The teratogenic effects of methylmercury on early development of the zebrafish, Danio rerio

Aquatic Toxicology 48 (2000) 343–354

The teratogenic effects of methylmercury on earlydevelopment of the zebrafish, Danio rerio

Jennifer C. Samson *, Jonathan ShenkerDepartment of Biological Sciences, Florida Institute of Technology, 150 West Uni6ersity Bl6d., Melbourne, FL 32901, USA

Received 23 September 1997; received in revised form 12 February 1999; accepted 8 April 1999

Abstract

Chronic bioassays were used to evaluate the concentration and exposure duration of methylmercury that resultedin specific teratogenic defects in Danio rerio embryos exposed at different developmental stages. Embryos in differentstages of development (cleavage, blastula, gastrula, or segmentation) were exposed to 20 or 30 mg/l of methylmercuricchloride (CH3HgCl) for various exposure durations (8, 16, 32 h, or continuously to hatching). These exposuresfrequently caused two morphological defects, tissue abnormality in the median finfold and a flexure of the posteriortail region. The critical period of exposure for the production of both effects begins around 18–20 h after fertilization,with increased exposure resulting in more severe effects. These critical periods coincide with both tail and medianfinfold formation. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Danio rerio ; Methylmercury; Teratogenic; Toxicity

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1. Introduction

Methylmercury is an environmental toxicantthat is of particular concern to aquatic organismsbecause of its ability to pass through cell mem-branes. Methylmercury is bioaccumulated byaquatic organisms through direct uptake from thewater and ingestion of contaminated foods (Tian-ye and McNaught, 1992). This chemical is alsobiomagnified in the tissues of organisms at highertrophic levels (Zillioux et al., 1993). Tissue analy-

ses show that mercury accumulated in fish isalmost entirely in the form of methylmercury(Westoo, 1969; Bache et al., 1971; Grieb et al.,1990). Mercury contamination in adult and juve-nile fish has been shown to disrupt vital functionsincluding reproduction, osmoregulation, orienta-tion, searching for food, predator recognition,and communication (Zillioux et al., 1993).

Embryonic exposure to methylmercury causes avariety of teratogenic effects including cyclopia,tail flexures, and cardiac malformations in themedaka and mummichog (Weis and Weis, 1977a;Dial, 1978a,b) and jaw deformities, twinning, andaxial coiling in the rainbow trout (Birge et al.,1979). Investigations that focus on different stagesof development provide specific information

* Corresponding author. Present address: Department ofBiological Sciences, 101 Warren St., Rutgers University,Newark, NJ 07102, USA. Tel.: +1-973-3535387; fax: +1-973-2751054.

0166-445X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

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J.C. Samson, J. Shenker / Aquatic Toxicology 48 (2000) 343–354344

about the critical periods of exposure for theproduction of these abnormalities (Akiyama,1970; Weis and Weis, 1977b; Sharp and Neff,1982).

The zebrafish, Danio rerio is a small, freshwa-ter, aquarium species that is used extensively as abioassay organism (Jones and Huffman, 1957;Anderson and Battle, 1967; Kihlstrom and Hulth,1972; Niimi and LaHam, 1975; Thomas, 1975;Laale, 1977; Ozoh, 1979; Groth et al., 1993;Roche et al., 1994). The ease of culturing andspawning large numbers of fish, and their rela-tively large fertilized eggs, make this species anexcellent model to study teratogenesis induced bytoxic substances. Embryonic development in D.rerio is rapid (�72 h from fertilization to hatch-ing at 28°C) and readily visible through the trans-parent chorion and embryo (Hisaoka and Battle,1958). The complete embryonic development wasextensively described and staged (Hisaoka andBattle, 1958; Hisaoka and Firlit, 1960).

The purpose of this investigation was to iden-tify the stages of embryonic development in D.rerio that were most sensitive to methylmercuryexposure, as determined by the induction of ter-atogenic effects.

2. Materials and methods

Over 100 mature male and female D. rerio weremaintained in two 71-l aquaria at 28°C on a 14:10light/dark photoperiod. When eggs were needed,male and female pairs were transferred from thestock population the evening before an experi-ment, and placed in breeding traps in a separateaquarium. The fish spawned naturally at the be-ginning of the light cycle and fertilized eggs werecollected immediately after spawning.

Toxicity tests were designed to determine whichconcentration, developmental stage, and exposureduration induced specific teratogenic defects. Alltest solutions were made by the addition ofaliquots of a concentrated 100-mg/l methylmer-curic chloride (CH3HgCl) stock solution to a solu-tion of essential salts (embryo medium) describedby Westerfield (1993). Based on preliminary ex-periments, concentrations of 20 and 30 mg

CH3HgCl/l were used. Exposure began with em-bryos in one of four developmental stages: earlycleavage (45 min post-fertilization), blastula (2 h15 min post fertilization), early gastrula (5 h 15min post-fertilization), and segmentation (10 hpost-fertilization), as described by Westerfield(1993). Embryos were incubated at 28°C in glassfinger bowls containing 100 ml of solution, undera 14:10 light/dark photoperiod.

At least 360 embryos (two replicates/180 em-bryos each) were used for each of the four devel-opmental stages. Of the 180 embryos, 60 wereexposed to each concentration and 60 were usedas controls. A total of ten embryos were removedfrom each treatment after 8, 16, and 32 h ofexposure, while remaining embryos were left insolution for the test duration. Those removedfrom solution were transferred to fresh embryomedium and allowed to develop until most em-bryos hatched (�72 h post-fertilization). Conse-quently, exposures were initiated at either thecleavage, blastula, gastrula, or segmentation stageof development and embryos remained in thesolution for 8, 16, 32 h, or continuously throughto hatching. Mortalities were recorded and re-moved at each time interval, and all test solutionswere renewed at 32 h.

At test termination any viable, unhatched em-bryos were gently dechorionated. All larvae werepreserved in 10% formalin solution and subse-quently examined under an inverted-stage phasecontrast compound microscope for teratogenicdefects. All embryos were counted and anomalousembryos were categorized according to type andseverity of effect.

Data from microscopic examination of the em-bryos were used to test for significant differencesbetween concentrations, stages exposed, and dura-tion of exposure for each teratogenic effect usingthe Kormogorov-Smirnov two-sample test (K-Stest, P=0.05) (Sokal and Rohlf, 1981).

3. Results

There was 15–20% mortality of embryos ex-posed to methylmercury, with most mortality oc-curring within the first 8 h after fertilization.Control mortality was 10–12%.

J.C. Samson, J. Shenker / Aquatic Toxicology 45 (2000) 343–354 345

Exposure of D. rerio embryos to sublethal con-centrations of methylmercury consistently causedtwo types of morphological defects. The durationof the exposure, developmental stage at exposure,and concentration, all affected the frequency andseverity of teratogenic defects. The two effectswere not mutually exclusive, and many embryosexhibited both traits. The first defect was a tissueabnormality of the median finfold or tail fin pri-mordium. In normally developing embryos, themedian finfold is a clear, thin membrane aroundthe entire trunk region containing actinotrichia,or unsegmented collagenous fin rays (Fig. 1A).Portions of the median finfold ultimately developinto the dorsal, anal, and caudal fins. In affectedembryos, the tissue structure of the finfold wasdisorganized and in the more severe cases, theshape of the finfold and the developingactinotrichia were altered. Anomalous embryosshowed a range in the severity of the effect andwere ranked on a qualitative scale:

0 - Normal finfold structure (Fig. 1A)1 - Small portion of the finfold tissue structurewas disorganized (Fig. 1B)2 - Most tissue near the base of the tail wasabnormal and disorganized but effect did notextending to the finfold’s outer margin (Fig.1C)3 - Abnormal organization of tissues extendedfrom the base of the tail to the finfold’s outermargin. The shape of the structure was nolonger uniform and the overall size was reduced(Fig. 1D)4 - The tissue was abnormal and disorganizedthroughout the entire finfold. The finfold andactinotrichia were deformed and the finfold wasseverely reduced in size (Fig. 1E).Some degree of finfold abnormality occurred in

29% of embryos exposed to 20 mg CH3HgCl/l andin 35% of embryos exposed to 30 mg/l, but did notoccur in a single control embryo.

The second effect of exposure of D. rerio em-bryos to methylmercury was a flexure of the pos-terior tail region. All embryos with this secondeffect also showed tissue abnormalities of thefinfold. In normally developing embryos, thenotocord and spinal cord, along with surroundingmyomeres, are straight to the posterior-most tip

of the tail (Fig. 1A). In affected embryos, thesestructures were flexed to some degree. In the moresevere cases, the flexure was extreme and theoverall length of the tail was stunted. Anomalousembryos showed a range in the severity of theeffect, and were ranked on a qualitative scale:

0 - Normal tail development (Fig. 1A)1 - Very slight flexure in the posterior-most tipof the tail (Fig. 1D)2 - Kink in the posterior-most tip of the tail(Fig. 1E)3 - The entire tail region was strongly flexedand the length was stunted (Fig. 1F).Tail flexures occurred in 10% of embryos ex-

posed to 20 mg CH3HgCl/l and in 22% of embryosexposed to 30 mg/l, but did not occur in a singlecontrol embryo.

3.1. Tissue abnormalities of the finfold

Embryos exposed to 20 mg CH3HgCl/l did notproduce a significant number of finfold abnormal-ities until they had been in solution for 32 h,regardless of the initial stage at exposure (PB0.001; Fig. 2). Embryos initially exposed to 30 mgCH3HgCl/l at the cleavage (hour 0:45), blastula(hour 2:15), and gastrula (hour 5:15) stages alsorequired exposures of at least 16 h to produce aneffect (PB0.01; Fig. 3A). But if exposure toCH3HgCl began at the segmentation stage (hour10), the embryos exhibited significant effects withonly 8 h of exposure (PB0.05; Fig. 3B). Thesedata indicated that the later stages of develop-ment, beginning with the segmentation stage, weremost sensitive to the effects of methylmercury.

Compared to the lower exposure concentration,the 30-mg CH3HgCl/l concentration significantlyincreased the severity of effects with exposuredurations of 32 h or longer (PB0.001; Figs. 2and 3). Comparisons of the frequency distributionfor the severity of abnormalities indicated longerexposure periods induced more severe abnormali-ties, regardless of stage of initial exposure.

3.2. Tail flexures

The 30-mg CH3HgCl/l concentration was theonly one to consistently produce a significant


Fig. 1. Range in severity of morphological defects in the median finfold and posterior tail region of newly hatched D. rerio embryosafter exposure to methylmercury. Scale bar=0.1 mm. (A) Normal development; morphological score=0. (B) Slight disorganizationin the finfold tissue structure; morphological score=1. (C) Most tissue around base of tail is abnormal and disorganized;morphological score=2. (D) Tissue abnormality extends from base of tail to outer margin of the finfold; morphological score=3;photo also shows slight flexure at the posterior-most tip of the tail; morphological score=1 (tail flexure). (E) Tissues of the finfoldshow extensive disorganization and abnormality throughout posterior region, resulting in deformity and severe reduction in overallsize; morphological score=4; photo also shows a definite kink at the posterior most tip of the tail; morphological score=2 (tailflexure). (F) Severe flexure of the entire posterior-most tail region after exposure to methylmercury; morphological score=3.

number of tail flexures regardless of initial stageof exposure (PB0.05). Embryos exposed to 20 mgCH3HgCl/l only produced a significant number offlexed tails when exposure began at the blastula

stage and went through to hatching (PB0.01).Although the 20-mg/l exposures beginning at theother three stages of development (cleavage, gas-trula, and segmentation) showed an increase in


Fig. 1. (Continued)

the number of flexed tails with an increase inexposure duration, the distributions were notsignificantly different than the control (P]0.05).

Embryos initially exposed to 30 mg CH3HgCl/l at the cleavage and blastula stages of develop-ment required continuous exposure to hatchingto produce significant abnormalities in the tail

structure (PB0.001; Fig. 4A), while exposuresstarting at the gastrula stage required an expo-sure of at least 32 h to produce an effect (PB0.001; Fig. 4B). But if exposure to CH3HgClbegan at the segmentation stage, the embryosexhibited significant effects with only 16 h ofexposure (PB0.05; Fig. 4C). Again these dataindicated that the later stages of development,


Fig. 1. (Continued)

beginning with the segmentation stage, were mostsensitive to the effects of methylmercury.

Comparison of the frequency distributions forseverity of tail flexures indicated longer exposuredurations induced more severe abnormalitieswhen embryos were initially exposed at the blas-tula, gastrula, and segmentation stages of devel-opment (Fig. 4).

4. Discussion

D. rerio embryos proved to be sensitivebioassay organisms for assessing the toxicity ofmethylmercury. The embryonic development ofD. rerio has been divided into six major stages(cleavage, blastula, gastrula, segmentation,pharyngula, and hatching) and each is defined by


Fig. 2. The frequency of finfold abnormalities in embryonic D. rerio produced by exposure to 20 mg CH3HgCl/l at differentdurations of exposure and initially exposed at the cleavage stage of development. Similar frequency distributions were seen inembryos initially exposed at the blastula, gastrula and segmentation stages of development.

a variety of developmental events (Westerfield,1993). The test exposures, beginning at differentdevelopmental stages and continuing for variousperiods of time, identified which stages were vul-nerable to methylmercury-induced finfold abnor-malities and tail flexures. These critical periodscoincide with both tail and median finfold forma-tion (Westerfield, 1993).

4.1. Tissue abnormalities of the finfold

Embryos initially exposed to the 20-mgCH3HgCl/l concentration did not begin to showfinfold abnormalities until they had been exposedfor at least 32 h, while embryos exposed tomethylmercury to the 30-mg CH3HgCl/l concentra-tion at the cleavage, blastula, and gastrula stagesrequired a minimum of 16 h of exposure (Fig. 3A).But embryos initially exposed at the segmentationstage (10 h into development) responded to 8 h ofexposure (Fig. 3B). These data suggest the criticalperiod for finfold abnormalities begins approxi-mately 18 h after fertilization, with increased expo-sure resulting in more severe effects. The medianfinfold is beginning to develop at this time. Aki-menko et al. (1995) determined that some of the

genes important in unpaired fin development(msxA, msxB, msxC and msxD) are expressed inD. rerio embryos beginning at hour 16 of develop-ment; one or more of these genes, or their prod-ucts, may thus be affected by methylmercury.

All three exposure parameters (initial stage atexposure, duration of exposure, and concentra-tion) were critical in disrupting the development ofthe finfold (Figs. 2 and 3). Embryos removed fromsolution before reaching the period of develop-ment when the finfold was being produced, exhib-ited normal finfold development. Once embryonicexposure coincided with the critical stages offinfold development, exposure concentration dic-tated the frequency of the abnormalities produced(Figs. 2 and 3). The 20-mg CH3HgCl/l exposurerequired a longer duration of exposure to disruptfinfold production than the 30-mg CH3HgCl/l ex-posure (Figs. 2 and 3). The 30-mg CH3HgCl/lconcentration also increased the severity of thefinfold abnormality compared to the lower concen-tration (Figs. 2 and 3).

The production of median finfold abnormalitiesin fish embryos has been reported with exposure toother toxicants. Although the previous studies didnot define the embryonic periods of develop-


Fig. 3. The frequency of finfold abnormalities in embryonic D. rerio produced by exposure to 30 mg CH3HgCl/l at differentdurations of exposure and stages of development. (A) Embryos initially exposed at the cleavage stage (similar frequency distributionswere seen in embryos initially exposed at the blastula and gastrula stages); (B) embryos initially exposed at the segmentation stage.

ment, similar abnormalities were observed in: ze-brafish (D. rerio) exposed to 0.002–0.005% 2-acetylaminofluorene (Hisaoka, 1958); Atlantic cod(Gadus morhua), Atlantic herring (Clupea haren-gus), and plaice (Pleuronectes platessa) exposed tocrude oil dispersants (Kuhnhold, 1972); bluegill

(Lepomis macrochirus) exposed to 239 mg/l cad-mium (Eaton, 1974); gar pike (Belone belone)exposed to 1 mg/l cadmium (von Westerhagen etal., 1975); and kelp bass (Paralabrax clathratus)exposed to surface microlayer samples contami-nated with metals (Cross et al., 1987).


Fig. 4. The frequency of flexed tails in embryonic D. rerio produced by exposure to 30 mg CH3HgCl/l at different durations ofexposure and stages of development. (A) Embryos initially exposed at the cleavage stage (a similar frequency distribution was seenin embryos initially exposed at the blastula stage); (B) embryos initially exposed at the gastrula stage; (C) embryos initially exposedat the segmentation stage.


The production of median finfold abnormalitiesafter exposure to methylmercury have not beendescribed for any other fish embryo. Most re-search involving methylmercury reported moresevere effects at similar concentrations: cardiacand skeletal malformations and craniofacial de-fects in killifish (Fundulus heteroclitus) at 50 mg/l(Weis and Weis 1977a; Weis and Weis, 1977b),cardiac malformations in Japanese medaka(Oryzias latipes) at 15–30 mg/l (Heisinger andGreen, 1975), craniofacial and skeletal malforma-tion in Japanese medaka (O. latipes) at 40–80mg/l (Dial, 1978a,b) and skeletal abnormalitiesincluding twinning in rainbow trout (Oncho-rynchus mykiss) at 5 mg/l (Birge et al., 1983).

A possible explanation for the lack of reportingof tissue abnormalities of the finfold in themethylmercury research cited above may be thatthose studies focused on more severe developmen-tal defects. Such severe effects were also producedin D. rerio embryos exposed to 40–80 mg/l duringpreliminary range finding experiments, but mor-tality was extremely high, and thus these concen-trations were not appropriate for sublethal,chronic exposures.

4.2. Tail flexures

Embryos initially exposed to the higher concen-tration of methylmercury at the cleavage, blastula,and gastrula stages required a minimum of 32 hof exposure before exhibiting flexures of the tail,while exposures through to hatching producedhigh rates of tail flexures (Fig. 4A, B). Embryosinitially exposed at the segmentation stage beganto show tail flexures after 8 h of exposure (Fig.4C), suggesting that the critical period for tailflexures begins 18–26 h after fertilization, withincreased exposure resulting in more severe ef-fects. The tail is undergoing rapid development atthis time.

All three exposure parameters (initial stage atexposure, duration of exposure, and concentra-tion) were critical in consistently disrupting tailmorphogenesis (Fig. 4). The 20-mg CH3HgCl/lconcentration was not sufficiently teratogenic tocause a significant effect except for embryos ex-posed from the blastula stage through to hatch-

ing. Embryos initially exposed at the cleavage,gastrula, or segmentation stages did show an in-crease in the number of flexed tails with an in-crease in exposure duration but the increases werenot significant.

When embryos were exposed to 30 mgCH3HgCl/l for an extended period of time, tailproduction was disrupted. The duration of theexposure had to extend into the middle of tailmorphogenesis to affect normal development. De-pending on how early in development exposurebegan, embryos had to stay in solution longer toreach the crucial time period. Once the crucialperiod was reached, increasing the exposure dura-tion increased the severity of the tail flexure, withthe most severe effects produced by continuousembryonic exposure (Fig. 4).

The concentration of 30 mg CH3HgCl/l whichproduced tail flexures in D. rerio embryos wassimilar to concentrations of mercury used to pro-duce axial malformations in other fish embryos:50 mg/l in the killifish (F. heteroclitus ; Weis andWeis, 1977b) and 40–80 mg/l in the Japanesemedaka, (O. latipes ; Dial, 1978a). Rainbow trout(O. mykiss) appear to be more sensitive tomethylmercury, with effects produced at concen-trations of only 5 mg/l of methylmercury (Birge etal., 1983). Methylmercury appears to be effectivein disrupting normal axial morphogenesis in avariety of species. Further investigation intomethylmercury’s mode of action may help explainthe tail’s vulnerability to this toxicant.

D. rerio embryos were most susceptable to lowlevels of methylmercury during the segmentationstage and beyond. Previous research indicatedother fish species were more sensitive in earlierstages of development and resistance increasedwith development (Battle and Hisaoka, 1952; An-derson and Battle, 1967; Akiyama, 1970; Laale,1977). These experiments used concentrations thatinduced more severe abnormalities than were re-ported in this study. Early embryonic develop-ment may not be as susceptable to the lower,sublethal concentrations used in these experi-ments, which were adequate to disrupt late stageprocesses such as fin and tail development.


Acknowledgements

We are grateful to DB Enviromental Laborato-ries in Rockledge, FL for the use of their labs andequipment to conduct this research, and especiallyDr Roy Laughlin and Tom DeBusk for theirassistance and advice.

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The teratogenic effects of methylmercury on early development of the zebrafish, Danio rerio