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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:
16 Nov - 20 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, 3.75, 7.50, 15.0, 30.0, 60.0 mg/L
- Sampling method: Samples of each concentration were taken at the beginning and at the end of the study.
- Sample storage conditions before analysis: If necessary the samples were stored at 6 ± 2 °C until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Appropriate amounts of the test substance were added directly to the test vessels. The test media were stirred for 1 min at 17000 rpm with an ultra turrax.
- Eluate: no
- Differential loading: yes
- Controls: yes, test medium control
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Method of breeding: Holding was performed at the test facility at 23 ± 2 °C and diffuse light. The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine. Food was provided three times a week at an amount of 4% of the fish body weight per feeding day. Fish were not fed 24 h prior to test start (Sera vipan, Sera GmbH, Heinsberg, Germany).
- Feeding during test: none

ACCLIMATION
- Acclimation period: at least 12 d
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): < 5%
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
66 mg CaCO3/L
Test temperature:
22.3 - 23.0 °C
pH:
7.04 - 7.63
Dissolved oxygen:
94 - 100% oxygen saturation
Nominal and measured concentrations:
nominal: Control, 3.75, 7.50, 15.0, 30.0, 60.0 mg/L
measured 0 h: < LOQ, 3.12, 6.95, 14.3, 27.5, 54.8 mg/L
measured 96 h: < LOQ, 2.53, 6.17, 12.9, 24.5, 51.9 mg/L (last concentration analysed on 17 Nov 2015 due to 100% mortality)
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria of 64 L, fill volume: 40 L, covered with a floating lid
- Aeration: no
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.03 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: same as test
- Intervals of water quality measurement: measured at the start and every 24 h during test

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 8.5 h light / 15.5 h dark
- Light intensity: 0.1 - 10 µmol photons *m-2*s-1 (approx. 7-750 lux)

EFFECT PARAMETERS MEASURED
- Mortality and behavioural changes were observed after 2, 24, 48, 72 and 96 h
- Size and weight was determined at the end of the study.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 6 and 60 mg/L; testing the higher concentration was planned as a definitve test using the threshold approach.
- Results used to determine the conditions for the definitive study: At 6 mg/L no mortality was recorded whereas 100% mortality was recorded at 60 mg/L after 24 h.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
42.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CI: 30 - 60 mg/L
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
60 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: At 15 mg/L all fish missed the escape reflex. At 30 mg/L four fish were on lateral position, three fish were staggering and seven fish hyperventilated after 96 h. At 60 mg/L all fish were staggering and hyperventilated after 2 h, followed by exitus lethalis after 24 h.
- Observations on body length and weight: Average body length was 2.41 cm and 0.173 g after 96 h.
- Mortality of control: 0%
Reported statistics and error estimates:
The data obtained were inadequate for the use of non linear regression models. The highest concentration caused 100% mortality, the next lower concentration caused no mortality. The geometric mean of these two concentrations was used as LC50.

Table 1: Cumulative mortality [%]

Nominal test concentration [mg/L]

Cumulative mortality [%]

2 h

24 h

48 h

72 h

96 h

60

0

100

100

100

100

30

0

0

0

0

0

15

0

0

0

0

0

7.5

0

0

0

0

0

3.75

0

0

0

0

0

Control

0

0

0

0

0

Validity criteria fulfilled:
yes
Conclusions:
This study resulted in a LC50 (96 h) of 42.4 mg/L based on mortality of D. rerio (OECD 203, GLP).

Description of key information

LC50 (96 h): 42.4 mg/L based on mortality of D. rerio (OECD 203).

Key value for chemical safety assessment

LC50 for freshwater fish:
42.4 mg/L

Additional information

One experimental study is available investigating the toxicity of the substance to freshwater fish. The study was conducted according to OECD 203 and GLP under static conditions with Danio rerio as test species. Prior to the definitive test the threshold approach was followed by conducting a limit test with a concentration of 60 mg/L. Since all fish died after 2 h of exposure an additional range-finding test followed by the definitive test with concentrations of 3.75, 7.50, 15.0, 30.0, 60.0 mg/L were conducted. The test concentrations were analysed by HPLC-DAD at the beginning and the end of exposure. Since they remained within 80-120% of nominal for the relevant concentrations the effect concentrations were based on the nominal values. After 96 h 100% mortality was observed at 60 mg/L whereas 0% mortality was recorded in the remaining concentrations. The LC50 (96 h) was calculated at 42.4 mg/L (nominal). The LC50 was calculated using the geometric mean of 30 and 60 mg/L since no non-linear regression methods could be used.