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2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) is a high-production volume chemical used in paper, ink, pesticide, and adhesive industries as a wetting and anti-foaming agent. The physicochemical properties and slow biodegradation rate of TMDD indicate a low bioaccumulation potential but a high prevalence in the environment. As a consequence, TMDD has been detected in several European rivers in the nanogram per liter and lower microgram per liter range; however, its environmental risk to aquatic organisms is considered low. Recent studies almost exclusively focused on acute effects by TMDD, little is known about cytotoxic and genotoxic effects, reproduction and developmental toxicity, endocrine disruption, and any kind of long-term toxicity and carcinogenicity so far. The present study aims to provide more specific baseline information on the ecotoxicological effects of TMDD in fish. For this end, cyto- and genotoxicity assays were carried out in vitro with the permanent fish cell line RTL-W1; in addition, in vivo studies were conducted with the early life stages of zebrafish (Danio rerio) in order to fill the data gaps in developmental toxicity and endocrine disruption. TMDD showed a cytotoxic and slight genotoxic potential in fish cell lines; moreover, various sublethal and lethal effects could be detected in developing zebrafish embryos. There was no evidence of endocrine-disrupting effects by TMDD; however, mortality following prolonged exposure to TMDD during fish sexual development test was clearly higher than mortality in the fish embryo test after 96-h exposure. Our results thus confirmed previous findings of laboratory screening tests, suggesting short-term toxic effects of TMDD in the intermediate, and long-term effects in the lower milligram per liter range.

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture. The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

The toxicity of intact sediments and sediment extracts, from both an uncontaminated site and a site contaminated by pulp-mill effluents, was tested in a five months study. The deposit-feeding amphipod Monoporeia affinis was exposed in soft-bottom flow-through water microcosms. To examine potential toxicity a set of reproduction endpoints was used including fecundity and different embryo aberrations such as malformed eggs. Among extracts, the aliphatic/monoaromatic and diaromatic fractions along with the total extract were shown to cause the highest toxicity measured as malformed eggs, while the polyaromatic fraction caused toxicity at background levels. A comparison between sediment extracts and pulp mill contaminated intact sediment, however, showed no toxicity of the intact sediment. Thus, the extraction procedure seems to increase bioavailability and subsequently toxicity as compared to the intact sediments in situ. In toxicity testing using fractionated extracts of sediments in a toxicity identification evaluation (TIE) procedures, caution should therefore be taken when assessing bioavailable contaminants in contaminated areas. This should be taken in account both in determining remediation priorities as well as in ecological risk assessments.

This study evaluated the effects of bleached and unbleached kraft mill effluent on reproductive success of largemouth bass (Micropterus salmoides). Bass were exposed to effluent concentrations (0, 10, 20, 40, or 80%) for 28 and 56 d. Parameters measured included hepatosomatic index (HSI) and gonadosomatic index (GSI) and plasma concentrations of 17beta-estradiol (E2), 11-ketotestosterone (11-KT), and vitellogenin (VTG). At the end of the 56-d period, bass were moved to hatchery ponds to evaluate spawning success. Spawning mats with eggs either were brought indoors for evaluation of fecundities, hatchabilities, and egg and fry size (measured at age 3 d), or were left in ponds and fry number and size recorded (average age of 14 d). Effluent exposure was verified by measuring resin acids (isopimaric, abietic. and dehydroabietic acids) in bile. Compared to controls, exposed bass had greater concentrations of resin acids in bile. In general, exposed females had lower concentrations of E2 and VTG (or = 20% effluent), whereas males had lower concentrations of 11-KT (or = 20% effluent) and increased E2 (or = 20% effluent). The HSI values increased in females (or = 10% effluent), and GSI values decreased in both sexes (or = 40% effluent). Fecundity, egg size, and hatchability did not differ across treatments, but an increase in the frequency of fry abnormalities and a decrease in fry weights was observed at effluent exposures of 40% and higher. However, results from the pond study, revealed a significant reduction in fry growth and survival (or = 10%). This decline may have been caused by an increased frequency of deformities, in conjunction with alterations of growth. These changes could have resulted from alterations in egg quality because of failure of parental reproductive systems, from acute embryo toxicity after translocation of contaminants from the mother to the developing embryo, or from both.

Mouse oocytes, cleavage-stage embryos, and blastocyst-stage embryos were studied to show the distribution of both an immunoanalog to nonerythroid spectrin (p 230) and F-actin. Using antibodies to nonerythroid spectrin, diffuse, positive cytoplasmic fluorescence was regularly seen in oocytes and embryo cells. The presence of nonerythroid spectrin in oocytes was confirmed by immunoblotting. Oocytes usually exhibited an inconspicuous submembraneous layer of nonerythroid spectrin, which was more pronounced in the area of the polar body. Oocytes regularly exhibited a peripheral concentration of actin. Throughout the cleavage and blastocyst stages, a cortical layer of nonerythroid spectrin and actin was usually observed in embryo cells. These submembraneous layers on the outer surface of the embryo were relatively thin as compared to those in areas of intercellular contact. The contact areas regularly showed distinct positive staining, including a concentration of label at the most peripheral region of each contact area. This resulted in the presence of ring-like fluorescence around each blastomere. Nonerythroid spectrin and actin showed concentration to the contact area between the oocyte and the polar body. Although the general localization patterns of nonerythroid spectrin and actin were similar, double staining experiments revealed that slightly different planes of focus were necessary to obtain sharp definition of the fluorescence of these components in areas of intercellular contact: the ring-like concentration of nonerythroid spectrin appeared to be localized more peripherally than that of actin. The cells of preimplantation embryos show motile features that include actual cell movements and striking changes in cell shape (e.g., during compaction). The sub-membraneous layers of nonerythroid spectrin and actin may contribute to the regulation of the deformability and thus the shape of embryo cells. Cytoskeletal components are probably involved in the formation of cell contacts and regulation of contact areas between blastomeres, as well as the control of cell movement. Thus, nonerythroid spectrin and actin may play important roles in the regulation of cleavage-stage development and blastocyst transformation.