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Ecotoxicological information

Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2004-06-04 to 2004-12-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from all concentrations: 0 (control), 1, 1.8, 3.2, 5.6 and 10 mg/L
- Sampling method: Samples were measured at the start and at the end (96 h) of the test.
- Sample storage conditions before analysis: at room temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium (reconstituted water and test material) was freshly prepared. A stock preparation with a test material concentration of 100 mg/L was prepared. Therefore, the calibrated flask with test material and vehicle, reconstituted water, was stirred with a magnetic stirrer for one hour. The stock preparation was diluted with reconstituted water to the different test media concentrations.
- Sampling: Samples were taken from all concentrations. After preparation of the test media the analytical values were determined. The maintenance of the test material concentrations was confirmed by determination of further samples after a period of 96 hours.
- Controls: Blank control performed with the test solution without the test substance.
- Evidence of undissolved material: No
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Source: West-Aquarium Institute of Toxicology, Merck KGaA, Germany

ACCLIMATION
- Acclimation period: > 14 days
- Acclimation conditions: Same as the test. The zebra fish were acclimatized to the conditions of the laboratory and were kept in reconstituted water.
- Type and amount of food during acclimation: The zebra fish were fed with a commercial flake diet, JBL Novo Bel® (JBL GmbH & Co. KG, Neuhofen). Furthermore the fish got daphnia and frozen mosquitolarva
- Feeding frequency during acclimation: 2-3 times a week.
- Health during acclimation: No mortality was observed.

FEEDING DURING TEST
No feeding was performed during die test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
n.a
Test temperature:
24 to 26 °C
pH:
7.56 - 7.91
Dissolved oxygen:
The dissolved oxygen concentrations were > 60 % oxygen saturation throughout the study.
Salinity:
n.a.
Conductivity:
n.a
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 1, 1.8, 3.2, 5.6 and 10 mg/L
- Measured concentrations: The analytically determined concentrations were 1.0, 1.7, 3.1, 5.5 and 9.7 at the start of the test and 0.9, 1.6, 3.0, 5.3 and 9.5 at the end of the test. The measured concentrations were determined between 94 - 100 % at the start and 89 - 95 % at the end of the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass aquaria
- Type: open
- Aeration: yes
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstitued water according to OECD guideline 203 (1984)
- Culture medium different from test medium: No
- Intervals of water quality measurement: The temperature was measured continuously during the test. pH- values and oxygen concentrations were measured after 0, 24, 48, 72 and 96 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 hours light - 12 hours dark regime

EFFECT PARAMETERS MEASURED: The fish were observed for signs of toxicity or death after 24, 48, 72 and 96 hours,

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study : not performed
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
7.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % C.L. (6.0 -9.4 mg/L)
Details on results:
- Observations on body length and weight: not observed
- Behavioural abnormalities: yes. Signs of toxicity were seen in the concentrations 3.2, 5.6 and 10.0 mg/L. They started in the higher concentrations after 3 hours and lasted up to the end of the experimental part . These consisted of absence of escape reflex, swimming slightly irregular, or at the water surface, or even at the bottom of the aquarium, fish were lying on the side/back, showed temporary restlessness, and accelerated respiration.
- Mortality of control: no mortality occurred in the control test.
- Other adverse effects control: not observed
- Abnormal responses: yes
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
not performed

Table 1: Nominal and corresponding analytical concentrations in % during the study

Nominal concentration
[mg/l]

Analytical concentration in % during the test

Start

96 h

1.0

100

90

1.8

94

89

3.2

97

94

5.6

98

95

10.0

97

95

 

  

Table 2: pH values and oxygen concentrations

Nominal concentration [mg/l]

pH value

O2 [%]

0 h

24 h

48 h

72 h

96 h

0 h

24 h

48 h

72 h

96 h

Control

7.86

7.58

7.71

7.69

7.58

94.7

82.4

78.8

85.4

82.0

1.0

7.83

7.56

7.72

7.67

7.59

95.3

88.7

84.9

83.8

88.0

1.8

7.84

7.61

7.69

7.67

7.60

94.9

89.7

85.9

83.9

85.6

3.2

7.85

7.63

7.64

7.60

7.61

95.7

86.6

83.3

81.9

84.2

5.6

7.86

7.64

7.64

7.60

7.60

93.7

78.1

72.4

73.0

78.9

10.0

7.91

7.66

--

--

--

94.8

61.2

--

--

--

 

 

 Table 3: Nominal concentrations and number of deaths/exposed fish

Nominal concentration [mg/l]

Dead / exposed fish

24 h

48 h

72 h

96 h

Control

0 / 7

0 / 7

0 / 7

0 / 7

1.0

0 / 7

0 / 7

0 / 7

0 / 7

1.8

0 / 7

0 / 7

0 / 7

0 / 7

3.2

0 / 7

0 / 7

0 / 7

0 / 7

5.6

0 / 7

0 / 7

0 / 7

0 / 7

10.0

7 / 7

7 / 7

7 / 7

7 / 7

 

 

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test substance to Danio rerio was determined by a static test. Under the given conditions of this study the 96 h LC50 value for fish was in the range of 1 to 10 mg/L (7.5 mg/L). and the EC0 was < 1.8 mg/L.
Executive summary:

The objective of this study was to determine the acute fish toxicity of the test material using zebra fish (Danio rerio) according to OECD 203 (1992) under GLP conditions. For this purpose, 7 zebra fish per test material group and further 7 zebra fish in a control group were exposed over 96 hours, under defined conditions. The fish were observed for signs of toxicity or death. Zebra fish (Danio rerio) were exposed to aqueous test material solutions of nominal 1.0, 1.8, 3 .2, 5.6, and 10.0 mg/L in an open static system. Samples were taken from all concentrations. The samples were analyzed using HPLC with UV-detection and external calibration for quantification. The maintenance of the test material concentrations was confirmed by determination of further samples after a period of 96 hours. The analytically determined concentrations were between 94- 100 % at the start and 89 - 95 % at the end. During the experimental phase of the study, the test material concentrations could be maintained at > 80 % of the initial concentrations. Therefore, the LC50 values had to be calculated with the nominal concentrations. No signs of toxicity were seen in the control group and in the zebra fish exposed to concentrations of 1.0 and 1.8 mg/L. Signs of toxicity were seen in the concentrations 3.2, 5.6, and 10.0 mg/L. They started in the higher concentrations after 3 hours and lasted up to the end of the experimental part. These consisted of absence of escape reflex, swimming slightly irregular, or at the water surface, or even at the bottom of the aquarium, fish were lying on the side/back, showed temporary restlessness, and accelerated respiration. Deaths occurred only in the highest concentration, after an exposure period of about 6-23 hours. Under the given conditions of this study the 96 h LC50 value for fish was in the range of 1 to 10 mg/L (7.5 mg/L) and the EC0 was < 1.8 mg/L.

Description of key information

The acute toxicity of the test substance to Zebra fish (Danio rerio) was determined by static test according to OECD 203 under GLP conditions. The LC50 was determined to be 7.5 mg/L (reference 6.1.1-1).

Key value for chemical safety assessment

LC50 for freshwater fish:
7.5 mg/L

Additional information

The objective of this study was to determine the acute fish toxicity of the test material using zebra fish (Danio rerio) according to OECD 203 (1992) under GLP conditions. For this purpose, 7 zebra fish per test material group and further 7 zebra fish in a control group were exposed over 96 hours, under defined conditions. The fish were observed for signs of toxicity or death. Zebra fish (Danio rerio) were exposed to aqueous test material solutions of nominal 1.0, 1.8, 3.2, 5.6, and 10.0 mg/L in an open static system. Samples were taken from all concentrations. The samples were analyzed using HPLC with UV-detection and external calibration for quantification. The maintenance of the test material concentrations was confirmed by determination of further samples after a period of 96 hours. The analytically determined concentrations were between 94 - 100 % at the start and 89 - 95 % at the end. During the experimental phase of the study, the test material concentrations could be maintained at > 80 % of the initial concentrations. Therefore, the LC50 values had to be calculated with the nominal concentrations. No signs of toxicity were seen in the control group and in the zebra fish exposed to concentrations of 1.0 and 1.8 mg/L. Signs of toxicity were seen in the concentrations 3.2, 5.6, and 10.0 mg/L. They started in the higher concentrations after 3 hours and lasted up to the end of the experimental part. These consisted of absence of escape reflex, swimming slightly irregular, or at the water surface, or even at the bottom of the aquarium, fish were lying on the side/back, showed temporary restlessness, and accelerated respiration. Deaths occurred only in the highest concentration, after an exposure period of about 6 - 23 hours. Under the given conditions of this study the 96 h LC50 value for fish was in the range of 1 to 10 mg/L (7.5 mg/L) and the EC0 was < 1.8 mg/L.