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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance OECD 203 (Jul-1992) guideline without deviation. The substance was a multi-constiuent of varying solubilities and therefore a WAF approach has been used in accordance with OECD Series on testing and assessment No. 23. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
July 1992
Deviations:
no
Principles of method if other than guideline:
- Principle of test: Water accomOdated fraction (WAF) was used in accordance with OECD guidance on aquatic toxicity testing of difficult substances and mixtures (Document no. 23, 2002).
- Short description of test conditions: Each concentration is prepared sepArately, whereby the test item is added to the medium at a specific loading rate. The material is left until fully dissolved (or as dissolved as possible), then particulate matter is left to settle out of the water column. The remaining substance in the water column of the sample is transferred to the test vessels. This is done at each concentration. The concentration-response relationship is based on the initial loading rate of the vessel.
- Parameters analysed / observed: Tyndall's effect is monitored to ensure minimal particulate matter is transferred to the test system. Further, chemical analysis is conducted to confirm that the correct loading has occurred, presence of the test item and to confirm there is a series of varying concentrations.
- although the study was conducted in accordance with the OECD guideline 203 (Fish acute toxicity test, 1992) as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.  However, during study performance a deviation to the study design was noted. Here only the 100 mg/L test loading rate was prepared in accordance with the WAF strategy. All other concentrations were made by serial dilution, even though weighing will have been possible. As no effects were seen at the limit dose which was not prepared by serial dilution, i.e. in accordance with OECD series on testing and assessment no. 23, the study is considered valid and the deviation has not impacted the integrity of the study as a limit test.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: confidential
- Expiration date of the lot/batch: 01 Jan 2019
- Purity test date: N/D

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry, dark, cool (2-8 °C)
- Stability under test conditions: sufficient for the test purpose
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: N/A
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The test was conducted with a semi-static design with renewal of test medium every 24 hours. All tested concentrations were analysed at 0 fresh, 24 hours fresh and aged, 48 hours fresh and aged and 72 hours fresh and aged, 96 hours fresh and aged. The concentrations were 0.0, 4.64, 10.0, 21.5, 46.4 and 100 mg/L. There was only one replicate per test item concentration.
- Sampling method: direct from vessel using pipette
- Sample storage conditions before analysis: All samples were stored deep frozen and stabilised with formic acid until they were transferred to the analytical laboratory.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accomadated fractions were used as per the OECD guidance on aquatic toxicity testing of difficult substances and mixtures (Document no. 23, 2002) at the highest concentration of 100 mg/L (the guideline and regulatory limit). The prerequisite amount of test item (200 mg) was added to the test vessel containing the test medium 2000 mL of test medium for the concentration of 100 mg/L and the stock for the serial dilutions. The stock was stirred for 24 h in the dark at room temperature. The stock solution was then allowed to settle to allow non-dissolved test item to sediment or float to the surface for 20 hours, at this point no undissolved material was found by eye assessment and using the Tyndall effect. After this the necessary volumes for the test were withdrawn via a Teflon tube from the middle level of the stock solution. Each successive concentration was then prepared by serial dilution. The serial dilutions produced concentrations of 4.64, 10.0, 21.5 and 46.4 mg/L. Fish were added to each solution individually with 7 fish at each concentration. Approximately 1 L of the prepared solutions were transferred to each test vessel. See table 1 for details on test preparations.
- Eluate: N/A
- Differential loading: N/A
- Controls: The test medium reconstituted test water consisting of analytical grade salts dissolved in purified water. The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity with a conductivity of 665 µS/cm. In line with the OECD 203 (1992) guideline.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The presence of undissolved material was erradicted from the test sytem by stirring for 24 hours and then settling for 20 hours.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The species used for the test was Danio rerio Hamilton (Cypriniformes: Cyprinidae). This species is a recommended sensitive indicator species for testing the side effects of chemicals.

Fish from the laboratory stock were used.

Originally, the animals were purchased from Fraunhofer IME (Schmallenberg, Germany) in a healthy condition. According to IME, they had previously been bred from a wild strain of zebra fish. Since they have been purchased, the zebra fish are being continuously reared in the laboratory of the testing facility. Only fish in good health and free from any apparent malformation were used. The last feeding was performed two days prior to test start.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
guideline and regulatory recommended duration for acute fish toxicity test with this species.
Post exposure observation period:
none
Hardness:
13°dH corresponding to 232 mg CaCO3/L
Test temperature:
22.9 – 23.7 °C
pH:
7.10 – 8.48
Dissolved oxygen:
≥ 74 % of air saturation
Salinity:
N/A - freshwater
Conductivity:
665 µS/cm
Nominal and measured concentrations:
0.00, 4.64, 10.0, 21.5, 46.4 and 100 mg/L.

Test was performed using loading rates and it was only possible to measure one analyte, therefore measured concnetrations of the entire substance were not possible. For the measured analyte the measured concentrations were between 99 to 111 %.
Details on test conditions:
TEST SYSTEM
- Test vessel: 2 L glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Filled with 1 L of medium
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): every 24 hours
- No. of organisms per vessel: 7fish
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): Nn/A
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water
- Total organic carbon: N/D
- Particulate matter: None
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity:N/D
- Ca/mg ratio: 4:1
- Na:K ratio: 10:1
- Culture medium different from test medium: No
- Intervals of water quality measurement: Hardness at 0 hours. All others at 0 hours and every 24 hours thereafter, for the aged and fresh preparations.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours with 30 minute transition
- Light intensity: N/D

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality to fish

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ~2.16
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study : yes
- Test concentrations: 0, 0.04, 0.4, 4 and 40 mg/L using a WAF approach.
- Results used to determine the conditions for the definitive study: yes, no mortality observed.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: There was slight reduced activity in two fish at 100 mg/L, however, this was transient and did not last over 4 hours.
- Observations on body length and weight: The mean body weight of the fish were 0.0954, 0.0809, 0.0952, 0.0910, 0.1302 and 0.1052 at 0, 4.64, 10.0, 21.5, 46.4 and 100 mg/L.
- Other biological observations: none
- Mortality of control: None
- Other adverse effects control: None
- Abnormal responses: Only behavioural in the top concentration, but not biologically significant.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: water accomodated fraction
- Effect concentrations exceeding solubility of substance in test medium: None seen.

Furthermore, the data on the measured analyte showed sufficient stability in the test system and showed suitable dosing at each concentration (Table 7).
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
Not applicable as no effects were seen.

Table 3 % mortality at each test item concentration at x hours

Test item loading rate [mg/L]

Control

4.64

10.0

21.5

46.4

100

Time [h]

Mortality [%]

4

0

0

0

0

0

0

24

0

0

0

0

0

0

48

0

0

0

0

0

0

72

0

0

0

0

0

0

96

0

0

0

0

0

0

Table 4 Sub-lethal observations in fish during 96 hours exposure to the test item

Test item loading rate.

Control

4.64

10.0

[mg/L]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time [h]

0

°

*

#

+

0

°

*

#

+

0

°

*

#

+

0

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

4

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

24

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

48

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

72

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

96

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

Test item loading rate

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

21.5

46.4

100

[mg/L]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time [h]

0

°

*

#

+

0

°

*

#

+

0

°

*

#

+

0

7

0

0

0

0

7

0

0

0

0

5

2

0

0

0

4

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

24

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

48

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

72

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

96

7

0

0

0

0

7

0

0

0

0

7

0

0

0

0

0 = no clinical signs

° = unusual behaviour (reduced activity and/or orientation to bottom or surface of the test vessels, dark pigmentation)

* = difficulties with maintenance of equilibrium

# = fish upside down with loss of equilibrium, showing only movement of gills as a sign of life

+ = no sign of life

Table 5 Weight and length of fish

 

Test item loading rate [mg/L]

 

Control

4.64

10.0

21.5

46.4

100

 

Length [mm]

Mean

23.3

22.6

23.0

23.1

24.1

24.0

Std. dev.

2.2

1.5

2.0

1.8

1.5

1.5

Mean*

23.4

Std. dev.*

1.8

 

Weight [g]

Mean

0.0954

0.0809

0.0952

0.0910

0.1302

0.1059

Std. dev.

0.0260

0.0244

0.0256

0.0268

0.0198

0.0227

Mean*

0.0998

Std. dev.*

0.0277

Table 6 Temperature of the test system

Test item loading rate

Control

4.64

10.0

21.5

46.4

100

[mg/L]

 

Time [h]

Temperature [°C]

0 fresh

23.3

23.3

23.4

23.3

23.4

23.4

24 aged

23.5

23.5

23.6

23.5

23.6

23.6

24 fresh

23.0

23.0

22.9

23.0

23.0

23.1

48 aged

23.6

23.6

23.6

23.6

23.6

23.6

48 fresh

23.2

23.1

23.1

23.1

23.1

23.1

72 aged

23.5

23.6

23.6

23.6

23.7

23.7

72 fresh

23.1

23.1

23.1

23.1

23.1

23.1

96 aged

23.5

23.6

23.6

23.6

23.6

23.6

Mean

23.3

23.4

23.4

23.4

23.4

23.4

Std. dev.

0.2

0.3

0.3

0.3

0.3

0.3

Mean

23.4

Std. dev.

0.2

Table 7 Oxygen saturation of the test system

Test item loading rate

Control

4.64

10.0

21.5

46.4

100

[mg/L]

 

Time [h]

Oxygen [%]

0 fresh

103

102

102

102

102

103

24 aged

77

78

79

81

78

76

24 fresh

101

102

102

102

103

105

48 aged

77

80

77

82

74

75

48 fresh

100

101

102

102

102

103

72 aged

78

82

80

84

80

78

72 fresh

102

103

103

103

104

107

96 aged

75

79

78

82

77

74

Mean

89

91

90

92

90

90

Std. dev.

13

12

13

11

14

15

Mean

90

Std. dev.

12

Table 8 pH of the test system

Test item loading rate

Control

4.64

10.0

21.5

46.4

100

[mg/L]

 

Time [h]

pH

0 fresh

7.22

7.36

7.38

7.43

7.51

8.32

24 aged

7.26

7.34

7.36

7.37

7.34

7.10

24 fresh

7.37

7.42

7.46

7.51

7.67

8.48

48 aged

7.27

7.37

7.37

7.40

7.33

7.17

48 fresh

7.36

7.43

7.47

7.51

7.64

8.35

72 aged

7.29

7.36

7.36

7.39

7.35

7.15

72 fresh

7.38

7.44

7.46

7.50

7.63

8.45

96 aged

7.20

7.31

7.32

7.37

7.32

7.12

Mean

7.29

7.38

7.40

7.44

7.47

7.77

Std. dev.

0.07

0.05

0.06

0.06

0.16

0.68

Mean

7.46

Std. dev.

0.31

Table 10 measured concentration of the analyte dibutyl amine

Test item

nominal

[mg/L]

Sampling

Dibutylamine

Mean fresh

[%]


[mg/L]

% of

nominal

0

0 h fresh

< LOD

-

-

24 h aged

< LOD

-

24 h fresh

< LOD

-

48 h aged

< LOD

-

48 h fresh

< LOD

-

72 h aged

< LOD

-

72 h fresh

< LOD

-

96 h aged

< LOD

-

4.64

0 h fresh

5.04

109

108

24 h aged

4.89

105

24 h fresh

4.95

107

48 h aged

5.00

108

48 h fresh

5.02

108

72 h aged

5.15

111

72 h fresh

5.00

108

96 h aged

5.06

109

10.0

0 h fresh

10.9

109

105

24 h aged

10.6

106

24 h fresh

10.6

106

48 h aged

10.6

106

48 h fresh

10.1

101

72 h aged

10.5

105

72 h fresh

10.3

103

96 h aged

10.0

100

21.5

0 h fresh

22.2

103

102

24 h aged

22.0

102

24 h fresh

21.8

101

48 h aged

21.3

99

48 h fresh

22.0

102

72 h aged

22.2

103

72 h fresh

21.4

100

96 h aged

21.7

101

Validity criteria fulfilled:
yes
Conclusions:
No biologically significant sublethal effects were observed. According to the results of the test, the LL50 (96 h) of the test item was assumed to be > 100 mg/L test item (nominal). The corresponding NOELR (mortality) (96 h) was 100 mg/L test item (nominal).
Executive summary:

The objective of this study was to determine the effects of the test item on the short-term toxicity to fish (Danio rerio). Where possible the no observed effect loading rate (NOELR), the lowest observed effect loading rate (LOELR) and the effective loading rate which caused 50 % mortality in the population (LL50), were estimated.

The study was conducted in accordance with the OECD guideline 203 (Fish acute toxicity test, 1992) as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.  However, during study performance a deviation to the study design was noted. Here only the 100 mg/L test loading rate was prepared in accordance with the WAF strategy. All other concentrations were made by serial dilution, even though weighing will have been possible. As no effects were seen at the limit dose which was not prepared by serial dilution, i.e. in accordance with OECD series on testing and assessment no. 23, the study is considered valid and the devaition has not impacted the integrity of the study as a limit test as per OECD 203 guidance, nor the integrity of the result.

A range finding study was used to determine definitive test concentrations and optimal stirring and settling time for the creation of the WAFs at each concentration.

The definitive test concentrations were, 4.64, 10.0, 21.5, 46.4, and 100 mg/L. One WAF was prepared were prepared by adding a prerequisite amount of test item to the test vessel, stirring for 24 hours in the dark at room temperature and then leaving any particulate to settle for 20 hours. All other concntrations were prepared by serial diltuion. After 20 hour settling no undissolved test item was found in the test medium using eye and Tyndall effect assessment. Further, not undissolved material was seen in the diluted test concnetrations. Water quality parameters were measured at the start and end, and every 24 hours. Analytical confirmation of the test substance was performed at each concentration at 0 (fresh), 24 hours (aged and fresh), 48 hours (aged and fresh), 72 hours (aged and fresh) and 96 hours (aged and fresh). All water quality parameters and study validity criteria were met as per OECD guideline 203.  

No biologically significant sublethal effects were observed. According to the results of the test, the LL50 (96 h) of the test item was assumed to be > 100 mg/L test item (nominal). The corresponding NOELR (mortality) (96 h) was 100 mg/L test item (nominal).

Description of key information

96h LL50 >100 mg/L to Danio rerio, OECD 203 (1992) Anon., 2018,

Key value for chemical safety assessment

Additional information

The objective of this study was to determine the effects of the test item on the short-term toxicity to fish (Danio rerio). Where possible the no observed effect loading rate (NOELR), the lowest observed effect loading rate (LOELR) and the effective loading rate which caused 50 % mortality in the population (LL50), were estimated.

The study was conducted in accordance with the OECD guideline 203 (Fish acute toxicity test, 1992) as the test item is a multi-constituent of varying solubilities a WAF approach was used in accordance with OECD series on testing and assessment no. 23.  However, during study performance a deviation to the study design was noted. Here only the 100 mg/L test loading rate was prepared in accordance with the WAF strategy. All other concentrations were made by serial dilution, even though weighing will have been possible. As no effects were seen at the limit dose which was not prepared by serial dilution, i.e. in accordance with OECD series on testing and assessment no. 23, the study is considered valid and the deviation has not impacted the integrity of the study as a limit test as per OECD 203 guidance, nor the integrity of the result.

A range finding study was used to determine definitive test concentrations and optimal stirring and settling time for the creation of the WAFs at each concentration.

The definitive test concentrations were, 4.64, 10.0, 21.5, 46.4, and 100 mg/L. One WAF was prepared were prepared by adding a prerequisite amount of test item to the test vessel, stirring for 24 hours in the dark at room temperature and then leaving any particulate to settle for 20 hours. All other concentrations were prepared by serial dilution. After 20 hour settling no undissolved test item was found in the test medium using eye and Tyndall effect assessment. Further, no undissolved material was seen in the diluted test concentrations. Water quality parameters were measured at the start and end, and every 24 hours. Analytical confirmation of the test substance was performed at each concentration at 0 (fresh), 24 hours (aged and fresh), 48 hours (aged and fresh), 72 hours (aged and fresh) and 96 hours (aged and fresh). All water quality parameters and study validity criteria were met as per OECD guideline 203.  

No biologically significant sublethal effects were observed. According to the results of the test, the LL50 (96 h) of the test item was assumed to be > 100 mg/L test item (nominal). The corresponding NOELR (mortality) (96 h) was 100 mg/L test item (nominal).