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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:

14-02-2020 to 09-04-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Justification for type of information:

Information as to the availability of the in vivo study is provided in 'attached justification'.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidelines for the Testing of Chemicals, Effects on Biotic Systems, Version 2, 203 Fish Acute Toxicity Test, China Environmental Press, China (2013)

Version / remarks:

The test was generally conducted in accordance with or equivalent to the following: China HJ/T153 - 2004 Chemical Test Guideline stipulations and Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, effects on Biotic Systems, 203 Fish Acute toxicity test [M]. Second Edition. Beijing: China Environmental Press. 2013: 30-36. The test was also completed under OECD TG 203: Fish Acute Toxicity Test (1992).

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The geometric mean of measured concentrations of test item were 1.8 mg/L, 2.7 mg/L, 4.3 mg/L, 6.3 mg/L and 8.8 mg/L for the nominal concentrations of 10%SS (saturated solution), 14%SS, 20%SS, 28%SS and 40%SS, respectively

- Sampling method: Samples were taken for analysis at least 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 each test item treatment group (one sample was pre-treated and stored under 2-8°C in case further analysis was required) and one from blank control.
Pretreatment of Samples : 5 mL of sample was accurately taken and 2 mL of dichloromethane was added for extraction. Allow the mixture to stand until there was a sharp demarcation line between the two layers, the lay of dichloromethane was taken and filtered through 0.45 µm hydrophobic PTFE filter before analysis.

- Sample storage conditions before analysis: Samples were used on day of sampling and analysed as soon as possible after their preparation.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: : The test item with volume of 1.000 mL was pipetted and directly added to 10 L of test water. The containers were kept tight and stirring for 5 hours using a stirring rate such that a vortex height was kept within 5.6-5.7 cm. The solution was allowed to settle down for 20 hours. Avoid the surface insoluble test item, the middle and bottom layers of solutions were withdrawn to obtain the 100 % saturated solution (shorted as SS). The 100%SS was taken and diluted to 5.5 L (the headspace was kept as small as possible with a glass cap) to obtain the test solutions of 10%SS, 14%SS, 20%SS, 28%SS, 40%SS. The geometric mean of measured concentrations of test item were 1.8 mg/L, 2.7 mg/L, 4.3 mg/L, 6.3 mg/L and 8.8 mg/L for the nominal concentrations of 10%SS, 14%SS, 20%SS, 28%SS and 40%SS, respectively
- Controls: For blank control, no test item was added, 5.5 L of dilution water was added.

- Test concentration separation factor: 1.4

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra Fish (Danio rerio).
- Strain: Not reported.
- Source: Recognised supplier (recorded in full study report) located in China. Code number C20190715.
- Age at study initiation (mean and range, SD): Not reported.
- Length at study initiation (length definition, mean, range and SD): Not reported. At the start of the test, ten fish were randomly selected. The total length of the fish was 2.0 ± 0.19 cm (mean ± SD); the wet weight of fish was 214 ± 20 mg (mean ± SD). Details in the report.
- Feeding during test: the fish were not fed during the study, only during acclimatation.

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions (same as test or not): Yes. Same water quality, 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).
- Feeding frequency: The fish were fed daily until 24 hours before the test.
- Health during acclimation (any mortality observed): no mortality

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

10-250 mg/L (calculated as CaCO3)

Test temperature:

during testing : 22.5-23.9°C

pH:

7.66-8.24

Dissolved oxygen:

78.1 %-99.8 % ASV during the definitive test.

Nominal and measured concentrations:

- DEFINITIVE TEST performed on semi-static test system with renewal every 24 hour intervals:
The geometric mean of measured concentrations of test item were 1.8 mg/L, 2.7 mg/L, 4.3 mg/L, 6.3 mg/L and 8.8 mg/L for the nominal concentrations of 10%SS, 14%SS, 20%SS, 28%SS and 40%SS, respectively. Detailed results of concentration analysis are listed in Table 6.1.1.2 Measured Concentration of Exposure Solution

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): closed
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): Renewal every 24 hours.
- No. of organisms per vessel: Ten fish were used in each group and there was no replicate.
- No. of vessels per concentration (replicates): no replicate
- No. of vessels per control (replicates) : 1 – no preplicate
- Biomass loading rate:<1g fish/litre;
- Number of fish : 10/concentration

TEST MEDIUM
- Source/preparation of dilution water: Water used in the test were purified by the reverse osmosis equipment. (Certified annually).
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Every 24 hour intervals; before and after renewal

OTHER TEST CONDITIONS
- Photoperiod: 12 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortalities and abnormalities; measured at 24h, 48h, 72h and 96h as applicable.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.4
- DEFINITIVE TEST performed on semi-static test system with renewal every 24 hour intervals:
The geometric mean of measured concentrations of test item were 1.8 mg/L, 2.7 mg/L, 4.3 mg/L, 6.3 mg/L and 8.8 mg/L for the nominal concentrations of 10%SS, 14%SS, 20%SS, 28%SS and 40%SS, respectively. Detailed results of concentration analysis are listed in Table 6.1.1.1 Measured Concentration of Exposure Solution

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limit was 3.6-4.9 mg/L.

Details on results:

- Other abnormalities: At the end of exposure, there was no mortality and abnormal symptoms in the blank control group, and the mortality at the groups of 10%SS, 14%SS, 20%SS, 28%SS, and 40%SS was 0%, 10%, 40%, 100% and 100%, respectively. The abnormal behavior was erratic swimming, lethargy and lay to one side at the bottom of container

- Observations on body length and weight: 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 fish were healthy and free from any apparent malformation.

For details see tables Table 6.1.1.2 Accumulative Mortality in Definitive Test and Table 6.1.1.3 Sub-lethal Effects of Exposure in the Definitive Test

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 372 mg/L (95% confidential limit = 331-419 mg/L.

Reported statistics and error estimates:

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

Sublethal observations / clinical signs:

Table 6.1.1.2 Accumulative Mortality in Definitive Test

Groups	Accumulative mortality (initial fish number=10)					96h Mortality (%)
	3h	24h	48h	72h	96h
Blank control	0	0	0	0	0	0
10%SS	0	0	0	0	0	0
14%SS	0	0	0	0	1	10
20%SS	0	0	0	1	4	40
28%SS	0	5	10	10	10	100
40%SS	0	10	10	10	10	100

 

 

Table 6.1.1.3 Sub-lethal Effects of Exposure in the Definitive Test

Groups	Number of fish with sub-lethal effects (initial fish number=10)
	3h	24h	48h	72h	96h
Blank control	0	0	0	0	0
10%SS	0	0	0	0	0
14%SS	0	0	0	5D	7D
20%SS	5D	10D	3D,1L	2A,2D	2A,4D
28%SS	2A,8L	5L	/	/	/
40%SS	10L	/	/	/	/

_{Remark: “/“ means all test fish were mortal; “A” means erratic swimming; “D” means lethargy; ”L” means lay to one side at the bottom of container.}

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 for the test item to Danio rerio was determined to be 4.2 mg/L based on geometric mean of measured concentrations.

Executive summary:

The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour semi-static test according to OECD TG 203 and the China Guidelines for the Testing of Chemicals, Effects on Biotic Systems, Version 2, 203 Fish Acute Toxicity Test (2013) under GLP.

Based on the results of range finding tests the nominal concentrations of 0 (control), 10%SS (saturated solution), 14%SS, 20%SS, 28%SS and 40%SS were selected for exposure to fish groups. Ten fish were used in each group and no replicate. 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 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.

The mean measured concentrations of test item in the old solutions were in the range of 48%-82% of those in the fresh solutions. The effect concentration was based on the geometric mean of measured concentration. The geometric mean of measured concentrations of test item were 1.8 mg/L, 2.7 mg/L, 4.3 mg/L, 6.3 mg/L and 8.8 mg/L for the nominal concentrations of 10%SS, 14%SS, 20%SS, 28%SS and 40%SS, respectively.

At the end of exposure, there was no mortality and abnormal symptoms in the blank control group, and the mortality at the different concentration of 10%SS, 14%SS, 20%SS, 28%SS and 40%SS was 0%, 10%, 40%, 100% and 100%, respectively. The abnormal behavior was erratic swimming, lethargy and lay to one side at the bottom of container.

The 96 hour LC50 for ST 06 C 19 to Danio rerio was determined to be 4.2 mg/L based on geometric mean of measured concentrations.

Description of key information

LC50 (fish) =  4.2 (C.I. 3.6-4.9 ) mg/L based on geometric mean of measured concentrations, 96-hour, freshwater, OECD TG 203, 2020

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

4.2 mg/L

Additional information

Key Study : OECD TG 203, 2020