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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:
05 March 2018 to 09 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
OECD Guideline for Testing of Chemicals, No. 203, "Fish, Acute Toxicity Test", adopted July 17, 1992
Deviations:
no
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:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Version / remarks:
EPA Health Effects Test Guidelines, OCSPP 850.1075.
Deviations:
no
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:
no
Vehicle:
no
Details on test solutions:
Because the Test Item was very poorly soluble in water (See more details in study with Citoxlab study code: 17/080-345AN and 17/080-316AN), a test solution was 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 was prepared 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 about 24 hours at approximately 30°C and then was equilibrated for about 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 solution. 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: 2.5 – 2.8 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
Remarks on exposure duration:
The frequency of the renewal periods were 24 hours.
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
189 mg/L (as CaCO3)
Test temperature:
20.2–21.1°C
pH:
7.95–8.36
Dissolved oxygen:
74–100 % 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:
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.

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).
Reference substance (positive control):
not required
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test item loading rate
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test item loading rate
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test item loading rate
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 required for this study
Reported statistics and error estimates:
No statistical analysis was performed because of the lack of toxic effects.
The LC50, NOEC, LOEC and LC100 were determined directly from the raw data.
Sublethal observations / clinical signs:

Results of the Preliminary Range-finding Test

Nominal concentrations

[mg/L test item nominal loading rate]

Control

0.1

1

10

100

Number of treated animals

4

4

4

4

4

Number of dead animal

0

0

0

0

0

There were no clinical signs observed in fish at any of the applied concentrations during the study.

 

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 test item nominal loading rate

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

1.64

0.23

0.33

100 mg/L test item nominal loading rate

1.63

0.23

0.33

 

Temperature (°C)

Test Group

Measured

0h1)

24h2)

241)

482)

481)

722)

721)

962)

Control

21.1

20.3

20.8

20.2

20.9

20.3

20.8

20.2

100 mg/L test item nominal loading rate

20.8

20.3

20.8

20.2

20.9

20.3

20.8

20.2

 

Dissolved oxygen concentration (% of the air saturation value)

Test Group

Measured

0h1)

24h2)

241)

482)

481)

722)

721)

962)

Control

99

96

100

93

100

90

99

88

100 mg/L test item nominal loading rate

75

93

74

95

77

91

78

96

 

pH

Test Group

Measured

0h1)

24h2)

241)

482)

481)

722)

721)

962)

Control

8.33

8.07

8.14

8.33

8.32

7.95

8.09

7.96

100 mg/L test item nominal loading rate

8.36

8.12

8.16

8.33

8.34

8.07

8.14

8.06

 

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

2.7

2.5

2.7

2.7

2.8

2.6

2.6

100 mg/L nominal loading rate WAF

2.6

2.7

2.6

2.8

2.7

2.5

2.7

Remark: AT the end of the test, body length of the 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 study the observed endpoints for the effect of the Test Item were the following:

The 24h, 48h, 72h and 96h EC50 value: > 100 mg/L test item nominal loading rate
The 96h EC100 value: > 100 mg/L test item nominal loading rate
The 96h NOEC: 100 mg/L test item nominal loading rate
The 96h LOEC: > 100 mg/L test item nominal loading rate

Based on the results of this study, the Test Item had no toxic effect at saturation on fish; the EC50 results and the LOEC 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 one test concentration at 100% saturated solution 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.

 

Since the Test Item was very poorly soluble in water (solubility level in water was <LOQ in water results obtained under the following Citoxlab Hungary study code: 17/080-345AN) and due to that there was no toxic effect at the solubility level of the Test Item in the test medium during the Preliminary Range-Finding Test the experiment was carried out without analytical measurements in agreement with the Sponsor.

 

Under the conditions of this study the observed endpoints for the effect of the Test Item were the following:

 

The 24h, 48h, 72h and 96h EC50 value: > 100 mg/L test item nominal loading rate

The 96h EC100 value: > 100 mg/L test item nominal loading rate

The 96h NOEC: 100 mg/L test item nominal loading rate

The 96h LOEC: > 100 mg/L test item nominal loading rate

 

Based on the results of this study, the Test Item had no toxic effect at saturation on fish; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium.

Description of key information

The 24h, 48h, 72h and 96h EC50 value: > 100 mg/L test item nominal loading rate

The 96h EC100 value: > 100 mg/L test item nominal loading rate

The 96h NOEC: 100 mg/L test item nominal loading rate

The 96h LOEC: > 100 mg/L test item nominal loading rate

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 one test concentration at 100% saturated solution 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.

Since the Test Item was very poorly soluble in water and due to that there was no toxic effect at the solubility level of the Test Item in the test medium during the Preliminary Range-Finding Test the experiment was carried out without analytical measurements in agreement with the Sponsor.

 

Under the conditions of the study the observed endpoints for the effect of the Test Item were the following:

The 24h, 48h, 72h and 96h EC50 value: > 100 mg/L test item nominal loading rate

The 96h EC100 value: > 100 mg/L test item nominal loading rate

The 96h NOEC: 100 mg/L test item nominal loading rate

The 96h LOEC: > 100 mg/L test item nominal loading rate

Based on the results of this study, the Test Item had no toxic effect at saturation on fish; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium.

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