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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:
29-7 to 2-8-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
- To avoid volatility effects the test was carried out without aeration. - Temperature fluctuation exceeded 1 °C --> range 20-24°C. -oxygen content dropped below 60% twice (58 and 56 %)
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
yes
Remarks:
as above
Qualifier:
according to guideline
Guideline:
other: OECD 23: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Oxiteno Europe BVBA
- Purity test date: 99.3 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Examined several times by chemical analysis (GC) in the freshly prepared media and in the media after 24 hours of exposure
Analytical monitoring:
yes
Details on sampling:
- Sampling method: analyzed every 24 h
- Sample storage conditions before analysis: immediately analysed
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: To produce the test item concentrations, the calculated amounts of the test item were pipetted every day to the glass aquaria holding 5 L of dilution water. All solutions were then stirred for 0.5 h on magnetic stirrers.

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Source: Interaquaristik.de Shop (Germany)
- Length at study initiation: mean 3.97 cm (S.D. = 0.22 cm)

ACCLIMATION
- Acclimation period: stock fish acclimated for 4 months
- Acclimation conditions: same as test
- Type and amount of food during acclimation: Commercial fish food, daily. Feeding discontinued 24 h prior to test start.

FEEDING DURING TEST: no feeding
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
not applicable
Hardness:
13.9 °dH (= 248 mg/L CaCO3)
Test temperature:
20 - 24 °C
pH:
7.4-8
Dissolved oxygen:
5.3-9 mg/l
Salinity:
not applicable
Conductivity:
no data
Nominal and measured concentrations:
nominal concentrations: 1.9, 4.3, 9.4, 20.7, 45.5 and 100 mg/L
measured concentration at 96h (mean): 1.095, 2.679, 6.230, 7.394, 39.967, 100.594 mg/L. Effective concentrations ranged from 22.8 % to 111.0 % of nominal values in the freshly prepared media and from 7.9 % to 98.2 % of nominal values in the media after 24 hours of exposure according to the semi-static test conditions.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass aquaria holding 5 L of test media covered by glass plates
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): every 24h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.8 g body weight (wet weight) per litre

- Intervals of water quality measurement: every 24 h pH, temperature and dissolved oxygen

OTHER TEST CONDITIONS
- Photoperiod: 16 h light: 8 h dark

EFFECT PARAMETERS MEASURED: mortality, observation on mortality, incidence of sub-lethal effects compared with controls every 24h

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, all fish were dead at the highest test concentration of 100 mg/L while no dead fish at 10 and 1 mg/L. Since degradation of the test item was observed in the dose range finder, a semi-static test set-up has been used for the main study.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
11.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
2 h
Dose descriptor:
LC100
Effect conc.:
14.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
24 h
Dose descriptor:
LC100
Effect conc.:
10.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
48 h
Dose descriptor:
LC100
Effect conc.:
16.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
11.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Mortality of control: 14.3 % after 96h

Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The LC50 was calculated by probit analysis using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10).
Sublethal observations / clinical signs:

Geometric mean of measured concentrations over time

Date

Exposure time

Measuring time

Test item concentration [mg/L]

1.9

4.3

9.4

20.7

45.5

100

2019-07-29

0 h

0 h

1.129

3.863

8.698

4.718

42.791

103.008

2019-07-30

24 h

24 h

0.522

1.939

3.921

1.856

37.329

98.236

0 h

2.029

4.047

9.248

22.975

 

 

2019-07-31

48 h

24 h

1.085

1.988

4.396

11.393

 

 

0 h

2.025

2.909

8.768

21.597

 

 

2019-08-01

72 h

24 h

0.913

2.072

3.983

5.315

 

 

0 h

1.661

4.082

9.585

20.830

 

 

2019-08-02

96 h

24 h

0.520

1.791

4.892

1.630

 

 

Results

Geometric mean [mg/L]

0 h

1.129

3.863

8.698

4.718

42.791

103.008

 24 h

0.768

2.737

5.840

2.959

39.967

100.594

 48 h

1.067

2.786

6.102

6.919

39.967

100.594

72 h

1.157

2.671

6.037

8.005

39.967

100.594

96 h

1.095

2.679

6.230

7.394

39.967

100.594

Mortality data (cumulative)

Mortality

(initial population: 7)

2 h **

24 h

48 h

72 h

96 h

Control

0

0

1/7 (injured)

1/7

1/7

1.9 mg/L

0

0

0

0

0

4.3 mg/L

0

0

0

0

0

9.4 mg/L

0

0

0

0

0

20.7 mg/L

0

0

0

1/7 (fish removed due to moribund behaviour)

1/7

45.5 mg/L

7/7

 7/7

 7/7

 

7/7

 

 7/7

100 mg/L

7/7

7/7

7/7

7/7

 7/7

Validity criteria fulfilled:
yes
Conclusions:
Short-term exposure of zebra fish to the test item determined in a semi-static test. The EC50 for mortality based on mean measured concentration is 11.1 mg/L.
Executive summary:

A study was conducted exposing the fish, Danio rerio, to the test item for 96 h. The study as performed following the OECD guideline 203 and fulfilled the validation requirements. Measured concentrations confirmed that test item in the exposure concentrations from 22.8 % to 111.0 % of nominal values in the freshly prepared media and from 7.9 % to 98.2 % of nominal values in the media after 24 hours of exposure according to the semi-static test conditions. Results determined an EC50 for mortality based on mean measured concentration of 11.1 mg/L.

Description of key information

A key study was conducted exposing the fish, Danio rerio, to the test item for 96 h (Neuhahn, 2019). The study as performed following the OECD guideline 203 and fulfilled the validation requirements. Measured concentrations confirmed that test item in the exposure concentrations from 22.8 % to 111.0 % of nominal values in the freshly prepared media and from 7.9 % to 98.2 % of nominal values in the media after 24 hours of exposure according to the semi-static test conditions.Results determined an EC50 for mortality based on mean measured concentration of 11.1 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
11.1 mg/L

Additional information

Two other supporting studies are available:

1) Juhnke and Lüdemann (1978):

The differences for the results of LC0, LC50 and LC100 (48 h) delivered by the two research institutes were not commented in the scientific article (a reason for this is possibly that the scientific article contains results for 200 different substances and it would be an extensive task to comment each one of them). There is no info given, if the origin of the fish used by the two institutes was the same (this could be reason for observed differences). In general, for some of the 200 substances the results also appear to show a great variation, whereas for others the results are not greatly different. Based on this data, the NOEC (48 h) may be < 26 mg/L and the LC50 (48 h) 36 mg/L.

2) Munk, Kirsch and Gelbke (BASF) (1993):

The data was generated with an official test method (OECD TG 203); good documentation of methodology and results. The results are therefore regarded as meaningful: LC50 (96 h): > 22 and < 46 mg/L; NOEC (96 h) = 21.5 mg/L