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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:
04 March 2016 to 15 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 236 (Fish embryo acute toxicity (FET) test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: duplicate test media samples from all test concentrations and duplicate control samples from both sampling times
- Sampling method: One sample of the freshly prepared stock solution and duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test. Duplicate samples from the test media of all test concentrations and the control were collected at the end of the test, after 96 h (replicates pooled to obtain sufficient volumes). All samples were diluted by a factor of 2 with acetonitrile.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest nominal test concentration of 100 mg/L test substance was prepared by dissolving 25.8 mg of the test item in 258 mL of test water by intense stirring for 20 min and ultrasonic treatment for 5 min. The pH of the solution was adjusted from 6.8 to7.3 with 1 M NaOH. Appropriate volumes of this solution were then mixed into test water to give the remaining test concentrations.
- Controls: Reconstituted water (ISO Medium)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Not reported
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish (Danio rerio)
- Sex: Male and female
- Source: In-house breeding
- Age at study initiation: The cell stages of the embryos introduced at test start was 2 to 8
- Quality of the Eggs (fertilisation rate): The fertilisation rate of the egg batch was 100 %, determined from a sample size of 400 eggs.

ACCLIMATION: No

FEEDING DURING TEST: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
none
Hardness:
231 – 285 mg/L as CaCO3
Test temperature:
25.8 – 26.0 °C
pH:
7.3 - 7.7
Dissolved oxygen:
96 - 101%
Conductivity:
558 – 612 µS/cm
Nominal and measured concentrations:
- Nominal concentrations: 100, 45.5, 20.7, 9.4 and 4.3 mg/L
- Measured concentrations: At the start of the test 111% of the nominal test concentration was found (average of all test concentrations). After 4 days test duration, 116% of the nominal value was determined (average of all test concentrations). During the test the fish embryos were exposed to a mean of 114% of nominal test concentrations. Reported results refer to nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 24 well plate
- Fill volume: ≥ 2 mL test medium
- Aeration: No
- No. of organisms per vessel: 1 egg per well, 24 eggs per well plate
- No. of vessels per concentration (replicates): 1 well plate, 20 eggs
- No. of vessels per negative control (replicates): 1 well plate, 24 eggs
- No. of vessels per positive control (replicates): 1 well plate, 20 eggs
- Internal plate control: 4 eggs in test water on each plate for each test concentration and positive control

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium
- Composition (solution in deionised water):
CaCl2 × 2H2O : 2.0 mmol/L (= 294.0 mg/L)
MgSO4 × 7H2O : 0.5 mmol/L (= 123.0 mg/L)
NaHCO3 : 0.75 mmol/L (= 65.0 mg/L)
KCl : 0.075 mmol/L (= 5.8 mg/L)
- Alkalinity: 0.8 mmol/L
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: Not reported
- Intervals of water quality measurement: pH, dissolved oxygen concentration, conductivity and water hardness measured at the beginning and end of the test

OTHER TEST CONDITIONS
- Adjustment of pH: Not reported
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 582 to 642 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Apical observations: Coagulation, lack of somite formation and non-detachment of the tail recorded at test start and after 24, 48, 72 and 96 h. Lack of heartbeat was recorded after 48, 72 and 96 h.
- Hatching: Recorded after 48, 72, and 96 hours.
Reference substance (positive control):
yes
Remarks:
4.0 mg 3,4 Dichloroaniline/L
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Basis for effect:
coagulation of the embryo
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Basis for effect:
coagulation of the embryo
Details on results:
- Biological observations: Up to 45.5 mg/L no fish embryo died or showed any of the four apical observations (lack of heartbeat, coagulation, lack of somites, non-detachment of the tail from the yolk sac) after 96 hours. At the highest tested concentration, 100 mg/L, three embryos were coagulated after 96 hours, though this was not calculated to be statistically different from the control treatment.
- Mortality of control: 4%
- Other adverse effects control: None reported
- Abnormal responses: None reported
- Any observations (e.g. precipitation) 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):
- Results with reference substance valid? Yes
- Mortality: 95%
- LC50: Not derived, as only one concentration was tested
- Other: Hatching success was 5%
Reported statistics and error estimates:
The 96-hour LC50 the corresponding LC20 and LC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis. The 96-hour LOEC and the 96-hour NOEC, were determined by Chi2 2x2 table test with Bonferroni correction. The software used to perform the statistical analysis was ToxRat Professional (Version 3.2.1, ToxRat Solutions GmbH).
Sublethal observations / clinical signs:

The mortality and apical observation data and estimated LC50 values are shown in the table below:

Effects of the test substance on the growth and survival of Danio rerio embryos

Nominal concentration (mg/L)

Hatching success (%)

Mortality and apical observations (%)

 

48 h

72 h

96 h

24 h

48 h

72 h

96 h

Control

0

13

96

0

0

4*

4*

4.3

0

20

100

0

0

0

0

9.4

0

50

100

0

0

0

0

20.7

0

70

100

0

0

0

0

45.5

0

75

95

0

0

0

0

100

0

65

85

15**

15**

15**

15**

96-hour LC50(mg/L)

>100

95% confidence interval

n.d.

96-hour NOEC (mg/L)

≥ 100

* One coagulated fish embryo observed in the control

** Three coagulated embryos observed at 100 mg/L

n.d. = not determined

Validity criteria fulfilled:
yes
Conclusions:
Based on nominal concentrations, the 96-hour LC50 for the test substance to zebrafish (Danio rerio) embryos was > 100 mg/L and the 96-hour NOEC was 100 mg/L.
Executive summary:

The acute toxicity of the test substance to embryos of zebrafish (Danio rerio) was determined under static conditions according to OECD 236 (2013) Fish Embryo Acute Toxicity Test. Fish embryos were exposed to a range of nominal concentrations of 4.3, 9.4, 20.7, 45.5 and 100 mg/L, alongside a test water control. The following endpoints were recorded as indicators of acute lethality in fish embryos: the coagulation of the embryo, lack of somite formation, the non-detachment of the tail-bud from the yolk sac and the lack of heartbeat. Furthermore, the hatching rate was recorded.

At 4.3 mg/L test substance and up to and including the concentration containing 45.5 mg/L of test substance no fish embryo died or showed any of the four apical observations (lack of heartbeat, coagulation, lack of somites, non-detachment of the tail from the yolk sac) after 96 hours. In the control one fish embryo coagulated after 96 hours and in the highest test substance concentration of 100 mg/L of test substance three embryos were coagulated after 96 hours, though this was not calculated to be statistically different from the control treatment. Hatching success in the control was 96%. At 4.3 mg/L of test substance up to and including 20.7 mg/L hatching success was 100%. At 45.5 mg/L of test substance hatching success was 95% and at 100 mg/L 85%.

Based on nominal concentrations, the 96-hour LC50for the test substance to zebrafish (Danio rerio) embryos was > 100 mg/L and the 96-hour NOEC was 100 mg/L.

Description of key information

The acute toxicity of the test substance to embryos of zebrafish (Danio rerio) was determined in the fish embry acute toxicity test. Fish embryos were exposed to a range of nominal concentrations of 4.3, 9.4, 20.7, 45.5 and 100 mg test substance, alongside a test water control. Based on nominal concentrations, the 96 hour LC50 was >100 mg test substance.

Key value for chemical safety assessment

Additional information