival, when controlling for potentially confounding variables. Moreover, there was no evidence for interactions between pollutants and vole abundance suggesting no synergistic effects of food stress and pollutants on these life-history traits. There was, however, some evidence for a non-linear negative association between p,p'-DDE (1,1-dichloro-2,2-bis (p-chlorophenyl) ethylene) and eggshell thickness. This suggests that the concentrations of pollutants in this ecosystem were too low to affect reproduction and survival in an owl predominantly consuming prey at low trophic levels, but may be sufficient to cause eggshell thinning at the highest concentrations.     

Characterisation of human exposure pathways to perfluorinated compounds--comparing exposure estimates with biomarkers of exposure

Commercially used per- and polyfluorinated compounds (PFCs) have been widely detected in humans, but the sources of human exposure are not fully characterized. The objectives of this study were to assess the relative importance of different exposure pathways of PFCs in a group of Norwegians and compare estimated intakes with internal doses obtained through biomonitoring. Individual PFC intakes from multiple exposure sources for a study group of 41 Norwegian women were estimated using measured PFC concentrations in indoor air and house dust as well as information from food frequency questionnaires and PFC concentrations in Norwegian food. Food was generally the major exposure source, representing 67-84% of the median total intake for PFOA and 88-99% for PFOS using different dust ingestion rates and biotransformation factors of 'precursor' compounds. However, on an individual basis, the indoor environment accounted for up to around 50% of the total intake for several women. Significant positive associations between concentrations of PFCs in house dust and the corresponding serum concentrations underline the importance of indoor environment as an exposure pathway for PFCs. For breast-fed infants, breast milk was calculated to be the single most important source to PFCs by far. The estimated intakes were confirmed by comparing serum concentrations of PFOA and PFOS calculated using PK models, with the corresponding concentrations measured in serum. Even though food in general is the major source of exposure for PFCs, the indoor environment may be an important contributor to human exposure. This study provides valuable knowledge for risk assessment of PFCs and control strategies.     

Evidence for life history changes in high-altitude populations of three perennial forbs

Relatively little is known about how the life histories of perennial forb species, and especially their lifetime patterns of growth, vary across environmental gradients. We used a post hoc approach (herb-chronology) to determine plant age and previous growth (width of successive annual rings in roots) in three species of perennial forb (two long-lived species [Penstemon venustus, Lupinus laxiflorus] and one short-lived [Rudbeckia occidentalis]) along a 1000-m altitudinal gradient in the Wallowa Mountains (northeast Oregon, USA). Plants from the highest altitude tended to be considerably older and produced up to five times as many flowering shoots as lowland plants. In addition, mean ring widths of high-altitude plants were about half those of lowland plants. In plants from low and intermediate altitudes, ring width either decreased linearly or varied inconsistently during the life of the plant. In contrast, ring widths of high-altitude plants increased at first and later decreased, resulting in curvilinear growth trajectories that were highly consistent among species. Together, these data for three ecologically distinct forb species provide evidence of a consistent shift toward more conservative and strongly constrained life histories at higher altitudes. More generally, the results indicate the possible importance of changes in selection pressures across strong environmental gradients on life history strategies within a single species.     

Integral assessment of estrogenic potentials of sediment-associated samples

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.