The zebrafish embryo model in environmental risk assessment—applications beyond acute toxicity testing

BACKGROUND, AIM, AND SCOPE: The use of fish embryos is not regulated by current legislations on animal welfare and is therefore considered as a refinement, if not replacement of animal experiments. Fish embryos represent an attractive model for environmental risk assessment of chemicals since they offer the possibility to perform small-scale, high-throughput analyses. MAIN FEATURES: Beyond their application for determining the acute toxicity, fish embryos are also excellent models for studies aimed at the understanding of toxic mechanisms and the indication of possible adverse and long-term effects. Therefore, we have reviewed the scientific literature in order to indicate alternative applications of the fish embryo model with focus on embryos of the zebrafish. RESULTS AND DISCUSSIONS: The analysis of the mode of action is important for the risk assessment of environmental chemicals and can assist in indicating adverse and long-term effects. Toxicogenomics present a promising approach to unravel the potential mechanisms. Therefore, we present examples of the use of zebrafish embryos to study the effect of chemicals on gene and protein patterns, and the potential implications of differential expression for toxicity. The possible application of other methods, such as kinase arrays or metabolomic profiling, is also highlighted. Furthermore, we show examples of toxicokinetic studies (bioconcentration, ABC transporters) and discuss limitations that might be caused by the potential barrier function of the chorion. Finally, we demonstrate that biomarkers of endocrine disruption, immune modulation, genotoxicity or chronic toxicity could be used as indicators or predictors of sub-acute and long-term effects. CONCLUSIONS: The zebrafish embryo represents a model with an impressive range of possible applications in environmental sciences. Particularly, the adaptation of molecular, system-wide approaches from biomedical research is likely to extend its use in ecotoxicology. RECOMMENDATIONS AND PERSPECTIVES: Challenges for future research are (1) the identification of further suitable molecular markers as indicators of the mode of action, (2) the establishment of strong links between (molecular) effects in short-term assays in embryos and long-term (toxic) effects on individuals, (3) the definition of limitations of the model and (4) the development of tests that can be used for regulatory purposes.     

Information system for monitoring environmental impacts of genetically modified organisms

Background, aim and scope  

European legislation stipulates that genetically modified organisms (GMO) have to be monitored to identify potential adverse environmental effects. A wealth of different types of monitoring data from various sources including existing environmental monitoring programmes is expected to accumulate. This requires an information system to efficiently structure, process and evaluate the monitoring data.     

Modelling changes in nitrogen emissions into the Oder River System 1875–1944

Studies of nutrient emissions into surface waters are usually only performed for years in recent decades. However, estimating nutrient emissions for the more distant past enables us to identify the main factors responsible for the increasing nutrient contamination since the end of the nineteenth century. We focussed on the Oder River System for 1875–1944, divided into 10-year periods. Nutrient emissions into surface waters were calculated with the model MONERIS (MOdelling Nutrient Emissions in RIver Systems). For seven different pathways and eight sources, the total nitrogen (TN) emissions were quantified. The TN-emissions into the surface waters for 1880 amounted to 25,300 t?year^?1, and by 1940, this value had almost doubled to 46,600?t?year^?1. In 1880, 57% of TN-emissions into the surface waters derived from urban systems, due to the high amount of untreated waste water. In 1940, only 34% of TN-emissions into surface waters derived from urban systems, despite a population growth of about 27% since 1880; point sources via newly constructed waste water treatment plants (WWTPs) increased from 4% (1880) to 26% (1940). During the study period, the main changes in diffuse TN-emissions from agriculture were caused by inorganic fertilizer application and nitrogen deposition, while TN-emissions via urban sources were shifted to point sources due to population growth and the construction of new WWTPs. Furthermore, estimated TN-concentrations could make a contribution to construct benchmarks for nutrient concentrations according to the physiochemical properties to implement the European Water Framework Directive (WFD 2000).     

Biological effect monitoring in dab (<Emphasis Type="Italic">Limanda limanda</Emphasis>) using gene transcript of CYP1A1 or EROD—a comparison

BACKGROUND, AIM, AND SCOPE: Gene expression analyses with real-time (RT)-polymerase chain reaction (PCR) gains importance in marine monitoring. This new technique has to be compared to the classical approaches like the well known biomarker ethoxyresorufin-O-deethylase (EROD) to test their suitability for monitoring programmes. The goal of the present study is to compare EROD activity and CYP1A1 mRNA expression in the important monitoring fish species dab (Limanda limanda) and to answer the question of whether these parameters reflect the polycyclic aromatic hydrocarbon (PAH) contamination of the fish. Further on, glyceraldehyd-3-phosphate dehydrogenase (GAPDH) was investigated as a potential housekeeping gene. MATERIALS AND METHODS: Female dab were caught in the summer of 2004 in the North Sea and in the Baltic. EROD activity was determined in liver samples by a kinetic fluorimetric assay according to a standard protocol. The gene expression of CYP1A (cytochrome P450 1A) and GAPDH were determined by means of RT-PCR. Results were compared to gonado somatic index and to the concentration of PAH metabolite 1OHPyr (1-hydroxypyrene) analysed in the bile fluids of the fish, respectively. RESULTS: Dab from all stations showed a considerable individual variation in the levels of both CYP1A mRNA and EROD. Highest mean values for CYP1A mRNA and EROD were detected in the northern part of the sampling area. In contrast, the PAH metabolite 1OHPyr was found at the highest concentration in fish caught near the German coast. CYP1A mRNA and EROD showed only a minor but significant correlation (r = 0.32, p < 0.05, n = 123). 1OHPyr in bile correlated significantly (p < 0.05) with the amount of GAPDH mRNA content in the liver. DISCUSSION: The significant but low correlation of CYP1A mRNA and EROD activity on an individual basis illustrates that these two parameters are apparently not closely linked. However, maximum EROD values correspond with maximum CYP1A mRNA concentrations when station means are regarded. Because EROD and CYP1A mRNA in dab follow different physiological principles, their application will lead to related but not identical monitoring results. This should be taken into account when future marine monitoring programmes are designed. The results also indicate that PAH are not the crucial factor for CYP1A and EROD levels in dab from the off-shore areas in the North Sea. This is remarkable because the PAH metabolism is known to be CYP1A-dependent and the widely used biomarker EROD has been recommended for monitoring PAH-related effects in fish from the North Sea. Due to a correlation between GAPDH and 1OHPyr, GAPDH was not suitable as housekeeping gene for dab. CONCLUSIONS: Neither the results from EROD nor from CYP1A1 mRNA measurements in dab reflected their exposure to PAH as measured by the PAH metabolite 1OHPyr. Thus, the question arises of whether EROD or CYP1A mRNA is a suitable biomarker at all to indicate PAH exposure in dab from the open North Sea. RECOMMENDATIONS AND PERSPECTIVES: For future biological effect monitoring, it is advisable to measure more and predominately independent parameters by RT-PCR and to incorporate more components of the detoxification system.     

Entwicklung eines Sedimentbiotests mit der ZwergdeckelschneckePotamopyrgus antipodarum (Gastropoda: Prosobranchia)

Currently, only few organismic biotest systems are available for the assessment of effects on reproduction, which consider an exposure toward whole sediments. A well-suited test organism is the parthenogenetic mudsnailPotamopyrgus antipodarum. This ovoviviparous prosobranch snail lives in the upper layers of freshwater and brackish sediments. The number of (unshelled) embryos turned out to be a very sensitive parameter for the indication of sediment components affecting reproduction. Additionally, this endpoint allows to signal possible estrogenic sediment compounds. With this 28-day bioassay, laboratory tests on the effects of various chemicals onP. antipodarum were performed, e.g. triphenyltin and bisphenol A, in relevant environmental concentration ranges. Furthermore, the suitability of our test system for the investigation of complex and multiple level ranged contaminations was examined by means of various natural sediments from the Neiße and Odra rivers.     

Isolation and characterization of 1,2,4-trichlorobenzene mineralizing Bordetella sp. and its bioremediation potential in soil

A soil which has been polluted with chlorinated benzenes for more than 25 years was used for isolation of adapted microorganisms able to mineralize 1,2,4-trichlorobenzene (1,2,4-TCB). A microbial community was enriched from this soil and acclimated in liquid culture under aerobic conditions using 1,2,4-TCB as a sole available carbon source. From this community, two strains were isolated and identified by comparative sequence analysis of their 16S-rRNA coding genes as members of the genus Bordetella with Bordetella sp. QJ2-5 as the highest homological strain and with Bordetella petrii as the closest related described species. The 16S-rDNA of the two isolated strains showed a similarity of 100%. These strains were able to mineralize 1,2,4-TCB within two weeks to approximately 50% in liquid culture experiments. One of these strains was reinoculated to an agricultural soil with low native 1,2,4-TCB degradation capacity to investigate its bioremediation potential. The reinoculated strain kept its biodegradation capability: (14)C-labeled 1,2,4-TCB applied to this inoculated soil was mineralized to about 40% within one month of incubation. This indicates a possible application of the isolated Bordetella sp. for bioremediation of 1,2,4-TCB contaminated sites.     

Chemical carcinogenesis — the price for DNA-repair?

This essay examines the possibility of merging the mutation theory of cancer with the hypothesis that cancer is a change in the state of the differentiation of cells. It is suggested that during normal development DNA rearrangements occur, concerning genes which code for differentiation specific cell communication proteins. These proteins are responsible for the proper functioning of growth control in a multicellular organism. DNA-damaging agents — mutagens — induce DNA repair enzymes, some of which may catalyse illegitimate genome rearrangements, thus leading to a change of the balance between growth and differentiation. A cell with a selective advantage may arise and become the origin of a tumor.     

Canopy structure versus physiology effects on net photosynthesis of mountain grasslands differing in land use

The present paper aims at investigating how changes in canopy structure and species physiology associated with the abandonment of mountain meadows and pastures affect their net photosynthesis. For this purpose, a multi-layer vegetation–atmosphere transfer (VAT) model is employed, which explicitly takes into account the structural and functional properties of the various canopy components and species. Three sites differing in land use are investigated, a meadow, a pasture and an abandoned area. Model simulations agree reasonably with measured canopy net photosynthetic rates, the meadow featuring the highest daily net photosynthesis, followed by the pasture and, finally, the abandoned area. A detailed process analysis suggests this ranking to be mainly due to bulk canopy physiology, which decreases from the meadow to the pasture and the abandoned area, reflecting species composition and species-specific photosynthetic capacities. Differences between the canopies with regard to canopy structure are found to be of minor importance. The amounts of green, photosynthetically active plant matter are too similar at the three sites to be a major source of variation in net photosynthesis. Large differences exist between the canopies with regard to the amount of photosynthetically inactive phytoelements. Even though a model analysis showed them to be potentially important, most of them are accumulated close to the ground surface, where they exert little influence on canopy net photosynthesis.