Environ. Sci. Technol. 2004, 38, 4763-4767
Aquatic Hypoxia Is a Teratogen and Affects Fish Embryonic Development EVA H. H. SHANG AND RUDOLF S. S. WU* Centre for Coastal Pollution and Conservation and Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, People’s Republic of China
Hypoxia occurs over large areas in aquatic systems worldwide, and there is growing concern that hypoxia may affect aquatic animals, leading to population decline and changes in community by elimination of sensitive species. For the first time, we report that sublethal levels of hypoxia can significantly increase (+77.4%) malformation in fish embryonic development. Disruption of apoptotic pattern was clearly evident at 24 h post-fertilization, which may be a major cause of malformation. Furthermore, embryonic development was delayed, and balance of sex hormones (testosterone and estradiol) was disturbed during embryonic stages, implicating that subsequent sexual development may also be affected. Overall, our results imply that hypoxia may have a teratogenic effect on fish and delay fish embryonic development, which may subsequently impair species fitness leading to natural population decline.
Introduction The severity, frequency of occurrence, and spatial scale of aquatic hypoxia have increased in the last few decades. Due to rapid human population growth and global warming, the situation is likely to worsen in coming years (1). Hypoxia has been shown to retard growth in bivalves (2), polychaetes (3), and fish such as plaice and dab (4). A recent study in our lab (5) demonstrated that hypoxia is an endocrine disruptor and significantly retarded gonad development, impaired fertilization success and reproductive output of adult carp (Cyprinus carpio), and also reduced hatching success and larval survival. It is generally accepted that embryonic and larval development are much more sensitive to environmental challenges than adult stages (6) since normal histogenesis and organogenesis depend on a series of highly intricate, programmed processes. Disturbance of any of these processes is likely to result in malformation and birth defects, which may ultimately affect the survival and fitness of adults. In vitro and in vivo studies based on mammalian systems provide evidence that hypoxia can induce apoptosis (7-11). Since apoptosis is a key process in modulating histogenesis and organogenesis (12-14), hypoxia may potentially disrupt these vital developmental processes through alteration of apoptosis. Surprisingly, effects of hypoxia on embryonic and larval development of nonmammalian species, including fish, remain completely unknown, except that delayed development was reported in mussel (Mytilus edulis) embryos exposed to 0.6-1.3 mg of O2 L-1 for 60 h (15), and modified cardiac activity and distribution of blood to various tissues * Corresponding author telephone: +852-2788-7401; fax: +8522788-7406; e-mail:
[email protected]. 10.1021/es0496423 CCC: $27.50 Published on Web 08/10/2004
2004 American Chemical Society
was found in zebrafish (Danio rerio) larvae during their development when exposed to 10 KPa (equivalent to 0.83 mg of O2 L-1) for 7 d (16). Balance of sex hormones plays a pivotal role in regulating sex differentiation and development of fish (17, 18). Our earlier study (5) showed that hypoxia can cause endocrine disruption in adult fish. However, it is not known whether the same would also occur during embryonic and larval stages and in what way this may affect development. In this study, we use zebrafish as a model species to test the hypothesis that hypoxia has a teratogenic effect on fish embryonic development. Specifically, we hypothesize that hypoxia may (a) affect the normal development of zebrafish embryos through apoptosis, leading to malformation; (b) retard embryonic development and hence growth; and (c) disturb the balance of sex hormones during embryonic development. All of these effects will eventually reduce the fitness of individuals and affect natural populations.
Materials and Methods Zebrafish Maintenance and Embryos Collection. Mature zebrafish were reared and maintained according to conditions described before (19). Briefly, fish were kept at 28.5 °C in aerated water (60 mg of “Instant Ocean”/dH2O) and subjected to a 14-h light:10-h dark cycle. Fertilized eggs were collected by placing a plastic box (12 × 24 cm) at the bottom of each tank after group mating during the first 30 min of the light period. They were washed and transferred to either hypoxic or normoxic aquaria within 1 h and incubated at 28.5 °C in embryo medium (19.3 mM NaCl, 0.23 mM KCl, 0.13 mM MgSO4‚7H2O, 0.2 mM Ca(NO3)2, and 1.67 mM HEPES, pH 7.2). The developmental stage of the embryos was described as hour post-fertilization (hpf) and classified according to the morphological characteristics described in an earlier study by Kimmel et al. (20). At 5 hpf, embryos were examined under a (60×) dissecting stereomicroscope (Zeiss). Only those embryos that developed normally and reached the blastula stage (30% epiboly) were selected for subsequent experiments. During the experimental period, 50% of the culture medium was changed daily. Hypoxia Exposure. A constant level of desirable oxygen level was established and maintained by bubbling a constant flow of premixed air and nitrogen mixture into water. Dissolved oxygen was monitored with a YSI model 52 dissolved oxygen meter. Viability Assay. To choose a suitable, sublethal level of hypoxia for experiment, a pilot study was first carried out to determine the viability of zebrafish embryos under four oxygen levels (5.8, 1, 0.8, and 0.5 mg of O2 L-1). Embryo viability was determined for each level of oxygen at 24, 48, 72, 96, 120, and 168 hpf, and dead embryos were removed promptly from the experimental tanks. Heart Rate. At different developmental stages (48, 72, 96, 120, 168, and 288 hpf), 10 embryos each were randomly selected from the control and hypoxic groups (0.8 and 0.5 mg of O2 L-1). The heart rate (beats min-1) of each individual was counted under a dissecting stereomicroscope and recorded by the same operator throughout the whole project. Malformation Assessments. Results of the viability experiment showed that 67% of embryos can survive and develop for at least 168 hpf when exposed to 0.8 mg of O2 L-1. Subsequently, fish embryos were exposed to 0.8 mg of O2 L-1 and normoxia (5.8 mg of O2 L-1) to study the sublethal effects of hypoxia on development. Embryogenesis of zebrafish is completed within the first 72 h, and major development of most internal organs VOL. 38, NO. 18, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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including the cardiovascular system, gut, liver and kidney occur in the first 24-48 h. At 48, 72, 96, 120, and 168 hpf, 10 embryos/ larvae each were sampled from the normoxic control and the hypoxic treatment groups. Body length of each embryonic fish was measured to the nearest 0.1 mm using a dissecting microscope fitted with a calibrated scale in one eyepiece. Because embryos were normally curled within the egg, the chorions were removed under a (60×) dissecting stereomicroscope (Zeiss) before measurement. Only spinal and cardiac malformations were noticeable, and any other form of malformation was not obvious. Number of individuals with spinal and/or cardiac malformation were counted and recorded to provide an estimate on percent of malformation embryos in each of the normoxia and hypoxia groups. Developing stages were determined according to the morphological characteristics under microscope. Hormone Assays. Testosterone (T) and 17β-extradiol (E2) were assayed using enzyme-linked immunoassay (ELISA). The estradiol enzyme immunoassay test kit was bought from ICN Pharmaceuticals, and the testosterone EIA kit was from Cayman Chemical Company. Three hundred zebrafish embryos each were randomly sampled from the hypoxic (0.8 mg L-1) treatment and normoxic control (5.8 mg L-1) at 48 and 120 hpf. Embryos were homogenized in 500 µL of 0.5 M potassium phosphate buffer (pH 7.4) and then centrifuged for 2 min at 5000g at 4 °C. Supernatant was collected and stored at -20 °C for hormone assay. To avoid possible changes in steroid levels due to different developing stages, samples were collected at the same time from all replicates, and all preparation was completed within 2 h. The detection limits of T and E2 were 6 and 1 pg mL-1, respectively. Apoptosis. In this study, apoptotic patterns in hypoxic and normoxic embryos were compared at 24 hpf, when significant extent of apoptosis occurs (21). Ten embryos each were randomly collected from the normoxic (5.8 mg L-1) and hypoxic (0.8 mg L-1) groups at 24 hpf, and chorion was removed using a pair of forceps under a (60×) dissecting stereomicroscope (Zeiss). Embryos were then stained with 5 µg mL-1 of vital dye acridine orange (AO, acridinium chloride hemi-(zinc chloride), Sigma) and incubated for 1520 min at room temperature. The specimens were washed twice for 1 min in 30% Danniean’s solution. After anaesthetization in 0.03% tricaine (3-aminobenzoic acid, ethyl ester methanesulfonate salt) for 3 min, fluorescence in embryos was observed and pictures of each individual were taken immediately using a confocal laser scanning microscope (Carl Zeiss LSM 510) connected to a cooled CCD camera. The output of images was exported as.TIFF files for image analysis using the software Metamorph 3.0 Universal Imaging (West Chester, PA). Apoptotic cells appeared as bright spots, and the total number of apoptotic cells was automatically counted by the software. Statistical Analysis. A Student’s t-test was used to test the null hypothesis that there was no significant difference between the mean of each parameter measured in the hypoxic group and the normoxic group. Percentage data were arc sin square root transformed before analysis. Differences were considered significant if p < 0.05.
Results Viability Assay. Almost 100% mortality was found when fertilized embryos were exposed to anoxia for more than 24 h. A dose-response relationship was demonstrated between oxygen concentration and mortality. Percentage of dead zebrafish embryos in the 5.8 (normoxic), 1.0, and 0.8 mg of O2 L-1 groups were 12.3 ( 1.53%, 17.0 ( 2.0%, and 28.3 ( 2.08%, respectively, after 120 h exposure. Mortality increased drastically to 89.7 ( 1.53% when oxygen level was further lowered to 0.5 mg of O2 L-1 (Figure 1). In the 0.8 mg of O2 4764
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FIGURE 1. Cumulative mortality in zebrafish embryos exposed to different oxygen levels (5.8, 1.0, 0.8, and 0.5 mg of O2 L-1) during different developmental stages (24, 48, 72, 96, 120, and 168 hpf). Values significantly different from the normoxic control are indicated by asterisks (n ) 100, mean ( SD) (t-test: *, p < 0.05; **, p < 0.01; ***, p < 0.001).
FIGURE 2. Change of heart rate in zebrafish embryos at different developmental stages (48, 72, 96, 120, 168, and 288 hpf) upon exposure to 5.8 and 0.8 mg of O2 L-1. Values significantly different from the normoxic control are indicated by asterisks (n ) 10, mean ( SD) (t-test: **, p < 0.01; ***, p < 0.001). L-1 hypoxic group, 22% mortality occurred within 48 h of exposure, and mortality was low thereafter. Heart Rate. Compared with the normoxic control, heart rate of embryos exposed to 0.8 mg of O2 L-1 changed with time in a similar pattern (Figure 2). Heart rate in the 0.5 mg of O2 L-1 group was highly variable, and no consistent pattern could be found, indicating a loss in synchronization of cardiac development upon exposure to hypoxic stress. Embryos exposed to 0.8 mg of O2 L-1 showed an increase in heart rate (138 ( 11.6 beats min-1), which was significantly higher than that of normoxic control (122 ( 4.6 beats min-1) at 96 hpf (p < 0.001). A decline was observed thereafter and was significantly lower (105 ( 8.0 beats min-1) than that of control embryos (117 ( 7.8 beats min-1) at 288 hpf (p < 0.01). Malformations. Development was clearly delayed when zebrafish embryos were exposed to hypoxia. Embryos exposed to 0.5 mg of O2 L-1 took twice as long to develop when compared to the normoxic embryos (Figure 3). Embryos exposed to hypoxia developed lost normal synchronization in their development, with their tails developing much faster than heads. External abnormalities such as spinal deformity (predominantly manifested as altered axial curvature) were clearly evident in the hypoxic fish. Many embryos failed to develop their vascular systems after several days and died. After 168 h, the percentage of fish with malformation in the hypoxic group (18.3 ( 2.31%) was significantly higher than that of the control (10.3 ( 1.15%) (Student’s t-test, p < 0.01) (Figure 4). Of the remaining
FIGURE 5. Body length of zebrafish embryos at different developmental stages (48, 72, 96, 120, and 168 hpf) upon exposure to 5.8 and 0.8 mg of O2 L-1. Values significantly different from the nromoxic control are indicated by asterisks (n ) 10, mean ( SD) (t-test: *, p < 0.05; ***, p < 0.001).
FIGURE 3. Retardation of development caused by severe hypoxia (0.5 mg of O2 L-1).
FIGURE 4. Percentage malformation in zebrafish embryos during different developmental stages (48, 72, 96, 120, and 168 hpf) upon exposure to 5.8 and 0.8 mg of O2 L-1. Values significantly different from the normoxic control are indicated by asterisks (n ) 100, mean ( SD) (t-test: *, p < 0.05; **, p < 0.01). embryos showing no visible signs of malformation, the headto-tail body length was measured at 48, 72, 96, 120, and 168 hpf. At 168 hpf, the average body length of embryos in hypoxic group (3.13 ( 0.13 mm) was significantly shorter than that of the control group (3.57 ( 0.16 mm) (p < 0.001) (Figure 5). Sex Hormones. At 48 hpf, testosterone level in hypoxic embryos showed a significant increase from 25.6 ( 0.56 to 34.4 ( 4.23 pg mL-1 (p < 0.01), while estradiol was significantly reduced from 8.03 ( 1.41 to 2.36 ( 0.10 pg mL-1 (p < 0.001) (Figure 6A). A reversed pattern was observed at 120 hpf: testosterone in hypoxic embryos was significantly reduced from 36.1 ( 1.43 to 27.3 ( 2.75 pg mL-1 (p < 0.01), while estradiol was significantly increased from 4.61 ( 1.28 to 12.5 ( 4.10 pg mL-1 (p < 0.05) (Figure 6B). Apoptosis. Compared with the normoxic control, apoptotic cells in the tail of hypoxic embryos were significantly reduced (-63.7%). In contrast, a significantly higher percentage (+116%) of apoptotic cells was found in the brain of hypoxic embryos as compared with control embryos (Figure 7). This clearly indicated that the apoptotic pattern in zebrafish embryos was altered by hypoxia.
FIGURE 6. Testosterone and estradiol (pg mL-1) in zebrafish embryos at 48 (A) and 120 hpf (B) upon exposure to 5.8 and 0.8 mg of O2 L-1. Values significantly different from the normoxic control are indicated by asterisks (n ) 4, mean ( SD) (t-test: *, p < 0.05; **, p < 0.01; ***, p < 0.001).
Discussion An earlier study (22) showed that development of zebrafish embryos was arrested by hypoxia for up to 24 h after fertilization without deleterious effect. In our study, however, almost 100% mortality was recorded when zebrafish were subjected to anoxia for longer than 24 h. In general, the critical dissolved oxygen concentration for supporting the survival of most aquatic organisms is around 2.8 mg of O2 L-1, while certain species could tolerate 0.5-1 mg of O2 L-1 for several days to weeks (23). Zebrafish are comparatively quite tolerant to hypoxia (24). Their embryos are transparent, and embryonic development is external and rapid, thus offering an excellent vertebrate model for developmental studies. We found that more than 70% of zebrafish embryos survived for VOL. 38, NO. 18, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 7. Number of apoptotic cells at 24 hpf in zebrafish embryos upon exposure to 5.8 and 0.8 mg of O2 L-1. Values significantly different from the control are indicated by asterisks (n ) 10, mean ( SD) (t-test: *, p < 0.05; **, p < 0.01). at least 168 h under hypoxia (0.8 mg of O2 L-1), which level is not uncommon in the natural environment. There are few published studies on the effects of hypoxia on development of fish embryos, but a higher occurrence of skeletal deformation was generally found in fish from polluted areas (25). Our results provide, for the first time, clear evidence that sublethal oxygen levels affect the developmental rate as well as the normal development of zebrafish embryo. These impairments at the earlier stages of the life cycle may subsequently reduce the fitness and therefore chance of survival of individuals in natural populations. A significantly higher percentage of malformation was accompanied with a loss in synchronization of development when zebrafish embryos were exposed to severe hypoxia (0.5 mg L-1). The heart rate of embryos at this oxygen concentration was highly variable, ranging from
