No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Toxicity of Binary Chemical Mixtures: Effects on Reproduction of Zebrafish ( Brachydanio rerio )
Abstract
A complete life-cycle test with zebrafish was carried outwith different concentrations of the binary mixture 3,4-dichloroaniline andlindane under flow-through conditions. Length and weight of fish of theF1-generation were reduced, even in the lowest test concentrationof 2 microg/L 3,4-dichloroaniline and 40 microg/L lindane. The same effects werefound in the early life stage test for the F2-generation. In themixture of 100 microg/L 3,4-dichloroaniline and 40 microg/L lindane, fish whichwere exposed for their whole life time stopped spawning, irreversibly; thefish population will become extinct. In an additional experiment, fish wereexposed to the same xenobiotic concentrations after reaching maturity. Inthis case, egg production was reduced. Cessation of egg production occurs ina concentration of 200 microg/L 3,4-dichloroaniline and 40 microg/L lindane.Nevertheless, effects on spawning are influenced by duration of exposure andthe life stages of exposure.
... An increase in the amount of exposure time typically results in quantification of effects at lower concentrations ͑McKim 1977; Bresch et al. 1990;Holcombe et al. 1995;Ensenbach and Nagel 1997;Nagel et al. 1998;Parrott et al. 2000͒. The age of the fish during an exposure can also impact the results of a study. ...
... Full life-cycle studies have been conducted over 25 years with sheepshead minnow, fathead minnow, and zebrafish ͑Carlson 1972; Nebeker et al. 1974;Ensenbach et al. 1997;Shinomiya et al. 1997;Nakamura et al. 1998;Kawahara and Yamashita 2000;Yasuda et al. 2000;Metcalfe et al. 2000͒. Many full lifecycle studies using the fathead minnow, and to a lesser extent the sheepshead minnow, have been conducted in the support of pesticide registrations. ...
This paper presents a detailed overview of endocrine-disrupting chemicals in which the writers discuss the disrupter agent modes of action, the impact evidence on humans, the different methods of analysis, and the difficulties met with biological screening tests. A discussion of exposure duration follows, on the deficiencies of both long-term and single-generation experiments, which will be important to future study. The present review also discusses how EDCs are discharged into municipal and industrial wastewater and can pass through wastewater-treatment plants and enter the aquatic environment (into rivers and seas). In the end, the writers present different alternatives and techniques, which several researchers continue to develop, and adapt to treat the aqueous solutions and mainly the wastewater.
... Kooijman and Bedaux [7] argue that length measurements are preferable when assessing growth because they require less handling of fish even though weight measurements are usually more accurate. Ensenbach and Nagel [25] found significant reduction in growth as indicated by changes in weight and length when exposing zebrafish to different binary mixtures of up to 100 g/L 3,4-dichloroaniline and up to 20 g/L lindane, although no effects on growth were observed when exposing the fish to up to 150 g/L lindane and 20 g/L 3,4-dichloroaniline separately. Ö rn et al. [26] found a decrease in body weight when exposing zebrafish to food containing a mixture of 20 polychlorinated biphenyls after 13 weeks of exposure but not after 4 weeks. ...
... Because zebrafish can be easily bred and reared in the laboratory, partial, full, and multiple life-cycle tests are increasingly popular in assessing the effects of xenobiotics. Ensenbach and Nagel [25] found a significant reduction in the number of eggs produced by zebrafish after exposure of adult animals to different binary mixtures of up to 100 g/L 3,4-dichloroaniline and 40 g/L lindane. However, they reported no influence on hatching rates and there was no increase in developmental abnormalities and no differences in survival rates of juveniles. ...
Toxic exposure of organisms interferes with organismal integrity at the biochemical level and ultimately gives rise to effects at the individual level. These effects may result in reductions in ecologically relevant characteristics such as growth, reproduction, and survival. A chronic toxicity test with zebrafish (Danio rerio) was conducted where fish were exposed to 50, 75, and 100% effluent for 28 d under flow-through conditions. Effects of effluent exposure were determined using endpoints of physiological (respiration during swimming), growth (condition, length, and weight), and reproductive (spawning and hatching) processes within the same population. Results clearly indicate that the condition and growth of zebrafish is depressed by exposure to the effluent. Also, increased oxygen consumption was found after 14, 21, and 28 d of exposure. Reproduction proved to correlate well with the condition of the motherfish in the control, and spawning and hatching were significantly depressed by effluent exposure. These results indicate that the evaluation of endpoints describing different ecologically relevant processes provides a rational assessment of the cause-effect relationships of effluent toxicity. This approach can quantify effects on different biological processes and can determine the interactions that occur between these different processes.
... Reduced fertilization rates in the exposed group as compared with controls may have different causes: direct effect of the substance on spermatogenesis; effect on the released sperm only; effect on the eggs by changes in the egg membrane that hinder or prevent penetration of intact spermatozoa. Ensenbach and Nagel (1997) reported that one of the most important effects of xenobiotics on fish is the impairment of reproduction. Finally, more recently, Kemadjou Njiwa and Mu¨ller (2002) found that sperm release by zebrafish during mating events decreased significantly when they were exposed to high DDT levels for a long time. ...
... More recently, Schettler (1998) reported that in general wildlife studies demonstrated a relationship between exposures to endocrine-disrupting substances and abnormal thyroid function, sex alterations, poor hatching success, decreased fertility and growth, and altered behavior. On the other hand, the reduction in egg production and decreased fertility reported by Ensenbach and Nagel (1997) during their investigation of the effect of dichloroaniline and lindane on the reproduction of zebrafish could have been due to the negative effects of the chemicals on the sperm released by the male fish as found in the present experiment. This study now shows that these negative effects occur earlier at the 5 and 50 mg/L concentrations of the chemical mixture. ...
A long-term toxicity test with zebrafish was carried out with different concentrations of DDT, Arochlor1254 (A54), and their 1:1 mixture under flow-through conditions. By collecting and counting the number of sperm released during separate mating events we observed that gametes are released asynchronously. Sperms are released in the form of sperm trails laid on the nest surface; subsequently active spermatozoa leave the trails and move in the water for several minutes. Sperm trails consist of bands of viscous material in which sperm are embedded. The water samples for the estimation of sperm presence were collected gradually within 180 min after 24 h, 2 weeks, 1 month, and 2 months of exposure. It was established that the reductions in count, activity of sperm, and average life span of sperm trails were significant (P<0.05) at the tested concentrations, 5 and 50 microg/L after 1 month and 2 weeks of exposure to each chemical tested and to their mixture, respectively. In conclusion, this study demonstrates that DDT and A54 could react synergically and increase the alteration in sperm release and activity as well as the life span of their trails.
... The average fecundity of this wild type strain of zebrafish was around 13 eggs/female, which is about 50% lower than that in some other inbred lines that have been selected for growth and reproductive output (Eaton and Farley 1974; Ensenbach and Nagel 1997 ). We found considerable variation in the numbers of eggs spawned daily in the zebrafish populations (Figure 1A ,B), highlighting the need for extensive replication in studies of this nature. ...
... treatments. This rate of survival is high for cultured zebrafish embryos (Ensenbach and Nagel 1997 ) and was higher than a comparable study (50%; Hill and Janz 2003). Most (98%) of this mortality occurred during the first stages of exogenous feeding and was not related to treatment. ...
Heightened concern over endocrine-disrupting chemicals is driven by the hypothesis that they could reduce reproductive success and affect wildlife populations, but there is little evidence for this expectation. The pharmaceutical ethynylestradiol (EE2) is a potent endocrine modulator and is present in the aquatic environment at biologically active concentrations. To investigate impacts on reproductive success and mechanisms of disruption, we exposed breeding populations (n = 12) of zebrafish (Danio rerio) over multiple generations to environmentally relevant concentrations of EE2. Life-long exposure to 5 ng/L EE2 in the F1 generation caused a 56% reduction in fecundity and complete population failure with no fertilization. Conversely, the same level of exposure for up to 40 days in mature adults in the parental F0 generation had no impact on reproductive success. Infertility in the F1 generation after life-long exposure to 5 ng/L EE2 was due to disturbed sexual differentiation, with males having no functional testes and either undifferentiated or intersex gonads. These F1 males also showed a reduced vitellogenic response when compared with F0 males, indicating an acclimation to EE2 exposure. Depuration studies found only a partial recovery in reproductive capacity after 5 months. Significantly, even though the F1 males lacked functional testes, they showed male-pattern reproductive behavior, inducing the spawning act and competing with healthy males to disrupt fertilization. Endocrine disruption is therefore likely to affect breeding dynamics and reproductive success in group-spawning fish. Our findings raise major concerns about the population-level impacts for wildlife of long-term exposure to low concentrations of estrogenic endocrine disruptors.
... As the most common species in aquatic ecosystem, the antioxidant enzyme defense system of fish is extremely sensitive to environmental pollutants, so many researchers focus on the toxic effects of nano-ZnO on fish using antioxidant enzymes as biomarkers ( Ahmad et al., 2006;Oliveira et al., 2008). Zebrafish is currently recommended by the International Organization for Standardization as a model organism (Ensenbach and Nagel, 1997) and is widely used in toxicology, environmental protection and developmental biology ( Ma et al., 2013). Li et al. showed that the 96-h LC 50 of nano-ZnO to zebrafish was 1.79 mg/L in embryonic stage and 4.62 mg/L in adult zebrafish ( Li et al., 2017). ...
The release of nano-zinc oxide (nano-ZnO) into the environment may lead to unpredictable risks, thus it
is necessary to study its potential harm to organisms. In this study, zebrafish exposed to nano-ZnO were
analyzed through cDNA microarrays to provide insight into the toxic effect of nano-ZnO on aquatic organisms at the molecular level. Results found that nano-ZnO inhibited the normal growth and development of zebrafish and other life activities by affecting the process of cell cycle. The nano-ZnO inhibited
the expression of the cyclins (Cycs), cyclin-dependent kinases (CDK) and the minichromosome maintenance (MCM), making the activation of Cyc/CDK complexs (CycD/CDK4, 6; CycE/CDK2; CycA/CDK2) and
MCM fail and resulting in DNA replication disorder in different periods (G1, M and G2 phase). Therefore,
the normal activities of individual organism such as cell division, differentiation and proliferation and the
functions of DNA binding and intracellular transfer were disturbed. These findings contribute to our
understanding of the toxicity of ZnO NPs to aquatic organisms, and also provide an evaluation basis for
assessing the environmental impact of nano materials
... Three in vivo fish models are commonly used to indicate the estrogenic potency of chemicals in simulated solutions, such as natural water or in sewage treatment plant effluents. These fish models include fathead minnows (Pimephales promelas), Japanese medaka (Oryzias latipes), and zebrafish (Danio rerio) ( Ensenbach and Nagel, 1997;Giesy et al., 2000;Metcalfe et al., 2001). In this study, zebrafish were used to assess the chronic sub-lethal toxicity effect. ...
Several ecological restoration projects have been constructed along urban rivers in North China to purify reclaimed municipal wastewater and improve the water quality of urban rivers. These projects attempt to address several environmental issues, including treating water contamination that is not fully remediated through standard wastewater treatment. This study investigated the efficiency of reducing endocrine disrupting compounds (EDCs) and estrogenic activity in reclaimed municipal wastewater along an 18.5 km field-scale ecological restoration project in Jialu River. The river only receives reclaimed municipal wastewater without natural effluent in North China. Data show that the chemical oxygen demand (COD) and ammonia nitrogen (NH3-N) of reclaimed municipal effluent improved when compared to the Chinese surface water standard, and natural estrogens, such as estrone (E1) and estradiol (E2), were effectively removed during ecological restoration purification processes. The estradiol activity based on measured EDCs concentrations (ΣEEQEDC) was less than 0.01 ng/L after the ecological purification of restoration river; however, synthetic endocrine disrupting compounds in reclaimed municipal wastewater, such as octylphenol (OP), bisphenol A (BPA), and dibutyl phthalate (DBP), were difficult to eliminate. The bioassays of MDA-kb2 cells and recombinant yeast in vitro showed no direct androgen response and estrogen effect in reclaimed municipal effluent after the purification processes. However, a chorionic long-term (21d) exposure in vivo test showed that exposure to the reclaimed municipal effluents, even after river purification, still significantly induced yolk protein vitellogenin (Vtg) in male zebrafish, leading to abnormal expression of testosterone (T) and E2. This indicates continued potent estrogenic activity to aquatic organisms, even after treatment and purification.
... A commercial-grade liquid endosulfan (6,7,8,9,10-hexachloro-1,5,5a,9, 9a-hexahydro-6,9-methano-2,4,3-benzodioxithepin-3-oxide; 35% EC) marketed by Jaipan, India 12 , was used throughout the experiment; from a 1.75 μl/l stock solution, the desired nominal concentrations were prepared 13 . ...
To study the effects of endosulfan, a mild estrogen-mimic, on survival, growth and reproduction of the model ze- brafish Danio rerio, the hatchlings were discretely immersed at selected doses (44-1400 ng/l) for 14 h. Immersion at the dose of 350 ng/l resulted in 12 and 27% mortality at the end of the treatment and at the age of sexual maturity respectively, as well as in the production of 72% females. The frequency of 'juveniles' increased from 17% at 44 ng/l to 44% at 1400 ng/l. Endosulfan acted as a growth suppressant and the mag- nitude of its suppression increased in the order of female > male > juveniles. During the 240-day experi- ment, it also postponed sexual maturity in females from the 120th dph (day post-hatching) in the control to the 181st dph in those treated at 1400 ng/l, reduced the spawning frequency (16-3 times) and cumulative fecundity (1424-159 eggs) by affecting the processes of maturation of eggs and vitellogenesis. In the treated but persisting males, it postponed sexual maturity (120-129th dph) and reduced fertilizability (91-13%) by reducing the motility duration of the sperm.
... This reduction in sperm released could therefore be due to the influences of the xenobiotic on the endocrine system as stated Mattison and Thomson,17) Donaldson and Scherer 18) More recently, Schettler 11) reported that in general wildlife studies demonstrated a relationship between exposures to endocrine disrupting substances and abnormal thyroid function, sex alterations, poor hatching success, decreased fertility and growth, and altered behavior. On the other hand, the reduction in egg production and decreased fertility reported by Ensenbach and Nagel 22) during the investigation of the effect of Dichloroaniline and Lindane on the reproduction of zebrafish could have been due to the negative effects of the chemicals on the sperm released by the male fish as found in the present experiment. How far these modifications in hormone concentrations lead to the decrease of sperm released by the male zebrafish is not known. ...
This study describes a long-term toxicity test within a period of 2 months using zebrafish (Brachydanio rerio) as the test species and concentrations of 0.05, 0.5, 5 and 50 μg/l DDT as a model substance. By collecting and counting the number of sperm released during separate mating events we observed that gametes are released asynchronously. Sperm is released in the form of sperm trails laid on the nest surface; subsequently active spermatozoa leave the trails and move in the water for several minutes. Sperm trails consists of bands of viscous material in which sperm is embedded. The water samples for the estimation of sperm presence were collected gradually within 180 min after 24 hr, 2 weeks, 1 month and 2 months of exposure. It was established that the reduction in count, activity of sperm and the average life span of sperm trails were significant (p < 0.05) at the tested concentrations 5 μg/l and 50 μg/l DDT after 1 month of exposure. In conclusion, this study demonstrates that long exposure time and higher DDT tested levels accelerate the occurrence of negative effect on the number and activity of sperm released as well as the life span of their trails.
... Reproductive effects included a significant decrease in the number of young produced by lindane-exposed females. The exposure of zebrafish to a mixture of lindane (0.04 ppm) and 3,4-dichloroaniline (0.1 ppm) had adverse effects on reproduction in a life-cycle test (Ensenbach and Nagel, 1997). ...
... The early life stage test, on the other hand, is not subject to much ethical considerations and also offers various toxicological endpoints for determining adverse effects. The gathered data could then be used to estimate pollutant toxicity both at the individual and fish population levels (Ensenbach and Nagel 1997). ...
The present study investigated the impact of fish cage aquaculture on the quality of lake water and sediment using a contact assay with zebrafish (Danio rerio) embryos. Fertilized zebrafish embryos were exposed to surface waters and whole sediments collected from three sites of varying levels of stress from Taal Lake, Philippines. Gonzales was the reference site, while Bañaga and Balakilong were sites of heavy aquaculture. No significant developmental differences were observed in water samples collected from all sites. Very high survival rates, high hatching success rates, and few abnormalities were observed in all water samples. All values did not differ significantly from the negative control. Similarly, zebrafish embryos exposed to sediments from the reference site and from sediment control also developed normally and showed no significant defects. However, a significant reduction in both survival and hatching success rates, and a considerable increase in rates of abnormalities, were recorded from sediments obtained from the aquaculture sites. The observed embryotoxic responses were discussed in relation to the presence of contaminants such as ammonia and copper which have settled in the sediments from unconsumed feeds. Results of the study clearly demonstrate that fish cage culture has a harmful impact on lake quality (particularly the sediment phase). We also recommend the use of an effect-directed analysis as a next-level approach to identify specifically other individual toxicants responsible for the teratogenic effects.
... The early life stages of fish provide an array of developmental parameters, which could serve as biomarkers of toxicant effects (Nagel 2002;Hallare et al. 2004;Braunbeck et al. 2005). The impact of toxicants on embryo growth and ontogeny could be extrapolated to assess effects at population levels (Ensenbach and Nagel 1997). Apart from its sensitivity, this test offers practical advantages over fish acute toxicity tests. ...
PurposeSediments serve as integral and dynamic parts of our aquatic systems. Within the last 15 to 20years, however, the scientific
community has begun noticing deterioration of sediment quality at an alarming rate worldwide. Sediments are now harboring
hazardous pollutants that can directly influence water quality, thereby creating very stressful conditions for aquatic life.
As a consequence, global efforts were initiated in the early 1970s, to find ways to assess sediment quality. Because of their
obvious ecological and economic significance, fish have remained a major taxonomic group for appraising the general quality
of aquatic systems. However, for sediment risk assessment, fish have lagged behind invertebrates due to their mobility and
generally, pelagic lifestyle. To our knowledge, this is the first paper that comprehensively presents and reviews the versatile
role of fish in assessing the state of health of aquatic sediments.
Main featuresThrough a literature search of the more relevant and/or more recent studies, this review attempted to trace the development
of the various approaches as well as to describe the future prospects of using fish as sentinels for sediment quality assessment.
Initially, the use of whole fish (juveniles or adults) bioassays contributed immensely to our understanding of sediment contamination
and ecotoxicology. But due to economic as well as ethical issues linked to the use of live vertebrates for toxicity testing,
the approach has shifted to using fish cell cultures and fish embryos. Much newer approaches involving receptors and gene
arrays in fish cells to elucidate the mode of action of sediment-borne contaminants are very promising. The review paper also
lists and explores some of the issues associated with the use of juvenile or adult fish, fish cell cultures, fish embryos,
and fish gene expression profiles in sediment toxicity evaluations to stimulate further discussions, and hopefully, to serve
as benchmark for future handling of similar or related aspects of fish utilization in sediment risk assessment.
Conclusions and perspectivesOverall, the present review has comprehensively explored the changing and progressing roles of fish in sediment toxicity evaluation.
Indeed the usefulness of this taxon as test organisms has provided a significant contribution to the advancement of sediment
toxicology. However, despite the quite optimistic and bright future for these current procedures, a number of issues and problems
remain. Therefore, efforts to develop new technologies and to refine current methods and approaches continue to challenge
many laboratories worldwide.
KeywordsAquatic toxicity-Biomarker-Fish-Sediment toxicity
... In early life stage test, toxicant effects on ontogenesis and growth can be examined through many diverse endpoints. The gathered data could then be used for estimating pollutant toxicity not only at individual but also at the level of fish populations (Luckenbach et al., 2001;Triebskorn et al., 2001;Ensenbach and Nagel, 1997). In addition to developmental parameters, other biomarkers can also be investigated in the growing embryos. ...
To determine the interactions between temperature and cadmium on zebrafish (Danio rerio) development, fertilized eggs were exposed to combinations of three temperature levels (21 °C, 26 °C, and 33 °C) and six cadmium concentrations (0, 0.25, 0.5, 2.0, 5.0, and 10.0 mg/L). Endpoints used included LC50 value (48 h), developmental rate, mortality, heart rate, hatching success, liver histopathology, embryo abnormalities, and heat shock protein (hsp) induction. Results showed a significant acceleration in the developmental rate with increasing temperature and irrespective of the presence of cadmium. Data on LC50 and ELS-test revealed that simultaneous exposure to both cadmium ions and cold stress (21 °C) was highly detrimental to growing embryos, causing a pronounced mortality and a significant reduction in average heart rate and embryo hatchability. In contrast, no similar reactions to cadmium were observed in pre-hatched embryos exposed to both control (26 °C) and high temperature (33 °C), and this can be explained by the significantly higher expression of hsp (hsp70) in embryos at these temperatures. Upon hatching, however, the larvae showed increased sensitivity to cadmium. The severity of malformations in the post-hatched larvae was in the order: hot cadmium stress>cold cadmium stress>cadmium stress alone>no stress at all. Liver histopathology as well as depletion in glycogen reserves exhibited greater severity with increasing cadmium concentration, irrespective of temperature. The present study confirms that temperature effectively confounds cadmium toxicity and needs to be considered for the accurate prediction and assessment of cadmium-induced toxicity in fish.
... Several studies revealed that the sensitivity of fish embryos and larvae to some chemicals is greater than that of adults (Lange et al., 1995;Nagel and Isberner, 1998;Nagel, 2002;Strmac et al., 2002). In early life-stage tests, toxicant effects on ontogenesis and growth can be examined through many diverse endpoints (Ensenbach and Nagel, 1997;Luckenbach et al., 2001). In addition, the direct exposure of a test organism to an environmental sample closely resembles natural conditions and can therefore be of high ecological relevance. ...
... The zebrafish was used as a test model because it is easy to grow, has a short generation time and breeds all year round. Developmental stages have been described extensively (Hisoaka and Battle, 1958;Westerfield, 1998) and there is extensive literature on ecotoxicology of this species (Laale, 1977;Groth et al., 1993;Ensenbach and Nagel, 1997;Hallare et al. 2004;Lammer et al. 2009). ...
Frequently measured pharmaceuticals in environmental samples were tested in fish embryo toxicity (FET) tests with Danio rerio, based on the draft OECD test protocol. In this FET test 2-h-old zebrafish embryos were exposed for 72 h to carbamazepine, diclofenac and metoprolol to observe effects on embryo mortality, gastrulation, somite formation, tail movement and detachment, pigmentation, heartbeat, malformation of head, otoliths and heart, scoliosis, deformity of yolk, and hatching success at 24, 48 and 72 h. We found specific effects on growth retardation above 30.6 mg/l for carbamazepine, on hatching, yolk sac and tail deformation above 1.5mg/l for diclofenac, and on scoliosis and growth retardation above 12.6 mg/l for metoprolol. Scoring all effect parameters, the 72-h-EC(50) values were: for carbamazepine 86.5mg/l, for diclofenac 5.3mg/l and for metoprolol 31.0mg/l (mean measured concentrations). In conclusion, our results for carbamazepine and metoprolol are in agreement with other findings for aquatic toxicity, and also fish embryos responded in much the same way as rat embryos did. For diclofenac, the FET test performs comparably to Early Life Stage testing.
... Although their use has been progressively banned worldwide (Weinhold, 2001;US EPA, 2002), they are still commonly used in countries such as Mexico, Guatemala, Nicaragua, Salvador, and in several African nations. Numerous studies with fish have shown that a chronic exposure to lindane may cause physiological effects, which include negative effects on development and reproduction, behavioral changes (Ensenbach and Nagel, 1997;Hanson et al., 2007), changes in the lipid metabolism (Singh, 1992;Singh et al., 1994), altered enzymatic activity (Ferrando et al., 1991(Ferrando et al., , 1992Betoulle et al., 2000), as well as changes in the immune and endocrine systems (Cossarini-Dunier et al., 1987;Hart et al.,1997;Arukwe et al., 2000;Singh and Canario, 2004). Furthermore, studies with yeasts have also shown that lindane elicits the differential expression of genes involved in mitochondrial functioning, organization and biogenesis, oxidative stress, and ionic homeostasis (Parveen et al., 2003). ...
In order to assess the effect of lindane exposure on gene expression in tilapia (Oreochromis niloticus), twenty male fish were individually weighted and injected intraperitoneally with a single dose of lindane (19.09 mg/kg bw) using corn oil as a carrier vehicle, while a second group of twenty male fish (controls) was only injected with the carrier vehicle. Groups of four fish each were then sacrificed at 3, 6, 12, 18 and 24h after treatment application and total RNA was extracted from liver tissue. The differential display (DD) technique was then used to identify differentially expressed cDNA fragments between treatment and control fish. A total of fifty cDNA fragments were isolated and sequenced, from which only four showed homology with genes previously described in other fish species, namely the immunoglobulin heavy chain (IgH), coagulation factor V (FV), casein kinase 2 alpha (CK2a), and the receptor protein-tyrosine-like phosphatase (RPT-LP). The expression of such genes was confirmed using quantitative real time-polymerase chain reaction (QRT-PCR). Results showed that lindane exposure triggered the differential expression of these genes during the first 6, 18 and 24h subsequent to treatment application, suggesting that lindane exposure can trigger a rapid immune system response in tilapias.
... This ''estrogenic pattern'' differs from developmental and reproductive changes as induced by nonestrogenic xenobiotics. In zebrafish life cycle experiments with compounds such as 4-chloroaniline (Bresch et al., 1990), 2,4-dichloroaniline (Ensenbach and Nagel, 1997 ), 2,3,7,8-tetrachlorodiben- zo-p-dioxin (Wannemacher et al., 1992 ), or 1,2,3- trichlorobenzene (Roex et al., 2001), the reproductive parameter most affected was the number of eggs produced per female, while fertilization success or the time of first spawning were not altered. Thus, a reduced fertilization success, particularly in combination with elevated vitellogenin levels and modifications of gonadal differentiation, appears to be indicative of an estrogenic mode of action of the test compound on zebrafish. ...
The EU-funded project IDEA aimed to evaluate (a) what parameters and endpoints allow the detection of endocrine-mediated developmental and reproductive effects of (xeno)estrogens in life cycle- and life stage-specific toxicity tests with the zebrafish Danio rerio, a small laboratory fish used in many ecotoxicity test guidelines, and (b) whether substances that act as estrogens in vertebrates may also adversely affect the development, differentiation, and reproduction of aquatic invertebrates. The invertebrate species investigated included Hydra vulgaris, Gammarus pulex, Chironomus riparius, Hyalella azteca, and Lymnaea stagnalis. The animals were exposed to the model estrogenic chemicals ethynylestradiol (EE2), bisphenol A (BPA), and octylphenol (OP), which exert their endocrine activity in vertebrates through the estrogen receptor. As endpoints, developmental and reproductive parameters at the organism level as well as molecular and cellular parameters were measured. Life cycle exposure of zebrafish to (xeno)estrogens induced a specific, partly irreversible response pattern, consisting mainly of (a) induction of vitellogenin (VTG), (b) alterations of gonad differentiation, (c) delay of first spawning, and (d) reduced fertilization success. The effects of EE2 on zebrafish were expressed at environmentally realistic concentrations, while BPA and OP became effective at concentrations higher than those usually found in the environment. The vitellogenic response was equally sensitive as the reproductive parameters in the case of EE2, but VTG was more sensitive in the case of BPA. Partial life cycle exposure of zebrafish had lasting effects on fish development and reproduction only when the fish were exposed during the stage of juvenile bisexual gonad differentiation. In (partial) life cycle and multigeneration studies with invertebrates, (xeno)estrogenic impact was assessed by a range of developmental and reproductive parameters including hatching, growth, moulting, mating behavior, and egg number. Several parameters were found to be responsive to (xeno)estrogens; however, most effects were induced only at higher, probably nonphysiological concentrations. Low-dose effects were observed in full life cycle experiments, particularly in the second generation. It remains to be established whether the estrogen-induced alterations in the invertebrate species indeed do result from disturbances of the endocrine system. The findings of the present research project support the development of appropriate testing methodologies for substances with estrogenic activity.
... Rarely are pesticides found as single compounds in the environment but instead are often found as mixtures. Studies have examined the effects of chemical mixtures on aquatic organisms (nontarget species) (Bailey et al. 1997;Ensenbach and Nagel 1997;Carder and Hoagland 1998;Jin-Clark et al. 2000;Anderson and Lydy 2002;Denton et al. 2003); however, no studies have examined the mixture effects of the pesticides used in this study or the effects of pre-exposures to OCs on subsequent effects of current-use pesticides. Therefore, the objectives of this research were (1) to measure the acute toxicity of nine commonly detected pesticides found in the Sacramento-San Joaquin Delta to the aquatic midge Chironomus tentans as single compounds and binary mixtures and (2) to test potential interactions of pre-existing DDE body residues with current pesticide exposure. ...
This study examined the effects of nine commonly detected pesticides in the Sacramento-San Joaquin Delta on the aquatic midge Chironomus tentans. Pesticides were chosen from a variety of chemical classes including organophosphate (OP) insecticides as well as triazine, triazinone, and substituted urea herbicides. Both single toxicant and binary mixture bioassays were performed. In addition, midges were pre-exposed to DDE at environmentally relevant concentrations and then challenged by exposing them to chlorpyrifos or diazinon in single-toxicant acute bioassays. Results indicate that most of the binary mixtures elicited additive responses in C. tentans, whereas OP insecticides in combination with various herbicides caused greater-than-additive responses. Pre-exposures with DDE did not have a significant impact on subsequent OP challenges at DDE concentrations of 0.2, 2, and 20 microg/kg. This study represents an important first step in understanding the interactions among various pesticides commonly detected throughout the Delta.
... Aside frorn being the most sensitive stage of life (Hutchinson et al., 1998;Kristensen, 1995;Birge et al., 1985;McKim, 1985), embryos offer many diverse endpoints to determine sublethal effects. The effects of toxicants on embryo growth and ontogeny could be extrapolated to assess effects at population levels (Ensenbach and Nagel, 1997). The ability of growing embryos to discriminate between varying levels of pollution in aquatic systems has been weIl documented in recent works (Luckenbach et al., 2001(Luckenbach et al., , 2003Klurnpp et al., 2002). ...
In order to achieve a more substantial appraisal of lake water quality, the assessment must not be based only on chemical measurements and analyses of the water itself, but even more so on the impact of existing conditions on aquatic biota. This is possible with the use of biotests or biomarkers, e.g. investigations of the developmental parameters (96-h embryotoxicity evaluate) or of the induction of heat shock proteins (proteotoxicity evaluate). To evaluate the suitability of these tests for environmental screening, fertilized zebrafish eggs were exposed to water samples collected from five sites of varying levels of stress from Laguna Lake, Philippines. Reconstituted water was used as laboratory control while water samples from a highly polluted freshwater source was used as positive control. Developmental parameters were noted and described within 48 and 96 h of exposure. Dilution experiments of the positive control were also done to further assess and compare toxicity potentials of Laguna Lake waters with those originating from a polluted freshwater. After the 96-h exposure, the levels of stress proteins (hsp 70) were determined in embryos from all exposure groups. Results showed 100% mortality in embryos exposed to undiluted positive control (PC) within only 12 h. Increasing dilution levels, however, resulted in lower mortality and lower abnormality rates. No detectable developmental differences were noted among embryos exposed to either the laboratory control or Laguna Lake waters at the end of 96 h, regardless of the source. Very high survival rates and high hatching success rates were observed in embryos exposed to lake waters as well as laboratory control, and the data did not differ significantly among the groups. Likewise, no significant malformations were noted among all developing embryos throughout the exposure period. However, the levels of heat shock proteins in the two sites located closest to Manila, the Philippine capital (Northern West Bay and Central West Bay), showed a pronounced elevation relative to the control, indicating that these stress proteins protect the embryos from the detrimental effects of pollutants in the water. Based on the 96-h early life stage (ELS) test, the water quality of the lake is good for fishery propagation despite the current levels of pollutants in the water. This finding is in accordance with the Class C status (i.e. suitable for fish growth and propagation) as given to the lake by the local environmental agency. On the other hand, data on proteotoxicity showed that the fish are under stress, presumably deriving from pollutants. This calls for a continuous monitoring and improvement of the lake water. The present study indicates that the two biomarker methods are very easy to use, practical, rapid, and sensitive for assessing water quality in a tropical lake and recommends for their incorporation into the future monitoring program of Laguna Lake.
Aromatic nitro and amino compounds have various commercial, governmental, and public applications. Their uses include manufacturing of dyes, pesticides, drugs, and explosives. These substances are typically considered high‐nitrogen compounds and as such share many of the same chemical and biological properties. Many have amines and/or nitrate groups on the ring and because of this exhibit properties where they are not highly water or lipid soluble and many tend to form crystals. When released to the environment, nitrate‐containing moieties are often reduced to amines by bacteria in wet soils and sediments. Acute effects typically target the nervous system; however, sublethal effects from repetitive exposures include anemia, methemoglobinemia, and often male reproductive effects. Cyanosis has been described from relatively high repetitive exposures. Oral exposures can affect the liver. Many are absorbed through the skin where dermal exposures can significantly contribute to systemic dose. Skin sensitization and irritation have been observed from exposure to some compounds. Many are eliminated via the urine; however, conjugated metabolites can be eliminated via the feces. Nitroso metabolites are suspected as having influence in observation of cancer, particularly in the bladder. Other similar nitramine compounds are also briefly described.
This datasheet on Aquatic toxicology covers Identity, Description, Further Information.
Logically, aromatic nitro and amino compounds should be discussed together because their toxic responses are often similar due to a common metabolic intermediate. Synthetically, amines are generally derived from nitro compounds, but in some cases nitro compounds can be prepared through amines when other methods fail to afford specific compounds. There are good and bad attributes to these types of compounds. Some act as sensitizers and contingent on physical properties, may be absorbed through the skin or mucous membranes. They may also cause methemoglobinemia, depending on such factors as the structure and the particular organism. Some members of this class are known as animal and human carcinogens; for humans the urinary bladder is the most prominent target organ. Nevertheless, these compounds and their derivatives have enlivened our world through their use as dyestuff intermediates or as photographic chemicals, they alleviate pain as components of widely used analgesics, and they cushion or insulate us through their use in flexible and rigid foams. Other important uses include production of pesticides, including herbicides and fungicides, as ingredients in adhesives, paints and coatings, antioxidants, explosives, optical brighteners, rubber ingredients, and as intermediates in many other products.
The effect of soil exogenous ciprofloxacin and copper combined pollution on the early growth of wheat was studied by the toxicological diagnosis method of higher plant. The potential ecological risks of soil contamination derived from livestock manure were investigated. Results indicated the early growth indexes of wheat expressed different degrees of responses to single copper or ciprofloxacin exposure. The wheat shoot and root length were mainly inhibited, while the seed germination rate was unaffected. The sensitivities of the three indexes to the two pollutants were in order of shoot length > root length > germination rate. Ciprofloxacin and copper combined pollution showed different effects on the wheat roots and shoot length. Compared with single copper exposure, inhibition effect on wheat root length was enhanced by the combined treatment of 20 mg·kg-1 copper and 80 mg·kg-1 ciprofloxacin, showing a synergistic effect, while the inhibition effect on wheat shoot length by the addition of 200 mg·kg-1 of copper eased after ciprofloxacin addition, showing an antagonistic effect. The complex, produced by the interaction of ciprofloxacin and copper, may be the cause of various biological effects.
Benzo(a)pyrene (BaP) and dichlorodiphenyltrichloroethane (DDT) are persistent organic pollutants and environmental estrogens (EEs) with known toxicity towards the green mussel, Perna viridis. In this study, the toxic effects of BaP (10 μg/L) and DDT (10 μg/L) and their mixture were assessed in green mussel gills with proteomic and metabolomic approaches. Metabolic responses indicated that BaP mainly caused disturbance in osmotic regulation by significantly decrease in branched chain amino acids, dimethylamine and dimethylglycine in gills of male green mussels after exposure for 7 days. DDT mainly caused disturbance in osmotic regulation and energy metabolism by differential alteration of betaine, dimethylamine, dimethylglycine, amino acids, and succinate in gills of male green mussels. However, the mixture of BaP and DDT didn't show obvious metabolite changes. Proteomic analysis showed different protein expression profiles between different treatment groups, which demonstrated that BaP, DDT and their mixture may have different modes of action. Proteomic responses revealed that BaP induced cell apoptosis, disturbance in protein digestion and energy metabolism in gills of green mussels, whereas DDT exposure altered proteins that were associated with oxidative stress, cytoskeleton and cell structure, protein digestion and energy metabolism. However, the mixture of BaP and DDT affected proteins related to the oxidative stress, cytoskeleton and cell structure, protein biosynthesis and modification, energy metabolism, growth and apoptosis.
Logically, aromatic nitro and amino compounds should be discussed together because their toxic responses are often similar due to a common metabolic intermediate. Synthetically, amines are generally derived from nitro compounds, but in some cases nitro compounds can be prepared through amines when other methods fail to afford specific compounds. There are good and bad attributes to these types of compounds. Some act as sensitizers and contingent on physical properties may be absorbed through the skin or mucous membranes. They may also cause methemoglobinemia, depending on such factors as the structure and the particular organism. Some members of this class are known as animal and human carcinogens; for humans, the urinary bladder is the most prominent target organ. Nevertheless, these compounds and their derivatives have enlivened our world through their use as dyestuff intermediates or as photographic chemicals, they alleviate pain as components of widely used analgesics, and they cushion or insulate us through their use in flexible and rigid foams. Other important uses include production of pesticides, including herbicides and fungicides, as ingredients in adhesives, paints and coatings, antioxidants, explosives, optical brighteners, rubber ingredients, and as intermediates in many other products.Keywords:Air pollutants;aromatic amino compounds;aromatic nitro compounds;bladder cancer;chloro compounds;databases;inventories
A long-term toxicity test with zebrafish was carried out with different concentrations of DDT, Arochlor1254 (A54), and their 1:1 mixture under flow-through conditions. By collecting and counting the number of sperm released during separate mating events we observed that gametes are released asynchronously. Sperms are released in the form of sperm trails laid on the nest surface; subsequently active spermatozoa leave the trails and move in the water for several minutes. Sperm trails consist of bands of viscous material in which sperm are embedded. The water samples for the estimation of sperm presence were collected gradually within 180 min after 24 h, 2 weeks, 1 month, and 2 months of exposure. It was established that the reductions in count, activity of sperm, and average life span of sperm trails were significant (P<0.05) at the tested concentrations, 5 and 50 μg/L after 1 month and 2 weeks of exposure to each chemical tested and to their mixture, respectively. In conclusion, this study demonstrates that DDT and A54 could react synergically and increase the alteration in sperm release and activity as well as the life span of their trails.
Zebrafish have been used predominantly in developmental biology and molecular genetics, but their value in toxicology as well as drug discovery has been recognized. To evaluate the toxicity of a chemical, it is essential to identify the endpoints of toxicity and their dose-response relationships, elucidate the mechanisms of toxicity, and determine the toxicodynamics of the chemical. In addition to detailed toxicological investigations of a single chemical, there also is a need for high-throughput large- scale screening for toxicity of several hundreds of chemicals at a time. In both cases, the zebrafish has numerous attributes. More is probably known about ''what is normal'' in the zebrafish than any other fish species. This includes morpho- logical, biochemical, and physiological information at all stages of early development and in juveniles and adults of both sexes. This makes using the zebrafish ideal for toxicology research where the objective is to identify adverse effects of chemical exposure.
To study the effects of endosulfan on survival, growth and reproduction of the obligate air-breathing male heterogametic fighting fish Betta splendens, posthatchlings of the fighting fish were discretely immersed for 3 h/day during the labile period on the 2(nd) , 5(th) , and 8(th) day posthatching (dph) at selected concentrations of commercial grade endosulfan ranging from 175 to 1400 ng/L. The immersions at 1,400 ng/L led to 21% mortality, among the 79% of surviving fry, 80% developed into females. The endosulfan reduced the air-breathing frequency of 5- and 8-day old hatchlings, and the reduction in the frequency persisted even after a depuration period of 172 days. In the ovary of the treated females, reduced number of vitellogenic oocytes with increased vacuolar cavities was observed. In the testis of the treated males, the reduced number of spermatogonia with increased vacuolar cavities was observed. The treated male induced the female to spawn a fewer eggs, which were subsequently incubated in his smaller bubble nest. The control females attained puberty on the 138(th) dph and spawned 120 eggs once in every 15 days, the females, which were previously treated at 1400 ng/L, postponed puberty to the 179(th) dph and spawned 70 eggs once in every 32 days. During the 240-day experiment, endosulfan is found to reduce significantly the cumulative progeny production from 760 to 144, reducing significantly to 19% of the control. © 2012 Wiley Periodicals, Inc. Environ Toxicol, 2012.
Induction of specific proteinsProtein degradationPhysiological effects: whole body responses/regulation
This chapter explains the utility of zebrafish as a model for toxicological research. Zebrafish (Danio rerio) have long been the genetic model of choice for vertebrate developmental biologists, as it provides several advantages for investigating organ and tissue development. Zebrafish have become a powerful model organism for investigating the molecular and cellular mechanisms by which environmental chemicals disrupt normal developmental processes. The utility of zebrafish as a laboratory model organism also makes it an excellent system for studying processes in juvenile and adult organisms, including reproductive development, carcinogenesis, aging, and the influence of environmental chemicals on these processes. The advent of modern genetic tools and genome sequencing projects has elevated zebrafish as a suitable model to effectively study human disease and pathophysiology, ushering in a new era of comparative biology and medicine. The chapter reviews the mechanistic toxicology and advantages of the zebrafish model system.
This chapter contains section titled:
Rainbow trout (Oncorhynchus mykiss) were implanted intraperitoneally with 0.5ml.100g–1 body weight of coconut oil alone (controls) or coconut oil contaning 0.05mg of -HCH (Lindane). After 18 days, changes in brain serotonin and GABA metabolism, as well as in serum cortisol and thyroxine levels, were measured. A lower final body weight was observed in -HCH treated fish when compared with control fish. No significant differences were found for serum thyroxine levels between control and treated fish, but a significantly higher cortisol level was found in the -HCH-implanted trout. Although GABA levels did not differ significantly in any brain region in the two treatment groups, the activity of the serotonergic system was significantly altered by the pesticide in both the hypothalamus and the telencephalon.
Zearalenone (ZON) is one of the worldwide most common mycotoxin and exhibits estrogenic activity in the range of natural steroid estrogens. The occurrence of ZON has been reported in soil, drainage water, wastewater effluents, and rivers, but its ecotoxicological effects on fish have hardly been investigated. The consequences of continuous long-term ZON exposure, including a subsequent depuration period, as well as transgenerational effects of F0 short-term exposure on F1 generation were investigated. Effects on growth, reproduction activity, physiology, and morphology of zebrafish (Danio rerio) were examined in a 182 day live-cycle experiment. Life-long exposure to ZON for 140 days increased wet weight, body length, and condition factor of female fish at 1000 ng/L, and sex ratio was shifted toward female from 320 ng/L ZON. Only females at 1000 ng/L ZON revealed a 1.5-fold induction of plasma vitellogenin (VTG). Relative fecundity at 1000 ng/L recovered significantly during the depuration period. An increased condition factor in adult female F1 fish implies that exposure of F0 generation to 1000 ng/L ZON affected growth of F1 generation. A negative correlation between relative fecundity in the F1 generation (all groups exposed to 320 ng/L ZON) and the nominal ZON concentrations of the F0 exposure might indicate an influence of F0 exposure on reproductive performance of F1 generation. No exposure scenario affected fertility, hatch, embryo survival, and gonad morphology of zebrafish. Evaluating the environmental relevance of this data, the risk for fish to be harmed by exposure to ZON solely seems rather marginal, but ZON might contribute to the overall estrogenicity in the environment. © 2011 Wiley Periodicals, Inc. Environ Toxicol 2011.
Graduation date: 2010 Pesticides are ubiquitous, with more than one billion tons of pesticide products used in the United States annually. These compounds are characterized by their toxic effects to the target organism; however, pesticides are also well known for their deleterious effects to non-target species. Because pesticides have ecological and human health effects, it is important to investigate their prevalence in the environment, as well as their bioavailability and toxicity. Passive sampling devices (PSD) are commonly used to evaluate contaminants, including pesticides, found in water, air and soil. The PSD used for this research is an aquatic sampler that sequesters freely dissolved non-polar and semi-polar contaminants by diffusion into and adsorption to a lipid-free polyethylene membrane tubing (LFT). Thus, PSD can act reasonably as a biological surrogate, mimicking non-dietary bioavailability. Additionally, PSD extracts are proposed to be amenable to investigate toxicity of biologically available environmental mixtures utilizing the embryonic zebrafish (Danio rerio) model. To investigate this potential application, experiments were performed to evaluate the toxicity of non-deployed blank PSD extracts and non-deployed extracts spiked with individual pesticides or pesticide mixtures. For this proof of concept study, embryonic zebrafish were static waterborne exposed before the initiation of organogenesis. Two time points were monitored for mortality and alterations in development. The blank extracts did not result in any adverse developmental effects, relative to non-exposed controls. Embryos exposed to a comprehensive fourteen compound pesticide mixture extract produced an increase in adverse developmental responses as concentration increased. Experiments investigating the toxicity of individual compounds and partial pesticide mixtures were also performed. These preliminary studies indicate that the coupling of PSD extracts and in vivo toxicological assessments is realistic.
Thesis (Ph. D.)--Texas Tech University, 1999. Includes bibliographical references.
Trier, Univ., Diss., 2002. Computerdatei im Fernzugriff.
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.
Chemicals with estrogen-like activity have drawn increasing attention during recent years because of the possible consequences for human and wildlife development and reproduction. Among wildlife species, research has been focused on animals from aquatic habitats receiving sewage and industrial effluents and agricultural runoff. For teleost fish, there exist a number of both laboratory and field reports on adverse health effects related to or induced by exposure to xenoestrogens (e. g. van der Kraak et al., 1992; Flouriot et al., 1995; Sumpter, 1995; Folmar et al., 1996; Nimrod and Benson, 1996).
Isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) were exposed to different concentrations (1x, 10x, 50x) of a complex mixture of 20 environmentally relevant contaminants (PAHs, PCBs, pesticides) typical of the chemical burden of surface waters and sediments of small rivers in southwest Germany to investigate sublethal cytological and biochemical alterations. Results document that all concentrations clearly induced biochemical and morphological changes. The activities of enzymes such as lactate dehydrogenase and catalase as well as the rate of lipid peroxidation were significantly increased, whereas acid phosphatase activity decreased. Cytopathological effects included deformation of cellular shape, deformation and dilation of the nuclear envelope, increase in heterochromatin, nuclear inclusions, heterogeneity of mitochondria, vesiculation of RER cisternae, as well as increases in myelinated bodies, lysosomes, and peroxisomes. If compared with cytological alterations induced in isolated rainbow trout hepatocytes after exposure to sediment extracts from rivers of corresponding contamination, cytological effects of exposure to the complex chemical mixture displayed numerous similarities.
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.
In this study, the impact of an exposure of zebrafish embryos (Danio rerio) until 3 months post fertilization to 17alpha-ethynylestradiol (EE2: 0; 0,1; 1; 10; 25 ng/l) was evaluated for growth and development, gonad development and body vitellogenin (VTG) content. After a recovery period of 5 months, the female reproductive success was evaluated. The results demonstrated a significant reduction in total body length, body weight, whole body Ca and P content and an increase in morphological abnormalities for fish exposed to 25 ng/l EE2 as a function of exposure time. An increase in total body VTG content was observed for fish exposed for a period of 3 months down to levels of 1 ng/l EE2. At the age of 3 months, a dose-dependent increase of the number of fish with no macroscopic recognizable gonads was observed (up to 100% at 25 ng/l EE2). After a recovery period on clean tap water for 5 months, all fish had developed either ovaries or testis with a gonadosomatic index not different from control fish and the sex ratio was similar in EE2 treatment groups and controls. Nevertheless, a reduced number of spawning females and a reduced egg production were found for the female fish exposed to 10 or 25 ng/l EE2 for 3 months and which were allowed to recover for 5 months. Although the underlying mechanism could not be elucidated, these findings did indicate that the reproduction potential of fish populations might be disturbed by a long-term exposure to EE2 (> or =10 ng/l) from fertilization until sexual maturity.
One of the most frequently detected pharmaceuticals in environmental water samples is the anti-rheumatic drug, diclofenac. Despite its increasing environmental significance, investigations concerning the effects of this drug on the early developmental stages of aquatic species are lacking up to now. To determine the developmental toxicity and proteotoxicity of this drug on the growing fish embryos, eggs of zebrafish were exposed to six concentrations of diclofenac (0, 1, 20, 100, 500, 1000, and 2000 microg l(-1)) using DMSO as solvent. Early life stage parameters such as egg and embryo mortality, gastrulation, somite formation, movement and tail detachment, pigmentation, heart beat, and hatching success were noted and described within 48- and 96-h of exposure. After the 96-h exposure, the levels of stress proteins (hsp 70) were determined in both the diclofenac-treated and respective DMSO controls. Results showed no significant inhibition in the normal development until the end of 96 h for all exposure groups. However, there was a delay in the hatching time among embryos exposed to 1000 and 2000 microg l(-1). Late-hatched embryos (108 h) did not differ morphologically from normally hatched embryos. The mortality and average heart rate data did not show significant differences for all embryos in both diclofenac-treated and DMSO control groups. No significant malformations were likewise noted among all developing embryos throughout the exposure period. The levels of heat shock proteins in diclofenac-treated and control embryos did not differ significantly. DMSO control embryos, on the other hand, showed a concentration-dependent increase in hsp 70 levels. We suggest possible modulating effect of diclofenac in DMSO-triggered expression of stress proteins and this might have a possible repercussion on the use of DMSO as solvent in any toxicity assay. Since the present data indicate no significant embryotoxicity and proteotoxicity induced by diclofenac and due to the fact that the concentrations of diclofenac used in the present study is up to 2000-fold higher than the concentrations detected in the environment, it is unlikely that this drug would pose a hazard to early-life stages of zebrafish.
Zebrafish (Danio rerio) has been a prominent model vertebrate in a variety of biological disciplines. Substantial information gathered from developmental and genetic research, together with near-completion of the zebrafish genome project, has placed zebrafish in an attractive position for use as a toxicological model. Although still in its infancy, there is a clear potential for zebrafish to provide valuable new insights into chemical toxicity, drug discovery, and human disease using recent advances in forward and reverse genetic techniques coupled with large-scale, high-throughput screening. Here we present an overview of the rapidly increasing use of zebrafish in toxicology. Advantages of the zebrafish both in identifying endpoints of toxicity and in elucidating mechanisms of toxicity are highlighted.
Heat shock proteins (HSPs) play a central role in cell protection and repair upon stresses, such as that caused by heat and heavy metals. Copper sulfate inducibility of a pHhsp70 construct expressing the enhanced green fluorescent protein (EGFP) gene under the control of the exogenous human hsp70 promoter was tested in transfected CHSE 214 cells and transgenic zebrafish (Danio rerio). We developed a transient expression system, using mosaically transgenic zebrafish, which allows rapid analysis of transgenic expression. Transfected CHSE 214 cells which had been exposed to 250 nM and 2.5 microM copper sulfate for up to 24h showed increased EGFP expression in a dose-dependent manner. The 1.5 microM copper sulfate caused stronger EGFP fluorescence than the 1.0 microM copper sulfate in transgenic zebrafish. Most of the expression was spotty and was detected in the gills, dorsal and ventral retina, myotubes of the trunk, and skin epithelium. Transgenic zebrafish exposed to copper sulfate exhibited gross dysmorphogenesis, edema and trunk abnormalities, such as spinal lordosis, in vertebral development 5 days after fertilization. This transgenic zebrafish system was sensitive enough to detect copper sulfate at doses below the median lethal concentration (the LC50 was calculated to be 1.2 microM (95% confidence interval of 0.6-1.9 microM)). These results indicate that zebrafish could be useful transgenic biosensor systems for the detection of xenobiotic toxicants in the environment.
Single pairs of zebrafish, Brachydanio rerio, 12 months old, spawned at an interval of 1.9 days when the male and female were left continuously together. Three months later the spawning interval had increased to 2.7 days, suggesting that age is a factor in determining the spawning cycle. Hard water had no effect on spawning cycle in these experiments, nor did grouping the fish with three males for each female. An experiment was done in which prolongation of the spawning cycle was enforced by separating the male from the female for random intervals of 2-9 days. In 93% of the trials spawning occurred on the morning following reintroduction of the male after a 2-day separation. Similar results were obtained upon reintroduction of the male after 3-9 days of separation. The greatest number of eggs per day can be obtained by leaving the pair continuously together. In two experiments in which this was done the mean number of eggs produced per day per pair was 23.1 and 60.4. In the experiment where male and female were separated for 2-9 days, and then spawning was allowed to occur, egg production varied from 45 eggs per day per pair with 2 days of separation to 10 eggs per day per pair with 9 days separation. Evidence was obtained that egg development is triggered by interaction with the male. Eggs usually could not be stripped from the female without the male having been present. Introduction of the male for 7 hours at the end of one day enabled eggs to be stripped from the female the next morning. The precise stimulus for egg development was not determined, but it is suggested that it may be the chasing of the female by the male, behavior which goes on almost continuously when the two are together.
Reproductive parameters and accumulation of chlorophenolic compounds by white sucker (Catostomus commersoni) populations exposed to bleached kraft mill effluent (BKME) were studied in the St. Maurice River, Quebec. Compared with unexposed populations from the same waterway, exposed white sucker showed obvious effects of chemical exposure as far as 100 km downstream of the mill, as revealed by a strong induction of ethoxyresorufin O-deethylase (EROD) activity. In males, circulating plasma testosterone levels were the same at all sites, while 11-ketotestosterone levels were significantly lower at exposed stations. In females, testosterone and 17beta-estradiol levels were significantly reduced at the two exposed stations relative to the reference station. Despite different hormone levels during sexual maturation, gonad weight as a proportion of carcass weight was similar at all sites for both sexes during early gonadal development. The relationship between carcass weight and fecundity was more variable at the station immediately downstream of the mill. Biochemical and physiological parameters measured in this study do not allow us to clearly relate perturbations in plasma steroid levels to impaired reproduction as measured by gonad weight and fecundity.
Successful human reproduction is a complex process which requires normal function of 2 individuals. Reproductive toxicants can impair reproduction by acting in the male, female or both. Reproductive toxicants can produce their adverse effects by several direct and indirect mechanisms. The mechanisms by which reproductive toxicants impair reproduction are reviewed.
In this study the acute toxicity of two carbamate pesticides, Carbaryl and Aldicarb, to the Guppy and the Zebrafish has been separately assessed with 96-h toxicity tests, performed using a semistatic procedure according to EEC guidelines. There was no significant difference in the toxicity of Aldicarb and Carbaryl to the Zebrafish, but Aldicarb was ∼ 4 times more toxic than Carbaryl to the Guppy. Moreover, the toxicity of both carbamates was higher to the Guppy than to the Zebrafish. Values for the 96-h LC50 obtained with Aldicarb were 52.9 μmol/l for the Zebrafish and 3.5 μmol/l for the Guppy. LC50 values obtained with Carbaryl were 46.0 μmol/l for the Zebrafish and 12.5 μmol/l for the Guppy. Analysis of test substance concentrations in the test system demonstrated, for each substance, a loss during each 24-h exposure period. In spite of the variability observed, the losses of Carbaryl appeared independent of time, whereas the losses of Aldicarb were higher during the initial exposure days. In addition, there was an indication that the rate of loss observed with both substances was greater in the presence of the Guppy than in the presence of the Zebrafish.
Female zebrafish (Brachydanio rerio) were exposed with their food to various doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1, 5, 10 and 20 ng/fish) to investigate effects on oogenesis and reproduction. Doses of ≥5 ng/fish led to dose-related reduction of egg numbers and to lethal anomalies of their offspring (edema and malformations of the notocord). Histology of the ovaries revealed severely impaired development of previtellogenic to vitellogenic oocytes. At doses of 1 ng TCDD/fish no significant toxic effects were observed. The model appears to be useful to study mechanisms of TCDD-induced reproduction toxicity in fish.
The responses of liver microsomal monooxygenases in three teleost species, croaker (Micropogon undulatus), rainbow trout (Salmo gairdneri) and scup (Stenotomus chrysops) to polynuclear aromatic hydrocarbon (PAH) induccrs were compared. In all three species PAH induced higher raies of ethoxyresorufin and/or benzo[a]pyrene (B[a]P) metabolism and levels of cytochrome P-450, but the species differed in some other responses. The induction of P-450 appears to be associated with the synthesis of new (or additional) microsomal protein at 57000 daltons in trout and 54000 daltons in scup, identifiable by SDS-polyacrylamide gel electrophoresis. Further da.a suggest that ethoxyresorufin metabolism in scup may be catalyzed by a minor form of P-450 distinct from the major induced protein. All scur examined thus far from natural populations exhibit properties of hepatic microsomes characteristically associated with the induced state. B[a]P is metabolized by control and untreated scup with a high regiospecificity normally associated with the production of reactive and mutagenic metabolites. which may have implications for animals in the field.
Field studies on ovarian development in English sole from urban and nonurban sites in Puget Sound, Washington, USA, demonstrated that animals with elevated levels of fluorescent aromatic compounds (FACs) in bile were less likely to enter vitellogenesis and had lower plasma concentrations of estradiol than female sole with low levels of contaminant exposure. Biliary FACs were positively correlated with hepatic P450 activity, which was also elevated in sole showing inhibited ovarian development. These findings suggest that contaminant exposure may disrupt vitellogenesis in female fish. Results of the field study were supported by laboratory experiments showing that pretreatment of gravid female English sole with extracts of contaminated sediment decreased levels of endogenous estradiol. A similar response was observed in rock sole and flathead sole treated with Prudhoe Bay crude oil. However, exposure of English sole to sediment extracts had little effect on the activity of hepatic-steroid-metabolizing enzymes, indicating that some mechanism other than enhanced steroid metabolism may be responsible for reductions in endogenous circulating-steroid levels. Our recent studies suggest that reductions in endogenous estradiol levels may result from depressed ovarian steroidogenesis, as contaminant-associated reductions in in-vitro ovarian estradiol production were observed in English sole, rock sole, and flathead sole. Preliminary studies on winter flounder and white croaker indicate that they may also experience reproductive impairment as a result of exposure to contaminants, but the phase of the reproductive cycle that is most severely impacted varies from species to species.
With the modernization of agricultural operations and the rapid growth of industrial activity, there has been much increase in the manufacture and utilization of insecticides, pesticides and herbicides which ultimately find their way into the rivers, lakes and ponds. Pesticides have been round to be highly toxic not only to fishes (TOOR et al., 1973; TOOR and Kaur, 1974; KAMALDEEP and TOOR, 1977), but also to the organisms which contribute to the food of fishes (ANDERSON, 1960; LOOSANOFF, 1960; DAVOES, 1961; BUTLER, 1969; BUTLER et al., 1970). In earlier studies in this laboratory, the effect of several pesticides has been seen on the survival of developing eggs and hatchlings, percentage hatchability, frequency of morphological and physiological deformities at different en[bryonic stages, and survival and behavioral re~~™ of the fry/fingerlings of Cyprinus carpio communis Linn. (TOOR and KAUR, 1974; KAMALDEEP and TOOR, 1977). These studies have indicated that fenitrothion is one of the very toxic pesticides being used at present. The present study deals with the effect of fenitrothion on the reproduction of Cyprinus carpio communis Linn., especially with regard toits effect on the activity of enzymes indicative of steroidogenesis in the gonads. The results regarding the histopathological effects of this pesticide on the gonads, liver, digestive tract and skin shall be presented in a separate paper.
Female three-spined sticklebacks were fed freeze-dried chironomids contaminated with polybrominated diphenyl ethers (PBDE) (Bromkal 70-5DE), polychlorinated naphthalenes (PCN) (Halowax 1014) and polychlorinated biphenyls (PCB) (Clophen A50) in low or high doses in the following combinations: Halowax 1014 + Clophen A50, Bromkal 70-5DE + Clophen A50, Halowax 1014 + Bromkal 70-5DE, and Halowax 1014 + Clophen A50 + Bromkal 70-5DE. The control group was fed uncontaminated freeze-dried chironomids. After 2.5 months of exposure, the fish were dissected for chemical, biochemical and morphological analyses. Hepatic cytochrome P450-dependent ethoxyresorufin-O-deethylase (EROD) activity was induced (3-to 7-fold) in all high-dose groups, but not in the low-dose groups. There were no significant differences between the control and exposed groups in liver somatic index (LSI), gonadosomatic index (GSI), progesterone 17α-hydroxylase activity or the mean proportion of atretic oocytes per total number of oocytes. The mean gonadal progesterone 6β-hydroxylase activity in the control group was 0.3 ± 0.2 pmol mg−1 protein min−1. All groups except the one that received a high dose of Halowax 1014 + Clophen A50 + Bromkal 70-5DE had significantly higher gonadal progesterone 6β-hydroxylase activities compared with the control group. The mean activities in the significantly induced groups ranged from 9.4 ± 3.3 to 20.6 ± 5.0 pmol mg−1 protein min−1. Rainbow trout yolk-sac fry were injected with the same substance blends as the sticklebacks, except that Halowax 1014 + Clophen A50 + Bromkal 70-5DE was not included in the study. Furthermore, each substance alone was injected into the fry. The substances were dissolved in dimethyl sulfoxide (DMSO), and three concentrations were prepared. Halowax 1014. Clophen A50, and Halowax 1014 + Bromkal 70-5DE significantly induced the EROD system. In conclusion, the study showed that Halowax 1014 and Clophen A50 were potent inducers of the cytochrome P450 enzyme system in fish, whereas Bromkal 70-5DE was not. Furthermore, the differences in progesterone hydroxylase activities recorded between the control and exposed groups point to the importance of future thorough studies of organohalogen influence on the hormonal cycle.
The LC50 (96 h) values and the bioconcentration factors (BCF) for nine anilines (aniline; 2-, 3-, 4-chloroaniline; 2-, 3-, 4-nitroaniline; 2,4- and 3,4-dichloroaniline) in the zebrafish (Brachydanio rerio) were determined. Biotransformation products of anilines in the zebrafish were analyzed by HPLC. The aim of the investigations was to find relationships between accumulation/elimination/metabolism and toxicity on the one hand and between chemical structure and biotransformation on the other. We found a good correlation of log BCF and of log Pow with log LC50. This concurs with the assumption that the internal dose determines the toxicological effect. All anilines investigated, with the exception of 2- and 4-nitroaniline, were transformed into the corresponding acetanilides. The toxicity of the compounds was not altered by biotransformation. A comparison of the extent of acetylation confirms the supposition that substituents in the ortho position sterically hinder acetylation.
This study describes a long-term test over three generations, using zebrafish (Brachydanio rerio) as the test species and concentrations of 1, 0.2, and 0.04 mg/L 4-chloroaniline (CA) as a model substance. The effect of the compound on the ecologically important parameters reproduction and growth was the focus of interest. Reduction in egg release by fish raised under CA was the most sensitive parameter in the test. Compared to the toxic threshold concentration for growth (0.4 mg/L), egg release was affected by a ten-fold lower concentration (0.04 mg/L). This study demonstrates that a long-term test is still the most appropriate method to assess the chronic toxicity of a substance on fish. A chronic toxicity test is proposed which comprises two generations, with the zebrafish as test species.
Juvenile lake trout (Salmo trutta m. lacustris) were exposed for 7 weeks to 0.05X and 0.2X 96-hr LC50 concentrations of simulated bleached kraft pulp mill effluent (KME - Sa + CP). A sulfate soap preparation, composed mainly of resin and fatty acids, with added chlorophenols (CP, tri-, tetra-, and penta-CP) was used as the toxicant mixture. Concentrations of free CP in plasma and free and conjugated CP in bile were proportional to their concentrations in the water. The greatest total gradient between bile and water CP was 5.2 X 10(4) for pentachlorophenol. The activity of a liver polysubstrate monooxygenase (PSMO) system, assayed with three model substrates, increased 40 to 67% due to KME - Sa + CP. However, the increase was not directly dependent on the exposure concentration. In contrast to PSMO, activities of conjugating enzymes (p-nitrophenol UDP-glucuronosyl and glutathione transferases) were decreased in the liver. Increased concentration of glutathione was noted in the liver and kidney. In addition, a small (9%) but significant decrease in blood hemoglobin concentration was observed at the higher exposure concentration. Although growth rate of lake trout was markedly decreased due to KME - Sa + CP, hydromineral balance and carbohydrate metabolism in fish were unaffected, indicating possible physiological compensation. On the other hand, lethality tests with lake trout preexposed to KME - Sa + CP at 0.2 X LC50 revealed decreased tolerance, whereas at the lower exposure concentration it was unchanged. We therefore conclude that various physiological adjustments in trout during subchronic exposures were not adaptive in terms of short-term tolerance.
Conclusions Our results suggest that chronic exposure to Aroclor 1254 increased susceptibility of test pinfish and spot to disease, and also appeared to be toxic to these fish. This PCB is rapidly stored by pinfish and spot, and persists in tissues for approximately three months. Our findings emphasize the need for further study on the effect of chronic exposure of aquatic organisms to polychlorinated biphenyls.
Influence of estradiol and testosterone on cytochrome P-450 and monooxygenase activity in Toxicity of Binary Chemical Mixtures on Zebrafish immature brook trout (Salvelinus fontinalis)
- Jj
- Pajor
- Am
- Thomas
JJ, Pajor AM, Thomas P (1982) Influence of estradiol and testosterone on cytochrome P-450 and monooxygenase activity in Toxicity of Binary Chemical Mixtures on Zebrafish immature brook trout (Salvelinus fontinalis). Biochem Pharmacol 31:3979–3989
Untersuchungen u ¨ber die Ausscheidung polychlori-erter Biphenyle in das Hü und ihre Verteilung zwischen Eiweiß und Dotter
- R Nagel
- Seitz
E (1974) Untersuchungen u ¨ber die Ausscheidung polychlori-erter Biphenyle in das Hü und ihre Verteilung zwischen Eiweiß und Dotter. Dissertation thesis, University of Mü Schä C, Nagel R, Seitz A (1989) Verhalten, Reproduktion und Populationsdynamik des Zebrabä (Brachydanio rerio) in einem naturnahen Laborsystem. Fischö 1(2):45–59
Statistical analysis Fundamentals of Aquatic Toxicology Chronic toxicity, uptake and retention of aroclor 1254 in two estuarine fishes
- Rd Lavin Pt
- Metha Cr
- Schoenfeld Da Hansen Dj
- Pr Parrish
- Ji Lowe
- Wilson
- Aj
- Wilson
- Pd
RD, Lavin PT, Metha CR, Schoenfeld DA (1985) Statistical analysis. In: Rand GM Petrocelli SR (eds) Fundamentals of Aquatic Toxicology. Hemisphere Publishing Corp, NY, pp 110– 123 Hansen DJ, Parrish PR, Lowe JI, Wilson AJ, Wilson PD (1971) Chronic toxicity, uptake and retention of aroclor 1254 in two estuarine fishes. Bull Environ Cont Toxicol 6(2):113–119
Chronic toxicity of lindane to selected aquatic invertebrates and fishes On a test of whether one or two random variables is stochastically larger than the other
- Kj
- Ks Buxton
- Derr
- Sk
- Jw Dean
- Sauter
KJ, Buxton KS, Derr SK, Dean JW, Sauter S (1976) Chronic toxicity of lindane to selected aquatic invertebrates and fishes. USEPA, Duluth, MN, EPA 600/3-76-046, 49 pp Mann HB, Whitney DR (1947) On a test of whether one or two random variables is stochastically larger than the other. Ann Math Statist 18:50
Longterm effects of zinc exposure on brook trout (Salvelinus fontinalis)
- Gw
- Benoit
- Da
- Leonard
- En
GW, Benoit DA, Leonard EN (1979) Longterm effects of zinc exposure on brook trout (Salvelinus fontinalis). Trans Am Fish Soc 108:76–87
Methods to test and assess effects of chemicals on reproduction in fish Methods for assessing the effects of chemicals on reproductive functions
- Em
- Scherer
EM, Scherer E (1983) Methods to test and assess effects of chemicals on reproduction in fish. In: Vouk VB, Sheehan PJ (eds) Methods for assessing the effects of chemicals on reproductive functions. Wiley and Sons, Chichester, pp 365–405
The effects of polychlorinated biphenyls on plasma steroid levels and hepatic microsomal enzymes in fish The ABC's of pollutant assay using fish
- K Franklin
- Cs
- Williams
- Wp
K, Franklin CS, Williams WP (1978) The effects of polychlorinated biphenyls on plasma steroid levels and hepatic microsomal enzymes in fish. J Fish Biol 13:401–409 Sprague JB (1973) The ABC's of pollutant assay using fish. In: Biol. methods for the assessment of water quality. ASTM special technical publication No. 528, pp. 6–30