Toxicity of lindane, atrazine, and deltamethrin to early life stages of zebrafish (Brachydanio rerio)

FCOI0SI(‘OLO<;Y

4ND

t’WIRONMth’l

AL SAFETY

20, 246-255

( 1990)

Toxicity of Lindane, Atrazine, and Deltamethrin to Early Life Stages of Zebrafish (Brachydanio rerio) GERHARD

GORGEANDROLANDNAGEL

Received

Fehruar~~

2, 1990.

Fertilized eggs of zebrafish were exposed under flow-through conditions to several concentrations of the following pesticides: lindane 40. 80, 110, 130, and 150 pg/liter; atrazine 300. 1300, and 9100 &liter: deltamethrin 0.5. 0.8. and 1.2 pg/liter. Hatching. abnormalities in development (external deformations, edema, etc.), and mortality were recorded over a period of 35 days. At the end of the experiment, the body lengths of the fish were measured. Survival of juvenile fish after 35 days was reduced by increasing concentrations of all xenobiotics tested: lindane enhanced the mortality from 1 IO rg/liter and atrazine from 1300 pg/liter. and deltamethrin showed an effect even at the lowest test concentration (0.5 &liter). Other parameters were affected differently: hatching rate was reduced only by deltamethrin (from 0.8 fig/liter); lindane caused a decrease in growth (40 &liter) but had no effect on the other parameters. Atrazine increased the number of deformations and edema (I 300 pg/liter) but did not influence hatching rate and growth. The sensitivity of the early life stages to the pesticides was compared with acute toxicity data (LCsO) of adult zebrafish. The early life stages were less sensitive to lindane (118 versus 75 &/liter). whereas in the case of atrazine ( 1300 versus 37,000 pg/liter) and deltamethrin (0.5 versus 2 pg/ liter; 0.5 pg/liter was the lowest concentration tested) larvae were more sensitive. cJ 1990 A~W&IIUC Press. Inc.

INTRODUCTION The results of life cycle toxicity tests on freshwater fish indicate that embryos and larvae are the most sensitive stages (Pickering and Gast, 1972; McKim et al., 1975, Sauter et al., 1976). Subsequently. the early life stage test was proposed as a toxicity test for fish (McKim, 1977). Starting with fertilized eggs, the early life stages should be exposed to toxicants for 28-32 days (60 days posthatch for salmonids). Toxic effects on survival, morphological changes, and growth should be measured. The aim of the present study was to determine whether an early life stage test with zebrafish is practicable and expressive. For this purpose the effects of lindane, atrazine, and deltamethrin on the embryonic and larval development of zebrafish were examined. Fertilized eggs were exposed under flow-through conditions to the test substance at different concentrations in two parallel groups. Two untreated groups were kept under flow-through conditions for control. The parameters recorded in this study were hatching, morphological abnormalities, mortality, and growth. In some stages of development, oxygen consumption was also determined, The eff‘ects on embryo survival and on survival and growth of larvae allow an estimation of the subchronic toxicity of the compounds. MATERIALS

AND

METHODS

Zebrafish eggs were obtained according to the method of Nagel ( 1986), and 2.50 were exposed to each vessel about 2 or 3 hr after spawning. After 24 hr the number O147-6513190

$3.00

Copyrighl 0 1990 by Academic Press. Inc All rights of reproduction in any form reserved.

246

TOXICITY

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247

of eggs in each vessel was reduced to 100 fecundated eggs (Nagel, 1986). The comparison of the fecundation rate between the controls and the exposed groups indicated that the xenobiotics caused no effects within the first 24 hr. Until Day 14 of development breeding took place in glass petri dishes (8 cm in height, 15 cm in diameter; 500 ml water). Subsequently, fry were transferred into glass vessels (20 X 15 X 30 cm, HxWxL; 6 liters of water). Water exchange was 3.6 and 30 liters/day, respectively. Beginning with Day 6, larvae were fed yolk powder two or three times daily. By Day 8, newly hatched brine shrimp (Arzemia sp.) and pulverized dry food (TetraMinj were added. Exposure chambers were cleaned daily. Hatching, malformed fry, and the number of dead embryos and larvae were recorded once a day. Body lengths of fish were measured at Day 35. Oxygen consumption of embryos, larvae, and juvenile fish was measured using a biological oxygen monitor (Yellow Springs Instr.). According to developmental stage, different numbers of animals (e.g., 30 embryos in the eggshell, 15 yolk sac larvae, 2-7 juveniles) were transferred to the test cup filled with 3 ml water. After 5 min of equilibration, the oxygen concentration of the water was measured over a period of 10 min. The flow-through test system consisted of a water tank in which filtered tap water was aerated and the temperature was regulated. The toxicant stock solutions were added via mixing chambers by a dispenser (Hamilton MicroLab 400). All pipes were made of glass and light-protected by aluminum foil. Saturated solutions of lindane and atrazine were prepared as stock solutions and stored at room temperature in the dark for at most 4 days. Because of the low water solubility of deltamethrin, acetone (35 mg/liter) was used as organic solvent. In a preliminary test over 14 days it was shown that 150 mg acetone/liter had no effect on the development of embryos and larvae. Atrazine (98%) was obtained from Riedel de Haen (Hannover. FRG) and lindane (99.8%) and deltamethrin (>98%) were kindly provided by CelaMerck (Ingelheim, FRG) and Roussel Uclaf (France), respectively. The concentrations of pesticides in the test chambers were checked twice a week (Table l-3). Lindane and deltamethrin were extracted with hexane, and atrazine was extracted with diethylether. Analyses were done by gas chromatography using an electron capture detector (5890 workstation, Hewlett-Packard; capillary column: crosslinked 5% phenyl methyl silicone , 25 m X 0.32 mm X 0.52 pm). Toxicant concentrations were quantified by linear regression of external standards. Exposure water was analyzed daily for dissolved oxygen (6 t 0.8 mg/liter), pH (8 f 0.5) and temperature (27 + 1“C); values are expressed as means t SD. Twice a week. additional determinations of total hardness (24 + 1“dH), carbonate hardness ( 14 f 1“dH), nitrate (lo-35 mg/liter), nitrite (0- 1.3 mg/liter), and ammonium (O-O.2 mg/liter) were carried out.

Statistical Analysis The procedure of Williams (197 1) was used to identify differences (P = 0.05) from controls regarding hatching rate, developmental anomalies, and survival. The LCsO values and 95% confidence limits were determined by a computer program (Tallarida and Murray, 198 1) based on the method of Litchfield and Wilcoxon ( 1949). Comparison of body length was done by the rank test of Mann and Whitney (1947) which is based on the Wilcoxon test.

248

GiiRGE

AND NAGEL. TABLE

I

EFFECTS OF LINDANE ON THE DEVELOPMENT OF ZEBRAFISH Concentration +- SD (pg/liter)” 40 k 3.7

Control Hatch (%) External deformities v ) Edema (?k) Survival 35 days (q) Length 35 days (mm)” Standard deviation

80 + 7.3

97: 97

99. 100

100.98

3; 3 0: 3 81: 76 17.0; 17.4 2.0: I.8

2. 3 0: 3 I: 0 0: I 92; 82 89: 82 16.2: 17.0* 16.1; 16.4** 1.5: I.6 2.1: I.7

I IO k 6.8

130 k 13.0 150h f 16.3

95: 99

100: 100

99

2: 4 0: 2 76; 71 16.1; 16.4** 2.2; 2.3

I: 6 0: I 22; 9** 16.1: 14.2 3.4; 3.2

4 0 2++ 14.7 I .4

Norc Start with 100 fecundated eggs each. Duration: 35 days. Each test was performed with two parallel group!;. ’ Mean values. ” Technical problems: the experiment could be finished with only one group. * P < 0. I. **P < 0.05 significantly different from controls.

RESULTS The hatching rate of embryos exposed to lindane and atrazine varied from 95 to 100%(Tables I and 2) and wasnot different from that of the controls. For deltamethrin, the hatching rate was significantly reduced (P = 0.0 1) concentrations 0.8 pug/literand higher (Table 3). However, when the early life stageswere treated with 130 and 150 pg/liter lindane, 9 100 pg/liter of atrazine. and deltamethrin even at the lowest test concentration (0.5 pg/liter), survival ofjuveniles after 35 days wasreduced significantly. The reduction in survival of juveniles at Day 35 at the concentration of 1300 /lg atrazine/liter could be proved only with low statistical significance (P < 0.1).

TABLE

7

EWFC‘TS OF AI‘RAZINE ON THE DEvELopMEN-r

0~ ZERRAFISH

Concentration Control Hatch (%) External deformities ( ‘K,) Edema (%) Survival 35 days (a.) Length 35 days (mm)“ Standard deviation

300 +- 40

+ SD (pg/liter)”

1300 + 220

9100 t 1300

100: 100

100: 99

100: 100

100: 100

4; 2 0; I 70: 68 14.4: 14.5 2.5: 3. I

5: 8 1: I 73: 67 14.5: 13.9 2.6; 2.4

12: 4 3; 3** 38: 55* 15.9: 14.2 2.5; 2.7

9: 7* 6; 5 *** I; s*** 16.0: 14.1 -: 2.8

h’f~c Start with 100 fecundated eggs each. Duration: 35 days. Each test was performed with two parallel groups. ’ Mean values. * P < 0.1. ** P < 0.05. *** I’ i 0.01 significantly ditferent from controls.

TOXICITY

SENSITIVITY TABLE

EFFECTS OF DELTAMETHRIN

3

ON THE DEVELOPMENT OF ZEBRAFISH Concentration

Control Hatch (%‘) External deformities (% ) Edema (%) Survival 35 days (%) Length 35 days (mm)” Standard deviation

249

OF ZEBRAFISH

2 SD (pg/liter)”

0.5 i 0.00

0.8 t- 0.15

1.2 ix 0.25

99: 98

97; 98

81: 59***

49: 45 ***

0: 0 3; 3 81: 72 16.3: 17.7 2.1: 2.1

6: I** 5; 8 33; 49 ** 17.4: 16.8 3.8: 2.8

7; I** 6; 7 37; l8*** 16.6: 17.2 3.3: 1.6

2: 5** 2; 8 14: 14*** 18.4: 17.0 3.0: 3.2

:‘lofc’.Start \\ith IO0 fecundated eggs each. Duration: 3.5 days. Each test was pcrthrmed with two parallel groups. a Mean \alucs. ** I’ < 0.05 and ***I’ < 0.01 signilicantl) difkrcnt t’rom controls.

The calculated LCso values at 35 days (95% confidence limits) of lindane and deltamethrin were I 18 ( 1IS- 121) pg/liter and 0.52 (0.46-0.58) pg/liter, respectively. No LC5,,/35 days ofatrazine could be calculated with the present data. The value yielded by approximation was 1200 pg/liter. The development oflarvae wasinfluenced by atrazine and deltamethrin. The number of edema was significantly increased by concentrations of 1300 and 9 100 &liter of

FIG. I. Spine malformation in zebrafish embryo (5 days): 25X

250

CiiRGE

AND NAGEL

atrazine. The number of external deformities was increased at all deltamethrin concentrations. However, this effect could not be correlated with increasing concentrations. It must be accentuated that the observed morphological developmental abnormalities were also found within the control groups of the lindane and atrazine tests (Figs. 1 and 2). Kimmel et al. ( 1989) described a zygotic recessivemutation in zebrafish which led to external malformations in the region of trunk and tail and caused death of mutants within the first week of development. Thus, it must be taken into consideration that the significance of the results depends on the rate of spontaneous abnormalities. It is obvious that lindane at the concentrations tested seemsto have no influence on the occurrence of external deformities and edema in zebrafish, whereas atrazine and deltmethrin impair the ontogenesisof zebrafish. The body lengths of fish exposed to sublethal concentrations of lindane (80 and 110 pg/liter) were significantly reduced. Growth was also limited at 40 pg/liter of lindane although with low statistical significance (P = 0.1). For atrazine and deltamethrin, comparisons of body lengths either show no difference between exposed groups and controls or are not admitted because of great variance in the number of random samples. No concentration-dependent influence of the pesticides could be correlated with the oxygen consumption of any tested developmental stage (Tables 4-6). In some developmental stages,O2 consumption decreaseswith increasing pesticide concentration, whereas the result of the following measurement is contrary, or it alternates irrespective of differences in concentration. DISCUSSION The results of the early life stage (ELS) test show that the maximum acceptable toxicant concentration (MATC-defined as the hypothetical toxic threshold concen-

FIG. 2. Edema in zebrafish embryo (5 days): 25~.

TOXICITY

SENSITIVITY TABLE

OF

251

ZEBRAFISH

4

EFFECTOFLINDANEONTHEOXYGENCONSUMPTIONOFDIFFERENTDEVELOPMENTAL STAGESOF~EBRAFISH (fig O?/mg/hrl Age (days)

2 3 4 11 17 23 30 35

Developmental stage El?!2 Larvae Larvae Juvenile Juvenile Juvenile Juvenile Juvenile

Number 15 15 15 6 4 4 2 3

Control

40 lg/liter

80 fig/liter

130 pg/liter

0.415 2.39 2.45 2.75 2.14 1.39 1.39 1.13

0.354 2.03 2.34 2.13 2.03 1.26 1.67 1.56

0.443 2.35 I.95 2.41 1.60 I.38 1.65 1.28

0.365 2.99 2.45 2.64 1.47 1.47 1.50 1.94

iVote. Depending on developmental stage. different numbers of individuals were used because of technical reasons.

tration between the highest concentration tested having no observed effects and the next higher toxicant concentration having significant toxic effects(Mount and Stephan, 1967)) of lindane for zebrafish is in the range of 40 (NOEC)-80 pg/liter (LOEC). At 40 pg/liter no adverse effect on embryo survival and on larval-juvenile survival, development, and growth was observed. In fact, survival after 3.5days of exposure to 80 pg/liter did not differ from the controls. but growth of larvae was significantly reduced. Macek et al. (1976a) examined the influence of lindane exposure on reproduction of fathead minnow, brook trout, and bluegill by determining the effects on hatching, mortality, and growth of larvae. No effects were detectable at concentrations of 1.423.4 pg/liter for the fathead minnow. There were effects on the survival of larvae of bluegill, but they could not be related to the lindane concentration in the range of 0.6-4.4 pg/liter. Growth of larvae was also not affected. Likewise, for brook trout no

TABLE

5

EFFECTOFATRAZINEONTHEOXYGENCONSUMPTIONOFDIFFERENTDEVELOPMENTAL STAGESOFZEBRAFISH (pgO,/mg/hr) Age (days) 2 4 7 14 24 30 35

Developmental stage kg Larvae Juvenile Juvenile Juvenile Juvenile Juvenile

Number 30 15 7 4 2 2 2

Control

300 pg/liter

1300 pg/liter

9 100 pg/liter

0.360 2.56 2.25 3.27 1.91 2.16 1.21

0.412 1.56 2.72 2.32 1.97 1.29* 1.26

0.337 2.46 2.06 3.29 1.68 2.22 1.21

0.300 2.29 2.90 3.17 -** -** -**

Nole. Depending on developmental stage. different numbers of individuals were used because of technical reasons. * One individual very apathetic. ** Measurement was finished after Day 14.

CiijRGE AND NAGEI

252

TABLE EFFECT

Age (days)

OFDELTAMETHRIN ONTHEOXYGENCONSUMPTION OFDIFFERENT DEVELOPMENTAL STAGES OFZEBRAFISH (wg O,/mg/hr)

Developmental stage

2

Egg

4 1

Larvae Juvenile Juvenile Juvenile Juvenile Juvenile

IO I7 24 31

6

Number

Control

0.5 fig/litel

0.8 pg/liter

1.20 pg/liter

30 I5

0.317 2.03 2.39 3.26 2.97 1.36 1.81

0.392 2.05 3.24 I.29 2.71 1.68 2.24

0.373 ’ 33 -.3.04 1.37

0.41I

I .07

1.01

3.51 2.03

2.54 2.33

1

4 2 I I

2.4 I

3.18 1.86

R;IW Depending on developmental stage. different numbers of individuals wereusedbecause oftechnical

reasons.

effects on survival and growth were found. It was presumed that hatch of larvae was reduced at concentrations of 16.6 pg/liter and higher. Hirose (1975) reported that hatching of medaka was retarded up to 7 days at 5 to 20 iyg/liter of lindane. In contrast to our findings, subsequently even abnormalities in development appeared in some fish. The results of atrazine experiments indicate that the MATC for zebrafish is between 300 and 1300 pg/liter. Survival and growth of fish exposed to 300 pg/liter were not significantly different from those of control fish. Survival of fish exposed to 1300 pg/ liter was only 46% (mean), and this is distinctly lessthan survival of controls (mean 69%): likewise. the statistical significance is moderate (P < 0.1). The only available study on the chronic toxicity of atrazine to ELS of other fish specieswas conducted aspart of life-cycle testsperformed with fathead minnow, brook trout, and bluegill (Macek e/ nl.. 1976b). There was no effect on hatching rate, mortality, ancl growth of fathead minnow embryos and larvae at concentrations between 15 and 2 13 pg/liter. After 90 days. survival of bluegill was reduced significantly at 49 pg/liter and higher compared to lower concentrations (down to 8 pg/liter). Unfortunately, the survival of the control group was as low as that at concentrations between 49 and 95 pg/liter of atrazine. In the case of brook trout, mortality was increased from 240 pg/ liter of atrazine after 60 days. After 90 days, body length wasalso significantly reduced at concentrations of 240-720 cc.g/litercompared to the body length of control fish and to that of fish exposed to minor concentrations (65-129 kg/liter). No deltamethrin MATC can be reported for the zebrafish becausethere is no NOEC among the tested concentrations. Even at the lowest tested concentration of 0.5 pug/ liter, survival and ontogenesiswere affected. Hatchability of embryos was reduced in a dramatic way at 0.8 pg/liter and higher. There are no available studies on the subchronic toxicity of deltamethrin to the ELS of other fish species. However, there are some results obtained in short-term toxicity tests on eggsof brook trout (Lhoste et al.. 1979) and rainbow trout (Kulzer et t~l., 1986). Exposure to 25 pg/liter of deltamethrin for 24 hr had no effect on the hatching rate and development of brook trout embryos. Even concentrations of 0.25 to 1 mg/liter DECIS (formulated product of deltamethrin) did not reduce the hatching

TOXICITY

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ZEBRAFISH

253

rate of embryos of rainbow and brook trout when they were exposed for a period of 1 hr. Nevertheless, the number of malformations and the mortality rose slightly during ontogenesis compared to those of the controls. Spehar ef al. (1983) reported that concentrations up to 1.4 pg/liter of permethrin had no effect on hatching rate nor on ontogenesis of embryos and larvae of fathead minnow during 5 weeks. Only survival of fish exposed to 1.4 pg/liter was significantly less than survival of the controls. Similar results were achieved by Curtis and Seim ( 1985) with fenvalerate at the ELS of steelhead trout. The hatching rate was not affected at concentrations between 0.02 and 0.5 yglliter. Three weeks later, the mortality of larvae rose and weight declined at concentrations of 0.5 pg/liter. After 70 days all fish at 0.5 pg/liter had died and mortality was significantly elevated even at 0.14 pg/liter. It is evident from the reported data that the early life stages of zebrafish are sufficiently sensitive to demonstrate detrimental effects of xenobiotics. With regard to the lack of corresponding data. it is not possible to draw a comparison between the sensitivity of the early life stages of zebrafish and that of other fish species. The hatching rate seems to be quite an insensitive parameter. Among the compounds tested. only deltamethrin caused detectable effects. However, this effect was observed at a range of concentrations beyond the LCso of ELS. Similar results were obtained with various fish species and a wide range of xenobiotics. With special regard to zebrafish, Niimi and LaHam (1975) stated that hatching rate is not a reliable toxicological parameter because of the short lifetime of embryos within the eggshell, lasting only 4 to 5 days. The significance of malformations during ontogenesis as indications of the danger of xenobiotics is also problematic in the case of zebrafish. The spontaneous rate of malformations in control groups varies over a wide range and renders it difficult to prove the statistical significance of observed effects. Nevertheless, every hint of a possible potential of a xenobiotic to cause morphological or teratogenic aberrations must be considered. Assessment of the parameter “growth” in fish early life stage tests is questionable, too. The test concept does not include the conditions necessary to achieve valid results. Feeding and available space must be controlled carefully and adapted to the size and the number of fish in each vessel. This implies an emphasis on time and expense for the ELS test. Therefore, investigations of the influence of toxicants on growth should be left to specific tests (Woltering, 1984). Oxygen consumption seems to be the most insensitive parameter of all, since none of the tested pesticides affected the oxygen consumption of any developmental stage (embryo, yolk sac fry, larvae. juvenile). Measurement of oxygen consumption results from the assumption that toxic influences of xenobiotics affect the activity of metabolism and are attendent with that of the O2 consumption. Murty (1986) has reported stimulation of oxygen uptake induced by sublethal concentrations of organochloro compounds, whereas lethal concentrations result in the opposite phenomenon. lncreasing concentrations of organophosphates are suspected of effecting a continual reduction of oxygen consumption. Nevertheless, itz riro experiments often become problematic, because the ventilation rate is elevated as a result of the stress situation imposed by the experimental procedure. Thus. it follows that if the procedures are not refined. one must expect faulty findings in general (Hughes, 1985). It can be concluded that fry mortality is the clearest response to xenobiotic exposure. Embryos are moderately sensitive to toxic effects of pesticides. Susceptibility to toxicants increases during larval development and it seems that the most critical stage of de-

254

GijRGE

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NAGEI

velopment is the late yolk sac fry stage, when the change to external nutrition occurs. This is supported by 173 toxicity tests in fish which show that fry survival is the most sensitive response (Woltering, 1984). The toxicological data of zebrafish early life stages should be compared to corresponding data obtained from adult zebrafish. When the L&/35 day of ELS are compared to acute toxicity data ( LC,0/96 hr) of adult zebrafish, the differences in sensitivity can be judged: ELS react to lindane in a less sensitive manner than adults ( 118 versus 75 pg/liter (Kuhnen-Clausen, 1984)), whereas to atrazine (1300 versus 37,000 pg/liter (Korte and Greim, 1981)) and deltamethrin (0.5 versus 2 pg/liter (L’Hotellier and Vincent, 1986)) fry react in a more sensitive manner. Comparison to results from prolonged fish tests (14 days) with adult zebrafish shows no difference. Survival of adults exposed to 53 pg/liter of lindane and to 20.000 pg/liter of atrazine was significantly reduced (Friesel et al., 1984). No comparable test with adult zebrafish exposed to deltamethrin is available. CONCLUSION It can be concluded that in reference to the toxicity of the tested pesticides, the early life stagesof zebrafish react in a manner at least assensitive as that of the adults. Indeed, it is feasible in the course of an early life stagetest to obtain much information about the impact of xenobiotics which goesbeyond the findings obtainable in an acute toxicity test. ACKNOWLEDGMENT This work

was supported

by the Umweltbundesamt

(UBA)

Berlin.

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3. 171-182.

WILLIAMS, D. A. ( 197 I). A test for differences between treatment means when severaldose levels are compared wrth zero dose control. BiotwIr~cs 27, IO3- I 17. WOI TERING, D. M. (1984). The growth response in tish chronic and early life stage toxicity tests: A critical review. .&/rrmt. To.vico/. 5, I-1 1.