Ecotoxicological soil evaluation by FETAX

The frog embryo teratogenesis assay-Xenopus (FETAX) is a powerful and flexible bioassay that makes use of the embryos of the anuran Xenopus laevis. FETAX satisfies the requirements of low cost, reliability and reproducibility and, thanks to its three endpoints (i.e., mortality, teratogenicity and growth inhibition) can detect the xenobiotics that affect embryonic development. In this paper, we have used FETAX to evaluate samples of soils collected in an oil-contaminated area. Embryos were exposed directly to the soil to be tested. Particular attention was devoted to provide a statistical procedure for analysing mortality and malformation data as well as growth retardation.     

Multi-bioassay approach for assessing the potency of complex mixtures of polycyclic aromatic hydrocarbons

The chick embryotoxicity screening test (CHEST) and the Salmonella/microsome bioassay were used to evaluate embryotoxic and mutagenic endpoints from crude coal tar (CT) and its fractionated polycyclic aromatic hydrocarbon (PAH) mixtures (designated as A, B, C, D and E). In the CHEST assay, CT and PAH mixtures were injected into the egg yolk. A dose-dependent increase in embryo mortality was observed for all fractions. The E fraction resulted in 47% embryo mortality at a dose of 0.125 mg/kg and was more toxic than CT. At a dose of 1 mg/kg, 85-100% embryonic deaths occurred in fractions C and D and these two fractions were more potent than fractions A and B. The main visual toxic manifestations were liver lesions, discoloration of the liver, and edema. Both CT and fractionated PAH mixtures were also tested in the Salmonella/microsome plate incorporation assay with Salmonella typhimurium strain TA98 and were evaluated with and without metabolic activation at five dose levels. In the presence of S9, the CT and fractions C, D and E induced a dose-dependent positive response. Results from the Salmonella/microsome assay were in good agreement with findings from the CHEST assay suggesting that these two bioassays in combination may facilitate the rapid detection and ranking of complex PAH mixtures.     

Influence of pH and aluminum on developing brook trout in a low calcium water

Eyed embryos of brook trout (Salvelinus fontinalis) were exposed to nominal pHs of 4.5, 5.5 and 7.5 with and without aluminum (300 microg liter(-1)) in extremely soft water (hardness <9mg liter (-1)) at 12 degrees C. Embryo mortality exceeded 80% at pH 4.5, averaged 15 to 18% in the pH 5.5 treatments and was less than 2% in the pH 7.5 treatments. Aluminum significantly reduced embryo mortality (85.3% vs 99.5%) at pH 4.5 but did not affect mortality at pH 5.5 or pH 7.5. Percent hatch and poor hatch were pH dependent and were not significantly influenced by aluminum. Brook trout larvae cumulative mortalities were 100% within 30 days at pH 4.5, with or without aluminum; 69% after 60 days at pH 5.5; 100% in 15 days at pH 5.5 with aluminum and 20% after 60 days at pH 7.5 with or without aluminum. Fish that survived the pH 5.5 treatment showed decreased growth and behavioral impairments compared to the controls (pH 7.5 without aluminum).     

Toxicity of waters from two streams to early life stages of brown trout (Salmo trutta f. fario L.), tested under semi-field conditions.

The development of brown trout (Salmo trutta f. fario L.) in water of two differently polluted streams and in a control situation was monitored in order to get insights into the impact of anthropogenic chemical stressors on the reproductive success of this fish species indigenous to both streams. The test streams, situated in the south of Stuttgart, Germany, were the complexly polluted K?rsch stream and the less polluted Kr?henbach stream. Bypass systems connected to the streams and a laboratory control system were used for continuous exposure of early brown trout stages shortly after fertilisation up to the end of the embryonic development. Temperature and oxygen conditions were standardised in all test series in order to minimise unspecific effects. The examined endpoints were: (1) mortality, (2) developmental rate, (3) time course of hatching, (4) malformations, and (5) growth. A retarded development, reduced growth rates and higher mortality rates of K?rsch stream water exposed embryos indicated an embryotoxic potential for the more polluted stream. High infection-related mortality rates of embryos suggested the presence of confounding factors also in the less polluted Kr?henbach stream. In parallel to the exposure experiment, physicochemical and limnochemical parameters as well as concentrations of organic contaminants and heavy metals were monitored. Analytical data confirm the different degrees of pollution of both streams.     

Acute effects of Fe₂O₃, TiO₂, ZnO and CuO nanomaterials on Xenopus laevis

Metal oxide nanomaterials have exhibited toxicity to a variety of aquatic organisms, especially microbes and invertebrates. To date, few studies have evaluated the toxicity of metal oxide nanomaterials on aquatic vertebrates. Therefore, this study examined effects of ZnO, TiO₂, Fe₂O₃, and CuO nanomaterials (20-100 nm) on amphibians utilizing the Frog Embryo Teratogenesis Assay Xenopus (FETAX) protocol, a 96 h exposure with daily solution exchanges. Nanomaterials were dispersed in reconstituted moderately hard test medium. These exposures did not increase mortality in static renewal exposures containing up to 1000 mg L⁻¹ for TiO₂, Fe₂O₃, CuO, and ZnO, but did induce developmental abnormalities. Gastrointestinal, spinal, and other abnormalities were observed in CuO and ZnO nanomaterial exposures at concentrations as low as 3.16 mg L⁻¹ (ZnO). An EC₅₀ of 10.3 mg L⁻¹ ZnO was observed for total malformations. The minimum concentration to inhibit growth of tadpoles exposed to CuO or ZnO nanomaterials was 10 mg L⁻¹. The results indicate that select nanomaterials can negatively affect amphibians during development. Evaluation of nanomaterial exposure on vertebrate organisms are imperative to responsible production and introduction of nanomaterials in everyday products to ensure human and environmental safety.     

Embryonic development assay with Daphnia magna: application to toxicity of aniline derivatives.

An assay system using Daphnia magna embryos was applied to investigate the adverse effects of aniline derivatives. The data were compared with our previous data for chlorophenols. This new assay provides useful information to evaluate the toxicity of chemicals and the differences in sensitivity between the life stages. The effects of 15 aniline derivatives on embryonic development of D. magna embryos were determined. At the start of exposure, 2-6-h old eggs (between stages 1 and 2, round in shape, diameter approx. 400 microm), were used. In control and solvent control groups, embryonic development from an egg to a free-swimming animal proceeded completely within 3 days with more than 90% hatchability. Median effective concentrations (EC50s) to reduce the numbers hatched were determined and gross morphological abnormalities of hatched animals recorded. Anilines induced no obvious morphological abnormalities and no developmental delay although premature deaths occurred. However, they affected the number of embryos hatching in a dose-dependent manner. In addition, this embryo assay was more sensitive to aniline derivatives (except for aniline) than acute juveniles immobilization assay. Ratios of 48-h EC50 (juvenile)/3-day EC50 (embryo) for eight anilines were greater than 5.0. Particularly, the ratios of 4-methyl-, 4-ethyl- and 3-methylaniline were 77, 23 and 11, respectively. EC50s for embryos and juveniles were poorly correlated (r = 0.41). This indicated that the sensitivities of the two life stages were different to the effects of anilines. EC50s were poorly correlated (r = -0.097) with the log Kow (1-octanol/water partition coefficient). These results were compared with previous results for phenols.     

The effects of engineered nanoparticles on survival, reproduction, and behaviour of freshwater snail, Physa acuta (Draparnaud, 1805)

Increasing uses of engineered nanoparticles (ENPs) in commercial products and industrial applications has eventually resulted to their releases into atmospheric, terrestrial, and aquatic environments. However, knowledge gaps in ENPs toxicity, fate, and behaviour currently limit our ability to quantify risk assessment of materials with nanoscale dimensions, and therefore, the extent of the resultant environmental impacts remains unknown. In the present study, we evaluated the effects of γ-alumina, α-alumina, modified TiO(2) (M-TiO(2)), and commercial TiO(2) (C-TiO(2)) ENPs on the survival, behaviour, and early life stages of the freshwater snail Physa acuta (Draparnaud). The toxicity evaluation was carried out after spiking commercial sand with ENPs concentrations of 0.005, 0.05, or 0.5 gk g(-1). Our findings suggest that increases of γ-alumina and α-alumina concentrations at sub-lethal level concentrations caused significant reduction in the embryo growth rate and embryo hatchability. In addition, these ENPs induced observable developmental deformities of the embryos. In addition, toxicity evaluations using acute 96-h and chronic 28-d tests showed exposure duration may be a significant factor in ENPs-induced toxicity. Therefore, long-term exposure of aquatic organisms to ENPs - potentially can alter certain ecological populations at different trophic levels - and may compromise the entire aquatic ecological functionality. The percentage hatchlings in test chambers containing 0.5 gk g(-1) γ-alumina and α-alumina concentration was 50% less to those observed in the controls. Our results suggest the embryonic growth and hatchability tests are useful endpoints in chronic sediment toxicity tests for determining the toxic thresholds of ENPs in sediment environment. Although no snail mortalities were observed during the static 96-h test containing sediment spiked with different concentrations of M-TiO(2), C-TiO(2), γ-alumina and α-alumina - the antioxidant enzymatic assay results indicated a significant change in antioxidant levels which altered peroxidation at 0.05 or 0.5 gk g(-1)concentrations for both γ-alumina and α-alumina.     

Chronic toxicity of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in soil determined using the earthworm (Eisenia andrei) reproduction test

The sublethal and chronic effects of the environmental contaminant and explosive octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in artificial soil were assessed using the earthworm (Eisenia andrei). Based on various reproduction parameters (total and hatched number of cocoons, number of juveniles and their biomass), fecundity was reduced at the different concentrations of HMX tested (from 280.0 +/- 12.3 to 2502.9 +/- 230.0 mg kg-1 dry soil) in spiked artificial soil (LOEC: 280.0 +/- 12.3 mg kg-1 dry soil). The growth of adult E. andrei was also reduced at the different concentrations tested, though no mortality occurred, even at the highest tested concentrations. The number of juveniles produced was correlated with the number of total and hatched cocoons, and the biomass of juveniles was correlated with the number of cocoons. Pooled results of these and earlier studies on explosives (TNT, RDX) using the E. andrei reproduction test confirm that effects of HMX on cocoon production are indicative of some reproductive consequences (number of juvenile and their biomass), whereas adult growth, in general, does not correlate strongly with change in reproduction capacity.     

Relation between mortality of young walleye (Stizostedion vitreum) and recruitment with different forms of compensation

The relation between mortality of young fish and recruitment is important for assessment of the environmental effects of facilities that kill large numbers of young fish, such as electric power stations and hydropower plants. A simulation model with a bioenergetic growth component was applied to examine the relation between mortality of young and recruitment for walleye (Stizostedion vitreum) with different forms of population regulation, including: food limited growth, food limited growth with size-dependent mortality, and food limited growth with age at maturity dependent on size. With food limited growth small increases in mortality of young reduced recruitment considerably, but the population slowly approached a new equilibrium. If mortality of young increased when growth was food limited, the population approached a new equilibrium of natality and mortality because with fewer individuals there was more food per individual, and individuals were larger in size and produced more eggs; this feedback adjusted natality to equal mortality. With either mortality or age at maturity dependent on size, large increases in mortality of young resulted in only small decreases in recruitment.     

The influence of engineered Fe2O3 nanoparticles and soluble (FeCl3) iron on the developmental toxicity caused by CO2-induced seawater acidification

An embryo development assay using a common test organism, the edible mussel (Mytilus galloprovincialis), exposed to both Fe₂O₃ nanoparticles and soluble FeCl₃ at 3 acidic pHs, has provided evidence for the following: (1) CO₂ enriched seawater adjusted to pH projections for carbon capture leakage scenarios (CCS) significantly impaired embryo development; (2) under natural pH conditions, no significant effect was detected following exposure of embryos to Fe, no matter if in nano- or soluble form; (3) at pH of natural seawater nano-Fe particles aggregate into large, polydisperse and porous particles, with no biological impact detected; (4) at pH 6 and 7, such aggregates may moderate the damage associated with CO₂ enrichment as indicated by an increased prevalence of normal D-shell larvae when nano-Fe was present in the seawater at pH 7, while soluble iron benefited embryo development at pH 6, and (5) the observed effects of iron on pH-induced development toxicity were concentration dependent.     

Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols

Polycyclic aromatic hydrocarbons (PAHs) have been detected in some commercial teas around the world and pose a threat to tea consumers. However, green tea polyphenols (GTP) possess remarkable antioxidant and anticancer effects. In this study, the potential of GTP to block the toxicity of the model PAH phenanthrene was examined in human embryo lung fibroblast cell line HFL-I. Both GTP and phenanthrene treatment individually caused dose-dependent inhibition of cell growth. A full factorial design experiment demonstrated that the interaction of phenanthrene and GTP significantly reduced growth inhibition. Using the median effect method showed that phenanthrene and GTP were antagonistic when the inhibitory levels were less than about 50%. Apoptosis and cell cycle detection suggested that only phenanthrene affected cell cycle significantly and caused cell death; GTP lowered the mortality of HFL-I cells exposed to phenanthrene; However, GTP did not affect modulation of the cell cycle by phenanthrene.     

Effect of a heavy metal model mixture on biological parameters of rainbow trout <Emphasis Type="Italic">oncorhynchus mykiss</Emphasis>

The effects of a model mixture (HMMM) of seven heavy metals (Cu, Zn, Ni, Cr, Pb, Cd, Mn) on the rainbow trout Oncorhynchus mykiss at all stages of development (embryos, larvae, adults) were investigated based on the annual average concentrations of these metals in cooling waste waters discharging from Ignalina Nuclear Power Plant (Lithuania) into the Drūk?iai lake. According to mortality parameters, the most sensitive to HMMM were larvae, although no significant differences between the sensitivity of embryos and adult fish to HMMM were found. Maximal toxic effect of HMMM was observed during the hatching period. Long-term exposure to sublethal concentrations of HMMM affected embryo development, growth of larvae, their cardio-respiratory and behavioural responses, induced significant changes in morphological, morpho-physiological, physiological and haematological parameters of adult fish. Respiratory responses and growth parameters of fish were found to be the most sensitive to low concentrations of HMMM. Adult fish were capable of detecting and avoiding low, sublethal concentrations of HMMM. Heavy metals in a mixture at low concentrations were more toxic than single ones. According to the background of the damages induced by HMMM, after-effects in a fish organism, as well as in a whole population, can be predicted.     

Seeds’ physicochemical traits and mucilage protection against aluminum effect during germination and root elongation as important factors in a biofuel seed crop (Ricinus communis)

We determined the length, volume, dry biomass, and density in seeds of five castor bean cultivars and verified notable physicochemical trait differences. Seeds were then subjected to different toxic aluminum (Al) concentrations to evaluate germination, relative root elongation, and the role of root apices’ rhizosphere mucilage layer. Seeds’ physicochemical traits were associated with Al toxicity responses, and the absence of Al in cotyledons near to the embryo was revealed by Al-hematoxylin staining, indicating that Al did not induce significant germination reduction rates between cultivars. However, in the more sensitive cultivar, Al was found around the embryo, contributing to subsequent growth inhibition. After this, to investigate the role of mucilage in Al tolerance, an assay was conducted using NH₄Cl to remove root mucilage before or after exposure to different Al concentrations. Sequentially, the roots were stained with hematoxylin and a quantitative analysis of staining intensity was obtained. These results revealed the significant contribution of the mucilage layer to Al toxicity responses in castor bean seedlings. Root growth elongation under Al toxicity confirmed the role of the mucilage layer, which jointly indicated the differential Al tolerance between cultivars and an efficient Al-exclusion mechanism in the tolerant cultivar.     

Atrazine effects on in vitro maturation and in vitro fertilization in the bovine oocyte

The effect of low levels of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on in vitro oocyte maturation, in vitro capacitation of sperm, or in vitro fertilization of bovine oocytes and on the quality of blastocyst formation was studied. Bovine oocytes collected from abattoir ovaries were matured, fertilized, and developed to the blastocyst stage in vitro. Embryos that reached a morula or blastocyst stage were stained with Hoechst 33258 stain to determine the number of blastomeres per embryo. Three bulls whose fertilization rates were proven consistent among straws were used for this study. Atrazine was tested at concentrations of 0.01, 0.1, 1, and 10 microM in either the maturation medium, sperm capacitation medium, or the fertilization medium. Because atrazine was dissolved in ethanol, an ethanol control was used to determine any possible effects of ethanol on the in vitro process. The addition of atrazine to both the maturation and fertilization media did not result in any significant difference in fertilization rates between the controls and the treatments. In the capacitation medium, a significant difference between the controls and the atrazine levels of 0.1, 1, and 10 microM was noted for one bull. Atrazine did not affect the number of blastomeres per embryo. There was not a significant difference (p>0.05) in the number of blastomeres per embryo between the controls and the different levels of atrazine in each medium. This study indicates that low levels of atrazine do not have an effect on in vitro fertilization rates or the number of blastomeres per embryo produced in vitro.     

Reproductive success of barn swallows nesting near a selenium-contaminated lake in east Texas, USA

Reproductive success and contaminant levels in 1986 and 1987 were compared between Barn Swallows nesting at selenium-contaminated Martin Lake, Texas, USA, and swallows nesting at a reference site. Nests were initiated about the same time or earlier at Martin Lake than at the reference site and clutch size was similar between the two locations. Nest success was significantly higher at Martin Lake than at the reference site and no embryo or chick deformities were documented. Selenium concentrations in 14 of 20 eggs from Martin Lake were above background (> 3 ppm, dry weight); two of 20 eggs contained > 5 ppm, a concentration associated with a 20% embryo mortality/deformity rate in some bird species. Selenium concentrations in the kidneys of adult swallows were higher at Martin Lake (mean = 14 ppm dry weight) than at the reference site (5.8 ppm). DDE, the only detected organochlorine compound, was in two of 10 eggs from Martin Lake; these concentrations were below those associated with chronic poisoning and reproductive problems. The maximum mercury concentration in livers of adult Barn Swallows (0.83 ppm, dry weight) was within the range for background levels (< 5 ppm).     

Triadimefon Causes Branchial Arch Malformations in Xenopus laevis Embryos (5 pp)

- DOI: http://dx.doi.org/10.1065/espr2006.01.014 Background, Aims and Scope Triazole-derivatives are potent antifungal agents used as systemic agricultural fungicides and against fungal diseases in humans and domestic animals. They act by inhibiting the cytochrome P-450 conversion of lanosterol to ergosterol, thus resulting in faulty fungal cell wall synthesis. Some data have been published about the teratogenic activity of triazoles on rodent embryos: Hypoplasias, abnormal shape, agenesis of the branchial arches, for example, were reported as typical induced malformations. Unfortunately, no data are available on the embryotoxicity of these compounds in amphibians, despite the increasing concern among the scientific community about the phenomenon of global amphibian population declines. The aim of the present work is to evaluate the embryo-lethal and teratogenic potentials of Triadimefon (FON), a triazolederivative widely used as an antimycotic in agriculture, by the test FETAX (Frog Embryos Teratogenic Assay, Xenopus) with particular attention being paid to the analysis of branchial arch malformations. Methods Xenopus laevis embryos were exposed continuously from stage 9 to increasing concentrations of FON and analyzed at stage 47 for mortality and teratogenicity (group I) to determine the median lethal (LC50) and teratogenic (TC50) concentrations. Another two pools of larvae were exposed to FON for a 2 hour period at early gastrula (Group II) or neurula (Group III) stages to verify which period of development is the most sensitive to FON. The malformations observed were further investigated by histological section and cartilage staining with Alcian blue. Results and Discussion The assay has estimated LC50 and TC50 values of 63.8 μM and 2.73 μM, respectively; the resulting TI (Teratogenic Index = LC50/TC50) value of 23.4 has underlined the very high teratogenic risk associated with this compound. Neurulation was more sensitive to FON exposure than gastrulation, since the TC50 estimated values for group III (neurula exposed) specimens was 7.6 times lower than those of group II (gastrula exposed). Interestingly, for each group analyzed, 100% of malformed embryos showed alterations at branchial arch derived cartilages: Anterior cartilages were reduced, missing, fused or incorrectly positioned while gill cartilages were altered only in the most severely affected specimens. In some cases these malformations were associated with hyperpigmentation. Our results support the hypothesis that FON can interfere with Neural Crest Cell (NCC) migration, since craniofacial components and melanophores are derived from neural crest material. Conclusion In conclusion, our data show Triadimefon to be a potent teratogen able to induce specific craniofacial malformation in Xenopus laevis embryos, probably interfering with the NCC migration into the branchial mesenchyme. These results are also interesting for ecotoxicological reasons as FON, as well as other pesticides, are likely to be present in water systems near agricultural or urban areas which may serve as habitats for developing amphibians and fishes. Recommendation and Outlook Our results are in agreement with the data obtained on in vitro cultured rat embryos suggesting that the FON mechanism of action involves strongly conserved molecules. The choice of Xenopus laevis as the model organism allows us to extend the toxicological and teratological observations to a molecular level, in order to search for novel genes regulated by FON exposure.     

Effects of 17α-trenbolone and melengestrol acetate on Xenopus laevis growth, development, and survival

The synthetic growth-promoting hormones trenbolone and melengestrol acetate have been detected in the environment near beef cattle feedlots and are reportedly transported via wind-borne particulate matter. Therefore, movement of synthetic hormones from beef cattle feedlots to water bodies via particulate matter is possible. Our objective was to evaluate potential effects of 17α-trenbolone (17α-TB), melengestrol acetate (MGA), and combinations of both on growth, development, and survival of Xenopus laevis larvae. On post-hatch day 2 (stage 33/34), X. laevis larvae were exposed to three nominal concentrations of 17α-TB (10, 100, and 500 ng/L), MGA (1, 10, and 100 ng/L), a combination of both (1/10, 10/100, and 100/500 ng/L MGA/17α-TB), frog embryo teratogenesis assay-Xenopus medium, or a solvent control. Significant increases in all X. laevis growth metrics were observed among larvae in the 1 ng/L MGA?+?10 ng/L 17α-TB and 10 ng/L MGA?+?100 ng/L 17α-TB treatments. Stage of development was increased among larvae in the 1 ng/L MGA?+?10 ng/L 17α-TB treatment group and significantly decreased among those in the 500 ng/L 17α-TB treatment. Total body mass and snout–vent length of X. laevis larvae were significantly reduced in the 100 ng/L MGA and 100 ng/L MGA?+?500 ng/L 17α-TB treatment groups. Larvae exposed to 500 ng/L 17α-TB had decreased total body mass, snout–vent length, and total length. In general, growth measurements decreased with increasing concentration of MGA, 17α-TB, or a combination of both. Survival among all treatments was not significantly different from controls. Amphibians exposed to MGA and 17α-TB in the environment may experience alterations in growth and development.     

Ecotoxicological Assessment of Sediment, Suspended Matter and Water Samples in the Upper Danube River. A pilot study in search for the causes for the decline of fish catches (12 pp)

Goals, Scope and Background Fish populations, especially those of the grayling (Thymallus thymallus), have declined over the last two decades in the upper Danube River between Sigmaringen and Ulm, despite intensive and continuous stocking and improvement of water quality since the 1970s. Similar problems have been reported for other rivers, e.g. in Switzerland, Great Britain, the United States and Canada. In order to assess if ecotoxicological effects might be related to the decline in fish catch at the upper Danube River, sediment, suspended matter and waste water samples from sewage treatment plants were collected at selected locations and analyzed in a bioanalytical approach using a battery of bioassays. The results of this pilot study will be used to decide if a comprehensive weight-of-evidence study is needed. Methods Freeze-dried sediments and suspended particulate matters were extracted with acetone in a Soxhlet apparatus. Organic pollutants from sewage water were concentrated using XAD-resins. In order to investigate the ecotoxicological burden, the following bioassays were used: (1) neutral red assay with RTL-W1 cells (cytotoxicity), (2) comet assay with RTLW1 cells (genotoxicity), (3) Arthrobacter globiformis dehydrogenase assay (toxicity to bacteria), (4) yeast estrogen screen assay (endocrine disruption), (5) fish egg assay with the zebrafish (Danio rerio; embryo toxicity) and (6) Ames test with TA98 (mutagenicity). Results and Discussion The results of the in vitro tests elucidated a considerable genotoxic, cytotoxic, mutagenic, bacteriotoxic, embryotoxic and estrogenic burden in the upper Danube River, although with a very inhomogeneous distribution of effects. The samples taken from Riedlingen, for example, induced low embryo toxicity, but the second highest 17β-estradiol equivalent concentration (1.8 ng/L). Using the fish egg assay with native sediments, a broad range of embryotoxic effects could be elucidated, with clear-cut dose-response relationships for the embryotoxic effects of contaminated sediments. With native sediments, embryotoxicity was clearly higher than with corresponding pore waters, thus corroborating the view that – at least for fish eggs – the bioavailability of particle-bound lipophilic substances in native sediments is higher than generally assumed. The effect observed most frequently in the fish egg assay was a developmental delay. A comparison of our own results with locations along the rivers Rhine and Neckar demonstrated similar or even higher ranges of ecotoxicological burdens in the Danube River. Conclusions The complex pattern of ecotoxicological effects caused by environmental samples from the Danube River, when assessed in an in vitro biotest battery using both acute and more specific endpoints, showed that integration of different endpoints is essential for appropriate hazard assessment. Overall, the ecotoxicological hazard potential shown has indeed to be considered as one potential reason for the decline in fish catches at the upper Danube River. However, based on the results of this pilot study, it is not possible to elucidate that chemically induced alterations are responsible for the fish decline. Recommendations and Perspective . In order to confirm the ecological relevance of the in vitro results for the situation in the field and especially for the decline of the grayling and other fishes, further integrated investigations are required. For linking the weight of evidence obtained by in vitro assays and fish population investigations, the application of additional, more specific biomarkers (e.g. vitellogenin induction, EROD and micronucleus assay) has been initiated in fish taken from the field as well as in situ investigations.     

Resistance to teratogenesis by F1 and F2 embryos of PAH-adapted Fundulus heteroclitus is strongly inherited despite reduced recalcitrance of the AHR pathway

Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Superfund site on the Elizabeth River (Portsmouth, VA, USA) are exposed to a complex mixture of polycyclic aromatic hydrocarbons (PAHs) from former creosote operations, but are resistant to the acute toxicity and cardiac teratogenesis caused by PAHs. The resistance is associated with a dramatic recalcitrance to induction of cytochrome P450 (CYP1) metabolism enzymes following exposure to aryl hydrocarbon receptor (AHR) agonists, along with an elevated antioxidant response and increased expression of several other xenobiotic metabolism and excretion enzymes. However, the heritability of the resistance in the absence of chemical stressors has been inconsistently demonstrated. Understanding the heritability of this resistance will help clarify the nature of population-level responses to chronic exposure to PAH mixtures and aid in identifying the important mechanistic components of resistance to aryl hydrocarbons. We compared the response of Atlantic Wood F1 and F2 embryos to benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), and a mixture of BkF and fluoranthene (Fl) to that of F1 embryos of reference site killifish. Resistance to cardiac teratogenesis and induction of CYP mRNA expression and CYP activity was determined. We found that both Atlantic Wood F1 and F2 embryos were highly resistance to cardiac teratogenesis. However, the resistance by Atlantic Wood F2 embryos to induction of CYP mRNA expression and enzyme activity was intermediate between that of Atlantic Wood F1 embryos and reference embryos. These results suggest that resistance to cardiac teratogenesis in Atlantic Wood fish is conferred by multiple factors, not all of which appear to be fully genetically heritable.     

Effects of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in zebrafish: general and reproductive toxicity

Mixed-sex populations of young adult zebrafish (~2-month-old) were exposed to measured RDX concentrations of 0, 1 or 9.6 ppm for up to 12 weeks followed by a 15-day rearing period in untreated water. RDX caused high mortality at 9.6 ppm, with most deaths occurring within the first 8 weeks of exposure. RDX at 9.6 ppm caused lower body weights at 4 and 8 weeks of exposure; and at 1 ppm, lower body weight was observed only at 4 weeks. Fish length was not affected by treatment at any time during the exposure period. The bioconcentration factor for RDX seemed to be influenced by time of exposure but not by water RDX concentration; its overall values were 1.01+/-0.13, 0.91+/-0.06 and 2.23+/-0.04 at 4, 8 weeks and 12 weeks, respectively. RDX was not detected in fish collected after the 15-day recovery period. In a separate experiment, adult females and males were separately exposed to RDX at measured concentrations of 0, 0.5 and 3.2 ppm for a period of 6 weeks. Reproductive performance was evaluated by biweekly breeding of the fish and measuring packed-egg volume (PEV) as index of fecundity. At 0.5 ppm, RDX caused elevated PEV levels relative to the control value at 2 weeks but not at 4 or 6 weeks, whereas no significant effects were noted at 3.2 ppm. Egg fertilization and embryo hatching rates were not affected by RDX at any of the concentrations tested. In conclusion, RDX at sublethal concentrations causes short-term negative effects on growth and, at 0.5 ppm, positive effects on fecundity.