Including non-dietary sources into an exposure assessment of the European Food Safety Authority: The challenge of multi-sector chemicals such as Bisphenol A.

In the most recent risk assessment for Bisphenol A for the first time a multi-route aggregate exposure assessment was conducted by the European Food Safety Authority. This assessment includes exposure via dietary sources, and also contributions of the most important non-dietary sources. Both average and high aggregate exposure were calculated by source-to-dose modeling (forward calculation) for different age groups and compared with estimates based on urinary biomonitoring data (backward calculation). The aggregate exposure estimates obtained by forward and backward modeling are in the same order of magnitude, with forward modeling yielding higher estimates associated with larger uncertainty. Yet, only forward modeling can indicate the relative contribution of different sources. Dietary exposure, especially via canned food, appears to be the most important exposure source and, based on the central aggregate exposure estimates, contributes around 90% to internal exposure to total (conjugated plus unconjugated) BPA. Dermal exposure via thermal paper and to a lesser extent via cosmetic products may contribute around 10% for some age groups. The uncertainty around these estimates is considerable, but since after dermal absorption a first-pass metabolism of BPA by conjugation is lacking, dermal sources may be of equal or even higher toxicological relevance than dietary sources.

Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase I).

We have developed a new in vitro skin irritation test based on an open source reconstructed epidermis (OS-REp) with openly accessible protocols for tissue production and test performance. Due to structural, mechanistic and procedural similarity, a blinded catch-up validation study for skin irritation according to OECD Performance Standards (PS) was conducted in three laboratories to promote regulatory acceptance, with OS-REp models produced at a single production site only. While overall sensitivity and predictive capacity met the PS requirements, overall specificity was only 57%. A thorough analysis of the test results led to the assumption that some of the false-positive classifications could have been evoked by volatile skin-irritating chemicals tested in the same culture plate as the non-irritants falsely predicted as irritants. With GC/MS and biological approaches the cross-contamination effect was confirmed and the experimental set-up adapted accordingly. Retesting of the affected chemicals with the improved experimental set-up and otherwise identical protocol resulted in correct classifications as non-irritants. Taking these re-test results into account, 93% overall sensitivity, 70% specificity and 82% accuracy was achieved, which is in accordance with the OECD PS. A sufficient reliability of the method was indicated by a within-laboratory-reproducibility of 85-95% and a between-laboratory-reproducibility of 90%.

Prevalidation of the ex-vivo model PCLS for prediction of respiratory toxicity.

In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1h. After 23 h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For all endpoints IC50 values were calculated if feasible. For each endpoint test acceptance criteria were established. This report provides the final results for all 20 chemicals. More than 900 concentration-response curves were analyzed. Log10[IC50 (µM)], obtained for all assay endpoints, showed best intra- and inter-laboratory consistency for the data obtained by WST-1 and BCA assays. While WST-1 and LDH indicated toxic effects for the majority of substances, only some of the substances induced an increase in extracellular IL-1α. Two prediction models (two-group classification model, prediction of LC50 by IC50) were developed and showed promising results.

Approaching the evolutionary advantage of ancillary types of haemoglobin in Daphnia magna by simulation of oxygen supply.

The planktonic crustacean Daphnia magna synthesizes haemoglobin (Hb) macromolecules of variant subunit composition and oxygen affinity. This is one of the strategies by which the animals cope with variations in environmental conditions such as ambient oxygen tension. The enrichment of high-affinity Hb molecules in the haemolymph of hypoxia-exposed animals is thought to reduce Hb synthesis costs due to an enhanced transport efficiency of these molecules in comparison to the low-affinity Hb molecules. How great this economic advantage is, and under which conditions this benefit disappears, is still not fully understood. Here we implemented a rigorously simplified model of the daphnid body and described the transport of oxygen from the environment via the haemolymph to the tissues in terms of the convection-diffusion-reaction equation. The model was validated by comparing various model predictions with experimental data. A sensitivity analysis was used to evaluate the influence of parameter uncertainties on the model predictions. Cost-benefit analysis revealed in which way at the system's level the increase in Hb oxygen affinity improves the oxygen loading at the respiratory surfaces and impairs the release of oxygen to the tissues. The benefit arising from the improved oxygen loading exceeds the disadvantage of impaired unloading only under conditions where the ambient oxygen tension is critically low and the Hb concentration is high. The low-affinity Hb, on the other hand, provides an advantage given that the Hb concentration is low and the ambient oxygen tension is well above the critical level. Computer-aided modelling and simulation therefore provide valuable mechanistic insights into the driving forces that could have shaped the evolution of globin genes in daphnids.

Effects of hypoxia acclimation on morpho-physiological traits over three generations of Daphnia magna.

The mechanisms, dynamics and effects of hypoxia acclimation were studied in the water flea Daphnia magna over three successive generations (parental, first and second filial generation: P, F1 and F2). The P generation was raised under normoxic conditions at 20 degrees C and became exposed to environmental hypoxia (10-19% air saturation) at maturity. Their progenies (F1 and F2) experienced hypoxia from birth onwards. Controls were kept under normoxic conditions. Individuals were successively sampled in a 3-day interval from each acclimation group to determine morpho-physiological parameters relevant in oxygen transport and regulation. Hypoxia acclimation induced adjustments at the haemoglobin (Hb) and metabolic level (within 3 days) but none at the systemic level. The convective performance and oxygen-sensitive control of the ventilatory and circulatory systems were the same in both acclimation groups. The Hb concentration and oxygen affinity increased by 266% and 32%, respectively. The 22% decrease in mass-specific oxygen consumption rate reduced the energy allocation to somatic growth without greatly affecting reproduction. The onset and duration of hypoxic exposure during ontogenesis have had a significant influence on Hb oxygen affinity and body size. Transgenerational effects of hypoxia acclimation could not be observed. The adjustments at the Hb and metabolic levels in combination with the smaller body size, which is advantageous to diffusive oxygen transport, reduced the critical ambient oxygen tension by approximately 50%.

Crater landscape: two-dimensional oxygen gradients in the circulatory system of the microcrustacean Daphnia magna.

Oxygen transport processes in millimetre-sized animals can be very complex, because oxygen molecules do not exclusively follow the pathway predetermined by the circulating fluid but may also simultaneously move from the respiratory surfaces to the tissues along different paths by diffusion. The present study made use of the oxygen-sensitive phosphorescence probe Oxyphor R2 to analyze the internal oxygen pathway in the transparent microcrustacean Daphnia magna. Oxyphor R2 was injected into the circulatory system and the distribution of oxygen partial pressure (P(O(2))) in the haemolymph was measured by phosphorescence lifetime imaging in the P(O(2)) range 0-6 kPa (0-30% air saturation). There were substantial differences in the shape of the two-dimensional P(O(2)) profiles depending on the concentration of haemoglobin (Hb) in the haemolymph. A steep global gradient, from posterior to anterior, occurred in animals with low concentrations of Hb (90-167 micromol l(-1) haem). In contrast, animals with a five- to sixfold higher concentration of Hb showed flat internal P(O(2)) gradients which, however, were only present under reduced ambient oxygen tensions (P(O(2)amb)=3-1 kPa), when Hb was maximally involved in oxygen transport. Under these conditions, the presence of Hb at high concentrations stabilized the unloading P(O(2)) in the central body to 0.9-0.4 kPa. Independent of Hb concentration and body size, the loading P(O(2)) was always 0.5 kPa below the P(O(2)amb). From these P(O(2)) profiles, it was possible (i) to follow the track of oxygen within the animal, and (ii) to visualize the shift from a diffusion-dominated to a convection-dominated transport as a result of increased Hb concentration.

Control of oxygen transport in the microcrustacean Daphnia: regulation of haemoglobin expression as central mechanism of adaptation to different oxygen and temperature conditions.

The pathway for oxygen, the control of oxygen transport and the role of haemoglobin expression for the physiological adaptation to different oxygen and temperature conditions were studied in the ecological model organism Daphnia magna. Ventilation of the inner walls of the carapace as the main gas exchange area as well as of the embryos in the brood pouch are controlled, oxygen-dependent processes. The P(O2)-dependent increase of heart rate as well as perfusion rate during short-term, progressive hypoxia improves the circulatory oxygen transport within the body. The regulation of haemoglobin (Hb) expression is the central mechanism for a medium-term adaptation to hypoxia. Genetic control elements and oxygen conditions near the two Hb synthesis sites (fat cells, epipodite epithelial cells) determine, which types of Hb subunits and, accordingly, hetero-multimeric Hb macromolecules are produced. One synthesis site may respond mainly to internal, the other one to external oxygen conditions. Depending on environmental condition, either higher quantities of macromolecules of unchanged functionality (P50) or increasing amounts of macromolecules with higher oxygen affinity are synthesized. The Hb subunit DmHbA is probably of considerable importance for this functional change. The physiological benefits of haemoglobin in Daphnia are discussed. Physiological adaptation of Daphnia to different temperatures is also related to the control of oxygen transport processes with the regulation of haemoglobin expression again as a central mechanism.

The dichotomous oxyregulatory behaviour of the planktonic crustacean Daphnia magna.

The dual function of appendage movement (food acquisition, ventilation) proved to be the key to explaining the peculiar oxyregulatory repertoire of the planktonic filter feeder Daphnia magna. Short-term hypoxic exposure experiments with normoxia-acclimated animals under varying food concentrations revealed a dichotomous response pattern with a compensatory tachycardia under food-free conditions and a ventilatory compensation prevailing under food-rich conditions. Food-free, normoxic conditions resulted in maximum appendage beating rates (fA) and half-maximum heart rates (fH), which restricted the scope for oxyregulation to the circulatory system. Food-rich conditions (10(5) algal cells ml(-1)), on the contrary, had a depressing effect on fA whereas fH increased to 83% of the maximum. In this physiological state, D. magna was able to respond to progressive hypoxia with a compensatory increase in ventilation. A conceptual and mathematical model was developed to analyse the efficiency of ventilatory and circulatory adjustments in improving oxygen transport to tissue. Model predictions showed that an increase in perfusion rate was most effective under both food-free and food-rich conditions in reducing the critical ambient oxygen tension (PO2crit) at which oxygen supply to the tissue started to become impeded. By contrast, a hypothetical increase in ventilation rate had almost no effect on PO2crit under food-free conditions, indicating that appendage movement is driven by nutritive rather than respiratory requirements. However, the model predicted a moderate reduction of PO2crit by hyperventilation under food-rich conditions. Since the regulatory scope for an adjustment in fH was found to be limited in D. magna under these conditions, the increase in ventilation rate is the means of choice for a fed animal to cope with short-term, moderate reductions in ambient oxygen availability. Under long-term and more severe hypoxic conditions, however, the increase in the concentration and oxygen affinity of haemoglobin represents the one and only measure for improving the transport of oxygen from environment to cells.

Circulatory oxygen transport in the water flea Daphnia magna.

To determine the contribution of circulatory convection to tissue oxygen supply in animals of Daphnia magna, heart rate ( f(H)), in-vivo Hb oxygen-saturation ( S(Hb)) and NADH fluorescence intensity ( I(NADH)) as a measure of the tissue oxygenation state were simultaneously measured using digital motion analysis, microabsorption spectroscopy and fluorescence microscopy. In addition, the relationship between stroke volume and body size was established. Groups of differently sized animals (small: 1.4-1.6 mm, medium: 2.7-2.9 mm, large: 3.3 mm) with either low (Hb-poor) or high Hb concentration (Hb-rich) in the hemolymph were exposed to a gradual decrease in ambient oxygen partial pressure ( P(O2amb)) between normoxia and anoxia. In all groups, f(H) increased in response to progressive hypoxia. The hypoxic maximum in f(H) was highest in medium-sized Hb-poor animals, whereas perfusion rate increased continuously with increasing body size in Hb-poor and Hb-rich animals. The P(O2amb) at which Hb in the heart region was half-saturated (in-vivo P(50)) was higher in medium-sized (Hb-poor: 3.2 kPa, Hb-rich: 2.0 kPa) than in small (Hb-poor: 2.1 kPa, Hb-rich: 1.5 kPa) and large animals (Hb-poor: 1.9 kPa). The in-vivo P(50) was always lower in Hb-rich than in Hb-poor animals. The I(NADH) indicated an impairment of tissue oxygenation starting at higher critical P(O2amb) with increasing body size and with lower Hb concentration. Model calculations suggest that at the respective critical P(O2amb), circulatory convection delivers less than half of the oxygen demand in Hb-poor animals. In contrast, in Hb-rich animals, the contribution of circulatory convection to tissue oxygen supply at respective critical P(O2amb) was much greater due to the higher concentration of Hb.

Benefits of haemoglobin in the cladoceran crustacean Daphnia magna.

To determine the contribution of haemoglobin (Hb) to the hypoxia-tolerance of Daphnia magna, we exposed Hb-poor and Hb-rich individuals (2.4-2.8 mm long) to a stepwise decrease in ambient oxygen partial pressure (P(O(2)amb)) over a period of 51 min from normoxia (20.56 kPa) to anoxia (<0.27 kPa) and looked for differences in their physiological performance. The haem-based concentrations of Hb in the haemolymph were 49 micromol l(-1) in Hb-poor and 337 micromol l(-1) in Hb-rich animals, respectively. The experimental apparatus made simultaneous measurement of appendage beating rate (fA), NADH fluorescence intensity (I(NADH)) of the appendage muscles, heart rate (fH) and in vivo Hb oxygen-saturation possible. In response to progressive, moderate hypoxia, both groups showed pronounced tachycardia and a slight decrease in fA. The fA and fH of Hb-rich animals were generally 4-6 % lower than those of Hb-poor animals. In addition, Hb-rich animals showed a significant decrease in the P(O(2)amb) at which the Hb in the heart region was half-saturated and a striking reduction in the critical P(O(2)amb) of appendage-related variables. In Hb-poor animals, the I(NADH) signal indicated that the oxygen supply to the limb muscle tissue started to become impeded at a critical P(O(2)amb) of 4.75 kPa, although the high level of fA was largely maintained until 1.77 kPa. The obvious discrepancy between these two critical P(O(2)amb) values suggested an anaerobic supplementation of energy provision in the range 4.75-1.77 kPa. The fact that I(NADH) of Hb-rich animals did not rise until P(O(2)amb) fell below 1.32 kPa strongly suggests that the extra Hb available to Hb-rich animals ensured an adequate oxygen supply to the limb muscle tissue in the P(O(2)amb) range 4.75-1.32 kPa. This finding illustrates the physiological benefit of Hb in enabling the animal to sustain its aerobic metabolism as the energetically most efficient mode of fuel utilization under conditions of reduced oxygen availability.

Respiratory adaptations to running-water microhabitats in mayfly larvae Epeorus sylvicola and Ecdyonurus torrentis, Ephemeroptera.

The mayfly larvae Epeorus sylvicola and Ecdyonurus torrentis inhabit either fast-flowing or, for the latter species, calm zones of running water. We studied (1) mechanisms and limitations of oxygen transport in single individuals (oxygen consumption rate, occurrence and rate of gill movements, and heartbeats) in running water of different oxygen concentrations and (2) capacities for anaerobiosis (L-lactate production). Our aim was to look for specific adaptations in the two species to slightly different microhabitats. Epeorus sylvicola, whose immovable gills are not able to generate ventilatory convection, proved to be an oxyconformer at both test temperatures (11 degrees and 15 degrees C). Ecdyonurus torrentis showed a progressively stronger oxyregulatory behavior at higher temperatures. In this species an onset of gill beating was found at moderate hypoxia (below 16 kPa). Ventilating individuals reached maximum rates (300 min-1) of 5-14 kPa. In the case of a further reduction of oxygen partial pressure, the ventilatory rate started to decrease. Ventilatory activity, however, was maintained down to very low oxygen concentrations. Neither in E. sylvicola nor in E. torrentis was experimental evidence found to confirm the hypothesis of a respiratory function of hindgut movements. During hypoxia, the heart rate was constant in both species (E. sylvicola: 80 min-1; E. torrentis: 60 min-1): bradycardia occurred either below 1.5 kPa or below 4 kPa. Anaerobiosis, that is, lactate production, was not detected in either species.

The sites of respiratory gas exchange in the planktonic crustacean daphnia magna: an in vivo study employing blood haemoglobin as an internal oxygen probe

Recent studies on Daphnia magna have revealed that the feeding current is important for uptake of oxygen from the ambient medium. Respiratory gas exchange should therefore mainly occur within the filtering chamber, whose boundaries are formed by the trunk and the extended carapace shell valves. The precise site of gas exchange in the genus Daphnia is, however, a matter of conjecture. We have developed a method of imaging the haemoglobin oxygen-saturation in the circulatory system of transparent animals, which provides an opportunity to localize oxygen uptake from the environment and oxygen release to the tissues. Experiments were carried out at 20 degrees C on 2.8-3.0 mm long parthenogenetic females maintained in hypoxic culturing conditions, which had resulted in an increased haemoglobin content in the haemolymph. In lateral views of D. magna, the highest values of haemoglobin oxygen-saturation occurred near the posterior margin of the carapace and, surprisingly, in the rostral part of the head. The ambient oxygen partial pressures at which haemoglobin was half-oxygenated were 15 mmHg (2.0 kPa) for the posterior carapace region and 6 mmHg (0.8 kPa) for the rostrum. Although not all parts of the circulatory system could be analyzed using this technique, the data obtained from the accessible regions suggest that the inner wall of the carapace is a major site of respiratory gas exchange. Taking the circulatory pattern and the flow pattern of the medium in the filtering chamber into consideration, it becomes clear that the haemolymph, after passing from the limbs to the carapace lacuna, becomes oxygenated while flowing through the ventral part of the double-walled carapace in a posterior direction. The laterally flattened rostral region, where sensory and central nervous system structures are located, seems to have direct diffusive access to ambient oxygen, which could be especially advantageous during severe hypoxia when the convective transport systems fail to supply enough oxygen to that region.

Epipodite and fat cells as sites of hemoglobin synthesis in the branchiopod crustacean Daphnia magna.

In contrast to the malacostracan crustaceans that use hemocyanin as the oxygen carrier, a number of branchiopod crustaceans, such as the water flea Daphnia magna, utilize hemoglobin (Hb) as the respiratory protein. By means of in situ hybridization (ISH) techniques with subsequent signal amplification using catalyzed reporter deposition, sites of Hb synthesis were localized in Daphnia magna. Based on a previously reported Hb-cDNA sequence, a specific ISH probe was designed and hybridized with the Hb-mRNA in histological sections of adult D. magna. The detection of Hb-mRNA was tissue specific and revealed that Hb is synthesized in fat cells, which play a role in fat and glycogen metabolism, and in epithelial cells of the epipodites, which are involved in osmoregulation. Sites of Hb synthesis have been identified in several invertebrate phyla, including Annelida and Nematoda. However, this is the first example in the class Crustacea, and only the second in the phylum Arthropoda.

The importance of the feeding current for oxygen uptake in the water flea daphnia magna

In the planktonic crustacean Daphnia magna (Branchiopoda, Cladocera), different views exist on the mechanism of respiratory gas exchange, ranging from gill breathing to general integumentary respiration. The presumed structures for specialized gas exchange are located ventrally within the filter chamber, which is continuously perfused with the ambient medium for food gathering. To localize respiratory gas exchange in D. magna, we determined the contribution of the feeding current to total oxygen transport. Combining microscopy with special optical techniques, we used a phosphorescent oxygen-sensitive dye for oxygen partial pressure (PO2) measurements and applied fluorescent microspheres for flow-rate analysis. Appendage beat rate was determined by digital image-processing. All experiments were carried out on hypoxia-adapted animals with a body length ranging from 2.3 to 2.7 mm at 20 degreesC. External PO2 measurement revealed oxygen depletion in the ventral body region but essentially no change at posterior, lateral and dorsal positions. The PO2 difference between the inflow and outflow of the feeding current was 13.0 mmHg (1.73 kPa). The flow rate of the feeding current ranged from 1.2 to 5.2 ml h-1 and showed a close correlation with appendage beat rate, which varied from 310.4 to 460.7 beats min-1. Model calculations based on the Fick principle suggest that oxygen extraction from the feeding current satisfies most of the total of oxygen requirement of D. magna.

Development of heart rate rhythmicity in Muscovy duck embryos.

The heart rate (HR) of Muscovy duck embryos (Cairina moschata f. domestica) was continuously recorded from as early as the 21st day of incubation (D21) until hatching (D34/35). The aim of the study was to investigate the influence of phonoperiods consisting of different acoustic stimuli on the course of HR and the development of HR periodicities during this period. Incubation was carried out at a constant temperature and in constant darkness. Until D25 HR was dominated by decelerative fluctuations only, indicating a main input from the parasympathetic system on the heart. Later sympathetic influences increased progressively. HR periodicity was investigated by means of chi 2-periodogram and fast Fourier transformation. Between D26 and D30 statistically significant and stable HR periodicities developed gradually. They had periods in the range from 5 to 38 h. Ultra-, circa- and infradian rhythms (< 20, 24 +/- 4 and > 28 h, respectively) occurred in parallel in some cases in the same embryo. The for the HR course important periods were dissimilar between individual embryos and had different intensities. There was no indication that acoustic stimulation (phonoperiods) had any effect on the development of HR periodicities.

A method for noninvasive, long-term recording of the avian embryo heart rate.

A technique for obtaining a noninvasive electrocardiogram (ECG) from avian embryos is described. Accounts of electronic signal processing and computer-aided long-term recording are included. Furthermore, an algorithm is demonstrated for extracting the relevant information on heart rate from noisy data samples. The method provides the basis for monitoring the heart rate of the avian embryo on different time scales and under different influences.