A synchronized amphibian metamorphosis assay as an improved tool to detect thyroid hormone disturbance by endocrine disruptors and apolar sediment extracts

Amphibian metamorphosis assays are used to evaluate potential effects of endocrine disrupting compounds on the thyroid hormone axis. In this study, Xenopus laevis tadpoles are kept in a solution of 0.2% thiourea (TU) to arrest and synchronise them in their development. The advantage of this synchronized amphibian metamorphosis assays is that synchronised tadpoles are available at any time to start metamorphosis experiments, and experimental groups are much more homogenous at the start of experimental exposure compared with groups selected from an untreated pool of animals. The water volume per animal was kept constant throughout the experimental period to overcome the influence of declining numbers of animals per aquarium due to metamorphosis and mortality on the density dependent development of the remaining tadpoles. Clophen A50 (a technical PCB mixture), the single congener 3,3',4,4'-tetrachlorobiphenyl (PCB 77) and apolar sediment extracts that were previously tested positive in the T-Screen, an in vitro proliferation assay for thyroid hormone disruption, were tested in the Synchronized Amphibian Metamorphosis Assay. Endpoints studied were mortality, malformations, body weight, and percentage of metamorphosed froglets at the end of the 60-day experimental period, percentage of tadpoles in different developmental stages, and developmental stage-dependent awarded penalty points. Dietary exposure to Clophen A50 (0.2-50mg/kg food) resulted in a significant increased percentage of tadpoles that did not pass metamorphosis at concentrations higher than 2mg/kg food. Time until metamorphosis in those animals that were able to metamorphose after the 60-days experimental period was significantly decreased. Dietary exposure to PCB 77, a congener that can be readily metabolised, did not result in significant effects in any exposure group (2-500 microg/kg food). Apolar sediment extracts from two of the three sites that are contaminated with a wide variety of chemicals significantly decreased the percentage of metamorphosed animals and significantly increased the number of tadpoles that remained in early and late metamorphic stages. These effects already occurred when the extracts where diluted more than 1000 times (on an organic carbon base) compared to environmental concentrations. The rank of potency was comparable to results obtained with the T-screen. This suggests the presence of thyroid hormone disrupting compounds in the aquatic environment and possible effects of such compounds on animal development in the wild.     

Occurrence of glucocorticogenic activity in various surface waters in The Netherlands

Considering the important role that surface waters serve for drinking water production, it is important to know if these resources are under the impact of contaminants. Apart from environmental pollutants such as pesticides, compounds such as (xeno)estrogens have received al lot of research attention and several large monitoring campaigns have been carried out to assess estrogenic contamination in the aquatic environment. The introduction of novel in vitro bioassays enables researchers to study if – and to what extent – water bodies are under the impact of less-studied (synthetic) hormone active compounds. The aim of the present study was to carry out an assessment on the presence and extent of glucocorticogenic activity in Dutch surface waters that serve as sources for drinking water production. The results show glucocorticogenic activity in the range of <LOD – 2.4 ng dexamethasone equivalents L⁻¹ (dex EQs) in four out of eight surface waters. An exploratory time-series study to obtain a more complete picture of the yearly average of fluctuating glucocorticogenic activities at two sample locations demonstrated glucocorticogenic activities ranging between <LOD – 2.7 ng dex EQs L⁻¹. Although immediate human health effects are unlikely, the environmental presence of glucocorticogenic compounds in the ng L⁻¹ range compels further environmental research and assessment.     

Advances in pollination ecology from tropical plantation crops

Although ecologists traditionally focus on natural ecosystems, there is growing awareness that mixed landscapes of managed and unmanaged systems provide a research environment for understanding basic ecological relationships on a large scale. Here, we show how tropical agroforestry systems can be used to develop ideas about the mechanisms that influence species diversity and subsequent biotic interactions at different spatial scales. Our focus is on tropical plantation crops, mainly coffee and cacao, and their pollinators, which are of basic ecological interest as partners in an important mutualistic interaction. We review how insect-mediated pollination services depend on local agroforest and natural habitats in surrounding landscapes. Further, we evaluate the functional significance of pollinator diversity and the explanatory value of species traits, and we provide an intercontinental comparison of pollinator assemblages. We found that optimal pollination success might be best understood as a consequence of niche complementarities among pollinators in landscapes harboring various species. We further show that small cavity-nesting bees and small generalist beetles were especially affected by isolation from forest and that larger-bodied insects in the same landscapes were not similarly affected. We suggest that mixed tropical landscapes with agroforestry systems have great potential for future research on the interactions between plants and pollinators.     

Disruption of thyroid hormone-mediated Xenopus laevis tadpole tail tip regression by hexabromocyclododecane (HBCD) and 2,2',3,3',4,4',5,5',6-nona brominated diphenyl ether (BDE206)

Thyroid hormone regulates amphibian metamorphosis, including the transformation of a tadpole into a froglet and regression of the tail. Xenopus laevis tadpole tail tips in organ culture (ex vivo) undergo regression when exposed to 3,3',5-triiodo-l-thyronine (T(3)) and interference by chemicals with this process was utilized as a bioassay to detect thyroid hormone disruption. In the present study the bioassay was further validated by investigating its response to compound induced T(3)-antagonism and - potentiation. Tadpole tail tips were exposed to two brominated flame retardants (BFRs) in presence or absence of T(3) at its EC(50) (20 nM). T(3)-induced tail tip regression was antagonized by 2,2',3,3',4,4',5,5',6-nona brominated diphenyl ether (BDE206) and potentiated by hexabromocyclododecane (HBCD) in a concentration dependent manner, which was consistent with results obtained with a in vitro T(3)-dependent proliferation bioassay termed the T-screen. Neither compound induced any effect in the absence of T(3). The results indicate that studying possible hormone disrupting effects of agonistic, antagonistic or potentiating compounds should include combined exposure with the natural hormone at around its EC(50) concentration. The results obtained with the tail tip exposures were in accordance with the T-screen predictions, and occurred at BFR-concentrations that were only 5-50 times those of T(3). The bioassay proved to be suitable not only for detecting T(3)-agonism, but also for antagonism and potentiation.     

Shade tree management affects fruit abortion, insect pests and pathogens of cacao

The mortality of cacao fruits caused by early fruit abortion or insect and pathogen attacks was investigated in differently managed agroforestry systems in Central Sulawesi, Indonesia. Nine agroforestry systems shaded by three different types of tree stands were selected, which represented a decrease in structural heterogeneity: forest remnants, diverse planted trees and one or two species of planted leguminose trees. After standardized manual cross-pollination, the development of 600 fruits on 54 trees (6 trees per agroforest) was followed during 18 weeks of fruit development. In total, 432 of all fruits were lost before maturity, which seriously undermined yields. The proportion of harvested fruits per tree (overall average: 27 ± 4%) was not affected by canopy type. Although shade cover did not have a significant effect, losses due to fruit abortion were most likely under forest shade, where nitrogen-fixing leguminose shade trees were absent. Fruit losses due to pathogenic infections and insect attacks increased with the homogenization of the agroforests, supporting the hypothesis that agricultural homogenization increases risks of pest outbreaks. In conclusion, shade management may be improved to increase yields from cacao using highly diversified natural shade agroforestry systems.     

Caveats to quantifying ecosystem services: fruit abortion blurs benefits from crop pollination.

The recent trend to place monetary values on ecosystem services has led to studies on the economic importance of pollinators for agricultural crops. Several recent studies indicate regional, long-term pollinator declines, and economic consequences have been derived from declining pollination efficiencies. However, use of pollinator services as economic incentives for conservation must consider environmental factors such as drought, pests, and diseases, which can also limit yields. Moreover, "flower excess" is a well-known reproductive strategy of plants as insurance against unpredictable, external factors that limit reproduction. With three case studies on the importance of pollination levels for amounts of harvested fruits of three tropical crops (passion fruit in Brazil, coffee in Ecuador, and cacao in Indonesia) we illustrate how reproductive strategies and environmental stress can obscure initial benefits from improved pollination. By interpreting these results with findings from evolutionary sciences, agronomy, and studies on wild-plant populations, we argue that studies on economic benefits from pollinators should include the total of ecosystem processes that (1) lead to successful pollination and (2) mobilize nutrients and improve plant quality to the extent that crop yields indeed benefit from enhanced pollinator services. Conservation incentives that use quantifications of nature's services to human welfare will benefit from approaches at the ecosystem level that take into account the broad spectrum of biological processes that limit or deliver the service.