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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 February 2018 to 23 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
OECD Guideline for Testing of Chemicals, Section 2, No. 203, "Fish, Acute Toxicity Test", adopted July 17, 1992.
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
Commission Regulation (EC) No 761/2009 of 23 July 2009 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restrictions of Chemicals (REACH), Annex Part C, C.1 (published in the Official Journal of the European Union L 142 of 31 May 2008)
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Version / remarks:
EPA Guideline 712-C-96-118: OCSPP 850.1075, "Fish Acute Toxicity Test, Freshwater and Marine" April 1996
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
yes
Details on sampling:
Based on the agreement with the sponsor and due to the fact that the Test Item is an UVCB the analytical measurement was performed by TOC analysis. Only the sum of the carbon content of the Test Item could be measured this way (and this did not change by the end of the test, independently from the stability), measurement was sufficient only at the beginning and end of the renewal periods to justify that the Test Item is present in the test solution, since more accurate data cannot be provided by TOC. The LOQ for this method was about 3 mg carbon /L. The carbon concentration of the Test Item was measured at the test concentration at the start and at the end of each renewal period (from an additional vessel without the test organism which would affect the total carbon present in solution). Sample from the control were taken for analysis at the beginning and end of each renewal period.
All samples will be analyzed directly after sampling.
Vehicle:
no
Details on test solutions:
Because the Test Item is a UVCB (Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials), a test solution were prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. A saturated Test Item solution at 100 mg/L Test Item nominal loading rate (WAF) was prepared individually by dispersing/dissolving the needed amount of Test Item into the test medium (aquarium water) two days before the start of the experiment. This solution was shaken for approximately 24 hours at approximately 30°C and then was equilibrated for approximately 24 hours at approximately 20°C. The non-dissolved test materials were removed by filtration through a fine (0.22 µm) filter to give the 100% saturated solutions. As only limit test was carried out, further dilution of this saturated solution was not performed.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Species: Zebrafish (Brachydanio rerio)
Source: Szent István University, Department of Aquaculture 2100 Gödöllő, Páter Károly u. 1. – Hungary
Justification of species: Zebrafish (Brachydanio rerio) is one of the convenient species for acute fish toxicity test and it is recommended by the test guidelines.
Number of animals: 7 fish in test concentration and control groups
Body length of animals: 1.8 – 2.4 cm
Acclimatisation: > 12 days
Animal health: Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed during the acclimatisation and test periods; the fish were healthy. Mortality was less than 5% of the population for seven days before the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
189 mg/L (as CaCO3)
Test temperature:
20.1–21.5°C
pH:
7.01–7.62
Dissolved oxygen:
76–97 % of the air saturation value at the temperature used
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
No toxicity was observed in the preliminary study therefore 100 mg/L Test Item nominal loading rate (WAF) and one control was used in the Main test.
Details on test conditions:
ENVIRONMENTAL TEST CONDITIONS
The water temperature, oxygen concentration and pH were measured daily during the experiment in each test aquarium.
Test temperature: 20.1–21.5°C
Dissolved oxygen concentration: 76–97 % of the air saturation value at the temperature used
pH: 7.01–7.62
Hardness of the dilution water: 189 mg/L (as CaCO3)
The light-dark cycle: 16 hours light and 8 hours darkness during the test
Feeding: the fish were not fed during the test

PERFORMANCE OF THE TEST
Taking into account that the stability of the Test Item in aquarium water was not available the Definitive Test was carried out in semi-static system. The frequency of the renewal periods was 24 hours. The test duration was 96 hours. One aquarium was used for test group and control group, respectively. Each aquarium comprised 7 fish and 5 L test solution. The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment. The choice of the test concentrations was done on the basis of the results of the preliminary range-finding test.

Preliminary Range-Finding Test
A preliminary concentration range-finding test was conducted to determine the toxicity of the Test Item so that appropriate test concentrations could be selected for use in the Definitive Test. Four fish were exposed to each concentration of the Test Item plus a control for 96 hours under semi-static conditions. During the formulation procedure the test solutions were prepared individually.
There were no clinical signs observed in fish at any of the applied concentrations during the study.

Test item concentrations in the Definitive Test
No toxicity was observed in the preliminary study therefore 100 mg/L Test Item nominal loading rate (WAF) and one control was used in the Main test.

OBSERVATIONS
The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. Mortality or any sub-lethal effect was not observed during the study. All animals were clinically normal during the study.
The test conditions (pH, temperature, oxygen saturation) were examined daily during the test.
The body weights of the fish were recorded before the introduction of fish. The body weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights (g fish/litre testing liquid).

ANIMAL WELFARE
After the experiment, all fish were humanely killed. Euthanasia for all animals was applied by an overdose of an aqueous solution of 250 mg/L Ethyl 3-aminobenzoate methanesulfonate (MS 222; CAS No.: 886-86-2). This technique is considered to provide a humane euthanasia by direct depression of the brain and vital centres.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LOELR
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
mortality (fish)
Details on results:
VALIDITY
The achievable validity criteria were within acceptable limits and therefore the study can be considered as valid.

MORTALITY DATA
No mortality or any sub-lethal effects were observed.

BODY WEIGHT
There was no considerable difference observed concerning body weights between the groups.
Results with reference substance (positive control):
Reference substance not used in the study.
Reported statistics and error estimates:
No statistical analysis was performed because of the lack of toxic effects.
Sublethal observations / clinical signs:

Results of the Preliminary Range-Finding Test

Nominal concentrations

[mg/L Test Item nominal loading rate (WAF)]

Control

0.1

1

10

100

Number of treated animals

4

4

4

4

4

Number of dead animals

0

0

0

0

0

 

Cumulative mortality data in the Limit Test

Test Group

Cumulative Mortality (number of dead fish)

[initial population = 7 fish]

3h

6h

24h

48h

72h

96h

Control

0

0

0

0

0

0

100 mg/L nominal loading rate WAF

0

0

0

0

0

0

 

Measured and calculated data of body weight

Test Group

Measured weight of 7 fish

(g)

Calculated mean weight of 1 fish

(g)

Loading of testing aquarium

(g fish/L testing liquid)

Control

1.78

0.25

0.36

100 mg/L nominal loading rate WAF

1.61

0.23

0.32

 

Temperature (°C)

Test Group

Measured

0h1)

24h2)

241)

482)

481)

722)

721)

962)

Control

21.5

20.3

21.2

20.2

21.1

20.3

21.1

20.2

100 mg/L nominal loading rate WAF

20.7

20.2

20.8

20.2

20.9

20.3

20.8

20.1

 

Dissolved oxygen concentration (% of the air saturation value)

Test Group

Measured

0h1)

24h2)

241)

482)

481)

722)

721)

962)

Control

85

97

82

97

81

96

82

93

100 mg/L nominal loading rate WAF

80

94

78

94

76

93

78

93

 

pH

Test Group

Measured

0h1)

24h2)

241)

482)

481)

722)

721)

962)

Control

7.12

7.54

7.01

7.37

7.43

7.21

7.20

7.07

100 mg/L nominal loading rate WAF

7.17

7.62

7.12

7.46

7.51

7.29

7.37

7.14

 

1): measurement at the start of the renewal period in the fresh test medium

2): measurement at the end of the renewal period in the old test medium

 

Body length (total length) of test fish at the end of the test

Test Group

Body Length [cm]

1

2

3

4

5

6

7

Control

2.4

2.2

2.3

1.9

2.1

2.3

2.1

100 mg/L nominal loading rate WAF

1.8

2.3

2.2

2.2

2.3

2.1

2.0

Remark: At the end of the test, body length of test fish was measured in order to check their compliance with the size range recommended for the species by the test guideline [Commission Regulation (EC) No 440/2008; Annex Part C, C.1].

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed endpoints for the effect of the Test Item were the following:

The 24h, 48h, 72h and 96h LLR50 value: > 100 mg/L nominal loading rate WAF
The 96h LLR100 value: > 100 mg/L nominal loading rate WAF
The 96h NOELR: 100 mg/L nominal loading rate WAF
The 96h LOELR: > 100 mg/L nominal loading rate WAF

Based on the results of this study, the Test Item had no toxic effect at saturation on fish; the LLR50 results and the LOELR are higher than the solubility level of the Test Item in the test medium.
Executive summary:

The acute toxicity of the Test Item was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system. The frequency of the renewal periods were 24 hours.

 

Because no toxic effect was observed at the solubility level of the Test Item in the preliminary test, only this test concentration (100 mg/L Test Item nominal loading rate (WAF) at the solubility level of the Test Item in the test medium and one control group were tested in a limit test in the definitive study.

 

One aquarium was used in the test group and one in the control group. Each aquarium comprised 7 fish and 5 litre test solution.

Based on the agreement with the sponsor and due to the fact that the Test Item is an UVCB the analytical measurement was performed by TOC analysis. Only the sum of the carbon content of the Test Item could be measured this way (and this did not change by the end of the test, independently from the stability), measurement was sufficient only at the beginning and end of the renewal periods to justify that the Test Item is present in the test solution, since more accurate data cannot be provided by TOC.

 

Under the conditions of this acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed endpoints for the effect of the Test Item were the following:

 

The 24h, 48h, 72h and 96h LLR50 value:       > 100 mg/L nominal loading rate WAF

The 96h LLR100 value:       > 100 mg/L nominal loading rate WAF

The 96h NOELR:       100 mg/L nominal loading rate WAF

The 96h LOELR:       > 100 mg/L nominal loading rate WAF

 

Based on the results of this study, the Test Item had no toxic effect at saturation on fish; the EL50 results and the LOELR are higher than the solubility level of the Test Item in the test medium.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: No details of GLP or recogonised test guidelines
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No data
Analytical monitoring:
not specified
Details on sampling:
No data
Vehicle:
yes
Details on test solutions:
Aminox was made up in saturated aqueous extracts (SAE). This was accomplished by allowing the material to sit in distilled water after vigorous
shaking for at least 24 hours at room temperature. The saturated aqueous extracts were then filtered and then test.
Test organisms (species):
Lepomis microlophus
Details on test organisms:
Size: 1-3 inches in length
All fish obtained from a state licensed commercial fish hatchery and were retained in the laboratory for at least 10 days before use.
Test type:
not specified
Water media type:
not specified
Total exposure duration:
96 h
Post exposure observation period:
No data
Hardness:
44 ppm CaCo3
Test temperature:
22 °C
pH:
6.5-8.5
Dissolved oxygen:
> 4mg/L at all times during test
Salinity:
No data
Nominal and measured concentrations:
No data
Details on test conditions:
The dilution water was natural pond water with a hardness expressed as 44 ppm CaCo3. Test was conducted at 22°C, pH 6.5 - 8.5 and dissolved oxygen remained above 4mg/L at all times during the test. All fish were obtained fro a state licensed commercial fish hatchery and were retained in the laboratory for a least 10 days prior to use.
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
EC0
Effect conc.:
16.3 other: ppm
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
100% survival rate of fish exposed to 16.3 ppm of Aminox
Results with reference substance (positive control):
No data
Reported statistics and error estimates:
No data
Validity criteria fulfilled:
not specified
Conclusions:
100% survival rate of fish exposed to 16.3 ppm of Aminox.
Executive summary:

The aim of the study was to determine the acute aquatic toxicity of Aminox. Study was conducted in accordance to Standard Methods for the Examination of Water and Wastewater, 13th ed. American Publis Health Associate, 1971. Results from the study showed Lepomis macrochirus exposed to Aminox for 96 hours at a concentration of 16.3 ppm did not cause mortality.

Description of key information

The 24h, 48h, 72h and 96h LLR50 value: > 100 mg/L nominal loading rate WAF

The 96h LLR100 value: > 100 mg/L nominal loading rate WAF

The 96h NOELR: 100 mg/L nominal loading rate WAF

The 96h LOELR: > 100 mg/L nominal loading rate WAF

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

The acute toxicity of the Test Item was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system. The frequency of the renewal periods were 24 hours.

Because no toxic effect was observed at the solubility level of the Test Item in the preliminary test, only this test concentration (100 mg/L Test Item nominal loading rate (WAF) at the solubility level of the Test Item in the test medium and one control group were tested in a limit test in the definitive study.

One aquarium was used in the test group and one in the control group. Each aquarium comprised 7 fish and 5 litre test solution.

Under the conditions of the acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed endpoints for the effect of the Test Item were the following:

The 24h, 48h, 72h and 96h LLR50 value: > 100 mg/L nominal loading rate WAF

The 96h LLR100 value: > 100 mg/L nominal loading rate WAF

The 96h NOELR: 100 mg/L nominal loading rate WAF

The 96h LOELR: > 100 mg/L nominal loading rate WAF

Based on the results of this study, the Test Item had no toxic effect at saturation on fish; the EL50 results and the LOELR are higher than the solubility level of the Test Item in the test medium.