No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Effect of 3,4-dichloroaniline on the early life stages of the zebrafish (Brachydanio retio): Results of a comparative laboratory study
Abstract
An early life-stage (ELS) test was conducted with the zebrafish and 3,4-dichloroaniline (3,4-DCA) in eight laboratories. Based on the results from all eight laboratories, an LOEC of 200 micrograms 3,4-dichloroaniline/liter applies for the early life stages of the zebrafish. Effects observed were reduction of the survival rate and malformations. If the 100 micrograms/liter concentration additionally tested by one laboratory is included in the assessment, an LOEC of 100 micrograms 3,4-DCA/liter is obtained for the survival rate and increase in length. The NOEC is 20 micrograms/liter. The present results of a comparative laboratory study with the zebrafish show that a 42-day ELS test can be conducted with this species of fish, and affords meaningful results.
... This species is the model used in the FET (OECD 2013b), which specifies 3,4-DCA as a positive control in this test with the expectation that a concentration of 4000 μg L À1 should result in at least 30% mortality after 96 h. Nagel et al. (1991) exposed several groups of newly spawned zebrafish ova to 3,4-DCA at concentrations from 2 to 200 μg L À1 . They found significant mortality at 2 weeks and beyond at 200 μg L À1 and at 4 weeks and beyond at 100 μg L À1 . ...
... Differences between studies in onset of exposure, exposure duration and exposure solution renewal regime make direct comparisons difficult. Call et al. (1987), Nagel et al. (1991), Schiwy et al. (2020), Hansen et al. (2021), and this study verified 3,4-DCA concentrations through analytical chemistry, but in other experiments the nominal concentrations reported may vary from the actual concentration, also affecting comparison. Furthermore, the rates at which fish embryos develop and even the developmental stage at which morphological changes occur varies widely between species, as do the temperatures at which they are typically held in the laboratory. ...
... The clearest advantage of this system is its ability to image and measure more endpoints in many more samples, much more quickly, than is practically feasible with manual methods. Other toxicity studies reviewed in this article used as few as 2 replicates of 10 animals (Schiwy et al. 2020), and 800 embryos were measured in the largest study (Nagel et al. 1991), with the work divided over 8 laboratories. In most cases, few endpoints were measured, and typically only mortality was reported at multiple timepoints. ...
Early life stages of fish are widely used for regulatory toxicity testing, and marine fish display high sensitivity to pollutant exposure. Exposure to pollutants during embryogenesis causes acute effects on embryonic development and survival, but also sub‐lethal impacts manifested as maldeveloped larvae. Acquiring time‐ and exposure‐dependent responses to pollutant exposure and other stressors in small organisms is labor intensive and often subjective. This leads to studies obtaining small sample sizes, with measurements often made infrequently during development. Automated monitoring methods can maintain consistency between measurements and allow many more measurements to be made, improving the quantity and quality of such data. We exposed Atlantic cod embryos to 3,4‐dichloroaniline, a reference chemical widely used as a positive control agent in regulatory fish embryo toxicity testing. We monitored their growth through daily imaging with an automated flow‐through imaging system. Biologically relevant sublethal endpoints were estimated from these images with a neural network and traditional machine vision methods. We demonstrate the automated capture and analysis of tens of thousands of images, producing detailed morphometric data from hundreds of fish over a 10‐d study period, and assess the effectiveness of the automated system. The automated method presented allows measurements to be made frequently without sacrificing the sampled organisms, making detailed time series of development obtainable. We show dose‐dependent effects of the toxicant on development and capture nonlinear responses that would not be attainable under a conventional manual sampling regime.
... Two specific applications of regulatory interest have been extensively studied in fish embryos, particularly using zebrafish (Danio rerio) as a model. These approaches are the identification of environmental contaminants or drugs potentially teratogenic for humans [7][8][9][10][11][12][13] and the prediction of acute toxicity in environmental and human risk assessment [5,10,[14][15][16]. ...
... In the present study, we investigated whether the low toxicity of allyl alcohol in fish embryos may be caused by a reduced metabolic capacity of an Adh-catalyzed transformation to acrolein. In general, acute toxicity in adult fish and embryos exhibit a similar sensitivity and a high correlation [15,16]. For allyl alcohol, however, fish embryos exhibit an about 1000fold lower toxicity (LC 50 of 478 mg/L; 2-50 hpf) in the range of the baseline toxicity for adult fish (1919 mg/L, calculated with ECOSAR, [28]). ...
Fish embryos are widely used as an alternative model to study toxicity in vertebrates. Due to their complexity, embryos are believed to more resemble an adult organism than in vitro cellular models. However, concerns have been raised with respect to the embryo's metabolic capacity. We recently identified allyl alcohol, an industrial chemical, to be several orders of magnitude less toxic to zebrafish embryo than to adult zebrafish (embryo LC50 = 478 mg/L vs. fish LC50 = 0.28 mg/L). Reports on mammals have indicated that allyl alcohol requires activation by alcohol dehydrogenases (Adh) to form the highly reactive and toxic metabolite acrolein, which shows similar toxicity in zebrafish embryos and adults. To identify if a limited metabolic capacity of embryos indeed can explain the low allyl alcohol sensitivity of zebrafish embryos, we compared the mRNA expression levels of Adh isoenzymes (adh5, adh8a, adh8b and adhfe1) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for adh8a and adh8b expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of adh8a, but not adh8b mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC50 = 0.42 mg/L in adh8a mRNA-injected embryos). Furthermore, GC/MS analysis of adh8a-injected embryos indicated a significant decline of internal allyl alcohol concentrations from 0.23-58 ng/embryo to levels below the limit of detection (< 4.6 µg/L). Injection of neither adh8b nor gfp mRNA had an impact on internal allyl alcohol levels supporting that the increased allyl alcohol toxicity was mediated by an increase in its metabolization. These results underline the necessity to critically consider metabolic activation in the zebrafish embryo. As demonstrated here, mRNA injection is one useful approach to study the role of candidate enzymes involved in metabolization.
... Cette évolution de l'efficacité alimentaire durant l'essai est différente de celle rapportée par Agbohessi et al. (2014) chez les juvéniles de C. gariepinus, qui ont trouvé dans leurs études une faible efficacité alimentaire durant tous les 28 jours d'essai chez les poissons exposés au Thionex par rapport aux témoins. Par ailleurs, plusieurs études (Jarvinen et Tanney, 1982;Nagel et al., 1991;Huynh et Nugegoda, 2012) avaient également montré que l'inhibition de la croissance peut être le résultat de l'inhibition de l'acétylcholinestérase. En effet, l'Acétamipride est de la famille des Néonicotinoïdes connues pour leur effet d'inhibition de l'activité de l'acétylcholinestérase, prouvée par exemple chez Gambusa affinis (Cheghib et al., 2020). ...
Cette étude vise à évaluer en conditions contrôlées, les effets sur la croissance et la composition biochimique, d’une exposition
de Clarias gariepinus aux insecticides Emamectine benzoate et Acétamipride. Ainsi, des juvéniles de C. gariepinus (2,38 ± 0,1
g) ont été soumis à trois concentrations sub-létales d'un produit commercial contenant les deux insecticides (0; 0,25 et 0,50 μl/l)
pendant 28 jours. Les données collectées et les dosages au laboratoire réalisés sur des poissons entiers, ont permis de déterminer
les taux de survie, les performances de croissance, l’efficacité alimentaire et les paramètres nutritionnels. Les résultats de l’étude
ont montré que si les taux de survie sont semblables pour tous les traitements, la croissance des individus exposés aux insecticides
était relativement inférieure à celle des individus témoins. Cette faible croissance chez les individus contaminés est liée à la baisse
de la prise alimentaire et de l’efficacité alimentaire. Ceci est lié à l’utilisation préférentielle des protéines corporelles et d’une partie
des glucides pour produire de l’énergie afin de faire face au stress chimique et au coût énergétique des comportements inhabituels
(hyperactivité, nage rapide, perte d’équilibre, etc) induits par l’insecticide.
... 8 For the validation of the test, several parameters must be obeyed including the use of three different types of controls: negative (dilution water), internal (dilution water), and positive (3,4-dichloroaniline-DCA). 8 DCA, the reference compound in the FET test, is an aniline pesticide whose toxicity to fish species at early life stages is well documented. [11][12][13][14] Despite its use as a toxic reference compound and status as a potentially hazardous environmental pollutant, very little is known about the molecular effects of DCA. Targeted studies have shown changes in expression of oxidative-stress related genes in exposed embryos. ...
The zebrafish (Danio rerio) is a small teleost fish that is becoming increasingly popular in laboratories worldwide and several attributes have also placed the zebrafish under the spotlight of (eco)toxicological studies. Since the 1990s, international organizations such as ISO and OECD have published guidelines for the use of zebrafish in ecotoxicological assessment of environmental toxicants such as the Fish Embryo Acute Toxicity (FET) test, OECD n° 236 guideline. This protocol uses 3,4‐dichloroaniline (DCA), an aniline pesticide whose toxicity to fish species at early life stages is well known, as a positive control. Despite its use, little is known about its molecular mechanisms, especially in the context of the FET test. Therefore, this study aimed to investigate such changes in zebrafish larvae exposed to DCA (4 mg/L) for 96 hours using gel‐free proteomics. Twenty‐four proteins detected in both groups were identified as significantly affected by DCA exposure, and, when considering group‐specific entities, 48 proteins were exclusive to DCA (group‐specific proteins) while 248 were only detected in the control group. Proteins modulated by DCA treatment were found to be involved in metabolic processes, especially lipids and hormone metabolism (eg, Apoa1 and Apoa1b and vitelogenins), as well as proteins important for developmental processes and organogenesis (eg, Myhc4, Acta2, Sncb, and Marcksb). The results presented here may therefore provide a better understanding of the relationships between molecular changes and phenotype in zebrafish larvae treated with DCA, the reference compound of the FET test.
... When comparing the sensitivity of N. furzeri to 100 μg/L 3,4-DCA to that of other fish species, it appears to be relatively resistant to the compound. Nagel and colleagues did find effects of 3,4-DCA exposure on life history traits such as survival rate and growth in early life stages of zebrafish that were exposed to 100 μg/L during 4 weeks ( Nagel et al., 1991). Crossland and Hillaby (1985) set the NOEC to protect an invertebrate pond ecosystem at 10 μg/L ( Crossland and Hillaby, 1985), which is five times lower than the NOEC found in this study. ...
Although aquatic organisms are increasingly exposed to pollutants and abnormally high temperatures as a consequence of climate change, interactive effects between those stressors remain poorly assessed. Especially in ectotherms, such as fish, increases in ambient temperature are expected to affect fitness-related traits and physiology. We used the turquoise killifish Nothobranchius furzeri to study the effects of a range of 3,4-dichloroaniline concentrations (0, 50, 100 μg/L) in combination with two temperature conditions (control and control +4 °C) during four months of exposure. As part of an integrated multi-level approach, we quantified effects on classic life history traits (size, maturation time, body mass, fecundity), critical thermal maximum and physiology (energy reserves and stress-associated enzymatic activity). While no interactive effects of 3,4-DCA exposure and increased temperature emerged, our results do show a negative effect of 3,4-DCA on thermal tolerance. This finding is of particular relevance in light of increasing temperatures under climate change. Due to increases in pest species and faster degradation of 3,4-DCA under higher temperatures, increased use of the pesticide is expected under climate change which, in turn, could result in a decreased tolerance of aquatic organisms to high temperatures.
... More recently, the fish embryo acute toxicity (FET) test, which assesses acute toxicity in zebrafish embryos up to 96 h post-fertilization (hpf), was approved as OECD Test Guideline 236 (OECD, 2013b;Busquet et al., 2014). One of the groups (Nagel et al., 1991;Schulte, 1994;Nagel, 2002) also worked on developing the zebrafish-specific version of TG 203 (Fish Acute Toxicity Test (OECD, 1992)), known as the DarT. This test was further refined by the addition of rat liver microsomes to the test system to allow for the evaluation of proteratogens (Busquet et al., 2008;Weigt et al., 2010). ...
There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Literature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure medium (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.
... This 3,4-D concentration therefore fulfilled the criteria for the early life stage test based on the revised OECD guideline [41]. In addition, the observed effects of 3,4-D on the embryos was comparable to the effects on survival and development in fish embryos and larvae [42,43], although the zebrafish embryos in the present study showed less pronounced effect on the heart function and the skeletal development than the early life stages of the rare minnow (Gobiocypris rarus, [43]). ...
Ochratoxin A (OTA) is a known contaminant in fish feed but its effect on fish health remains rather unknown. A study was conducted to investigate the effects of different concentrations of ochratoxin on early life stages of zebrafish (Danio rerio). The tests with ochratoxin A showed a correlation between the exposure to mycotoxin and the amount of damage. The mortality rate and the incidents of embryonal damage was increased by increasing ochratoxin concentrations. The calculations resulted in a lethal concentration for 50% of the embryos (LC50) of 0.29 mg/L and a concentration at which 50% of the animals showed impairment (EC50) of 0.36 mg/L after 96 h of exposure. During the test, reduced heart rates were also observed revealing a clear dose-response relationship. The EC50 determination for this endpoint was 1.26 mg/L after 72 h of exposure. The measurement of oxidative stress was proven to be the most sensitive system to indicate OTA effects on the zebrafish embryos with an EC50 value of 0.067 mg/L after 72 h of exposure. The test validity was given because the control test with 3,4-Dichloroaniline (3,4-D) showed a LC50 value of 2.88 mg after 96 h of exposure which is comparable to the available reference values. According to the current knowledge, these experimental doses did not exceed the environmental concentrations of this ochratoxin A. However, this study raises concerns about the effects of ochratoxin on fish.
... Decline in fish growth may be due to low food intake and impact of the pesticide on metabolisation of feedstuff into somatic development (Lal et al., 2013). The weak growth of contaminated fish may also be due to acetyl-cholinesterase inhibition as reported by Jarvinen and Tanner (1982); Cleveland and Hamilton (1983), Nagel et al., (1991) and Huynh and Nugegoda (2012). Delay in growth could be the transformation into energy of a portion of nutrients from digestion of food consumed to cope with chemical stress that constitutes the exposure to agricultural pesticides. ...
The present study of 60 days was carried out to investigate the individual and combined effect of pesticides at various concentrations on the growth and behavior of Labeo rohita and Cirrhinus mrigala. Severe changes were observed regarding behavior of treated fish of the both species as compared to their respective control groups. The treated fishes while swimming exhibited jerky, restless and whirling movements with small resting periods along with frequent surface visiting for engulfing air. Changes in pigmentation and other apparently noticeable morphological aberrations like erosion, fin blackening, hemorrhage and body deformities were observed fortnightly. Growth performance decreased in both the species viz. L. rohita and C. mrigala as compared to control while an increase in Feed Conversion Ratio was observed in pesticide treated fishes.
... The lowest observed effect concentrations (LOECs) of different endpoints exposed to 3,4-DCA were reported in Scheil et al. (2009, while LC 50 after 72 h rare minnow (Gobiocypris rarus) were 4.1 mg/L (Zhu et al., 2013) EC 50 , no observed effect concentration (NOEC), and LOEC of 3,4-DCA after 48 h exposed to zebrafish embryos were estimated to 1.62 mg/L, 0.97 mg/L, and 1.46 mg/L, respectively (Lange et al., 1995). Nagel et al. (1991) estimated LOECs of 0.1 and 0.2 mg/L for survival rates in early life stages of zebrafish after 4 and 2 weeks of exposure to 3,4-DCA, respectively. Schiller et al. (2014) identified the EC 50 of propanil exposed to zebrafish embryo after 48 h was 1.8 mg/L. ...
... Lal et al. (2013) found a significant decline in plasma levels of GH and IGF-I in malathionexposed Asian catfish Heteropneutes fossilis and showed that this decline was related to reductions in fish growth, also due to low food intake and influence of the pesticide on metabolization of feed into somatic growth. The weak growth of contaminated fish could also be the result of inhibition of acetyl-cholinesterase as reported by other authors such as Jarvinen and Tanner (1982), Cleveland and Hamilton (1983), Nagel et al. (1991) and Huynh and Nugegoda (2012). Other important factor explaining the delay in growth could be the transformation into energy of a portion of nutrients from digestion of food consumed to cope with chemical stress that constitutes the exposure to agricultural pesticides. ...
The African catfish Clarias gariepinus is a widespread species in the Beninese cotton basin. In this study, the impacts of exposure to agricultural pesticides endosulfan [Thionex 350 EC (emulsifiable concentrate)] and Tihan 175 O-TEQ (oil toxicity equivalent) on growth, feed utilization and energy budget of C. gariepinus were investigated. Fingerlings (1.58 ± 0.02 g) were exposed to borehole water (control), 0.23 ppb (environmental concentration), 440 ppb (Lethal Concentration 50 %/20, LC50/20) and 880 ppb (LC50/10) of Tihan; and to 11 ppb (LC50/20), 22 ppb (LC50/10) and 29.40 ppb (environmental concentration) of Thionex for 28 days. Fish biomass was assessed weekly and fish samples were taken from different aquaria to determine the specific growth rate (effect on growth), feed efficiency rate and protein efficiency ratio (impact on feed utilization), and the biochemical composition of fish (impact on the energy budget). The results showed that endosulfan environmental concentration induced 100 % of mortality in catfish fingerlings while mortality rate was comparable between control fish and Tihan-treated fish over the 28-day period (p > 0.05). In contrast to survival, the two pesticide types tested induced a marked decrease in growth only during the first 2 weeks of exposure (p
... Equação, reta e limites de previsão 95%, da análise de regressão para os resultados dos ensaios crônicos com Danio rerio (em VCest) e Pimephales promelas (em VC). larvas de Danio rerio (D.r.)Valor Crônico (VC > 28d), em mg L -1 , para Pimephales promelas (P.p.)Tabela 3 -Validação da equação estabelecida para estimar o efeito tóxico a larvas do Danio rerio com base nos ensaios de duração prolongada com Pimephales promelas.Agente químico CENO ≥ 28 dias determinada experimentalmente* CENO ≥ 28 dias estimada para larvas do Danio rerio** Pimephales promelas Danio rerio 3,4-dicloroanilina (em µg L -Fonte: a)Call et al. (1987); b)Nagel et al. (1991); c) Macek et al. ...
... The zebrafish has been broadly used as a model in aquatic toxicology studies during the last two decades (Nagel et al. 1991; Zok et al. 1991). The use of the zebrafish as a toxicity model for mammals and humans is well established (McCollum et al. 2011 ). ...
The zebrafish (Danio rerio) is a freshwater teleost of the family Cyprinidae that is established as a model organism in many research fields. Here, we define the characteristics that a fish species should have to serve as a model for finfish aquaculture research and argue that the zebrafish fulfils essentially most of them. We first describe several aspects of the biology of the zebrafish including phylogenetic relationships, development and growth and reproduction, both in the wild and under laboratory conditions. Next, we review the work already carried out in zebrafish that is related to different aspects of aquaculture research (reproduction, stress, pathology, toxicology nutrition and growth). We assess critically the areas in which zebrafish still offers further potential as a model organism for aquaculture, which include, but are not limited to, development, immunology, genomics and reproduction. In other areas, however, limitations must be borne in mind and caution must be taken when extrapolating results. This is, for example, the case of some growth studies. Finally, we provide information on resources for research with zebrafish. Current general limitations of work with zebrafish come from the fact that in contrast to other laboratory animal models such as rodents, strict breeding protocols are generally not adopted. Nevertheless, as there is no major obstacle to overcome these limitations and due to its intrinsic advantages, we conclude that the zebrafish is likely to play an increasing role as a model organism in many areas of research for finfish aquaculture.
... The influences of toxicant on population growth rate differed significantly with developmental stages of fish. The poor growth rate of fish could be 11 the result of the effect of chemicals on enzyme activity (Woltering, 1984;Nagel et al., 1991).Gill et al. (1991)also correlated the expansion of fish liver to an increase in liver protein. The same results were found in the present study. ...
Catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and selenium-dependent glutathione peroxidase (Se-GPx) activities in liver of rainbow trout (Oncorhynchus mykiss; 116.88±21.69g) were evaluated after exposing fish to sublethal concentrations (25μg/L) of carbosulfan in flow-through tanks for 60 days. During the experiment activities of CAT, SOD, GST, and Se-GPx and histopathological effects were determined once a week and once at the end of the 21 days of recovery period. All enzymes were affected by carbosulfan when compared to control fish. Fish had intracellular oedema, cell necrosis, pycnotic nucleus, and increase of sinusoidal space in the liver. After 21 days of the recovery period, all enzyme activities had returned to control levels and fish had no histological lesions in liver. Therefore all the changes observed during exposure were reversible. Results indicate that the liver CAT, SOD and GST enzymes are highly sensitive to carbosulfan as their activities altered significantly, suggesting they could be useful in predicting sublethal pesticide toxicity and useful as an indicator for assessment of pesticides in contaminated water.
... Thus, environmental conditions allowing for an undisturbed early development period can be regarded as one key for successful recruitment, which is a prerequisite for a fish species to establish a stable population. Early life stage (ELS) toxicity tests have been established as an experimental approach to determine and quantify sublethal and lethal effects of xenobiotics on the early development of fish (Sprague, 1971;McKim, 1977;Birge et al., 1985;McKim, 1985;van Leeuwen et al., 1985;Nagel et al., 1991). Within the frame work of the Valimar project, ELS studies were performed to characterize the fish embryotoxic potentials of two differentially polluted streams. ...
Early life stage (ELS) studies with brown trout(Salmo trutta f. fario L.) andstone loach (Barbatula barbatula L.)were performed between 1995 and 2000 toevaluate embryotoxic potentials in twodifferently polluted streams in southwestGermany. With both species, semistatic exposureexperiments with water samples and sedimenteluates were conducted in the laboratory.Additionally, brown trout ELS tests wereperformed in flow-through systems in thesemi-field and in the field. Thus, differentlevels of complexity of environmentalconditions were addressed which allowed thestudy of effects of xenobiotic contamination,temperature, and sediments on the success ofembryonic development. Additionally, effects ofwater from the polluted stream on fertilizationof brown trout eggs were determined. In themore polluted stream, xenobiotics caused anembryotoxic potential for both brown trout andstone loach, and viability of exposed browntrout eggs was drastically reduced by suspendedsolids in the water which covered the eggs.Additionally, fertilization rates of browntrout eggs were significantly decreased inwater of the more polluted stream. In the lesspolluted stream, low water temperature andinfestations by protozoic ectoparasites causedmortality of embryos. In this stream, pollutionand sediment effects were not observed. Resultsmade evident that in the more polluted streamrecruitment of brown trout was drasticallyimpaired.
... Thus, spatial and temporal variations of propanil persistence in the Andreoni canal watercourse should not be disregarded. Propanil is easily degraded to 3,4-DCA, a well-known persistent endocrine disruptor to fish (Monteiro et al., 2006;Nagel et al., 1991) that has been found at the CM. This metabolite accumulates in water, produces carcinogen products (Weisburger and Weisburger, 1966) and together with propanil, has been included in the Community Strategy for Endocrine Disruptors (Commission of the European Communities, 2001). ...
Environmental water chemistry analysis and microcosm toxicity bioassays (MTB) were performed to assess lethality of herbicides on the mole crab Emerita brasiliensis in a sandy beach affected by a freshwater discharge (Andreoni canal) from rice crops. A 5-yr macrocosm field sampling (MFS) was conducted to evaluate freshwater effects on population abundance. Propanil was only detected at the inner portion of the Andreoni canal (IAC), whereas quinclorac and clomazone were found at the IAC and at the canal mouth (CM). A major propanil metabolite was detected at the CM. Herbicides were undetectable at 13km from the CM. MTB showed an increased susceptibility to propanil with decreasing crab sizes. The MFS showed a drastic decrease in abundance towards the freshwater discharge, concurrently with decreasing salinities. The triad approach that included water analyses, toxicological experiments and long-term field sampling allowed rejecting relationships between herbicide exposure and mole crab lethal effects.
... Compared with full life-cycle tests with ®sh (FFLC), the performance of ELS tests demands much less eort, still oering considerable sensitivity (Birge et al., 1985; Hutchinson et al., 1998). Results can be reproduced under standardised conditions in the laboratory (Nagel et al., 1991). In general, ELS tests are used as a tool to assess the toxicity of environmental pollutants in a prospective way. ...
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 Körsch stream and the less polluted Krähenbach 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 Körsch 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 Krähenbach 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.
The available alternative methods for testing developmental toxicity comprise either cellular models or whole embryos of rodents, fish, or amphibian. The simplest cellular models consider the use of human or animal embryonic stem cells (embryonic or of induced pluripotency) under differentiation and one of the most widely used endpoints in these methods is the alterations in differentiated beating cardiomyocytes, although determination of other molecular markers is also extended. More complex cellular models consider the use of cocultures or 3D cultures or the so-called organoids. These models mimic the physiological environment in a much better way than the simple monolayer cultures. The use of whole embryos allows the determination of which teratogenic effects are expectable after exposure to developmental toxicants, which is one of the main disadvantages of the cellular methods. The appropriate assessment of chemical safety for development needs of a battery of alternative methods applied. Integrated Approaches to Testing and Assessment (IATA) would allow in the close future to perform safe and reliable assessment of developmental toxicity based on alternative methods.
The fathead minnow, Pimephales promelas, a small cyprinid from North America, is widely used as laboratory test fish. In the present study, rearing and breeding conditions for the fat-head minnow were optimized in our laboratory to produce eggs on a regular basis for testing. Firstly, these eggs were observed and a timeline for normal development of the embryo was established. Results showed only minor differences to already existing studies (Devlin et al. 1996, Manner and Dewese 1974). Based on fathead minnow-specific developmental data, the embryo toxicity test was adapted and refined. Using the fathead minnow embryo test, LC50 data were generated for a heavy metal (cadmium chloride), a substituted aniline (3,4-dichloroaniline), which is currently used as the positive control in the fish embryo toxicity test with the zebrafish (ZFET) and a substi-tuted phenol (2,4-dichlorophenol). Fathead minnow embryos proved to be slightly less sensi-tive for cadmium chloride and clearly less sensitive for 3,4-dichloroaniline than adult fathead minnow (OECD TG 203), whereas for 2,4-dichlorophenol no difference in sensitivity could be detected. However, the comparison with the ZFET showed that zebrafish embryos are sig-nificantly more sensitive. For all three substances, toxicity increased after hatching (≥ 96 and 120 hpf). Differences in toxicity between the fathead minnow and the zebrafish embryo tests might be due to differences in the chorion. Examinations of the chorion with light, confocal laser scanning and transmission electron microscopy revealed significant differences to the zebrafish chorion. The fathead minnow chorion was thicker and had less and smaller pores on the outside of the chorion. Further-more, the TEM images showed a layer composition different to the zebrafish chorion. Histo-logical stains and a test with the fluorescent dye 2,7-dichlorofluorescein supported the as-sumption that at least the outer layer of the fathead minnow chorion is composed of a material different to the zebrafish. A preliminary attempt with the fathead minnow embryo instead of the zebrafish embryo in the newly developed in vivo EROD assay (Kais) was performed successfully. However, first sig-nals of EROD activity were detected at least 24 hours later than in the zebrafish, and the sig-nal intensity was weaker than in zebrafish.
There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21st Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in academic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Literature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure media (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.
The use of zebrafish for aquatic vertebrate (eco)toxicity testing allows the assessment of effects on a wide range of biological levels – from enzymes to sensory organs and behavioral endpoints. The present study investigated the effects of the insecticide diazinon and the herbicide diuron regarding the acute toxicity and behavior of zebrafish embryos and larvae. After conducting the fish embryo toxicity test, three concentrations (1, 2 and 3.5 mg L−1 for diazinon and 1, 2 and 3.8 mg L−1 for diuron) were evaluated for effects on embryonic spontaneous movement and heartbeat, larval light-dark transition response, and thigmotaxis. Although the modes-of-action are different, both pesticides proved to be moderately toxic to early life stages of zebrafish with 96 h LC50 of approximately 6.5 mg L−1 and similar EC50 values of approximately 4 mg L−1. Changes in behavioral endpoints were detected 24 h of exposure, suggesting that behavioral measurements can serve as sensitive and early indicators of pesticide exposure. Changes in behavior, such as decrease in spontaneous coiling movements of embryos and reduction of thigmotaxis in larvae, were pronounced for diuron, indicating the usefulness of the application of behavioral endpoints to assess the effects of other herbicides. In the case of diazinon, the effects were less prominent, but the detected changes in ratios between activity in light and darkness also point to the possibility of using behavioral changes for evaluation of insecticide effects. The obtained results support the usage of behavioral endpoints in zebrafish embryos and larvae for the detection of early effects of pesticides.
Risk assessments for compounds released to the environment typically rely on single-species toxicity studies to predict concentrations at which effects may be observed. These single-species toxicity studies are usually conducted with a few species, cultured under optimum conditions (diet, temperature, light, etc.) and tested in clean water with constant exposure to the compound of interest. Chronic toxicity data are then extrapolated to the ecosystem during risk assessments to predict concentrations that will not adversely impact the environment. Several approaches have been developed that apply statistical methods to estimate toxicant concentrations adversely affecting a small percentage of single species (e.g., 5%). There are several rarely stated, and infrequently tested, biological and statistical assumptions required to make this extrapolation. One test of the ability to use single-species toxicity data to protect ecosystems is to compare effects on single species with effects on experimental and natural ecosystems (e.g., microcosms, model ecosystems, field). Towards this end, we summarized the chronic single species and experimental ecosystem data on a variety of substances (n = 11), including heavy metals, pesticides, surfactants, and general organic and inorganic compounds. Single-species data were summarized as genus-specific geometric means using the NOEC or EC20 concentration. Genus mean values spanned a range of values with genera being affected at concentrations above and below those causing effects on model ecosystems. Geometric mean model ecosystem no effect concentrations corresponded to concentrations expected to exceed the NOEC of 10 to 52% of genera. This analysis suggests that laboratory-generated single species chronic studies can be used to establish concentrations protective of model ecosystem, and likely whole ecosystem, effects. Further, the use of the 5% of genera affected level is conservative relative to mean model ecosystem data but is a fairly good predictor of the lower 95% confidence interval on the mean model ecosystem NOEC.
This chapter discusses the study of the currently available models for testing developmental toxicity (embryotoxicity and teratogenicity). The main alternative models for testing developmental toxicity are described. These models are divided between validated models (whole-embryo culture test (WEC), micromass test (MM) and embryonic stem cell test (EST)) and those that are not currently validated (although have proven scientific validity) as is the case of zebrafish, frog embryo teratogenesis assay (FETAX), in silico models for predicting embryotoxicity, in vitro cellular models different from the EST method, and methods using fragments of embryos. The non-validated alternative models for testing developmental toxicity are also explained here. To date, only three in vitro methods (MM, EST and WEC) have been validated by an international agency (ECVAM) in order to be used for testing the embryotoxicity potential of chemicals, although other models such as FETAX and zebrafish have also proved their validity for this purpose. Methods based on the employment of embryos allow the specific malformation expected after exposure to the chemical to be determined, while methods based on cellular systems are more relevant in order to determine the mechanism underlying the adverse observed effect and still display a wide field for improving their prediction capability.
Cytopathological alterations in hepatocytes of fish following exposure to xenobiotic compounds represent a powerful tool to reveal sublethal effects of chemicals and to elucidate underlying modes of action. The present communication reviews the available information about ultrastructural changes in fish liver as well as isolated hepatocytes; whereas the discussion of in vivo effects is primarily focused on data from rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio), the presentation of in vitro data has been restricted to results from experiments with rainbow trout hepatocytes due to a lack of data from studies with hepatocytes from other species. Both in vivo and in vitro exposure to xenobiotics results in sensitive, selective, and, especially in in vitro experiments, extremely rapid responses of hepatocytes, which, however, may be confounded by internal parameters (species, sex, age, hormonal status) and external parameters (temperature, nutrition, duration of exposure). Thus, transfer of results and conclusions from one experiment to another is usually not possible. Likewise, in vitro results may not necessarily be extrapolated to the situation in intact fish, and effects by acute toxic exposure cannot be translated into sublethal effects.
Zebrafish are ideal models for studying toxicological effects during development, due to ease of maintenance, translucent eggs, and a short reproduction cycle. The author discusses possibilities, prospects, and limitations of a method to maintain a zebrafish breeding stock for continuous embryo production and for performing and evaluating embryo tests.
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:
Base level:For ethical reasons,the conventional routine actute toxicity test with juvenile or adult fish should be replaced by a 48 h embryo test with zebrafish.
Level 1: At level 1, the early life-stage test (OECD guideline 210) deserves priority over the prolonged fish test according to OECD guideline 204.
Level 2: Only a complete life-cycle test fulfills the requirements of a chronic toxicity test. It can, therefore, not be substituted by an early life-stage test. Since extrapolation from acute-chronic ratio (ACR) data is not possible as well, there is no alternative to a complete life-cycle test.
The ecological risk assessment of toxicants in soil requires reproducible and relevant test systems using a wide range of species. To supplement present test methods from the Organisation of Economic Cooperation and Development (OECD) in artificial soil with earthworms and springtails, a toxicity test in OECD artificial soil has been developed using the bacterivorous nematode Plectus acuminatus (Bastian, 1865) (Nematoda; Plectidae). The juvenile to adult ratio was used as a test parameter since previous life-cycle studies pointed out that fitness of P. acuminatus was strongly determined by changes in both reproduction and juvenile survival. Optimal conditions for the performance of nematodes in OECD artificial soil were determined (pHKCl = 5.5, temperature = 20°C, and a moisture content of 70% dry wt. artificial soil), and tests were conducted with cadmium, copper, and pentachlorophenol. After an exposure period of 3 weeks the EC50 for cadmium was 321.0 ± 1.7 mg/kg dry wt., and the no-observed-effect concentration (NOEC) was 32 mg/kg dry wt. The EC50 for pentachlorophenol was 47.9 ± 1.2 mg/kg dry wt., and the NOEC was <10 mg/kg dry wt. For copper the EC50 was 162 ± 0.2 mg/kg dry wt., and the NOEC was 32 mg/kg dry wt. It is concluded that the nematode test may well supplement current soil test systems using earthworms and springtails.
The toxitity of 3,4-dichloroaniline to perch was investigated in order to compare the sensitivity of crucial life stages with common test fish like zebrafish. Although in the acute toxicity test perch were more sensitive by a factor of five, larval sensitivity was in the same range in both species as well as in other egg-laying species. In spite of different water temperature and size, the zebrafish is a good model to predict early life stage toxicity of 3,4-DCA to the compared European freshwater species. Perch larvae are not suited for early life stage tests, as they are sensible towards experimental conditions and perform early cannibalism.
Zebrafish (Brachydanio rerio) were evaluated as a small fish model for environmental carcinogenesis monitoring. Criteria used for evaluation included: (1) sensitivity to dose response exposures of 6 known carcinogens by 4 routes of exposure, (2) histopathologic evaluation of the resulting lesions and comparison with responses in other species, (3) response to promoters of neoplasia, (4) ability to conduct carcinogen metabolism studies to understand mechanisms of action, (5) determining the role of oncogenes in zebrafish carcinogenesis, and (6) success in developing transgenics that would increase the species' usefulness as a carcinogen monitoring model. Findings were (1) zebrafish did respond to known carcinogens with variable but overall acceptable sensitivity, (2) the histopathology of their responses was similar to that observed in other small fish species, (3) they did not respond well in limited promotional studies, (4) metabolism studies were very difficult due to small fish and organ size and results did not always support observed tumor responses, (5) ras and p53 genes were sequenced but lack of grossly observable tumors prevented the determination of mutations in these genes, and (6) although progress was made, the production of transgenic fish was not achieved. Although none of these results would disqualify zebrafish as environmental monitors, their strict requirement for tropical or subtropical water temperatures would make them completely unsuitable for temperate water use.
Modeling mathematically the effects of chemicals on aquatic population systems needs information about effect concentrations. Due to environmental protection aims and the complex nature of aquatic ecosystems, a diverse set of tests has been established. In this paper 31 tests are discussed, selected from a total of 55 tests, which should be suitable for mathematical modeling or show some ecosystemic relevance. We introduced eight criteria by which the tests were evaluated and used Hasse diagrams to present the results. We found out that there exists no superior test. However, eight tests with specific, useful abilities were identified. The concept of equivalence and order relations contributes to analyze such kind of complex information.The ranking may be influenced by the criteria used, therefore a sensitivity study was performed, which shows that the criterion concerning ecosystemic relevance was the most important. Many examples help to outline the use of Hasse diagrams. The role of equivalence classes is discussed and a newly introduced stability index is applied.
Risk assessments for compounds released to the environment typically rely on single-species toxicity studies to predict concentrations at which effects may be observed. These single-species toxicity studies are usually conducted with a few species, cultured under optimum conditions (diet, temperature, light, etc.) and tested in clean water with constant exposure to the compound of interest. Chronic toxicity data are then extrapolated to the ecosystem during risk assessments to predict concentrations that will not adversely impact the environment. Several approaches have been developed that apply statistical methods to estimate toxicant concentrations adversely affecting a small percentage of single species (e.g., 5%). There are several rarely stated, and infrequently tested, biological and statistical assumptions required to make this extrapolation. One test of the ability to use single-species toxicity data to protect ecosystems is to compare effects on single species with effects on experimental and natural ecosystems (e.g., microcosms, model ecosystems, field). Towards this end, we summarized the chronic single-species and experimental ecosystem data on a variety of substances (n = 11), including heavy metals, pesticides, surfactants, and general organic and inorganic compounds. Single-species data were summarized as genus-specific geometric means using the NOEC or EC20 concentration. Genus mean values spanned a range of values with genera being affected at concentrations above and below those causing effects on model ecosystems. Geometric mean model ecosystem no effect concentrations corresponded to concentrations expected to exceed the NOEC of 10 to 52% of genera. This analysis suggests that laboratory-generated single-species chronic studies can be used to establish concentrations protective of model ecosystem, and likely whole ecosystem, effects. Further, the use of the 5% of genera affected level is conservative relative to mean model ecosystem data but is a fairly good predictor of the lower 95% confidence interval on the mean model ecosystem NOEC.
The effects on newly fertilized eggs, embryos and larvae of zebrafish Danio rerio following exposure to sediment samples from the more heavily contaminated River Körsch, southern Germany, occurred earlier and were more prominent than in samples from the less contaminated Krähenbach. Dose- and time-related effects following exposure to Körsch sediment eluates and extracts included: (1) hatching failure and subsequent death of larvae exposed to undiluted aqueous sediment eluates and reduced hatching rates at sediment extract concentrations 0·0125%; (2) increased mortality after exposure to 25 and 50% dilutions of aqueous sediment eluates, and dilutions of 0·00625% sediment extracts; (3) reduction of heart beat frequency for 50% dilutions of sediment eluates and concentrations of 0·025% extracts; (4) increased frequency of heart and yolk sac oedema after exposure to 0·0125% sediment extracts. Since adverse effects of sediment extracts observed in zebrafish laboratory tests correlated with reproductive failure in natural populations of brown trout Salmo trutta f. fario in the severely polluted River Körsch, early life stages tests with zebrafish appear to be a suitable tool to assess the contamination rate of natural sediments.
Bioconcentration factors (BCF) and metabolism of 3,4-dichloroaniline in different life stages of guppy (Poecilia reticulata Peters) and zebrafish (Brachydanio rerio Hamilton-Buchanan) were investigated. The results prove that embryos of zebrafish, four-day-old yolk sac larvae, 17-day-old larvae and adult zebrafish are able to transform the chemical to 3,4-dichloroacetanilide. Bioconcentration factors in these life stages are between 30 and 42, exept yolk sac larvae showing a BCF of 86. 3,4-Dichloroacetanilide was also detected in extracts of newborn and adult guppies, but they are able to form an additional, yet unknown metabolite. The BCF varies from 34 in six-month-old guppies to 226 in newborns. Tissue distribution of 3,4-dichloroaniline and its metabolites in adult guppies demonstrates high concentrations in gallbladder and intestine. Metabolite pattern of embryos (one week before hatching) are nearly identical to those of adult female guppies. In zebrafish there are in any tissues or organs nearly same concentrations of the chemical.
Australian catfish, T. tandanus were exposed to a short term pulse of chlorpyrifos at 2 or 10 μg L(-1) and grown in optimal conditions to investigate the effect of the pesticide on fish growth. The final weight was lower in fish exposed to chlorpyrifos (8.54 ± 0.19 and 8.77 ± 0.44 g) respectively compared to the control fish (10.5 ± 0.72 g) while the hepatosomatic index in fish exposed to chlorpyrifos was a higher (1.86 ± 0.10 and 2.01 ± 0.12) than in the control fish (1.65 ± 0.14). Both bi-weekly growth rate and brain Acetylcholinesterase (AChE) activity increased with post-exposure time.
Acute exposure to certain dichloroaniline (DCA) isomers results in renal and hepatic toxicity in vivo. In the present study we examined whether dichloroaniline structural isomers were cytotoxic to liver and kidney slices in vitro and compared the toxicities of the different structural isomers. These studies were necessary in order to validate the use of an in vitro slice system for examination of the cellular mechanisms for toxicity. Renal cortical and hepatic slices were incubated for 90 min with 2,3-DCA, 2,4-DCA, 2,5-DCA, 2,6-DCA, 3,4-DCA or 3,5-DCA at a final concentration of 0-1 mm. Pyruvate-directed gluconeogenesis was measured following an additional 30-min incubation with 10 mM pyruvate. Cytotoxicity was also determined by measurement of lactate dehydrogenase (LDH) release 120 min after the addition of dichloroaniline isomers at a final concentration of 0, 0.5, 1 or 2 mM. Gluconeogenesis in renal cortical slices was inhibited by all of the isomers beginning at a concentration of 0.5 mM. Renal slice LDH leakage was elevated above control levels by 1-2 mM 3,4-DCA or 3,5-DCA. A final concentration of 2 mM was needed for 2,3-DCA, 2,4-DCA, 2,5-DCA or 2,6-DCA in order to detect a significant (P < 0.05) increase in renal slice LDH leakage. Hepatic slices incubated with 0.5-2 mM 2,3-DCA or 2 mM 2,5-DCA exhibited diminished pyruvate-directed gluconeogenesis. After exposure to 2,4-DCA, 2,6-DCA, 3,4-DCA or 3,5-DCA, pyruvate-directed gluconeogenesis was similar to that in the controls. LDH leakage was increased significantly (P < 0.05) above control values by exposure to 2 mM 3,4-DCA or 3,5-DCA. In conclusion, DCA structural isomers were toxic in vitro to liver and kidney slices. These results indicated that the kidney was more sensitive than the liver to DCA isomers, and that the most toxic isomer was 3,5-DCA. These results are similar to those previously observed in vivo.
Linuron is a globally used phenylurea herbicide, and a large number of studies have been made on the microbial degradation of the herbicide. However, to date, the few bacteria able individually to mineralise linuron have been isolated only from European agricultural soils. An attempt was made to isolate linuron-mineralising bacteria from Japanese river sediment using a uniquely designed river ecosystem model (microcosm) treated with (14)C-ring-labelled linuron (approximately 1 mg L(-1)).
A linuron-mineralising bacterium that inhabits river sediment was successfully isolated. The isolate belongs to the genera Variovorax and was designated as strain RA8. Strain RA8 gradually used linuron in basal salt medium (5.2 mg L(-1)) with slight growth. In 15 days, approximately 25% of (14)C-linuron was mineralised to (14)CO(2), with 3,4-dichloroaniline as an intermediate. Conversely, in 100-fold diluted R2A broth, strain RA8 rapidly mineralised (14)C-linuron (5.5 mg L(-1)) and more than 70% of the applied radioactivity was released as (14)CO(2) within 3 days, and a trace amount of 3,4-dichloroaniline was detected. Additionally, the isolate also degraded monolinuron, metobromuron and chlorobromuron, but not diuron, monuron or isoproturon.
Although strain RA8 can grow on linuron, some elements in the R2A broth seemed significantly to stimulate its growth and ability to degrade. The isolate strictly recognised the structural difference between N-methoxy-N-methyl and N,N-dimethyl substitution of various phenylurea herbicides.
A main objective of ecotoxicology is to assess the effect of chemicals on ecosystems and to evaluate their potential ecological risk (Moriarty, 1983). Consequently, many theoretical and experimental explorations have been made to provide an adequate basis for the effect assessment of toxicants at the ecosystem level. Much attention is paid to the development of statistical methodologies to extrapolate the results obtained from laboratory toxicity tests to the field situation (Van Straalen and Denneman, 1989; Wagner and Lake, 1991; Aldenberg and Slob, 1993). Basically these methods estimate safe environmental concentrations of hazardous compounds from distribution models of critical effect levels obtained from single-species toxicity tests for different organisms
1. It is widely assumed that stressors such as toxicants affect organisms by impairment of those life-cycle variables that are most sensitive to these toxicants. We tested this premise by contrasting a fitness assessment with the most sensitive life-cycle variable approach using cadmium and the nematode Plectus acuminatus as a case study. 2. Based on complete life-cycle experiments, a deterministic model was constructed relating changes in juvenile and adult variables, including a Weibull survival distribution, to fitness, which was defined as the intrinsic rate of population increase. 3. Based on a sensitivity analysis of the model it was indicated that impairment of the most sensitive trait, the reproductive period which was reduced by 45%, did not have any effect on fitness. However, a prolongation by cadmium of the juvenile period by 7.5%, the least sensitive trait, resulted in a fitness decrease of 5%. 4. Application of this approach to the evaluation of critical effect levels for cadmium implies that a hazard assessment based on the most sensitive life-cycle trait leads to erroneous predictions of the biological impact that toxicants cause.
Das Ziel der vorliegenden Arbeit bestand darin, durch Einsatz verschiedener biologischer Tests und Endpunkte ein Set von Biotests zu etablieren, welches zur ökotoxikologischen Diagnostik des Belastungszustands kleiner Fließgewässer einsetzbar ist. Zunächst wurde die akute Toxizität von freiem Wasser und wäßrigen Eluaten bzw. acetonischen Extrakten von Sedimenten aus zwei Modellfließgewässern in der Nähe von Stuttgart, dem Krähenbach und der Körsch, die sich in ihrer Belastung durch Schadstoffe deutlich unterscheiden, mit Hilfe der Dauerzellinie RTG-2 aus der Regenbogenforelle ermittelt. Im zweiten Teil dieser Arbeit wurden isolierte Hepatocyten der Regenbogenforelle eingesetzt, um Aussagen zur subakuten cyto- und gentoxischen Wirkung von Wasser- und Sedimentproben zu erhalten. Es folgte schließlich ein Vergleich von biochemischem und cytopathologischem Reaktionsmuster von Primärhepatocyten auf Belastung mit Wasser- und Sedimentproben sowie Schadstoffgemischen, die entsprechend der durchschnittlichen Belastung des stärker kontaminierten Gewässers (Körsch) zusammengesetzt waren. Im dritten Teil wurden Frühstadien des Zebrabärblings eingesetzt, um natives Wasser und Sediment der beiden Fließgewässer in einem Early Life Stage-Test auf ihre akute bzw. subakute Embryotoxizität zu untersuchen. Neben klassischen Parametern wie Mortalität und Schlüpfrate wurden auch cytopathologische Veränderungen in der Leber der Frühstadien berücksichtigt. Die Kombination von Zell- und Embryolarvaltests mit biochemischen, gentoxischen und ultrastrukturellen Untersuchungen erwies sich als geeignet, ein empfindliches Instrumentarium zur ökotoxikologischen Diagnose des Belastungszustands kleiner Fließgewässer bereitzustellen. Insbesondere im Kompartiment Sediment konnte so ein hohes cyto-, gen- und embryotoxisches Potential ermittelt werden.
In this project, an assay was developed and applied to identify hormone active substances in the aquatic environment. Laboratory fish were exposed during the reproductive and development phase to a range of established endocrine active compounds; these were estrogen (17 beta-estradiol), anti-estrogen (tamoxifen), androgen (methyldihydrotestosterone), anti-androgen (flutamide), and an antithyroid compound (propylthiouracil); also a field sample from a national field study (LOES) was tested. Parameters were fecundity, fertility, development, plasma vitellogenin, and histopathology with emphasis on gonads; for vitellogenin, (immuno)histochemistry detection methods were developed, as an alternative for the common ELISA methods. This approach enables the identification of a variety of endocrine effects within a relatively short period (9wk); histopathology is the most specific and sensitive parameter for the reference compounds used. Changes in sex ratio of offspring appears the most sensitive endpoint with relevance for reproduction fitness. The educational histopathology database from this project is available on the Internet (http://www.rivm.nl/fishtoxpat/). In dit project is een testmodel ontworpen en toegepast voor de identificatie van hormoonactieve stoffen in het aquatisch milieu. In het laboratorium zijn zebravissen gedurende de reproductie en ontwikkelingsfase blootgesteld aan een reeks bekende hormoonactieve stoffen, te weten een oestrogeen (17 beta-oestradiol) , een anti-oestrogen (tamoxifen), androgeen (methyldihydro-testosteron), anti-androgeen (flutamide), een schildklierremmer (propylthiouracil); tenslotte is een veldmonster getest in het kader van het landelijk onderzoek oestrogene stoffen (LOES). Onderzocht zijn parameters voor eiproductie, vruchtbaarheid en ontwikkeling van het eieren en nageslacht. Daarnaast is het plasmaeiwit vitellogenine bepaald, dat een maat is oestrogene activiteit. Hiervoor zijn (immuno)histochemische bepalingsmethoden ontwikkeld als alternatief voor de klassieke ELISA. Voorts zijn dieren histopathologisch onderzocht op afwijkingen in (ontwikkeling van) geslachtsorganen en endocriene organen. Uit de resultaten blijkt dat met het testsysteem binnen een betrekkelijk korte blootstellingsperiode (9 weken) inzicht verkregen kan worden in effecten van hormoonverstoring op het niveau van reproductie en ontwikkeling, waarbij met name het histopathologisch onderzoek een cruciale rol speelt, zowel betreffende gevoeligheid als specificiteit voor alle geteste referentiestoffen. Verstoring in de geslachtsverhouding blijkt het meest gevoelige voor de voortplanting relevante effect te zijn. De histopathologische bevindingen staan in de vorm van een educatieve atlas op Internet (http://www.rivm.nl/fishtoxpat/).
The zebrafish is a popular model for studies of vertebrate development and toxicology. However, in vitro approaches with this organism have not been fully exploited because cell culture systems have been unavailable. We developed methods for the culture of cells from blastula-stage diploid and haploid zebrafish embryos, as well as cells from the caudal and pelvic fin, gill, liver, and viscera of adult fish. The haploid embryo-derived cells differentiated in culture to a pigmented phenotype and expressed, upon exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin, a protein that was immunologically and functionally similar to rainbow trout cytochrome P450IA1. Zebrafish cultures were grown in a complex basal nutrient medium supplemented with insulin, trout embryo extract, and low concentrations of trout and fetal bovine serum; they could not be maintained in conventional culture medium containing a high concentration of mammalian serum. Using calcium phosphate-mediated transfection, a plasmid constructed for use in mammalian cells was introduced into zebrafish embryo cell cultures and expressed in a stable manner. These results indicated that the transfection procedures utilized in mammalian systems can also be applied to zebrafish cell cultures, providing a means for in vitro alteration of the genotype and phenotype of the cells.
The aim of the work presented in this paper was to compare toxic threshold concentrations of three substances obtained from growth test in rainbow trout (Salmo gairdneri) with data from early life-stages in zebrafish. The growth test was conducted over a period of 7 wk in case of 4-chloroaniline and 4 wk in case of 3,4-dichloroaniline and diazinon. The data from the experiment in zebrafish originate from life-cycle studies; here, only the results obtained within the first 6 wk of development after fertilization are considered. These time limits have been set, as in the FRG a growth test in rainbow trout extending over 4 wk and an early life-stage test in zebrafish extending over 6 wk are being discussed for the Chemical Act.
Acute and long-term toxicity of 3,4-dichloroaniline and lindane to zebrafish were examined in tap water and water from the Rhine River and the toxicity of the binary mixture 3,4-dichloroaniline/lindane in tap water was investigated. The acute toxicity of 3,4-dichloroaniline and lindane was not influenced by the complex matrix of river water compared with tap water. The binary mixture of 3,4-dichloroaniline and lindane demonstrated an additive effect in the acute test. The survival rate of early life stages in river water was reduced by lindane (80 micrograms/liter), whereas the effects of 3,4-dichloroaniline on survival and growth were visible but not significant. The binary mixture of 2 micrograms/liter, 3,4-dichloroaniline and 40 micrograms/liter lindane in tap water had an influence on growth on the early life stages of zebrafish.
1. Induction of zebrafish P450 by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was studied in liver tissue, primary liver cell culture and multipassage cell culture derived from zebrafish haploid and diploid embryos and liver. 2. TCDD induced two hepatic proteins (54 and 50 kDa) in vivo which were recognized by anti-trout P4501A1 IgG. The 54-kDa protein was induced by TCDD in primary and multipassage hepatocyte cultures and in haploid and diploid embryo-derived cells. The proteins in liver homogenates were not induced by aqueous exposure of zebrafish to beta-naphthoflavone (BNF). 3. Homogenates of zebrafish liver, cultured hepatocytes and embryo-derived cells also exhibited increased ethoxyresorufin O-deethylase (EROD) and 7-12-dimethyl-benz[a]anthracene (DMBA) hydroxylase activity following TCDD exposure.
In addition to survival and hatching parameters, cytological alterations in liver and kidney of 4- and 6-d old zebrafish larvae (Brachydanio rerio) following single microinjection of fertilized eggs at the germ-ring stage with 5, 12.5, and 25 ng 4-chloroaniline/egg were investigated by means of electron microscopy. Whereas survival remained unaffected, microinjection with 4-chloroaniline disturbed hatching of larvae. Hatching was delayed by microinjection of 12.5 ng 4-chloroaniline/egg and above when compared to controls. Cytological investigations revealed ultrastructural changes in both liver and kidney in a dose- and time-dependent fashion. In the liver, major cytopathological changes included fenestration, fragmentation, and vesiculation of the rough endoplasmic reticulum, proliferation of atypical mitochondria, and atypical lysosomes. Furthermore, myelin whorls, lipid inclusions, and cholesterol crystals were increased, whereas glycogen stores were reduced. Renal tubular cells displayed altered brush borders, proliferation of nucleoli, atypical mitochondria, fenestrated, fragmented, and vesiculated RER cisternae, as well as giant lysosomes. Most of these effects indicate cellular dysfunction (e.g., disturbance of lipid metabolism in the liver), whereas others illustrate general cellular stress-responses to chemical aggression. Comparisons of results with those of previous studies based on conventional fish exposure prove the suitability and sensitivity of microinjection bioassays with zebrafish eggs as an alternative to conventional early life-stage tests.
The zebrafish (Danio rerio) is assuming prominence in developmental genetics research. By comparison, little is known of tumorigenesis and nothing is known of carcinogen metabolism in this species. This study evaluated the ability of zebrafish to metabolize a well-characterized human carcinogen, aflatoxin B1 (AFB1), to phase I and phase II metabolites and assessed hepatic AFB1-DNA adduction in vivo. Fish i.p. injected with 50-400 micrograms [3H]AFB1/kg body wt displayed a linear dose response for hepatic DNA binding at 24 hr. AFB1-DNA adduct levels among treatments showed no statistical difference over the period from 1 to 21 days after injection, suggesting poor adduct repair in this species. DNA binding in female fish was 1-7-fold higher than that in males (p < 0.01). An in vitro AFB1 metabolism assay verified that zebrafish liver extracts oxidize AFB1 to the 8,9-epoxide proximate electrophile (Km = 79.0 +/- 16.4 microM, Vmax = 11.7 +/- 1.4 pmol/min/mg protein at 28 degrees C). The excretion of AFB1 and its metabolites was also examined by HPLC. As is typical of other fish studied, major metabolites excreted were aflatoxicol (AFL) and aflatoxicol-glucuronide (AFL-g), followed by unreacted AFB1. AFL appeared as early as 5 min after injection, whereas AFL-g was a significant metabolite after 18 hr. This study shows that in vivo administration of AFB1 to zebrafish results in moderate adduction of the carcinogen to liver DNA and that zebrafish have the capacity for both phase I and phase II metabolism of AFB1. The approximate fourfold difference between rainbow trout and zebrafish AFB1-DNA covalent binding index appears insufficient to explain the relative resistance of zebrafish to dietary AFB1 hepatocarcinogenicity.
Two collaborative research projects were designed to develop and validate methods for determining the chronic effects of xenobiotics in freshwater ecosystems. The work reported here focuses on the development of methods for measuring effects on fish, invertebrates, and algae in outdoor artificial streams. 3,4-Dichloroaniline (3,4-DCA), has been used as a reference xenobiotic in two artificial stream experiments. The first used five stream channels: a control and treatments ranging from 70 to 2400 microg/liter. The second used eight stream channels--a control and treatments ranging from 0.45 to 4700 microg/liter and four coupled, 510-liter-capacity, downstream ponds-a control and treatments of 1.7, 37, and 820 microg/liter. Effects on the biota of the stream channels and the downstream ponds were examined using a range of sampling techniques and in situ toxicity tests.
Evidence is provided that diets high in polyunsaturated fatty acids enhance fertilization and growth performance in zebrafish.
The sensitivity of the early life stage (ELS) toxicity test for two compounds with different modes of action was determined, and related to other toxicity tests with the same compounds. The zebrafish. Danio rerio, was used as a test organism, and the two model compounds were 1,2,3-trichlorobenzene (123TCB), a non-polar narcotic, and parathion, an acetylcholinesterase (AChE) inhibitor. Hatching and survival after 28 days were significantly reduced in the highest 123TCB treatment (263 microg/l), but not in any of the parathion treatments. Growth of the larvae was negatively affected at parathion concentrations above 20 microg/l, while AChE was only significantly inhibited at the highest concentration, 93 microg/l. No effects on growth were found in the 123TCB treatments. In comparison with acute and chronic studies with both compounds, the ELS test turned out to be less sensitive than chronic studies and more sensitive than acute studies. The difference in sensitivity between the tests systems seems however, to depend on the mode of action of the compound.
The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1-2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.
Behaviour, reproduction and population dynamics of zebrafish (Brachydanio rerio HAM.-BUCH.) in a complex experimental system
Keywords. Artificial ecosystem, bionomics, Brachydanio rcrio,
population dynamics, predation, reproduction, zebrafish.
Abstract. Two parallel examples of a brook microcosm with a small population of zebrafish (Brachydanio rerio) were installed. Courtship activity and egg production were at times higher than in small test vessels. Egg development was faster. The capacity of the system was already reached with 10 to12 adult fish in 500 litres of water . The zebrafish is an r-selectionist with a great demand for space for bringing up offspring. The development of water quality and invertebrate populations in the two parallel systems were constant and similar to each other. The parallel experimental populations showed qualitatively and quantitatively the same characteristics of courtship activity and egg production. The most effective regulation of population density was the predation on larvae by adults. The predation behaviour of the adults and the fitness of larvae are the most relevant parameters to assess the influence of xenobiotics on populations of zebrafish
The results so far obtained justify the planning of a long-term study with zebra fish which also covers the aspect of reproduction. Egg production should be studied over a period of seven weeks under the influence of foreign substances. Fish also should be bred under the influence of the test substances. A period of some weeks might be sufficient for breeding.
A protocol for determination of “no-effect” concentrations on hatch and survival in the zebrafish (Brachydanio rerio) has been evaluated at five laboratories for variability using chromium (K2Cr2O7) and zinc (ZnSO4 × 7H2O) as toxicants. Repeatability (r) and reproducibility (R), expressed as C.V. (coefficient of variation), for logarithmic median survival time ranged between 1% and 9% for r and between 1% and 29% for R depending upon concentration of toxicant. Variability increased at high concentrations, especially for zinc. For logarithmic median hatching time, C.V. for r ranged from 6% to 24% and for R between 11% and 27% for the various concentrations of chromium and zinc. Estimated logarithmic “no-effect” concentrations on survival of zinc and chromium had a C.V. for r below 10% and a C.V. for R below 15% for zinc and below 25% for chromium. Zinc delayed the time to hatch at lower concentrations than those that reduced survival time, for the estimated logarithmic “no-effect” concentrations of zinc on hatch r was around 75% and R around 100%. The “no-effect” concentrations based on all nine or ten tests from the five laboratories were 15 mg Cr/L and 0.5 mg Zn/L. Because of the cumulative toxicity of chromium in fish the chronic “safe” concentration may be considerably lower, but the “no-effect” concentration of 0.5 mg Zn/L for effects on hatching time is consistent with the chronic “safe” concentration found in other species of fish at the same hardness (100 mg/L expressed as CaCO3).
Die Untersuchungen zur Eiprodukcion beim Zebrabärbling zeigen, daß in der Zahl der abgelegten Eier erhebliche mdividuelle Unterschiede bestehen. Die Gruppengröße hat im Bereich von 2 Weibchen und 4 Männchen bis 8 Weibchen und 16 Männchen keinen Einfluß auf die Eizahl, die zwischen 40 und 100 Eier je Weibchen und Tag schwanken kann. Die Eiablage erfolgt in einem Zyklus von zwei Tagen. Eine Trennung der Geschlechter beeinflußt sowohl die Zahl als auch die Qualität der abgelegten Eier. Verwendet man die Eizahl als Parameter in einem Reproduktionstest, dann muß die Gesamteizahl, die Zahl der im Körper denaturierten und die Zahl der unbefruchteten Eier erfaßt werden.
Information on the biological activity of a substance is often obtained from experiments in which the treatments comprise a series of doses of the substance and a zero dose control. The aim of such experiments, particularly in toxicity studies, may be to determine the lowest dose, if any, at which there is activity. A new test procedure is proposed for this situation in the case where the activity of interest is a change in the mean of a single response variate. Tables are given whereby this procedure can be used when all treatments are equally replicated. The advantages of this test over other established tests are discussed.
Literaturrecherche und Auswertung zur Notwendigkeit chronischer Tests-lnsbesondere des Reproduktionstests-Am Fisch ftir die Stufe II nach dem Chemikaliengesetz. I 4. I .-97 3 16 Ring test of an embryo-larval toxicity test with zebrafish (Brachydanio rerio) using chromium and zinc as toxicants
- I Chorus
- Um-Weltbundesamt
- Geschlftszeichen
- G Dave
- B Damgaard
- M Grande
- J E Martelin
- Rosander B And Viktor
CHORUS, I. (1987). Literaturrecherche und Auswertung zur Notwendigkeit chronischer Tests-lnsbesondere des Reproduktionstests-Am Fisch ftir die Stufe II nach dem Chemikaliengesetz. I 4. I.-97 3 16/7. Um-weltbundesamt, Geschlftszeichen. DAVE, G., DAMGAARD, B., GRANDE, M., MARTELIN, J. E., ROSANDER. B., AND VIKTOR, T. (1987). Ring test of an embryo-larval toxicity test with zebrafish (Brachydanio rerio) using chromium and zinc as toxicants. Environ. To-w. Chem. 6, 61-71.
Deliberations and investigations of the DIN working group
- H Bresch
- P Code
- B Hamburger
- P-D Hansen
- I Juhnke
- F Krebs
- K Lillelund
- M Markert
- R Munk
- R Nagel
- E A Nljsch
- J Scheubel
- And
- Spieser
BRESCH, H., CODE, P., HAMBURGER, B., HANSEN, P-D., JUHNKE, I., KREBS, F., LILLELUND, K., MARKERT, M., MUNK, R., NAGEL, R., NLJSCH, E. A., SCHEUBEL, J., AND SPIESER, 0. H. (1986). Deliberations and investigations of the DIN working group " fish test " on a long-term test according to the German Chemicals Act.
Gkotoxikologie-Grundlagen fur die ijkotoxikologischen Bewertung von Umweltchemikahen nach dem Chemikaliengesetz, ecomed verlagsgesellschaft A test or differences between treatment means when several dose levels are compared with zero dose control
- R Nagel
- Umweltchemikalien
- Zu
- Bewertung
- Habilitationsschrift
- P Rudolph Maim
- R And Boje
- Landsberg
- Munich
- C Sch&ers
- R Nagel
- A And Seitz
NAGEL, R. (1988). Umweltchemikalien und F&he-Beitrage zu einer Bewertung. Habilitationsschrift, Maim. RUDOLPH, P., AND BOJE, R. (1986). Gkotoxikologie-Grundlagen fur die ijkotoxikologischen Bewertung von Umweltchemikahen nach dem Chemikaliengesetz, ecomed verlagsgesellschaft. Landsberg, Munich. SCH&ERS, C., NAGEL, R., AND SEITZ, A. (1989). Verhalten, Reproduktion und Populationsdynamik des Zebrabarblings (Brachydanio rerio) in einem natumahen Laborsystem. Fischiikologie l(2), 45-59. Water Research Centre (1987). Environmental effects of chemicals (CFM 9350 SLD) PRD 1388-M/2. WILLIAMS, D. A. (197 1). A test or differences between treatment means when several dose levels are compared with zero dose control. Biometrics 27, 103-117.
A comparative study of 1,1,2-dichloroaniline for marine and fresh water organisms
- Adema
REFERENCES ADEMA, D. M. M., AND VINK, G. J. (198 I). A comparative study of 1,1,2-dichloroaniline for marine and fresh water organisms. Chemosphere 10, 535-554.
Early life stage toxicity tests
- McKim