[No public or meaningful name is available]

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:

2021-02-24 - 2021-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also conducted according to GLP, and designed according to OECD GD 23.

Justification for type of information:

See attached justification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

June 18 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on testing and assessment, No.23 “Guidance document on aqueous phase aquatic toxicity testing of difficult test chemicals”,

Version / remarks:

February 2019

Qualifier:

according to guideline

Guideline:

ISO 7346/1-3 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)])

Version / remarks:

1996

GLP compliance:

yes (incl. QA statement)

Remarks:

2021-03-17

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and all test solutions at the start and every 24h thereafter in old and new solutions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: an excess of the test item was added to 10 L of water. The mixing vessel was closed and mixing was initiated with the vortex extending ca. 10% of the vessel depth. The solution was mixed for 24 hours in the dark at room temperature and then left 1h undisturbed before use.
- Test concentration separation factor: 1.5
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test solutions were observed to be clear. The Tyndall effect was verified and not observed.
- Other relevant information: The concentration of the stock solution was analytically verified and the required dilutions for the test concentrations were determined from this confirmed concentration.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Elevage de la grande rivière, le Fond Garel, 69490 Saint Forgeux, France
- Age at study initiation (mean and range, SD): Juveniles
- Length at study initiation (length definition, mean, range and SD): average 1.86 cm
- Weight at study initiation (mean and range, SD): average 0.130 g

ACCLIMATION
- Acclimation period: 9 days
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food during acclimation: flaked, powdered or live food (such as brine shrimp nauplii)
- Feeding frequency during acclimation: Daily until 24 hours before the start of the test
- Health during acclimation (any mortality observed): No mortality observed in the 7d period prior the start of the test

FEEDING DURING TEST: No feeding during test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

40 - 250 mg/L (as CaCO3)

Test temperature:

22.1 - 23.6 °C

pH:

Start: 8.00 - 8.31
End: 7.54 - 7.79

Dissolved oxygen:

Start: 96.6% - 97.8%
End: 83.0% - 90.2%

Conductivity:

< 10 µS.cm-1

Nominal and measured concentrations:

Nominal: 1.5, 2.2, 3.3, 5.0, and 7.5 mg.L-1
Geometric means of the analytically confirmed concentrations: 1.41, 1.95, 3.09, 4.76 and 6.59 mg.L-1

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessels
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 3.5 L capacity filled with minimum headspace (ca. 1 cm)
- Aeration: None
- Renewal rate of test solution (frequency): daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: < 0.8 g.L-1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as described in OECD TG 203
- TOC < 0.3 mg.L-1

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light : 8h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Fish were inspected 1 and 4 hours after the start of the exposure, and every day twice a day thereafter until the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Range finding study
- Test concentrations: 1.0, 2.5, 5.0 and 8.0 mg.L-1
- Results used to determine the conditions for the definitive study: After 96h 0% mortality at <= 2.5 mg.L-1; 100% mortality at >= 5.0 mg.L-1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3.785 mg/L

95% CI:

2.925 - 4.842

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

No mortality was observed in the control at the end of the test.
The dissolved oxygen concentration was higher than 60% of the air saturation value throughout the test in all vessels.
Since concentrations of the test substance were not maintained on the first day within 80% of the nominal concentrations, results were based on the geometric of the analytically confirmed concentrations.

Sublethal observations / clinical signs:

Table. Incidence of mortality and total mortality during the final test

	Nominal concentration mg.L-1 (Geometric mean of measured concentration mg.L-1)
	Control (0)			1.50 (1.41)			2.20 (1.95)			3.30 (3.09)			5.00 (4.76)			7.50 (6.59)
	Dead fish	Surviving fish with sublethal effects or abnormalities	Mortality (%)	Dead fish	Surviving fish with sublethal effects or abnormalities	Mortality (%)	Dead fish	Surviving fish with sublethal effects or abnormalities	Mortality (%)	Dead fish	Surviving fish with sublethal effects or abnormalities	Mortality (%)	Dead fish	Surviving fish with sublethal effects or abnormalities	Mortality (%)	Dead fish	Surviving fish with sublethal effects or abnormalities	Mortality (%)
1h	0	0	-	0	0	-	0	0	-	0	2	-	0	7	-	0	7	-
4h	0	0	-	0	0	-	0	0	-	0	3	-	0	5	-	0	7	-
24h	0	0	0	0	0	0	0	7	0	0	7	0	0	7	0	2	5	28.6
48h	0	0	0	0	0	0	0	7	0	0	7	0	1	6	14.3	7	0	100
72h	0	0	0	0	0	0	0	7	0	2	5	28.6	3	4	42.9	7	0	100
96h	0	0	0	0	0	0	0	7	0	3	4	42.9	4	3	57.1	7	0	100

Types of sublethal effects/abnormalities observed: abnormal swimming behavior, lighter skin pigmentation, loss of equilibrium, irregular ventilation, hypo/hyperactivity.

 

Table. Concentrations of the test item (mg.L-1) in test water

Nominal concentration (mg.L-1)	Measured concentration (mg.L-1)								Geometric mean analytically confirmed concentrations
	Start (0h)	24h (old)	24h (new)	48h (old)	48h (new)	72h (old)	72h (new)	96h (end)	mg.L-1	%Nominal
Control	< LOD	< LOD	< LOD	< LOD	< LOD	< LOD	< LOD	< LOD	N/A	N/A
1.5	1.53	1.32	1.52	1.25	1.59	1.20	1.62	1.31	1.41	94
2.2	2.04	1.51	2.25	1.81	2.37	1.78	2.20	1.80	1.95	89
3.3	3.24	2.37	3.47	2.79	3.89	2.92	2.91	3.37	3.09	94
5.0	4.46	3.89	5.90	4.33	5.88	4.42	4.79	4.76	4.76	95
7.5	7.31	5.22	7.15	6.91	-	-	-	-	6.59	88

Analysis were not performed after 48h at the highest concentration as all fish were dead.

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item to the zebrafish (Danio rerio) was investigated in a closed semi-static test. Under the experimental conditions and based upon analytically confirmed concentrations, the 96-hour LC50 value was 3.785 mg.L-1.

Executive summary:

The toxicity of the test item to Danio rerio was investigated in a study conducted according to the OECD TG 203 (2019) and in compliance with GLP. The design further included the recommendations of the OECD GD 23 (testing difficult substances).

Following a preliminary range-finding study, seven fish were exposed to an aqueous solution of the test item over 96 hours at the required nominal test concentrations 1.5, 2.2, 3.3, 5.0 and 7.5 mg.L-1, and to a control. The mortality of the fish was determined by visual observation after 1, 4, 24, 48, 72 and 96 hours. Samples taken from the control and all test concentrations were analysed at the start of the test and every 24 hours thereafter in old and new solutions with surviving fish.

The analytical results of this test showed that test item levels were overall maintained within 80% of the nominal concentrations throughout the duration of the test, except for the first day of the test where measured concentrations were slightly lower than 80%. Therefore, the evaluation of the effects on Danio rerio was based on the geometric of the analytically confirmed concentrations: 1.41, 1.95, 3.09, 4.76 and 6.59 mg.L-1. After the 96-hours exposure, mortality was 0% at 1.41 and 1.95 mg.L-1, 42.9% at 3.09 mg.L-1, 57.1% at 4.76 mg.L-1 and 100% at 6.59 mg.L-1.

Under experimental conditions and based upon analytically confirmed concentrations, the 96-hour LC50 value was 3.785 mg.L-1.

Description of key information

The toxicicity of the test item to the zebrafish (Danio rerio) was investigated in a closed semistatic test. Under experimental conditions and based upon analytically confirmed concentrations, the 96-hour LC50 value was 3.785 mg.L-1.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC50

Effect concentration:

3.785 mg/L

Additional information

The toxicity of the test item to Danio rerio was investigated in a study conducted according to the OECD TG 203 (2019) and in compliance with GLP. The design further included the recommendations of the OECD GD 23 (testing difficult substances).

Following a preliminary range-finding study, seven fish were exposed to an aqueous solution of the test item over 96 hours at the required nominal test concentrations 1.5, 2.2, 3.3, 5.0 and 7.5 mg.L-1, and to a control. The mortality of the fish was determined by visual observation after 1, 4, 24, 48, 72 and 96 hours. Samples taken from the control and all test concentrations were analysed at the start of the test and every 24 hours thereafter in old and new solutions with surviving fish.

The analytical results of this test showed that test item levels were overall maintained within 80% of the nominal concentrations throughout the duration of the test, except for the first day of the test where measured concentrations were slightly lower than 80%. Therefore, the evaluation of the effects on Danio rerio was based on the geometric of the analytically confirmed concentrations: 1.41, 1.95, 3.09, 4.76 and 6.59 mg.L-1. After the 96-hours exposure, mortality was 0% at 1.41 and 1.95 mg.L-1, 42.9% at 3.09 mg.L-1, 57.1% at 4.76 mg.L-1 and 100% at 6.59 mg.L-1.

Under experimental conditions and based upon analytically confirmed concentrations, the 96-hour LC50 value was 3.785 mg.L-1.