Roland Nagel

UV-Filtersubstanzen in Wasser und Fischen

ZusammenfassungZur Erfassung der UV-Filtersubstanzen wird eine Methode vorgestellt, die die simultane Bestimmung dieser Verbindungen und von Organochlorverbindungen wie PCB und DDT in unterschiedlichen Fischkompartimenten bis in den ng/kg-Bereich ermöglicht. Die Nachweisgrenzen liegen zwischen 40 und 90 ng/kg Filet und die Wiederfindungsraten bewegen sich zwischen 78 und 104%.Die Belastung von Wasser und Fischen des Meerfelder Maares, Eifel, mit UV-Filtersubstanzen wurde 1991 und 1993 exemplarisch untersucht. Insgesamt konnten in den Fischen des Meerfelder Maares 6 UV-Filtersubstanzen identifiziert und quantifiziert werden. Die im Sommer 1991 gefangenen Barsche waren mit 2,0 mg/kg Fett (Summe nachgewiesener UV-Filtersubstanzen) und die Rotaugen von 1993 mit 0,50 mg/kg Fett belastet. In beiden Fischarten lag die Belastung in der gleichen Größenordnung wie die der Organochlorverbindungen PCB und DDT. In den Rotaugen aus drei weiteren deutschen Seen konnte im Filet Methylbenzylidencampher, eine lipophile UV-Filtersubstanz, nachgewiesen werden. Diese Befunde zeigen, daß UV-Filtersubstanzen in deutschen Seen weir verbreitet und vermutlich als relevante Umweltchemikalien zu betrachten sind. Da im Wasser der Seen die Konzentrationen der UV-Filter substanzen meistens unter der Bestimmungsgrenze lagen, können Fische für diese lipophilen Verbindungen als Expositionsmonitor verwendet werden. Eine ökotoxikologische Bewertung ist zum jetzigen Zeitpunkt nicht möglich, da die Datenlage völlig unzureichend ist.AbstractA method was developed for the analysis of fish samples to detect sunscreen agents (SSA) together with organochlorides (PCB, DDT) in different fish tissues. Detection limits of the compounds analyzed are in the range of ng/kg and vary between 40 and 90 ng/kg fillet. Recovery rates range from 78 to 104%.The contamination of water and fish with SSA in Meerfelder Maar lake, the Eifel, Germany was investigated in 1991 and 1993 and allowed the identification and quantification of six different SSA in the fish. The sum of SSA concentrations in perch taken in the summer of 1991 was 2.0 mg/kg lipid and 0.50 mg/kg lipid was found in roach sampled during the summer of 1993. Both species were contaminated with SSA and organochlorides to the same ranges as PCB and DDT. Even in the fillet of roach taken from three other lakes in Germany, methylbenzylidene camphor, a lipophilic SSA, was detected. These results indicate that SSA are wide-spread in German lakes. Therefore, they can be seen as a new group of relevant environmental chemicals. In the investigated lakes, the concentrations of the SSA in water were mostly below the detection limits. Thus, fish can be used as a biomonitor for these lipophilic compounds. Due to the lack of toxicological data for aquatic organisms, an ecotoxicological assessment is impossible at the moment.

Comparison of testing acute toxicity on embryo of zebrafish, Brachydanio rerio and RTG-2 cytotoxicity as possible alternatives to the acute fish test

Abstract The present study compares two possible alternative methods to replace the acute fish test. Fertilized eggs of zebrafish, Brachydanio rerio were used to investigate the acute toxicity of chemicals. Different toxicological endpoints such as coagulation of the eggs, development of gastrulation, number of somites, development of organs, circulation, heartbeat, otolithanlage and pigmentation have been examined during the embryonic development of zebrafish within the first 48 h. Differences to the normal development were measured by these endpoints. Chemicals have been examined in a preliminary approach with respect to their cytotoxicity on the RTG-2 cells (Rainbow Trout Gonad). The vitality of cells was measured by Neutral Red-and MTT assay. Urea, sodium chloride, 2-nitroanisole, 3,4-dichloroaniline, 4-nitrophenol, phenol, 2,4-dinitrophenol, dinitro-ortho-cresole, carbaryl and malathion were used as compounds with different modes of actions. The results of the cytotoxicity tests with Neutral Red uptake (NR50) and MTT (MTT50) have been compared with data of zebrafish, Brachydanio rerio. We could show that the embryo test in most cases is more sensitive than the acute toxicity test with adult zebrafish and in all cases is more sensitive than the RTG-2 cell test. Therefore, considering the wide range of possibilities the embryo of zebrafish offers, we think that the embryo test is a very good candidate to replace the acute fish test. Whether the cytotoxicity test with RTG-2 cells is also an alternative requires further, more detailed studies.

Development of a New Screening Assay to Identify Proteratogenic Substances using Zebrafish Danio rerio Embryo Combined with an Exogenous Mammalian Metabolic Activation System (mDarT)

The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays, for example, in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were cocultured at 2 hpf (hours postfertilization) with the test material at varying concentrations, induced male rat liver microsomes and nicotinamide adenine dinucleotide phosphate (reduced) for 60 min at 32 degrees C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterward fish embryos were transferred individually into 24-well plates filled with fish medium for 48 h at 26 degrees C with a 12-h light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, that is, without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when coincubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the D. rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.

Bioaccumulation of lindane and hexachlorobenzene by tubificid sludgeworms (Oligochaeta) under standardised laboratory conditions

Abstract Lipophilic chemicals released into the aquatic environment tend to adsorb to suspended material and sediments. These compounds are therefore not bioavailable to many pelagic organisms, and their overall bioaccumulation is often not adequately covered by the currently prescribed measuring of bioconcentration in fish. The assessment of bioaccumulation in sediment-dwelling organisms shall give more detailed information on the hazard of chemicals for aquatic ecosystems. Therefore a standardised test design is required. The freshwater tubificids Tubifex tubifex and Limnodrilus hoffmeisteri were chosen as test organisms, while 14 C-Lindane (γ-Hexachlorocyclohexane, γ-HCH), and 14 C-Hexachlorobenzene served as test substances. The bioaccumulation studies were carried out in a static system consisting of artificial sediment and reconstituted water. Uptake as well as deputation phases were examined. Bioaccumulation factors (BAF) were determined from the ratio of concentration in worm tissue to concentration in sediment, and from the ratio of uptake rate constant (k s ) to elimination rate constant (k e ), respectively. Lindane concentrations were found to be about 4 times higher in the worms compared to the sediment, HCB was accumulated by a factor of approximately 7. The tubificid bioaccumulation factors of Lindane and HCB are discussed in comparison to corresponding fish bioconcentration data from lieterature. The presented data show that the assessment of bioaccumulation based exclusively on the extrapolation of fish-BCF to other organisms and environmental compartments is not appropriate.

Tubifex tubifex as a link in food chain transfer of hexachlorobenzene from contaminated sediment to fish

Sediments contaminated with poorly water-soluble organic chemicals pose a risk to aquatic food chains. Sediment-associated chemicals can be accumulated by endobenthic, sediment-ingesting invertebrates. Some tubificid species – or other benthic annelids – serve as food for benthivorous fish, which thereby ingest the sediment-borne chemicals and may accumulate contaminant concentrations far higher than from water exposure only, and transfer them to organisms of higher trophic levels. For measurement of biomagnification, a sediment based food chain was developed and established in the laboratory. The two-step food chain included the sediment-dwelling freshwater oligochaete Tubifex tubifex (Müller) as a representative species of benthic infauna. The three-spined stickleback (Gasterosteus aculeatus, Linné), a small teleost fish which often feeds primarily on benthic invertebrates, served as a model predator. Spiked artificial sediment and reconstituted water as the overlying medium were used. Experiments were performed using 14C-labelled hexachlorobenzene, a hydrophobic pollutant as a model compound. To examine the influence of benthic prey on the bioaccumulation of the test substance in the predator, fish were exposed to spiked water, spiked sediment, pre-contaminated prey organisms, or combinations of these exposure routes. The results of these experiments indicate that for hexachlorobenzene, the presence of contaminated Tubifex tubifex as a food source in combined exposure leads to significantly higher accumulation in fish than exposure to single pathways.

Intersexuality in Gammarus fossarum Koch: a common inducible phenomenon?

The occurrence of intersexuality was investigated in Gammarus fossarum populations from five different streams in Germany in the years 2000-2003. In the streams Lockwitzbach and Korsch sampling sites were upstream and downstream of sewage treatment plant effluents, in the three other streams an effluent was absent. In the stream Lockwitzbach the median frequency of intersexes at the two sampling sites was 7.1 % and 13 % over two years. In the streams Korsch and Zschonerbach the frequency of intersexes was lower with 0.2 % and 0.8 % at the two sampling sites at the Korsch, respectively, and between 0.3 % and 0.7% at the Zschonerbach. Because in the Goldersbach and the Friedrichsbach in the absence of effluents the frequency of intersexes was 3.2% and 24%, an impact of chemicals from municipal sewage treatment plants can be excluded. As the phenomenon occurred in three of five streams but can easily be overlooked, it may be much more common than previously assumed. At the upstream sampling site of the Lockwitzbach with a high frequency of intersex the affected specimens were larger compared to females. An exposure of gammarids from streams with low intersex frequency to water from streams with high intersex frequency led to a significant, time dependent increase of intersex frequency. Thus, although there are reports about induction of intersex by photoperiod, a factor which plays no role in the experiments performed here, it is concluded that some factor in the water - may be a chemical - is responsible for the induction of intersex in G. fossarum. At the moment it is unclear whether an anthropogenic or natural factor is responsible and which role environmental conditions play.

Ökotoxikologie soll endlich wissenschaftliches Fach werden

Umweltbundesamt, Bismarckplatz 1, 14193 Berlin 2 Universit~it Bremen, Zentrum f0r Umweltforschung und Umwelttechnologie (UFT), 28359 Bremen a Universit&t Bayreuth, Lehrstuhl f0r Umweltchemie und s 95440 Bayreuth 4 Fraunhofer-lnstitut Molekularbiologie und Angewandte Oekologie, 57392 Schmallenberg s Technische Universit&t Dresden, Institut for Hydrobiologie AG (3kotoxikologie, 01062 Dresden 8 UFZ-Umweltforschungszentrum Leipzig-Halle GmbH, Sektion Chemische s 04318 Leipzig

INTERSEXUALITY IN GAMMARUS FOSSARUM KOCH, 1835 (AMPHIPODA)

We present the first description of intersexes in Gammarus fossarum Koch, 1835 (Amphipoda). Intersexes were found in monthly samples collected in two streams in different regions of Germany. The frequency of intersexuality differed considerably between the populations from the two streams. The Lockwitzbach, located in Sachsen, showed a frequency of 8.8% intersexes, which is much higher compared to the K?rsch (located in Baden-W?rttemberg) with 0.6%. In contrast to the K?rsch, there were also differences between the two sampling sites in the Lockwitzbach. No seasonal pattern in intersex frequency was observed. Intersexes occurred in all length classes of the adolescent and adult gammarids examined. They were significantly larger than females, but showed no difference in body length compared to males. External sex characteristics of intersex individuals have been described in detail, while in histological analyses of internal sex characteristics ovarian tissue was found exclusively. Microsporidians, parasites often associated with intersexuality, were not detected. The intersexes seemed to be functional females, regarding their ability to breed.

Testing of chemicals with fish — a critical evaluation of tests with special regard to zebrafish

In this review, different test systems with fish according to the German Chemicals and Plant Protection Acts are introduced and evaluated. On the basis of a critical consideration of these test systems, the following test concept with zebrafish (Danio rerio) is proposed:

Artificial indoor streams as a method to investigate the impact of chemicals on lotic communities.

The potential hazard of chemicals on aquatic communities are generally evaluated by standardised single-species bioassays. Safety assessment is based on results gained from organisms adapted to lentic systems and biological interactions in ecosystems are neglected. While lotic communities are often at first in contact with chemicals, it is astonishing that microcosms with lentic communities are mainly used as a bridge between laboratory bioassays and outdoor aquatic systems. Hence, we established five artificial indoor streams to simulate abiotic factors of small rivers. The closed-circuit system was filled with nutrients added to tap water. Washed pebbles were used as sediment. The dynamics of a simple biocoenoses consisting of aufwuchs,Lumbriculus variegatus Asellus aquaticus andGammarus fossarum was investigated. The dynamic of aufwuchs and periphyton was determined as dry weight and chlorophyll-a, respectively and qualitatively by pigment pattern. The abundance of different developmental stages ofL. variegatus was determined at the end of the experiment as well as the population dynamics ofG. fossarum andA. aquaticus. Survival rates of gammarids and juveniles per female were investigated and data were used for modelling the population dynamics. The experiment was carried out to investigate the performance of the established artificial streams and the developed approaches to investigate effects of chemicals on a basic lotic community. The prime reason to establish this approach was to close a gap between complex artificial stream systems and laboratory single species tests to assess the impact of chemicals on the aquatic environment.