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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:
27 November 2017 to 01 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
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:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 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 Restriction of Chemicals (REACH), Annex Part C, C.1.
Deviations:
no
Qualifier:
according to
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. certificate)
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
yes
Details on sampling:
The concentration of the test item was measured at the test concentration at the beginning and at the end of the experiment.
Vehicle:
no
Details on test solutions:
Because the test item is poorly soluble in water, 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.
Saturated test item solution (100 mg/L nominal loading rate) was prepared by dispersing/dissolving the amount of test item into the test medium (aquarium water) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give appropriate 100 mg/L nominal loading rate. The solution was considered to be saturated.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Species: Zebrafish (Brachydanio rerio)
Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
Justification of species: Zebrafish (Brachydanio rerio) is one of the convenient species for acute fish toxicity test.
Number of animals: seven fish per test group
Body length of animals: 2.2 – 2.6 cm
Food and Feeding: The fish were not fed during the test
Acclimatisation: more than 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. There was no mortality of the population for seven days before the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
No post observation period specified.
Hardness:
The hardness of the dilution water was determined as 191 mg/L (as CaCO3).
Test temperature:
The test temperature was between 20.5 and 21.5 °C.
pH:
The pH was in the range of 7.38 – 8.13.
Dissolved oxygen:
The dissolved oxygen concentration was in the range of 67 – 99 % of the air saturation value at the temperature used.
Salinity:
Not specified.
Conductivity:
Not specified.
Nominal and measured concentrations:
Because no toxic response was observed during the preliminary range-finding test, only one test concentration at 100 mg/L nominal loading rate and one control group was tested in the definitive study in a static system (based on analytical results indicated).
Test item concentration could not be detected and therefore measured concentration was below the Limit of Detection (LOD = 0.01 mg/L) at each measurement during the experiment, consequently biological results were based on the nominal loading rate.
Details on test conditions:
Environmental test conditions
The water temperature, oxygen concentration and pH were measured daily during the experiment in each test aquarium.
The test temperature was between 20.5 and 21.5 °C.
The dissolved oxygen concentration was in the range of 67 – 99 % of the air saturation value at the temperature used.
Test solutions were aerated during the test to avoid decreasing of dissolved oxygen concentration below the accepted level.
The pH was in the range of 7.38 – 8.13.
The hardness of the dilution water was determined as 191 mg/L (as CaCO3).
The light-dark cycle during the test was 16 hours light and 8 hours darkness.

Description of the Test Procedure
The test duration was 96 hours. One aquarium was used for test group and for the control group, respectively. Each aquarium comprised 7 fish and 5 L test solution. The animals were not fed during the test. 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 made 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 approximate 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 (with 24-hour renewal in absence of stability data).
During the formulation procedure the test item stock solution was prepared by similar method described above. The test solutions were prepared by appropriate diluting of this stock solution.

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 effects were not observed during the experiment.
The test conditions (pH, temperature, oxygen saturation) were measured 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 (1.0 g fish/litre testing liquid).
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
MORTALITY DATA: No sublethal effect was observed.
BODY WEIGHT: There was no considerable difference observed concerning body weights between the groups.
Results with reference substance (positive control):
Reference substance not reported.
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.

Results of the Preliminary Range-Finding Test

Nominal concentration

[% saturated solution]

Untreated 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

0

0

0

0

0

0

 

Measured and calculated data of bodyweight

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

1.23

0.18

0.25

100 mg/L nominal loading rate

1.80

0.26

0.36

 

Temperature

Test Group

Temperature (°C)

Measuring

0h

24h

48

72

96

Control

21.5

20.8

20.6

20.5

20.6

100 mg/L nominal loading rate

21.5

20.8

20.6

20.6

20.5

 

Dissolved oxygen concentration

Test Group

Dissolved oxygen concentration (%)

Measuring

0h

24h

48

72

96

Control

87

86

98

97

99

100 mg/L nominal loading rate

67

87

97

96

98

 

Temperature

Test Group

pH

Measuring

0h

24h

48

72

96

Control

7.76

7.48

7.38

7.71

7.64

100 mg/L nominal loading rate

8.13

7.82

7.73

7.95

7.78

 

Body length of test fish at the end of the test

Test Group

Body length [cm]

1

2

3

4

5

6

7

Control

2.5

2.3

2.3

2.4

2.3

2.2

2.3

100 mg/L nominal loading rate

2.2

2.6

2.4

2.4

2.3

2.5

2.4

Remark: At the end of the test, body length of test fish were measured in order to check their compliance with the size range recommended for the species by the test guideline [OECD Guideline for Testing of Chemicals, No. 203, “Fish, Acute Toxicity Test”, adopted July 17, 1992].

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of NAUGARD® XL-1 was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a static system.

Under the conditions of the acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of NAUGARD® XL-1 were the following:

The 24h, 48h, 72h and 96h LC50 value: > 100 mg/L nominal loading rate
The 96h LC100 value: > 100 mg/L nominal loading rate
The 96h No-Observed Effect Concentration (NOEC): 100 mg/L nominal loading rate
The 96h Lowest Observed Effect Concentration (LOEC): > 100 mg/L nominal loading rate
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 static system.

 

Because no toxic response was observed during the preliminary range-finding test, only a Limit test was carried out using only one test concentration at 100 mg/L nominal loading rate (100 % saturated solution) and one control group in the definitive study under static conditions.

 

The test concentration was analytically determined at the start and at the end of the experiment. Test item concentration could not be detected and therefore measured concentration was below the Limit of Detection
(LOD = 0.01 mg/L) for all samples during the experiment. The biological results are based on the nominal loading rate.

 

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

 

All achievable validity criteria were met during this study.

 

Under the conditions of the acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of NAUGARD® XL-1 were the following:

 

The 24h, 48h, 72h and 96h LC50 value: > 100 mg/L nominal loading rate

The 96h LC100 value: > 100 mg/L nominal loading rate

The 96h No-Observed Effect Concentration (NOEC): 100 mg/L nominal loading rate

The 96h Lowest Observed Effect Concentration (LOEC): > 100 mg/L nominal loading rate

Description of key information

The 96h No-Observed Effect Concentration (NOEC): 100 mg/L nominal loading rate

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

The acute toxicity of the test item was assessed over an exposure period of 96 hours in a static system.

Under the conditions of the acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of NAUGARD® XL-1 were the following:

 

The 24h, 48h, 72h and 96h LC50 value: > 100 mg/L nominal loading rate

The 96h LC100 value: > 100 mg/L nominal loading rate

The 96h No-Observed Effect Concentration (NOEC): 100 mg/L nominal loading rate

The 96h Lowest Observed Effect Concentration (LOEC): > 100 mg/L nominal loading rate