Oestr-5(10)-ene-3,17-dione

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:

17 Dec 2019 - 29 Feb 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: Chemicals Fish Acute Toxicity Test (GB/T 27861-2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidelines for the Testing of Chemicals (HJ/T 153-2004)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

The study references to Procedure 203 of the Guidelines for the Testing of Chemicals of the OECD: Fish, Acute Toxicity Test (2019).

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

According to GLP standards of China.

Analytical monitoring:

yes

Details on sampling:

During the actual test, HPLC was used to analyse the test substance concentration in water samples at 0, 24, 48, 72, and 96 hours.
- Concentrations: 19.8%, 29.7%, 44.5%, 66.7%, and 100% saturation concentrations
- Sampling method: 10.0 mL test solution was taken from the blank, 19.8%, 29.7%, 44.5%, and 66.7% saturated solutions, passed through 0.22 μm aqueous membrane, the first 1 mL filtrate was discarded, then 1.00 mL filtrate was collected, and used in HPLC analysis. 1.00 mL of the 100% saturated solution was taken, diluted with acetonitrile to 10.0 mL (10 times dilution), passed through 0.22 μm organic membrane, the first 1 mL filtrate was discarded, then 1.00 mL filtrate was collected, and used in HPLC analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution dilution method was used to prepare the test substance solutions. For the pre-test, 1.2236 g was weighed and test water was used to dilute to 12 L; for the actual test, 3.0582 g was weighed, and test water used to dilute to 30 L. It was then stirred in an airtight agitator, continuously for 24 hours, at 500 rpm, at 30 °C, with the height of the vortex generated while stirring being approximately one-tenth the height of the solution surface in a static state. Then it was set aside at room temperature for one hour and, after the solution was filtered through a 0.45-μm aqueous membrane (the initial 100 mL was discarded), it served as the test substance 100% saturated solution.
- Controls:
Blank control test: Under the same test conditions as the test substance treatment groups, a test water blank control test was carried out simultaneously.
Reference compound test: Under identical test conditions, potassium dichromate was used as the reference toxin in a reference compound test.
- Test concentration separation factor: 1.36 - 1.74 (based on measured concentrations)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):The saturated solution was colourless, and no undissolved materials were observed under microscopy.

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

TEST ORGANISM
- Common name: Chinese rare minnow
- Source: The Gobiocypris rarus used in the test were produced and raised within the testing laboratory.
- Length at study initiation (length definition, mean, range and SD): 2.984 cm (relative standard deviation 4.89 %)
- Weight at study initiation (mean and range, SD): 0.2285 g (relative standard deviation 5.65 %)

ACCLIMATION
- Acclimation period: Prior to use in the test, the fish were acclimated in the test environment for seven days.
- Acclimation conditions (same as test or not): not the same:
Light: 12 hours light each day, intensity 877-926 lux;
Water quality: same water quality as the test water;
Temperature: 23.0-23.3 °C;
Dissolved oxygen: 103.8-107.0% of the air saturation value;
- Type and amount of food during acclimation as well as feeding frequency: They were fed newly hatched Artemia salina twice daily, terminating 24 hours prior to the start of the test.
- Health during acclimation (any mortality observed): The fish mortality rate in the seven-day acclimation period was 0.

FEEDING DURING TEST (as applicable)
No feeding.

Test type:

static

Water media type:

freshwater

Remarks:

The water used in the test was tap water aerated and treated for 24 hours or longer.

Limit test:

no

Total exposure duration:

96 h

Hardness:

149 mg CaCO3/L (measured in blank control at the start of the test)

Test temperature:

22.7 - 24.0 °C

pH:

7.03 - 7.36

Dissolved oxygen:

73.7 - 107.2 %

Nominal and measured concentrations:

The results indicated that, in water samples from saturated solution concentrations at 19.8%, 29.7%, 44.5%, 66.7%, and 100% preparation concentrations, the concentrations of test substance measured in water samples were 2.38, 4.15, 5.66, 8.71, and 13.0 mg/L, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: 15 L fish tanks (Haimen Haoqi Laboratory Instruments Factory)
- Material, size, fill volume: glass, 10 L
- Aeration: the water used in the test was aerated for 24 h or longer
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: Fish loading per unit of water was approximately 0.2285 g (wet weight)/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Particulate matter: < 4.00 mg/L
- Metals: Cadmium: < 0.5 µg/L; Lead: <2.5 µg/L; Cobalt: < 0.02 mg/L; Nickel: < 0.05 mg/L, Copper: 5.2 µg/L; Chromium6+: 0.004 mg/L; Iron: <0.01 mg/L; Zinc: <0.09 mg/L; Silver: <0.03 mg/L

OTHER TEST CONDITIONS
- Photoperiod: 12 hours daily
- Light intensity: 876 - 926 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 24 h-LC50 (reference substance), 96h-LC0, 96h-LC100, 96h-LC50 (test substance)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.36 - 1.74 (based on measured concentrations)
- Range finding study: Yes
- Test concentrations: 19.8%, 29.7%, 44.5%, 66.7%, and 100% saturated solution (corresponding to measured concentrations of 2.38, 4.15, 5.66, 8.71, and 13.0 mg/L, respectively)
- Results used to determine the conditions for the definitive study (determined in a pre-test): 10% and 100% saturated solution

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (purity: 99.9 %)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

6.6 mg/L

95% CI:

>= 5.44 - <= 8

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

13 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

2.38 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Other abnormalities: see 'Any other information on results incl. tables'
- Mortality of control: none
- Other adverse effects control: none
- Effect concentrations exceeding solubility of substance in test medium: none

Results with reference substance (positive control):

- LC50 after 24 h: 254 mg/L (CI 237 - 271 mg/L)

Sublethal observations / clinical signs:

Test Fish Fatalities (Actual Test)

Prepared concentration (mg/L)	Measured concentration (mg/L)	Number of fish (n)	Number of dead fish (n)
			3 h	6 h	24 h	48 h	72 h	96 h
0 (blank control)	ND^1)	10	0	0	0	0	0	0
19.8 % saturated solution	2.38	10	0	0	0	0	0	0
29.7% saturated solution	4.15	10	0	0	1	1	1	1
44.5% saturated solution	5.66	10	0	2	3	4	5	5
66.7% saturated solution	8.71	10	3	6	6	6	6	6
100% saturated solution	13.0	10	10	10	10	10	10	10
LC50 (mg/L)					7.10	6.85	6.60	6.60
95% confidence interval (mg/L)					5.89 - 8.56	5.65 - 8.29	5.44 - 8.00	5.44 - 8.00

ND: lower than the limit of quantitation (LOQ = 0.0300 mg/L).

 

Observation Results from Actual Test

Prepared concentration (mg/L)	Measured concentration (mg/L)	Observation results
		3 h	6 h	24 h	48 h	72 h	96 h
0 (blank control)	ND^1)	10^1) NB^1)	10 NB	10 NB	10 NB	10 NB	10 NB
19.8 % saturated solution	2.38	10 NB	10 NB	10 NB	10 NB	10 NB	10 NB
29.7% saturated solution	4.15	10 NB	9 NB & 1SR	9 NB & 1 death	9 NB & 1 death	9 NB & 1 death	9 NB & 1 death
44.5% saturated solution	5.66	10 NB	7 NB & 1 SR & 2 deaths	5 NB & 2 SR	5 NB & 1 SR & 4 deaths	5 NB & 5 deaths	5 NB & 5 deaths
66.7% saturated solution	8.71	5 NB & 2 SR & 3 deaths	4 NB & 6 deaths	4 NB & 6 deaths	4 NB & 6 deaths	4 NB & 6 deaths	4 NB & 6 deaths
100% saturated solution	13.0	10 deaths	10 deaths	10 deaths	10 deaths	10 deaths	10 deaths

NB¹⁾ – normal activity; SR²⁾ – fish lay facing up or sideways.

 

Validity criteria fulfilled:

yes

Remarks:

Validity was stated according to Chinese guidelines

Conclusions:

LC50 (96 h) = 6.60 mg/L (95% confidence interval was 5.44 - 8.00 mg/L (based on mean of actual measured concentrations))

LC0 (96 h) = 2.38 mg/L (based on mean measured concentration)

LC100 (96 h) = 13.0 mg/L (based on mean actual measured concentration)

Executive summary:

In accordance with Guidelines for the Testing of Chemicals (HJ/T 153-2004), Chemicals – Fish Acute Toxicity Test (GB/T 27861-2011), and Chemical Test Methods – Ecological System Effects Volume (second edition, Beijing: China Environment Press, 2013: 30-36), and with reference to Procedure 203 of the Guidelines for the Testing of Chemicals of the OECD: Fish, Acute Toxicity Test (2019), this project used the static method to measure the acute toxicity of the test substance in fish (Gobiocypris rarus). In the blank control group, the activity of the test fish was normal, and the 24 h-LC50 of the reference compound potassium dichromate in Gobiocypris rarus was 254 mg/L. In the 29.7% saturated solution concentration group (corresponding to 4.15 mg/L measured test substance) and higher concentration treatment groups, after the test fish came into contact with the test substance, they lay facing up or sideways. At the end of 96 hours of exposure, the test fish in the 100% saturated solution group (corresponding to 13 mg/L measured test substance) were all dead. 

Following LC-values were obtained after 96h:
LC₅₀ = 6.60 mg/L (95% confidence interval was 5.44 - 8.00 mg/L (based on mean of actual measured concentrations))
LC₀ = 2.38 mg/L (based on mean measured concentration)
LC₁₀₀ = 13.0 mg/L (based on mean actual measured concentration)

Description of key information

In accordance with Guidelines for the Testing of Chemicals (HJ/T 153-2004), Chemicals – Fish Acute Toxicity Test (GB/T 27861-2011), and Chemical Test Methods – Ecological System Effects Volume (second edition, Beijing: China Environment Press, 2013: 30-36), and with reference to Procedure 203 of the Guidelines for the Testing of Chemicals of the OECD: Fish, Acute Toxicity Test (2019), this project used the static method to measure the acute toxicity of the test substance in fish (Gobiocypris rarus). In the blank control group, the activity of the test fish was normal, and the 24 h-LC50 of the reference compound potassium dichromate in Gobiocypris rarus was 254 mg/L. In the 29.7% saturated solution concentration group (corresponding to 4.15 mg/L measured test substance) and higher concentration treatment groups, after the test fish came into contact with the test substance, they lay facing up or sideways. At the end of 96 hours of exposure, the test fish in the 100% saturated solution group (corresponding to 13 mg/L measured test substance) were all dead. 

Following LC-values were obtained after 96h:
LC₅₀ = 6.60 mg/L (95% confidence interval was 5.44 - 8.00 mg/L (based on mean of actual measured concentrations))
LC₀ = 2.38 mg/L (based on mean measured concentration)
LC₁₀₀ = 13.0 mg/L (based on mean measured concentration)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

6.6 mg/L