An in vitro/in vivo screening assay as a sensitive tool to assess endocrine disruptive activity in surface water

Adult male fathead minnow were exposed for 14 or 28-days under flow-through conditions to undiluted filtered water samples from the rivers Meuse and Rhine in the Netherlands. The experiment included two vessels per treatment each containing 10 fish and samples of five fish were taken after 14 and 28 days. Additional groups were exposed to 17alpha-ethinylestradiol (EE2) as a reference and untreated drinking water as a negative control. Major endpoints examined included induction of vitellogenin (VTG) synthesis, VTG mRNA activity, hepato- and gonadosomatic indices (HSI and GSI) and gonadal histology. No significant difference was recorded in body weight or mean GSI values between the various treatments. Only exposure to Meuse water resulted in significantly higher HSI means after 14 days. Histological examination showed no apparent effects on gonadal tissue except for eosinophilic blood plasma in fish exposed to Meuse water or EE2. After 14 and 28 days, elevated VTG and VTG mRNA levels were measured in most livers of the fish exposed to Meuse water, but not in the fish exposed to Rhine water. This was confirmed by measuring estrogenic responses in the in vitro ER CALUX assay. Induction of VTG synthesis proved to be the most sensitive endpoint in the Non Spawning Male Fish Assay for in vivo detection of bio-available estrogenic activity supplementary to a sensitive in vitro assay. The other endpoints examined varied too much and required a higher number of fish or replicates to achieve sufficient power for statistical testing making them less animal friendly.     

The use of a metallothionein-like-proteins (MTLP) kinetic approach for metal bioavailability monitoring in dredged material

Ecotoxicological effects associated with contaminants present in dredged material from Spain were determined using a marine biotest based on the determination of metallothionein-like-protein concentrations (MTLPs) in the shore crab Carcinus maenas. Intermoult female C. maenas were exposed in the laboratory to sediments from three Spanish ports, Ría de Huelva (SW, Spain), La Coru?a (NW, Spain) and Bahía de Cádiz (SW, Spain) per replicate during 21 days. Hepatopancreas samples from crabs were taken for metallothioneins analysis on days 0, 7 and 21. Furthermore, chemical analysis was performed in the stations to determine the degree and nature of sediment contamination (Cr, Ni, Cu, Zn, Cd, Pb, Hg, As, PAHs and PCBs). A significant increase (p<0.05) in metallothionein concentration was observed over time in individuals exposed to sediment from the port Ría de Huelva characterized by high concentrations of metals. A toxicokinetic approach is proposed in this study related to the use of this biomarker in C. maenas to evaluate bioavailability associated with metals present in dredged material. As a first step, this toxicokinetic approach might reveal as a sensitive tool for evaluating bioavailability of contaminants present in dredged material.     

A time-compressed simulated geomagnetic storm influences the nest-exiting flight angles of the stingless bee Tetragonisca angustula

Insects have been used as models for understanding animal orientation. It is well accepted that social insects such as honeybees and ants use different natural cues in their orientation mechanism. A magnetic sensitivity was suggested for the stingless bee Schwarziana quadripunctata, based on the observation of a surprising effect of a geomagnetic storm on the nest-exiting flight angles. Stimulated by this result, in this paper, the effects of a time-compressed simulated geomagnetic storm (TC-SGS) on the nest-exiting flight angles of another stingless bee, Tetragonisca angustula, are presented. Under an applied SGS, either on the horizontal or vertical component of the geomagnetic field, both nest-exiting flight angles, dip and azimuth, are statistically different from those under geomagnetic conditions. The angular dependence of ferromagnetic resonance (FMR) spectra of whole stingless bees shows the presence of organized magnetic nanoparticles in their bodies, which indicates this material as a possible magnetic detector.     

The antennal sensilla of Melipona quadrifasciata (Hymenoptera: Apidae: Meliponini): a study of different sexes and castes

The sensilla of insects are integumental units that play a role as sensory structures and are crucial for the perception of stimuli and for communication. In this study, we compared the antennal sensilla of females (workers and queens), males (haploid (n) and diploid (2n)), and queen-like males (QLMs, resulting from 2n males after juvenile hormone (JH) treatment) in the stingless bee Melipona quadrifasciata. Images of the dorsal antenna surfaces were acquired using a scanning electron microscope. As reported for other hymenopterans, this species exhibits a heterogeneous sensillar distribution along the antennae. Thirteen different types of sensilla were found in the antennae of M. quadrifasciata: trichodea (subtypes I to VI), chaetica (subtypes I and II), placodea, basiconica, ampullacea, coeloconica, and coelocapitula. Sensilla trichodea I were the most abundant, followed by sensilla placodea, which might function in olfactory perception. Sensilla basiconica, sensilla chaetica I, sensilla coeloconica, and sensilla ampullacea were found exclusively in females. In terms of the composition and size of the sensilla, the antennae of QLMs most closely resemble those of the 2n male, although QLMs exhibit a queen phenotype. This study represents the first comparative analysis of the antennal sensilla of M. quadrifasciata. The differences found in the type and amount of sensilla between the castes and sexes are discussed based on the presumed sensillary functions.     

Testing the influence of gravity on flower symmetry in five <Emphasis Type="Italic">Saxifraga</Emphasis> species

Flower symmetry is considered a species-specific trait and is categorized in asymmetry, actinomorphic symmetry, bisymmetry and zygomorphic symmetry. Here we report on the intra-individual variation of flower symmetry in the genus Saxifraga and the influence of light, gravity and intrinsic factors on the development of flower symmetry. We tested five species—Saxifraga cuneifolia, Saxifraga imparilis, Saxifraga rotundifolia, Saxifraga stolonifera and Saxifraga umbrosa—concerning six flower parameters—angles between petals, petal length, petal pigmentation, angular position of carpels, movement of stamens and (only for S. imparilis and S. stolonifera) the length of the two lower elongated petals in regard to their position towards the stem. Specimens of all species were tested on a vertical clinostat as a gravity compensator, on a horizontal clinostat as a light incidence compensator and on a stationary control. The results show that the angle of incident light has no apparent impact on flower symmetry, whereas gravity affects the angular position of petals in S. cuneifolia and S. umbrosa and the petal colouration in S. rotundifolia. In S. cuneifolia and S. umbrosa, the absence of directional gravity resulted in the development of actinomorphic flowers, whereas the corresponding control flowers were zygomorphic. The development of flowers in S. rotundifolia was not altered by this treatment. The length of the two elongated petals in S. stolonifera and S. imparilis was not affected by gravity, but rather was determined by position of the flower within the inflorescence and resulted in asymmetrical flowers.     

Effects of three pesticides on the avoidance behavior of earthworms in laboratory tests performed under temperate and tropical conditions

Little research has been performed on the impact of pesticides on earthworms under tropical conditions. Taking into consideration the often-limited resources in tropical countries, simple screening tests are needed. Therefore, it was investigated whether three pesticides relevant for the Brazilian Amazon (benomyl, carbendazim, lambda-cyhalothrin) affect the avoidance behavior of the earthworm Eisenia fetida. The tests were performed for two days according to ISO guideline 17512 but were adapted to tropical conditions (i.e. test substrate, test organism and temperature). The results indicate that this test gives reproducible and reliable results. Toxicity values (NOEC, EC50) are lower than those determined in 14 day-acute mortality tests and are approximately in the same range such as those found in 56 day-chronic reproduction tests with the same earthworm species, which were performed in parallel. Therefore, the use of the earthworm avoidance tests is recommended as a screening tool for the risk assessment of pesticides.     

Influence of tidal regime on the distribution of trace metals in a contaminated tidal freshwater marsh soil colonized with common reed (Phragmites australis)

A historical input of trace metals into tidal marshes fringing the river Scheldt may be a cause for concern. Nevertheless, the specific physicochemical form, rather than the total concentration, determines the ecotoxicological risk of metals in the soil. In this study the effect of tidal regime on the distribution of trace metals in different compartments of the soil was investigated. As, Cd, Cu and Zn concentrations in sediment, pore water and in roots were determined along a depth profile. Total sediment metal concentrations were similar at different sites, reflecting pollution history. Pore water metal concentrations were generally higher under less flooded conditions (mean is (2.32 ± 0.08) × 10⁻³ mg Cd L⁻¹ and (1.53 ± 0.03) × 10⁻³ mg Cd L⁻¹). Metal concentrations associated with roots (mean is 202.47 ± 2.83 mg Cd kg⁻¹ and 69.39 ± 0.99 mg Cd kg⁻¹) were up to 10 times higher than sediment (mean is 20.48 ± 0.19 mg Cd kg⁻¹ and 20.42 ± 0.21 mg Cd kg⁻¹) metal concentrations and higher under dryer conditions. Despite high metal concentrations associated with roots, the major part of the metals in the marsh soil is still associated with the sediment as the overall biomass of roots is small compared to the sediment.     

Detection and identification of baculovirus pesticides by multitemperature single-strand conformational polymorphism

The method of single-strand conformational polymorphism (SSCP) was modified in our laboratories for the characterization of baculoviruses, insect viruses with great potential for use as bioinsecticides in biological protection programs. A series of primers were synthesized after the comparison of the polyhedrin gene sequences of over 20 baculoviruses. Polyhedrin is a highly conserved protein which is responsible for the persistence of the virus in the environment. Universal primers were designed which could be used in polymerase chain reactions (PCR) containing genomic DNA from an array of nucleopolyhedrosis viruses (NPVs) including these which are used as biopesticides against important pests of forests and crops, such as Anticarsia gemmatalis, Spodoptera frugiperda, Lymantria dispar, Lymantria monacha and many others. PCR products were denatured and subjected to single-strand DNA electrophoresis at variable temperatures (MSSCP) where, after silver staining, they gave ssDNA band patterns characteristic for each baculovirus species. This technique can be potentially applied to detect baculoviruses in insects collected in the field, as well as to plant tissues and the excrements or bodies of predators without need for sequencing the PCR products. Sometimes MSSCP can be used not only for species determination but also as an indication of genomic variability which can be related to infectivity.     

Contribution of Spartina maritima to the reduction of eutrophication in estuarine systems

Salt marshes are among the most productive ecosystems in the world, performing important ecosystem functions, particularly nutrient recycling. In this study, a comparison is made between Mondego and Tagus estuaries in relation to the role of Spartina maritima in nitrogen retention capacity and cycling. Two mono-specific S. maritima stands per estuary were studied during 1 yr (biomass, nitrogen (N) pools, litter production, decomposition rates). Results showed that the oldest Tagus salt marsh population presented higher annual belowground biomass and N productions, and a slower decomposition rate for litter, contributing to the higher N accumulation in the sediment, whereas S. maritima younger marshes had higher aboveground biomass production. Detritus moved by tides represented a huge amount of aboveground production, probably significant when considering the N balance of these salt marshes. Results reinforce the functions of salt marshes as contributing to a reduction of eutrophication in transitional waters, namely through sedimentation processes.