Inter-laboratory trial of a standardized sediment contact test with the aquatic plant Myriophyllum aquaticum (ISO 16191).

A whole-sediment toxicity test with Myriophyllum aquaticum has been developed by the German Federal Institute of Hydrology and standardized within the International Organization for Standardization (ISO; ISO 16191). An international ring-test was performed to evaluate the precision of the test method. Four sediments (artificial, natural) were tested. Test duration was 10 d, and test endpoint was inhibition of growth rate (r) based on fresh weight data. Eighteen of 21 laboratories met the validity criterion of r ≥ 0.09 d(-1) in the control. Results from 4 tests that did not conform to test-performance criteria were excluded from statistical evaluation. The inter-laboratory variability of growth rates (20.6%-25.0%) and inhibition (26.6%-39.9%) was comparable with the variability of other standardized bioassays. The mean test-internal variability of the controls was low (7% [control], 9.7% [solvent control]), yielding a high discriminatory power of the given test design (median minimum detectable differences [MDD] 13% to 15%). To ensure these MDDs, an additional validity criterion of CV ≤ 15% of the growth rate in the controls was recommended. As a positive control, 90 mg 3,5-dichlorophenol/kg sediment dry mass was tested. The range of the expected growth inhibition was proposed to be 35 ± 15%. The ring test results demonstrated the reliability of the ISO 16191 toxicity test and its suitability as a tool to assess the toxicity of sediment and dredged material.

Function and regulation of the glutathione peroxidase homologous gene GPXH/GPX5 in Chlamydomonas reinhardtii.

When exposed to strong sunlight, photosynthetic organisms encounter photooxidative stress by the increased production of reactive oxygen species causing harmful damages to proteins and membranes. Consequently, a fast and specific induction of defense mechanisms is required to protect the organism from cell death. In Chlamydomonas reinhardtii, the glutathione peroxidase homologous gene GPXH/GPX5 was shown to be specifically upregulated by singlet oxygen formed during high light conditions presumably to prevent the accumulation of lipid hydroperoxides and membrane damage. We now showed that the GPXH protein is a thioredoxin-dependent peroxidase catalyzing the reduction of hydrogen peroxide and organic hydroperoxides.Furthermore, the GPXH gene seems to encode a dual-targeted protein, predicted to be localized both in the chloroplast and the cytoplasm, which is active with either plastidic TRXy or cytosolic TRXh1. Putative dual-targeting is achieved by alternative transcription and translation start sites expressed independently from either a TATA-box or an Initiator core promoter. Expression of both transcripts was upregulated by photooxidative stress even though with different strengths. The induction required the presence of the core promoter sequences and multiple upstream regulatory elements including a Sp1-like element and an earlier identified CRE/AP-1 homologous sequence. This element was further characterized by mutation analysis but could not be confirmed to be a consensus CRE or AP1 element. Instead, it rather seems to be another member of the large group of TGAC-transcription factor binding sites found to be involved in the response of different genes to oxidative stress.

Zebrafish (Danio rerio) neuromast: promising biological endpoint linking developmental and toxicological studies.

Aquatic toxicology is facing the challenge to assess the impact of complex mixtures of compounds on diverse biological endpoints. So far, ecotoxicology focuses mainly on apical endpoints such as growth, lethality and reproduction, but does not consider sublethal toxic effects that may indirectly cause ecological effects. One such sublethal effect is toxicant-induced impairment of neurosensory functions which will affect important behavioural traits of exposed organisms. Here, we critically review the mechanosensory lateral line (LL) system of zebrafish as a model to screen for chemical effects on neurosensory function of fish in particular and vertebrates in general. The LL system consists of so-called neuromasts, composed of centrally located sensory hair cells, and surrounding supporting cells. The function of neuromasts is the detection of water movements that is essential for the fish's ability to detect prey, to escape predator, to socially interact or to show rheotactic behaviour. Recent advances in the study of these organs provided researchers with a broad area of molecular tools for easy and rapid detection of neuromasts dysfunction and/or disturbed development. Further, genes involved in neuromasts differentiation have been identified using auditory/mechanosensory mutants and morphants. A number of environmental toxicants including metals and pharmaceuticals have been shown to affect neuromasts development and/or function. The use of the LL organ for toxicological studies offers the advantage to integrate the available profound knowledge on developmental biology of the neuromasts with the study of chemical toxicity. This combination may provide a powerful tool in environmental risk assessment.

Internal exposure of whitefish (Coregonus lavaretus) to estrogens.

Gonad malformations have been found in fish all over the world. Particularly in Lake Thun (Switzerland) a high prevalence of gonad deformations in whitefish has been observed. Very often, a link between exposure to endocrine disrupting compounds and altered gonad morphology exists. Hence, we analyzed the estrogenic burden in bile and muscle from whitefish (coregonids) from Lake Thun and linked it to observed gonad malformations. Estrogenicity in bile, measured with the yeast estrogen screen (YES) was exclusively caused by the natural steroids estrone and 17beta-estradiol. Estrogenicity determined in muscle tissue using YES was similar in cases and controls, and between the sexes. Furthermore, endocrine active compounds in the lake water were investigated using passive sampling devices to monitor tributaries and the main outflow of Lake Thun. Here, we found accumulated estrogenicity. With target chemical analysis small amounts of estrone and bisphenol A were determined. We conclude, that the whitefish from Lake Thun are not suffering from (xeno)estrogens. The present study contributed substantially to the search for the cause for gonad malformations in Lake Thun whitefish, even though the cause of the malformations remains yet to be discovered.

Estrogen receptor subtype beta2 is involved in neuromast development in zebrafish (Danio rerio) larvae.

Estrogens are known to play a role in both reproductive and non-reproductive functions in mammals. Estrogens and their receptors are involved in the development of the central nervous system (brain development, neuronal survival and differentiation) as well as in the development of the peripheral nervous system (sensory-motor behaviors). In order to decipher possible functions of estrogens in early development of the zebrafish sensory system, we investigated the role of estrogen receptor beta(2) (ERbeta(2)) by using a morpholino (MO) approach blocking erbeta(2) RNA translation. We further investigated the development of lateral line organs by cell-specific labeling, which revealed a disrupted development of neuromasts in morphants. The supporting cells developed and migrated normally. Sensory hair cells, however, were absent in morphants' neuromasts. Microarray analysis and subsequent in situ hybridizations indicated an aberrant activation of the Notch signaling pathway in ERbeta(2) morphants. We conclude that signaling via ERbeta(2) is essential for hair cell development and may involve an interaction with the Notch signaling pathway during cell fate decision in the neuromast maturation process.

Molecular multi-effect screening of environmental pollutants using the MolDarT.

Molecular effect detection is a useful approach for ecotoxicological screening of chemicals. We show here the application of the molecular DarT (MolDarT), where the expression of selected target genes is detected in short-term (120 h) exposed developing zebrafish (Danio rerio), thus allowing subacute multi-effect compound screening. The genes metallothionein 2 (mt2), cytochrome P450 1A1 (cyp1a1), and recombination activation gene 1 (rag1) are used as endpoints that describe detoxification/metal toxicity (mt2), detoxification/PAH toxicity (cyp1a1), and acquired immune system disruption (rag1). Each gene's developmental expression was studied in unexposed zebrafish during 4 to 120 h past fertilization (hpf), and all three genes were found to be expressed at 120 hpf. Furthermore, mt2 transcripts were present at high levels at 4 hpf, indicating a maternal transfer. For positive toxicity controls, freshly fertilized zebrafish eggs were exposed for 120 hpf to ZnSO(4), 1,5-dimethylnaphthalene (DMN) and CdCl(2). Exposure to 100 and 200 microM ZnSO(4) significantly induced mt2; 10 microM DMN and 20 microM DMN resulted in significantly increased cyp1a1 abundance; and 5 and 10 microM CdCl(2) significantly reduced rag1 expression levels. Furthermore, we analysed these target genes for their expression in zebrafish eggs from a previous exposure study. The eggs were exposed for 120 hpf to the environmental pollutants estradiol (E2), ethinylestradiol (EE2), nonylphenol (NP), atrazine, cyproconazol, and bisphenol A (BPA) and found differential expression of the three genes. Exposure to the (xeno-)estrogenic compound NP (0.75 microM) significantly lowered mt2 expression. This study shows the potential of short-term in vivo multi-effect screenings within one single subacute exposure using the MolDarT.