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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:
11 -15 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline No. 203. All validity criteria were fulfilled.
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
22 September 2015
Specific details on test material used for the study:
- Water solubility: 13.7 g/L at 20 °C [Experimental data (Safepharm, 2008), OECD 105 (flask method)]
- Vapour pressure: 0.178 Pa at 25 °C [Experimental data (Harlan, 2009), analogue data, OECD 104 (gas saturation method)]
- Log Kow: 1.95 at 30 °C [Experimental data (Safepharm, 2008), OECD 117 (HPLC method)]
- Relative density: 1.35 at 20°C [Experimental data (Safepharm, 2008), OECD 109 (pycnometer method)]
- Stability in water: Hydrolytically stable at environmentally relevant pHs (internal experimental Sponsor data)
Analytical monitoring:
yes
Details on sampling:
Analytical measurement was performed at the applied test concentration levels and from the control at the beginning and at the end of the renewal periods.
The samples were analysed by HPLC system.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution with a nominal concentration of 100 mg/L was prepared with the test item and aquarium water. The test solutions were prepared by the appropriate diluting of this stock solution prior to treatment of fish i.e., 100, 55.56, 30.86, 17.15 and 9.53 mg/L.
- Controls: The dilution water (circulated and filtered) was used without of addition of the test item.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: Black Molly” Fish Farm, Veszprém, Hungary.
- Length at study initiation: 2.7 – 3.3 cm
- Feeding during test: Fish were not fed during the test

ACCLIMATION
- Acclimation period: > 12 days
- 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:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
185 mg/L (as CaCO3)
Test temperature:
20.9-21.3 °C
pH:
7.77 – 8.24
Dissolved oxygen:
81 – 98 % of the air saturation value
Salinity:
None
Nominal and measured concentrations:
Nominal concentrations: 9.53, 17.15, 30.86, 55.56 and 100 mg/L
Measured concentrations: 8.29, 15.11, 27.66, 50.14 and 90.98 mg/L. See Table 6.1.1/1 in "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: One aquarium was used for test groups and control group respectively.
- Renewal rate of test solution: Frequency of the water renewal period was 48 hours.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- 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.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness

EFFECT PARAMETERS MEASURED
- The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. Mortality and any sub-lethal effect were determined in each observation period.
- The test conditions (pH, temperature, oxygen saturation) were measured at the start and at the end of the renewal periods during the experiment.
- 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).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- A range-finding test was not performed and five test concentrations in a geometric series with a separation factor of 1.8 and one untreated control were tested in the main experiment in a semi-static system.
The following nominal concentrations were tested: 9.53, 17.15, 30.86, 55.56 and 100 mg/L.
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 (fish)
Remarks on result:
other:
Remarks:
analytically confirmed nominal
Duration:
96 h
Dose descriptor:
other: Highest tested concentration without observed effect
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mortality and weight
Details on results:
- No mortality was observed in any of the treated or control groups.
- There was no considerable difference observed concerning body weights between the groups.
Reported statistics and error estimates:
The LC50 values of the test item could not be calculated due to the lack of effects. The NOEC and LOEC, the LC50 and LC100 values were determined from the raw data.
Sublethal observations / clinical signs:

Table 6.1.1/1: Geometric mean of the measured concentrations

Nominal concentrations, mg/L

Measured concentrations at the start of the 1st renewal period (t0h new), mg/L

Measured concentrations at the end of the 1st renewal period (t48h old), mg/L

Measured concentrations at the start of the 2nd renewal period (t48h new), mg/L

Measured concentrations at the end of the 2nd renewal period (t96h old), mg/L

Geometric mean measured concentrations, mg/L

Percentage of the nominal concentrations

Control

-

-

-

-

-

-

9.53

8.04

7.20

9.65

8.44

8.29

87.0

17.15

14.74

13.27

17.87

14.90

15.11

88.1

30.86

26.88

24.36

32.52

27.50

27.66

89.6

55.56

48.19

44.67

59.02

49.74

50.14

90.2

100

88.33

81.80

107.1

88.55

90.98

91.0

Table 6.1.1/2: Cumulative mortality data in the definitive test

Concentration

 

Cumulative mortality[number of fish]

(initial population = 7 fish)

Nominal

mg/L

Calculated

mg/L

% of

nominal

3 h

6 h

24 h

48 h

72 h

96 h

Control

 

0.0

-

0

0

0

0

0

0

9.53

8.29

87.0

0

0

0

0

0

0

17.15

15.11

88.1

0

0

0

0

0

0

30.86

27.66

89.6

0

0

0

0

0

0

55.56

50.14

90.2

0

0

0

0

0

0

100.0

90.98

91.0

0

0

0

0

0

0

 

Validity

The achievable validity criteria were within acceptable limits and therefore the study can be considered as valid.

Validity criteria fulfilled:
yes
Conclusions:
Under the experimental conditions, the 96 h LC50 value was determined to be > 100 mg/L, based on analytically confirmed nominal concentrations.
Executive summary:

A study was performed to assess the acute toxicity of the test item to Brachydanio rerio, under semi-static conditions according to OECD Guideline 203 with GLP compliance. 

At the request of the Sponsor a range-finding test was not performed and five test concentrations in a geometric series with a separation factor of 1.8 and one untreated control were tested in the main experiment. The nominal concentrations of test item used in the main experiment were: 9.53, 17.15, 30.86, 55.56 and 100.0 mg/L. The corresponding measured geometric mean test item concentrations, analytically determined at the start and at the end of the renewal periods, were: 8.29; 15.11; 27.66; 50.14 and 90.98 mg/L.

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

 

At the request of the Sponsor, as the geometric mean measured concentrations deviated not more than 20 per cent from the nominal, biological results are based on the analytically confirmed nominal concentrations.

 

No mortality was observed in any of the treated or control groups. There was no considerable difference observed concerning body weights between the groups.

 

All achievable validity criteria were met during this study.

 

Under the experimental conditions, the 96 h LC50 value was determined to be > 100 mg/L, based on analytically confirmed nominal concentrations.

Description of key information

OECD Guideline 203, GLP, key study, validity 1:

96h-LC50 (Brachydanio rerio) > 100 mg/L (analytically confirmed nominal concentration).

Key value for chemical safety assessment

Additional information

One key study is available to assess the 96h acute toxicity of the registered substance to the fish species Brachydanio rerio, under semi-static conditions, according to the OECD Guideline 203 with GLP compliance. The nominal concentrations of test substance used in the main experiment were: 9.53, 17.15, 30.86, 55.56 and 100.0 mg/L. The corresponding measured geometric mean test substance concentrations, analytically determined at the start and at the end of the renewal periods, were: 8.29; 15.11; 27.66; 50.14 and 90.98 mg/L. As the geometric mean measured concentrations deviated not more than 20 per cent from the nominal, biological results were based on the analytically confirmed nominal concentrations. No mortality was observed in any of the treated or control groups. There was no considerable difference observed concerning body weights between the groups. In conclusion, under the experimental conditions, the 96h-LC50 value was determined to be greater than 100 mg/L, based on analytically confirmed nominal concentrations.