In the drug discovery pipeline, safety pharmacology is a major issue. The zebrafish has been proposed as a model that can bridge the gap in this field between cell assays (which are cost-effective, but low in data content) and rodent assays (which are high in data content, but less cost-efficient). However, zebrafish assays are only likely to be useful if they can be shown to have high predictive power. We examined this issue by assaying 60 water-soluble compounds representing a range of chemical classes and toxicological mechanisms.
Over 20,000 wild-type zebrafish embryos (including controls) were cultured individually in defined buffer in 96-well plates. Embryos were exposed for a 96 hour period starting at 24 hours post fertilization. A logarithmic concentration series was used for range-finding, followed by a narrower geometric series for LC50 determination. Zebrafish embryo LC50 (log mmol/L), and published data on rodent LD50 (log mmol/kg), were found to be strongly correlated (using Kendall's rank correlation tau and Pearson's product-moment correlation). The slope of the regression line for the full set of compounds was 0.73403. However, we found that the slope was strongly influenced by compound class. Thus, while most compounds had a similar toxicity level in both species, some compounds were markedly more toxic in zebrafish than in rodents, or vice versa.
For the substances examined here, in aggregate, the zebrafish embryo model has good predictivity for toxicity in rodents. However, the correlation between zebrafish and rodent toxicity varies considerably between individual compounds and compound class. We discuss the strengths and limitations of the zebrafish model in light of these findings.
Citation: Ali S, Mil HGJv, Richardson MK (2011) Large-Scale Assessment of the Zebrafish Embryo as a Possible Predictive Model in Toxicity Testing. PLoS ONE 6(6): e21076. https://doi.org/10.1371/journal.pone.0021076
Editor: Ferenc Mueller, University of Birmingham, United Kingdom
Received: March 22, 2011; Accepted: May 18, 2011; Published: June 28, 2011
Copyright: © 2011 Ali et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Smart Mix Programme of the Netherlands Ministry of Economic Affairs and the Netherlands Ministry of Education, Culture and Science (M.K.R.) under grant number SSM06010, the University of Azad Jammu and Kashmir, Pakistan (S.A.) under project No. F-3/PD/Main & Mirpur/369/2007. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
There is an unmet need for low-cost, high-throughput animal models in some fields of biomedical research such as drug screening and toxicity assessment , . The zebrafish embryo is emerging as one such model . It has been proposed as a bridge between simple assays based on cell culture, and biological validation in whole animals such as rodents . The zebrafish cannot replace rodent models but is complementary to them, being particularly useful for rapid, high-throughput, low-cost assays, as for example in the early (pre-regulatory) stages of the drug development pipeline .
Among the attractive features of the zebrafish embryo model are its small size, small volume of compound consumed and rapid development. The organogenesis of major organs is completed at 5 days post fertilization (dpf) . Also, many fundamental cellular and molecular pathways involved in the response to chemicals or stress are conserved between the zebrafish and mammals . Genomic sequencing has shown extensive homology between zebrafish and other vertebrate species (including humans), and some aspects of brain patterning, structure and function are also conserved –. We have shown for example that the glucocorticoid receptor of the zebrafish is functionally closer to that of the human than is its mouse cognate . The availability of genomic tools in the zebrafish provides an advantage over other teleosts such as the fathead minnow (Pimephales promelas) used, for example, in environmental toxicity assessment in the United States . Indeed, zebrafish embryos may be a suitable replacement for some of these adult fish toxicity tests .
The zebrafish is increasing being used in toxicological studies [reviewed by: 13], . Example include the use of adult zebrafish for the testing of lead and uranium , malathion , colchicine , anilines , and metronidazole ; and the use of juveniles for testing agricultural biocides . Zebrafish embryos are also being used in toxicity studies [reviewed by: 21]. Examples include the use of zebrafish embryos for testing nanoparticles , .
Although the body plans of zebrafish are in many aspects similar to those of mammals, there are important differences. The fish is ectothermic, and lacks cardiac septa, synovial joints, cancellous bone, limbs, lungs and other structures –. Therefore, some toxic effects seen in humans are difficult to model in the zebrafish. Furthermore, the zebrafish embryo remains inside the chorion at least up to 48 hpf . In pre-hatching embryos, therefore, the chorion (a membrane perforated by channels of 0.5–0.7 µm in diameter), may provide a barrier to diffusion of compounds –.
The evolutionary divergence of zebrafish and mammals is around 445 million years ago  and so it is by no means certain that we will necessarily share the same sensitivity to toxic substances. Therefore, there is a need for validation of the model using compounds that have a known effect in other species . One study has reported, using 18 toxic compounds, that toxicity in zebrafish was well-correlated with values reported from rodent studies . The zebrafish embryo system has also been compared, as a toxicology screen, with the aquatic crustacean Daphnia magna . Such studies are an important step towards the kind of comparative toxicity database represented by the well-known ‘Registry of Cytotoxicity’ which examines the predictive power of cell assays .
Our aim here is to determine the toxicity of 60 compounds from diverse pharmacological and chemical classes, and examine the strength of correlation between zebrafish embryo LC50 and data from the literature on rodent LD50. Compounds are added to the water in which the embryos develop, and so we focus here on water soluble compounds to avoid any confounding effects of carrier solvents.
Materials and Methods
All animal experimental procedures were conducted in accordance with local and international regulations. The local regulation is the Wet op de dierproeven (Article 9) of Dutch Law (National) and the same law administered by the Bureau of Animal Experiment Licensing, Leiden University (Local). This local regulation serves as the implementation of Guidelines on the protection of experimental animals by the Council of Europe, Directive 86/609/EEC, which allows zebrafish embryos to be used up to the moment of free-living (approximately 5–7 days after fertilisation). Because embryos used here were no more than 5 days old, no licence is required by Council of Europe (1986), Directive 86/609/EEC or the Leiden University ethics committee.
Male and female adult zebrafish (Danio rerio) of AB wild type were purchased from Selecta Aquarium Speciaalzaak (Leiden, the Netherlands) who obtain stock from Europet Bernina International BV (Gemert-Bakel, the Netherlands). Fish were kept at a maximum density of 100 individuals in glass recirculation aquaria (L 80 cm; H 50 cm, W 46 cm) on a 14 h light: 10 h dark cycle (lights on at 08.00). Water and air were temperature controlled (25±0.5°C and 23°C, respectively). The fish were fed twice daily with ‘Sprirulina’ brand flake food (O.S.L. Marine Lab., Inc., Burlingame, USA) and twice a week with frozen food (Dutch Select Food, Aquadistri BV, the Netherlands).
Defined embryo buffer
To produce a defined and standardized vehicle for these experiments, we used 10% Hank's balanced salt solution (made from cell-culture tested, powdered Hank's salts, without sodium bicarbonate, Cat. No H6136-10X1L, Sigma-Aldrich, St Louis, MO) at a concentration 0.98 g/L in Milli-Q water (resistivity = 18.2 MΩ·cm), with the addition of sodium bicarbonate at 0.035 g/L (Cell culture tested, Sigma Cat S5761), and adjusted to pH 7.46. A similar medium has been used previously –.
Egg water was made from 0.21 g ‘Instant Ocean®’ salt in 1 L of Milli-Q water with resistivity of 18.2 MΩ·cm.
Eggs were obtained by random pairwise mating of zebrafish. Three adult males and four females were placed together in small breeding tanks (Ehret GmbH, Emmendingen, Germany) the evening before eggs were required. The breeding tanks (L 26 cm; H 12.5 cm, W 20 cm) had mesh egg traps to prevent the eggs from being eaten. The eggs were harvested the following morning and transferred into 92 mm plastic Petri dishes (50 eggs per dish) containing 40 ml fresh embryo buffer. Eggs were washed four times to remove debris. Further, unfertilized, unhealthy and dead embryos were identified under a dissecting microscope and removed by selective aspiration with a pipette. At 3.5 hpf, embryos were again screened and any further dead and unhealthy embryos were removed. Throughout all procedures, the embryos and the solutions were kept at 28±0.5°C, either in the incubator or a climatised room. All incubations of embryos were carried out under a light cycle of 14 h light: 10 h dark (lights on at 08.00). All pipetting was done manually, with an 8-channel pipetter.
Viability of early embryos
There are reports of an early “mortality wave” in zebrafish embryos cultured under certain conditions [for examples], [ see: 40,41]. In order to assess this mortality wave in our facilities, and to avoid taking embryos during such a die-off, we raised cleaned embryos in 92 mm Petri dish (60 eggs per dish) containing 40 ml Hank's buffer alone, or egg water alone. We scored the fertilisation rate and mortality of embryos at 4, 8, and 24 hpf (see below) in these two media.
Evaporation of buffer from 96-well plate
Evaporation rate of buffer from the 96-well plates (Costar 3599, Corning Inc., NY) was determined as follows. In each well of the plate, 250 µL of freshly prepared buffer was dispensed at 0 h. As for all 96-well plate experiments reported in this study, the lids were in place but were not sealed with a sealing mat or film (because preliminary studies indicated that all embryos die within sealed plates). The plates were kept at 28±0.5°C without refreshing the buffer (static non-replacement regime) and weighed at daily intervals on a digital balance. Results were calculated as mean from four different plates. Buffer volume from some individual wells in different regions of the plate were also weighed at 4 days to determine the impact of well location on the evaporation rate.
We used water-soluble compounds representing a range of different chemical classes and biochemical activities (Table S1). The required dilution was always freshly prepared in buffer just prior to assay on zebrafish embryos.
Mortality rate (Table 1) was recorded at 48, 72, 96 and 120 hpf in both logarithmic series and geometric series using a dissecting stereomicroscope. Embryos were scored as dead if they were no longer moving, the heart was not beating and the tissues had changed from a transparent to an opaque appearance.
To determine a suitable range of concentrations for testing, we performed range-finding using a logarithmic series (0.0, 1.0, 10.0, 100.0 and 1000 mg/L) as recommended in standard protocols . Zebrafish embryos of 24 hpf from Petri dish were gently transferred using a sterile plastic pipette into 96-well microtitre plates. A single embryo was plated per well, so that dead embryos would not affect others, and also to allow individual embryos to be tracked for the whole duration of the experiment. A static non-replacement regime was used. Thus there was no replacement or refreshment of buffer after the addition of compound. Each well contained 250 µL of either freshly prepared test compound; or vehicle (buffer) only as controls. We used 16 embryos for each concentration and 16 embryos as controls for each drug.
Geometric series and LC50 determination
After the range finding experiments, a series of concentrations lying in the range between 0% and 100% mortality were selected. The actual concentrations used are shown in Table S2. The concentrations were in a geometric series in which each was 50% greater than the next lowest value . Each geometric series of concentrations for each compound was repeated three times (in total 48 embryos per concentration and 48 embryos for vehicle for each drug). LC50 (expressed in mg/L of buffer) was determined based on cumulative mortality obtained from three independent experiments at 120 hpf using Regression Probit analysis with SPSS Statistics for windows version 17.0 (SPSS Inc., Chicago, USA). Thus the embryos are exposed to the drug for 96 h. The LC50 in mg/L was converted into LC50 mmol/L to make relative toxicity easier to examine.
The sources of LD50 data from rodents (rats and mice) are shown in Table 2.
Statistical analyses were performed using GraphPad Prism for Windows (version 5.03) or R (v. 2.12). One way ANOVA and Newman-Keuls Multiple Comparison test was employed for survival rate. Correlation and ANCOVA models were used to investigate the relationship between LC50 in zebrafish embryos and published LD50 values in rodents.
Results and Discussion
We have examined the toxicity, in zebrafish embryos, of a 96 h exposure (during the period 24 hpf to 5 dpf) to 60 compounds of differing biochemical classes. Our logarithmic and geometric concentration series both showed concentration-dependent mortality. LC50 values were determined, and compared with rodent LD50 values from the literature.
Infertility and spontaneous early mortality of eggs/embryos
We found that, in controls (buffer only), 5% of eggs were unfertilised, and a further 9% represented embryos that died spontaneously in the first 24 hpf. This is similar to the spontaneous mortality of 5–25% reported elsewhere for early zebrafish development . We find no significant difference between these values when Hank's buffer was used as the medium, and when egg water was used (Figure 1A, B). In order to avoid this natural early mortality we began our assays at 24 hpf. This also makes our study consistent with a previous one, in which the zebrafish was exposed to different compounds at 24 hpf to find the correlation between zebrafish and rodent toxicities .
Embryos were kept in 92 mm Petri dishes with 40 ml of either buffer or egg water, 60 eggs per dish. Each error bar represents ±SEM of N = 420 embryos each for buffer and egg water. A, cumulative infertility and early mortality in buffer. B, the same, in egg water. There is no significant difference between the two media in terms of survival and fertilization percentage.
It could be argued that, by beginning exposure at 24 h, we are missing out on early developmental toxicity effects, such as the action of compounds on gastrula stages. However, this is likely to be a trade-off because other compounds mainly cause embryo death at these early stages. For example, a recent study  showed that exposure of zebrafish embryos at early stages (dome to 26-somite) to ethanol resulted in high mortality, while exposure at later stages (prim-6 and prim-16) led to a high incidence of abnormal embryos. Other examples of compounds which are more toxic to larval stages than to embryonic and adult stages of freshwater fish species are copper and cadmium –. Finally, it is known that presence of chorion at early stages acts as a possible barrier to diffusion of compounds , , .
Rate of evaporation from 96-well plates at 28.0°C
In our study, we did not replace the buffer. Therefore, we decided to check how much water would be lost during this period by evaporation from the 96-well plate (with its lid in place). We found that, by 96 h of incubation at 28.0°C, 9.46% of the buffer had evaporated (Figure 2A). Further investigation showed that the rate of evaporation was higher in the external rows and columns, and highest of all in the four corner wells (Figure 2B). In view of this evaporation pattern, we filled all the 96-wells with buffer, but did not plate embryos into wells A1-H1 and A12-H12. A way of mitigating the effects of this rate of evaporation would be to use dynamic replacement of buffer, as in a microfluidic chip , or static replacement (e.g. daily refreshing). Nonetheless, static non-replacement, as used here, is a popular technique for zebrafish embryo culture, and was used in a recent toxicity study .
Buffer was dispensed in four different 96-well plates. A, cumulative average percentage buffer loss per plate. All wells were initially filled with 250 µL buffer. B, percentage buffer loss after 96 h, per well, as a function of well position. The letters A–H and the numbers 1–12 correspond to the standard coordinates embossed into 96-well plates. All wells were initially filled with 250 µL buffer. Only the wells with grey columns were measured.
Concentration response and LC50 of compounds
For all compounds, mortality at 5 dpf was concentration-dependent (Table 1). This was true for both logarithmic and geometric series. By contrast, controls (vehicle only) showed 0% mortality. The LC50 values are shown in Table 2.
Correlation between zebrafish embryo log LC50 and rodent log LD50
To examine the ability of zebrafish assays to predict toxicity in rodents, we analysed a correlation between our zebrafish embryo log LC50 values, and rodent log LD50 from the literature. The comparison is shown graphically in Figure 3. A correlation test produced Spearman's rank correlation of 0.7688 (p<0.001) and a Pearson's product-moment correlation 0.7832 (df = 178, p<0.001) between zebrafish embryo LD50 and rodent log LD50 for the whole set of compounds. These values of correlation indicate that zebrafish LC50 and rodent LD50 values co-vary. This is consistent with a previous report  that the toxicity of 18 compounds in zebrafish embryos was well-correlated with values reported from rodent studies. It is also in line with another study  suggesting that zebrafish embryos could be used as a predictive model for the developmental toxicity of compounds.
Zebrafish embryo LC50 was determined based on cumulative mortality after 96 h exposure of compounds from three independent experiments and rodent LD50 was taken from the literature. Key: blue, regression line; solid black lines, 0.25 and 0.75 quartiles; dashed line, perfect correlation line. The slope of the regression line (blue) is 0.73403.
Toxicity by compound class
We next developed a statistical model that examines the similarity between zebrafish and rodent toxicity values when the compounds are clustered into chemically similar groups. To do this, we mapped zebrafish values to rodent values, taking account of specific variances in intercept and slope, due to those groupings. The groupings were alcohols, alkaloids, amides, carboxylic acids, glycosides and the remaining compounds (others). We designed an ANCOVA with the values [zebrafish embryo log LC50] as dependent variables, and [rodent log LD50] and [compound type] as independent variables. Table 3 shows the statistics of our ANCOVA model, while the dataset is displayed graphically in Figure 4. As can be seen, there is a significant effect of compound type on intercept and slope.
The effect of the different compounds on the slope and intercept of the ANCOVA model. Although we must consider the effect of the unknown error in the rodent LD50 values, the different compound classes seem to cluster in different regions in the graph.
The slope for amides (Table 3) does not differ significantly from 1.0, indicating a very similar toxicity in zebrafish and rodents. By contrast, ‘others’ and alcohols have a slope significantly greater than 1.0, indicating that they are generally less toxic in zebrafish than in rodents. The groups carboxylic acids, glycosides and alkaloids have a slope significantly less than 1.0 indicating that they are more toxic in zebrafish than in rodents (Table 3).
If we look at the relative toxicity ([zebrafish LC50 mmol/L] ÷ [rodent LD50 mmol/kg]) of individual compounds we see the following examples of compounds that have a similar toxicity in the two sepcies: coumarin (0.95), benserazide hydrochloride (1.06), phenformin hydrochloride (1.11) and theobromine (1.11). Examples of compounds less toxic in zebrafish than in rodents are aconitine (0.01), ouabain octohydrate (0.02), tubocurarine hydrochloride (0.07), morphine hydrochloride (0.08) and colchicine (0.13). At the other extreme are compounds more toxic in zebrafish than in rodents including: Tween80 (103.01), sodium dodecyl sulfate (98.33), lead acetate trihydrate (29.49) and copper (II) nitrate trihydrate (19.40).
Among the alcohols, the general trend is a lower toxicity in zebrafish than in rodents. Tween 80 is an exception to this trend because it is much more toxic to zebrafish. This could be because of its surfactant properties, a suggestion supported by the comparably high relative toxicity to zebrafish (98.33) that we find for another surfactant tested, sodium dodecyl sulphate (SDS). Our LC50 for SDS in 96 h exposure to zebrafish embryos was 3.6 mg/L. This is similar to the dose of SDS that causes pathological changes in the gills of the teleost Thalassoma pavo . Copper also appears to interfere with ion transport in the gills [reviewed in: 48] as does lead . The lower relative toxicity of colchicine to zebrafish has been previously reported . The suggestion is that teleosts may have some protection by virtue of being unable to oxidise colchicine to the much more toxic oxycolchicine .
It is also possible that experimental methodology underlies some of the species differences found here. The standard error for the rodent LD50 values were not available in Toxnet or the Registry of Cytotoxicity. This is significant because error in the independent variable can have a significant effect on both slope and intercept. Other study-dependent influences on the data could include differences in exposure time, developmental stage, route of exposure between the zebrafish and rodent studies.
Our findings show that the zebrafish embryo is a tool that offers potential in the evaluation of drug safety. However, we show that the predictivity varies between the class of compound studied. More work is required to examine how the covariance of zebrafish and rodent toxicity is influenced by such factors as compound type, absorption, metabolism and mechanism of toxicity.
Summary of compounds used in this study for toxicity evaluation in zebrafish embryo.
We thank Peter J. Steenbergen for expert technical assistance and zebrafish breeding.
Conceived and designed the experiments: SA MKR HGJvM. Performed the experiments: SA. Analyzed the data: SA MKR HGJvM. Contributed reagents/materials/analysis tools: MKR. Wrote the paper: SA MKR.
- 1. Lieschke GJ, Currie PD (2007) Animal models of human disease: zebrafish swim into view. Nat Rev Genet 8: 353–367.
- 2. Bull J, Levin B (2000) Perspectives: microbiology. Mice are not furry petri dishes. Science 287: 1409–1410.
- 3. Redfern WS, Waldron G, Winter MJ, Butler P, Holbrook M, et al. (2008) Zebrafish assays as early safety pharmacology screens: paradigm shift or red herring? J Pharmacol Toxicol Methods 58: 110–117.
- 4. Rubinstein AL (2003) Zebrafish: from disease modeling to drug discovery. Curr Opin Drug Discov Devel 6: 218–223.
- 5. Voelker D, Vess C, Tillmann M, Nagel R, Otto GW, et al. (2007) Differential gene expression as a toxicant-sensitive endpoint in zebrafish embryos and larvae. Aquat Toxicol 81: 355–364.
- 6. Guo S (2004) Linking genes to brain, behavior and neurological diseases: what can we learn from zebrafish? Genes Brain Behav 3: 63–74.
- 7. Tropepe V, Sive HL (2003) Can zebrafish be used as a model to study the neurodevelopmental causes of autism? Genes Brain Behav 2: 268–281.
- 8. Veldman MB, Lin S (2008) Zebrafish as a developmental model organism for pediatric research. Pediatr Res 64: 470–476.
- 9. Postlethwait JH, Woods IG, Ngo-Hazelett P, Yan YL, Kelly PD, et al. (2000) Zebrafish comparative genomics and the origins of vertebrate chromosomes. Genome Research 10: 1890–1902.
- 10. Schaaf MJ, Champagne D, van Laanen IH, van Wijk DC, Meijer AH, et al. (2008) Discovery of a functional glucocorticoid receptor beta-isoform in zebrafish. Endocrinology 149: 1591–1599.
- 11. United States Environmental Protection Agency (1996) pp. 1–11. Ecological Effects Test Guidelines: OPPTS 850.1075: Fish Acute Toxicity Test, Freshwater and Marine.
- 12. Lammer E, Carr GJ, Wendler K, Rawlings JM, Belanger SE, et al. (2009) Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Physiol C Toxicol Pharmacol 149: 196–209.
- 13. Hill AJ, Teraoka H, Heideman W, Peterson RE (2005) Zebrafish as a model vertebrate for investigating chemical toxicity. Toxicol Sci 86: 6–19.
- 14. Teraoka H, Dong W, Hiraga T (2003) Zebrafish as a novel experimental model for developmental toxicology. Congenit Anom (Kyoto) 43: 123–132.
- 15. Labrot F, Narbonne JF, Ville P, Saint DM, Ribera D (1999) Acute toxicity, toxicokinetics, and tissue target of lead and uranium in the clam Corbicula fluminea and the worm Eisenia fetida: comparison with the fish Brachydanio rerio. Arch Environ Contam Toxicol 36: 167–178.
- 16. Kumar K, Ansari BA (1986) Malathion toxicity: effect on the liver of the fish Brachydanio rerio (Cyprinidae). Ecotoxicol Environ Saf 12: 199–205.
- 17. Roche H, Boge G, Peres G (1994) Acute and chronic toxicities of colchicine in Brachydanio rerio. Bull Environ Contam Toxicol 52: 69–73.
- 18. Zok S, Gorge G, Kalsch W, Nagel R (1991) Bioconcentration, metabolism and toxicity of substituted anilines in the zebrafish (Brachydanio rerio). Sci Total Environ 109–110: 411–421.
- 19. Lanzky PF, Halling-Sorensen B (1997) The toxic effect of the antibiotic metronidazole on aquatic organisms. Chemosphere 35: 2553–2561.
- 20. Gorge G, Nagel R (1990) Toxicity of lindane, atrazine, and deltamethrin to early life stages of zebrafish (Brachydanio rerio). Ecotoxicol Environ Saf 20: 246–255.
- 21. Truong L, Harper SL, Tanguay RL (2011) Evaluation of embryotoxicity using the zebrafish model. Methods Mol Biol 691: 271–279.
- 22. George S, Xia T, Rallo R, Zhao Y, Ji Z, et al. (2011) Use of a high-throughput screening approach coupled with in vivo tebrafish embryo screening to develop hazard ranking for engineered nanomaterials. ACS Nano 5: 1805–1817.
- 23. Bai W, Tian W, Zhang Z, He X, Ma Y, et al. (2010) Effects of copper nanoparticles on the development of zebrafish embryos. J Nanosci Nanotechnol 10: 8670–8676.
- 24. Dahm R, Geisler R (2006) Learning from small fry: the zebrafish as a genetic model organism for aquaculture fish species. Mar Biotechnol (NY) 8: 329–345.
- 25. Grunwald DJ, Eisen JS (2002) Headwaters of the zebrafish — emergence of a new model vertebrate. Nat Rev Genet 3: 717–724.
- 26. Hu N, Yost HJ, Clark EB (2001) Cardiac morphology and blood pressure in the adult zebrafish. Anat Rec 264: 1–12.
- 27. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203: 253–310.
- 28. Mizell M, Romig ES (1997) The aquatic vertebrate embryo as a sentinel for toxins: zebrafish embryo dechorionation and perivitelline space microinjection. Int J Dev Biol 41: 411–423.
- 29. Lee KJ, Nallathamby PD, Browning LM, Osgood CJ, Xu XH (2007) In vivo imaging of transport and biocompatibility of single silver nanoparticles in early development of zebrafish embryos. ACS Nano 1: 133–143.
- 30. Henn K, Braunbeck T (2011) Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio). Comp Biochem Physiol C Toxicol Pharmacol 153: 91–98.
- 31. Braunbeck T, Boettcher M, Hollert H, Kosmehl T, Lammer E, et al. (2005) Towards an alternative for the acute fish LC(50) test in chemical assessment: the fish embryo toxicity test goes multi-species — an update. ALTEX 22: 87–102.
- 32. Peterson KJ, Lyons JB, Nowak KS, Takacs CM, Wargo MJ, et al. (2004) Estimating metazoan divergence times with a molecular clock. Proc Natl Acad Sci U S A 101: 6536–6541.
- 33. McGrath P, Li CQ (2008) Zebrafish: a predictive model for assessing drug-induced toxicity. Drug Discov Today 13: 394–401.
- 34. Parng C, Seng WL, Semino C, McGrath P (2002) Zebrafish: A preclinical model for drug screening. Assay Drug Dev Technol 1: 41–48.
- 35. Martins J, Oliva TL, Vasconcelos V (2007) Assays with Daphnia magna and Danio rerio as alert systems in aquatic toxicology. Environ Int 33: 414–425.
- 36. Halle W (2003) The Registry of Cytotoxicity: toxicity testing in cell cultures to predict acute toxicity (LD50) and to reduce testing in animals. Altern Lab Anim 31: 89–198.
- 37. Irons TD, Macphail RC, Hunter DL, Padilla S (2010) Acute neuroactive drug exposures alter locomotor activity in larval zebrafish. Neurotoxicol Teratol 32: 84–90.
- 38. Macphail RC, Brooks J, Hunter DL, Padnos B, Irons TD, et al. (2009) Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol. Neurotoxicology 30: 52–58.
- 39. Wielhouwer EM, Ali S, Al-Afandi A, Blom MT, Olde Riekerink MB, et al. (2011) Zebrafish embryo development in a microfluidic flow-through system. Lab Chip 11: 1815–1824.
- 40. Fraysse B, Mons R, Garric J (2006) Development of a zebrafish 4-day embryo-larval bioassay to assess toxicity of chemicals. Ecotoxicol Environ Saf 63: 253–267.
- 41. Organisation for Economic Cooperation and Development (1998) OECD Guideline For Testing of Chemicals. OECD 212. Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages.
- 42. Ali S, Champagne DL, Alia A, Richardson MK (2011) Large-scale analysis of acute ethanol exposure in zebrafish development: a critical time window and resilience. PLoS ONE 6(5): e20037.
- 43. McKim JM, Eaton JG, Holcombe GW (1978) Metal toxicity to embryos and larvae of eight species of freshwater fish-II: copper. Bull Environ Contam Toxicol 19: 608–616.
- 44. Shazili NA, Pascoe D (1986) Variable sensitivity of rainbow trout (Salmo gairdneri) eggs and alevins to heavy metals. Bull Environ Contam Toxicol 36: 468–474.
- 45. Scudder BC, Carter JL, Leland HV (1988) Effects of copper on development of the fathead minnow, Pimephales promelas Rafinesque. Aquatic Toxicology 12: 107–124.
- 46. Selderslaghs IW, Van Rompay AR, De CW, Witters HE (2009) Development of a screening assay to identify teratogenic and embryotoxic chemicals using the zebrafish embryo. Reprod Toxicol 28: 308–320.
- 47. Brunelli E, Talarico E, Corapi B, Perrotta I, Tripepi S (2008) Effects of a sublethal concentration of sodium lauryl sulphate on the morphology and Na+/K+ ATPase activity in the gill of the ornate wrasse (Thalassoma pavo). Ecotoxicol Environ Saf 71: 436–445.
- 48. Eyckmans M, Tudorache C, Darras VM, Blust R, De BG (2010) Hormonal and ion regulatory response in three freshwater fish species following waterborne copper exposure. Comp Biochem Physiol C Toxicol Pharmacol 152: 270–278.
- 49. Rogers JT, Richards JG, Wood CM (2003) Ionoregulatory disruption as the acute toxic mechanism for lead in the rainbow trout (Oncorhynchus mykiss). Aquat Toxicol 64: 215–234.