Vaccine adjuvant: it makes the difference.

The use of protein or DNA in vaccination process rather than living or attenuated microorganism, aims at the increase of the vaccines safety. However, in these cases, the use of adjuvant is frequently required to improve their immunogenicity. In this study, we show the importance of the adjuvant in a vaccine formulation. Vaccines for tuberculosis provide an instructive example, based on the mycobacterial 65 kDa heat shock protein (hsp65). The same antigen can elicit completely different patterns of immune response depending on how it is administered. Thus, the same antigen might or not protect mice from challenge with Mycobacterium tuberculosis, depending on the formulation. These data suggest that, despite the name, the adjuvant plays a fundamental role on the vaccination process.

Ring-testing and field-validation of a terrestrial model ecosystem (TME)--an instrument for testing potentially harmful substances: effects of carbendazim on soil microbial parameters.

The effects of carbendazim on substrate induced respiration (SIR), dehydrogenase activity (DHA), phosphatase activity and thymidine incorporation by bacteria were evaluated in an experiment with an open intact Terrestrial Model Ecosystem (TME) and in a simultaneous field-validation study. Experiments were performed on four different European soils in Germany, The Netherlands, United Kingdom and Portugal. Data analysis focused on (i) detecting differences between experiments, especially in control values, (ii) checking similarity in data variability at each treatment level between experiments and (iii) analysing the resemblance of response to the model chemical in both experiments. Results obtained showed that control values from TME experiments were similar to those obtained on the respective field site, in most of the comparisons made for SIR, DHA and thymidine incorporation. Phosphatase activity revealed more differences, but values of both experiments had the same order of magnitude. At least part of the variation could be explained from the correlation of the microbial parameters with soil moisture content. Comparisons on data variability also revealed the absence of significant differences between experiments in all parameters in most cases, indicating that TMEs were able to represent the spatial variability found in the field. Effects of carbendazim, when occurring, were observed at treatment levels exceeding the highest recommended application rate of 0.36 kg a.i./ha. Effects on SIR and DHA were observed early in time, but effects on phosphatase activity and thymidine incorporation rate were found 8 or 16 weeks after chemical application. These effects were mild, and rarely a 50% inhibition on any of these parameters was seen at carbendazim dosages up to 87.5 kg a.i./ha. The response to the model chemical in TMEs and field plots was similar in most cases. These results give promising prospects for the use of TMEs as an integrative tool in higher tier levels of different assessment schemes.

Ring-testing and field-validation of a terrestrial model ecosystem (TME)--an instrument for testing potentially harmful substances: effects of carbendazim on nematodes.

The effects of the fungicide carbendazim (applied in the formulation Derosal) on nematodes was determined in Terrestrial Model Ecosystem (TME) tests and field-validation studies. TMEs consisted of intact soil columns (diameter 17.5 cm; length 40 cm) taken from a grassland or, in one case, from an arable site. The TMEs were taken from the same site where the respective field study was performed. The tests were performed in Amsterdam (The Netherlands), Bangor (Wales, England), Coimbra (Portugal) and Flörsheim (Germany). Differences concerning nematode overall abundance, the number of nematode families, the trophical structure of the nematode cenosis and the maturity index (MI) were not found between the controls of TME tests and the respective field-validation studies. Effects caused by the chemical treatment, however, were observed on the number of nematode families, on the trophical structure of the nematode cenosis and on the maturity index (MI). Effects on the relative abundance of the omnivorous nematodes were most pronounced, whereas the overall nematode abundance was not affected. The observed effects appear not to differ between the TME tests and the respective field-validation studies. All measurement endpoints in both TMEs and field, showed rather large variations. Therefore, NOEC-values were often equal or higher than the highest treatment level and EC50-values were calculated only for the omnivorous nematodes. NOEC- and EC50-values derived from the TME ring-test and the field-validation study indicate that the reproducibility (i.e. the variation between the partners) was reasonable, although different soils from different sites were used. The EC50-values determined for the effect of carbendazim on the relative abundance of the omnivorous nematodes ranged between 0.93 and 7.24 kg a.i./ha (1.24-9.63 mg/kg). Due to the higher sensitivity of the relative abundance of the omnivorous nematodes compared to the other measurement endpoints it is recommended to use this parameter as the main endpoint.

Ring-testing and field-validation of a terrestrial model ecosystem (TME)--an instrument for testing potentially harmful substances: effects of carbendazim on enchytraeids.

The effects of the fungicide carbendazim (applied in the formulation Derosal) on enchytraeids were determined in Terrestrial Model Ecosystem (TME) tests and field-validation studies. TMEs consisted of intact soil columns (diameter 17.5 cm; length 40 cm) taken from a grassland or, in one case, from an arable site. The TMEs were taken from the same site where the respective field study was performed. The tests were performed in Amsterdam (The Netherlands), Bangor (Wales, England), Coimbra (Portugal) and Flörsheim (Germany). Concerning the enchytraeid overall abundance and the number of species, differences between sampling points and differences between the TME tests and the respective field-validation studies were not found in the controls. Generally, a high variability of data which reduced the probability of determining statistically significant differences was determined. Effects of the model chemical carbendazim were determined for the overall abundance of enchytraeids and the number of enchytraeid species. The clearest dose-response relationship was found for the abundance of the genus Fridericia. The abundance of the genera Achaeta and Enchytraeus was not affected. Effects were most pronounced 8 and 16 weeks after application of the test chemical. The observed effects appeared not to differ between the TME tests and the respective field-validation studies. Due to the high variability of data NOEC-values could often not be determined. The EC50-values derived from the TME pre-test, TME ring-test and field-validation study indicate that the reproducibility (i.e. the variation between the partners) of the EC50-values was reasonable, although different soils were used at the different sites. The EC50-values for effects of carbendazim on enchytraeid abundance ranged between 0.5 and 28.4 kg a.i./ha (corresponding to 0.7-37.8 mg/kg), on the number of species between 7.2 and 87.4 kg a.i./ha (9.5-116.2 mg/kg) and on the abundance of Fridericia between 0.7 and 18.6 kg a.i./ha (0.9-24.7 mg/kg). Since one specific taxon has the potential to be more sensitive for a chemical stressor than other taxa, it is recommended to include investigations on the species level in the assessment of TME or field studies.

Ring-testing and field-validation of a terrestrial model ecosystem (TME)--an instrument for testing potentially harmful substances: fate of the model chemical carbendazim.

The fate of the fungicide carbendazim (applied in the formulation Derosal) in soil was determined in Terrestrial Model Ecosystem (TME) tests and corresponding field-validation studies, which were performed in four different countries (United Kingdom, Germany, Portugal, and The Netherlands). The tests used different soil types, and lasted for 16 weeks. On three of the four sites, grassland soils were used while the fourth site had an arable soil. TMEs consisted of intact soil columns (diameter 17.5 cm; length 40 cm) and were taken from the site where the field study was performed. In the first series of TME tests, carbendazim was applied at four dosages ranging between 0.36 and 77.8 kg a.i./ha, while in the second series of TME tests and the field-validation studies six dosages between 0.36 and 87.5 kg a.i./ha were applied. DT50 values for the dissipation of carbendazim in the TME and field tests were in most cases not significantly affected by the dosage used and ranged between 3.1 and 13.9 weeks in the top 15 cm soil layers. Corresponding DT90 values ranged between 10.1 and 46.1 weeks. DT50 and DT90 values tended to be higher in the more acidic soils of Amsterdam and Flörsheim (pH-KCl 4.8-5.1 and 5.3-5.9, respectively) than in the less acidic soils of Bangor and Coimbra (pH-KCl 5.8-6.6 and 6.4-7.1, respectively). Fate of carbendazim in soil showed similar patterns in the two TME tests and the corresponding field-validation study performed at each site. The only exception was Flörsheim, where the compound was significantly more persistent in the field probably due to different climatic conditions. Carbendazim was not recovered from leachates produced in the TME tests, nor was the compound detected in soil layers deeper than 15 cm. This demonstrates that no significant leaching occurred. This study demonstrates the the TME tests were quite successful in predicting the fate of carbendazim under field conditions.

Ring-testing and field-validation of a terrestrial model ecosystem (TME)--an instrument for testing potentially harmful substances: effects of carbendazim on earthworms.

The effects of the fungicide carbendazim (applied in the formulation Derosal) on earthworms (Lumbricidae) was determined in Terrestrial Model Ecosystem (TME) tests and field-validation studies. TMEs consisted of intact soil columns (diameter 17.5 cm; length 40 cm) taken from a grassland or, in one case, from an arable site. The TMEs were taken from the same site where the respective field-validation study was performed. The tests were performed in Amsterdam (The Netherlands), Bangor (Wales, UK), Coimbra (Portugal) and Flörsheim (Germany). The sites selected had an earthworm coenosis representative of the different land use types and regions. In addition, the differences between the coenosis found in the TMEs and the respective field sites were in general low. A high variability was found between the replicate samples, which reduces the probability of determining significant differences by the statistical evaluation of the data. Similar effects of the chemical treatment were observed on abundance as well as on biomass. Effects were most pronounced 16 weeks after application of the test chemical. The observed effects on earthworm abundance and biomass did not differ between the TME tests and the respective field-validation studies. Effects on earthworm diversity were difficult to assess since the number of individuals per species was low in general. However, the genus Lumbricus and in particular L. terrestris and L. rubellus seemed to be more affected by the chemical treatment than others. NOEC and EC50-values derived from the TME pre-test, the TME ring-test and the field-validation study indicate that the TMEs of the different partners delivered comparable results although different soils were used. Due to the high variability NOECs could often not be determined. The EC50-values for the effect of carbendazim on earthworm abundance ranged between 2.04 and 48.8 kg a.i./ha (2.71-65.2 mg/kg soil) and on earthworm biomass from 1.02 to 34.6 kg a.i./ha (1.36-46.0 mg/kg soil). These results indicate that the abundance and biomass of earthworms are suitable endpoints in ecotoxicological studies with TMEs.

Ring-testing and field-validation of a terrestrial model ecosystem (TME)--an instrument for testing potentially harmful substances: effects of carbendazim on nutrient cycling.

The effect of the fungicide carbendazim (applied in the formulation Derosal) on nutrient cycling in soil was determined in Terrestrial Model Ecosystem (TME) tests and corresponding field-validation studies, which were performed in four different countries (United Kingdom, Germany, Portugal, and The Netherlands). The tests used different soil types, and lasted for 16 weeks. On three of the four sites, grassland soils were used while the fourth site had an arable soil. TMEs consisted of intact soil columns (diameter 17.5 cm; length 40 cm) and were taken from the site where the field study was performed. In the first series of TME tests, carbendazim applied at dosages unto 77.8 kg a.i./ha did not affect sulfate and phosphate concentrations in the top 15 cm soil layers. These nutrients were therefore no longer included in the second series of TME tests and the field-validation studies. Ammonium concentrations in the top soil layers of TMEs and field plots, and in the leachates of the TME columns did not show any effect of carbendazim treatment. Nitrate concentrations in soils and leachates did show some reduction at the highest treatment levels (77.8 kg a.i./ha in the first TME tests, 87.5 kg a.i/ha in the second TME tests and the field-validation studies). Since nitrate concentrations in both soils and leachates were correlated with soil moisture content, these effects could however, mainly be attributed to variations in soil moisture contents, and in some cases also indirectly to effects on earthworm activity. It is concluded that carbendazim, even at dosages as high as 87.5 kg a.i./ha, does not have a significant impact on soil nutrient cycling processes. Nutrient levels in TME tests and the field-validation studies generally showed similar patterns, thus confirming the predictive value of the TME test system.

Ring-testing and field-validation of a terrestrial model ecosystem (TME)--an instrument for testing potentially harmful substances: effects of carbendazim on organic matter breakdown and soil fauna feeding activity.

Organic matter (OM) decomposition and soil fauna feeding activity were integrated as functional endpoints into ecotoxicological tests with intact-soil-core Terrestrial Model Ecosystems (TMEs). Cellulose filter paper served as standardized OM and was either inserted into the top soil or placed on the soil surface for a period of up to 16 weeks. Faunal feeding activity was assessed by the bait-lamina method. The fungicide carbendazim, applied at six dosages ranging from 0.36 kg/ha to 87.5 kg a.i./ha, served as a model chemical. To validate the results from the TME test, a field study was run in parallel. In TMEs the cellulose paper inserted into the soil was decomposed faster than under field conditions. The carbendazim-induced effects on OM decomposition in TMEs and in the field were comparable and followed a clear dose-response relationship. The calculated EC50 values after 8 weeks of incubation were 9.5, 7.1 and 2.1 kg carbendazim/ha for grassland TMEs, grassland field and arable TMEs, respectively. The feeding activity of the soil fauna showed a large variability. The EC50 values for the effect of carbendazim on bait-lamina consumption ranged between 2.0 and 56 kg a.i./ha. Effects on decomposition were correlated with effects on enchytraeids and earthworms but not with effects on bait-lamina consumption.