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Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 January 2019 to 26 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: National Standardization Technical Commitiee for the Management of Hazardous Chemicals. GB/T 27861-2011 Chemicals Fish Acute Toxicity Test [S]. Beijing: China Standard Press. 2012.

Deviations:

not specified

GLP compliance:

yes

Remarks:

CLP compliance statement in date of 26 April 2019.

Specific details on test material used for the study:

- Storage conditions: To be stored refrigerated (2-8°C) protected from light, stored under nitrogen after first opening.
- Vapour pressure: 1.3 Pa at 20°C and 2.4 Pa at 25°C (OECD Guideline 104, effusion method, LPL 2018)
- Water solubility: 39.7 mg/L (iSafeRat v1.7 prediction). Experimental study on-going at the date of the present study
- Log Kow: 3.93 (iSafeRat v1.7 prediction). Experimental study on-going at the date of the present study
- Stability: Stable (less than 10% loss) from pH 2-12 (Firmenich internal data)

Analytical monitoring:

yes

Details on sampling:

Samples were taken for analysis at the beginning of exposure (0 h), at the end of exposure (96 h) and 24, 48, 72 hours of exposure before and after test solution renewal.
On each occasion, three samples were taken from test item treatment group and one from blank control.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
For blank control group, no test item was added, 5L of test water was added to test container.
Test item treatment groups: 500 mg of test item (actual weighed range: 499.9-500.4 mg) was weighed and added to 10 L of test container to give the nominal concentration of 50 mg/L. The containers were kept tight and the solutions were kept stirring for about 2 hours with high stirring speed. After a period of about 3 hours settling, avoid the surface insoluble test item, the bottom layer of solutions was withdrawn to obtain the saturated solution. 2.5, 1.9, 1.5, 1.15, 0.9 L of saturated solution was taken and diluted to 5 L with test water to obtain the test solutions of 50%SS, 38%SS, 30%SS, 23%SS, 18%SS.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish (Danio rerio)
- Source: The Zebra fish was purchased from the supplier and then maintained in the facility of CTI-SET. The code number of fish used in this test was Zebra fish S20181030
- Age, length and weight at study initiation: Fish were of approximately the same size and age (i.e. the length of the largest fish was not more than twice of that of the smallest fish). The length of fish was in the range of 1.0-3.0 cm. The total length of the fish was 2.36 +/- 0.05 cm (mean +/- SD); the wet weight of fish was 170 +/- 5 mg (mean +/- SD).
The fish were healthy and free from any apparent malformation.

ACCLIMATION
The fish were acclimated in the water having the same quality as the test water. During the seven days acclimation before the test, the water temperature was maintained in the range of 21-25°C and the change was not more than 2°C. 12 hours light was given daily and dissolved oxygen concentration was above 80% of air saturation value (ASV). The fish were fed daily until 24 hours before the test. During the 7 days acclimation prior to the test, the total mortaility was 0%. The fish could be used.

FEEDING DURING TEST
The fish were not fed during the study

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Total hardness of the test water was measured as 180 mg CaCO3/L at the start of the test, in the allowed range of 10-250 mg CaCO3/L

Test temperature:

During the period of the exposure, water temperature was maintained in the range of 22.8-23.9°C, in the allowed range of 21-25°C +/-2°C

pH:

pH value of the water was in the range of 8.01-8.42, in the allowed range of 6.0-8.5

Dissolved oxygen:

Dissolved oxygen was in the range of 84.9-99.7% ASV, so not less than 60% of the air saturation value

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

- Nominal concentrations: 18%SS, 23%SS, 30%SS, 38%SS and 50%SS
- Measured concentrations: 6.2, 7.9, 11, 17 and 20 mg/L

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): closed
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: <1g fish/litre

TEST MEDIUM / WATER PARAMETERS
Tap water dechlorinated by aeration (also named as treated water) was used as test water, pH value of the water was in the range of 6.0-8.5 and total hardness was in the range of 10-250 mg/L (calculated as CaCO3).

OTHER TEST CONDITIONS
- Light intensity: 12 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
At the start of test, ten fish were randomly selected to measure the body length (full length) and weight (wet-weight).
Fish were inspected for mortalities or visible abnormalities after 3, 24, 48, 72 and 96 hours. Criteria of death are absence of respiratory movement and lack of response to external stimulation of the caudal peduncle. The dead fish were removed, stored temporarily in the refrigerator and then delivered to the approved waste disposal company for treatment.
Temperature, pH and concentration of dissolved oxygen of the dilution water and all test dilutions were measured every 24 hours. Total hardness of the test water was measured at the start of the study.

TEST CONCENTRATIONS
According to the result of the preliminary test, the saturated solution (shorted as SS) of the test item was prepared in the definitive test. The concentrations of the test item were set to be 50%SS, 38%SS, 30%SS, 23%SS, 18%SS and blank control was set in parallel. Ten fish were used in each group and there was no replicate. The semi-static exposure procedure was used and the test solutions were renewed every 24 hours. The test containers were kept tight during the exposure period.

Reference substance (positive control):

yes

Remarks:

Potassium bichromate (CAS N° 7778-50-9)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

8.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 7.6-9.1 mg/L

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

6.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Observations: At the end of expoure, there was no mortality and abnormal symptoms in the blank control group, and the mortality at the groups of 18%SS, 23%SS, 30%SS, 38%SS and 50%SS was 0%, 40%, 100%, 100% and 100%, respectively. The abnormal behavior was erratic swimming, lethargy and lay to one side, breathed but could not swim.
- Concentration analysis: The test item was not detected in blank control group. The mean measured concentrations of the test item in the test solutions were in the range of 76 -88% of initial mean measured concentration. The effect concentration was based on the geometric mean of measured concentrations, corresponding to 6.2, 7.9, 11, 17 and 20 mg/L for the nominal concentrations of 18%SS, 23%SS, 30%SS, 38%SS and 50%SS, respectively

Results with reference substance (positive control):

- Results with reference substance valid? YES
The result of the latest sensitivity test (Study 18ST009) conducted from 19 to 20 November 2018 indicated that the 24h-LC50 of potassium bichromate (CAS N° 7778-50-9) to Zebra fish was 353 mg/L and its 95% confidential limit was 290-430 mg/L. The 24h-LC50 was within the range of 200-400 mg/L.

Reported statistics and error estimates:

The EPA software TRIMMED SPEARMAN-KARBER METHOD (TSK) VERSION 1.5 was used to calculated the LC50 and its 95% confidence limits.

Sublethal observations / clinical signs:

Table 6.1.1/1: Accumulative mortality in the definitive test

Nominal concentration (mg/L)	Accumulative mortality (initial fish number = 10)					96h mortality (%)
	3h	24h	48h	72h	96h
Blank control 18%SS 23%SS 30%SS 38%SS 50%SS	0 0 0 0 0 0	0 0 0 5 10 10	0 0 2 9 10 10	0 0 2 10 10 10	0 0 4 10 10 10	0 0 40 100 100 100

Table 6.1.1/2: Sub-lethal effects of exposure in the definitive test

Nominal concentration (mg/L)	Number of fish with sub-lethal effects (initial fish number = 10)
	3h	24h	48h	72h	96h
Blank control 18%SS 23%SS 30%SS 38%SS 50%SS	10A 10A 10A 10A 10A 10A	10A 4B, 6E 5L, 2B, 3E 1B, 4L / /	10A 4B, 6E 4B, 4L 1L / /	10A 10E 6L, 2E / / /	10A 2L, 3B, 5E 5L, 1B / / /

Remark: "/" means all test fish were mortal; "A" mean normal; "B" means erratic swimming; "E" means lethargy; "L" mean lay to one side, breathed but could not swim.

Table 6.1.1/3: Measured concentrations of exposure solutions

Exposure time (hours)	Test solution	Mean measured concentration, mg/L (% to initial mean measured concentration)
		Blank control	18%SS	23%SS	30%SS	38%SS	50%SS
0	Fresh	ND	7.342	9.343	12.512	19.026	22.416
24	Old	ND	6.303 (86%)	8.112 (87%)	11.035 (88%)	15.988 (84%)	18.372 (82%)
	Fresh	ND	6.154 (84%)	7.870 (84%)	10.302 (82%)	/	/
48	Old	ND	5.841 (80%)	7.672 (82%)	9.679 (77%)	/	/
	Fresh	ND	6.414 (87%)	7.635 (82%)	10.723 (86%)	/	/
72	Old	ND	6.038 (82%)	7.518 (80%)	10.159 (81%)	/	/
	Fresh	ND	6.306 (86%)	7.598 (81%)	/	/	/
96	Old	ND	5.600 (76%)	7.562 (81%)	/	/	/
Geometric mean measured concentration (mg/L)		NA	6.2	7.9	11	17	20

Remarks: SS means saturated solution; "ND" means not detected; "NA" means not applicable; "/" means not determined.

Validity criteria fulfilled:

yes

Conclusions:

In this study, based on the geometric mean of measured concentrations, the 96h-LC50 of the test item to Zebra fish was determined at 8.3 mg/L and its 95% confidence limit was 7.6 -9.1 mg/L.

Executive summary:

The purpose of this study was to estimate the acute toxicity of the test item to Zebra Fish (Danio rerio). According to the result of the preliminary test, the definitive test was performed using semi-static exposure procedure. The test duration lasted 96 hours and the test solutions were renewed every 24 hours. The saturated solution (SS) of the test item was prepared in the definitive test. The concentrations of the test item were 50%SS, 38%SS, 30%SS, 23%SS, 18%SS and blank control was set in parallel. Ten fish were used in each group and no replicate was performed. The mortality and abnormal behaviour of the fish were observed and recorded at 3, 24, 48, 72 and 96 hours of exposure. According to the mortality of test fish, the LC50 was calculated. The concentrations of the test solutions were analyzed at the beginning of exposure (0 hour), at the end of exposure (96 hours) and at 24, 48 and 72 hours before and after test solution renewal.

At the end of exposure, there was no mortality and abnormal symptoms in the blank control group, and the mortality at the concentration of 18%SS, 23%SS, 30%SS, 38%SS and 50%SS was 0%, 40%, 100%, 100% and 100%, respectively. The abnormal behavior was erratic swimming, lethargy and lay to one side, breathed but could not swim. The test item was not detected in blank control group. The mean measured concentrations of the test item in the test solutions were in the range of 76 -88% of initial mean measured concentration. The effect concentration was based on the geometric mean of measured concentrations, corresponding to 6.2, 7.9, 11, 17 and 20 mg/L for the nominal concentrations of 18%SS, 23%SS, 30%SS, 38%SS and 50%SS, respectively.

In this study, based on the geometric mean of measured concentrations, the 96h-LC50 of the test item to Zebra fish was determined at 8.3 mg/L and its 95% confidence limit was 7.6 -9.1 mg/L. The 96h highest concentration causing 0% mortality (LC0) was 6.2 mg/L and the lowest concentration causing 100% mortality (LC100) was 11 mg/L.

Description of key information

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

96h-LC50 (Danio rerio) = 8.3 mg/L (95% CL: 7.6 - 9.1 mg/L), based on geometric mean measured concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

8.3 mg/L

Additional information

One experimental study is available to assess the acute toxicity of the registered substance to zebra fish Danio rerio, according to OECD Guideline 203 with GLP compliance.

According to the result of the preliminary test, the definitive test was performed using semi-static exposure procedure. The test duration lasted 96 hours and the test solutions were renewed every 24 hours. The saturated solution (SS) of the test item was prepared in the definitive test. The concentrations of the test item were 50%SS, 38%SS, 30%SS, 23%SS, 18%SS and blank control was set in parallel. Ten fish were used in each group and no replicate was performed. The mortality and abnormal behaviour of the fish were observed and recorded at 3, 24, 48, 72 and 96 hours of exposure. According to the mortality of test fish, the LC50 was calculated. The concentrations of the test solutions were analyzed at the beginning of exposure (0 hour), at the end of exposure (96 hours) and at 24, 48 and 72 hours before and after test solution renewal.

At the end of exposure, there was no mortality and abnormal symptoms in the blank control group, and the mortality at the concentration of 18%SS, 23%SS, 30%SS, 38%SS and 50%SS was 0%, 40%, 100%, 100% and 100%, respectively. The abnormal behavior was erratic swimming, lethargy and lay to one side, breathed but could not swim. The test item was not detected in blank control group. The mean measured concentrations of the test item in the test solutions were in the range of 76 -88% of initial mean measured concentration. The effect concentration was based on the geometric mean of measured concentrations, corresponding to 6.2, 7.9, 11, 17 and 20 mg/L for the nominal concentrations of 18%SS, 23%SS, 30%SS, 38%SS and 50%SS, respectively.

In this study, based on the geometric mean of measured concentrations, the 96h-LC50 of the test item to Zebra fish was determined at 8.3 mg/L and its 95% confidence limit was 7.6 -9.1 mg/L. The 96h highest concentration causing 0% mortality (LC0) was 6.2 mg/L and the lowest concentration causing 100% mortality (LC100) was 11 mg/L.