Hexahydro-2H-azepin-2-one, sodium salt

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apr. 2022 - Aug. 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

26. Jul. 2013

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: L. Brüggemann GmbH & CO. KG, Batch No. 32CS8F2413
- Purity: ≥ 99.95 % (m/m)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature ((20 ± 5 °C); keep under inert gas and wrap container lightproof
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: solubility in H2O > 1 g/L

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid, stock liquid or gel: 1000 mg/L
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): Stock solution with the concentration 1000 mg/L in test medium will be prepared. This stock solution will be used for preparation of the lower concentrated stock solutions at the concentrations of 100 mg/L and 10 mg/L. These stock solutions will be mixed with dilution water again. The mixture flows directly into the aquaria. Flow of stock solution and dilution water will be adjusted to reach the planned concentration of 10 mg/L and 1 mg/L in the treatments. Accordingly, the concentrations of the stock solutions are 10-fold higher than the nominal concentrations of the treatments. At the beginning of the test, the test concentrations in the aquaria (volume 12 L) will be prepared by direct addition of a stock solution with the concentration 1000 mg/L.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Solution

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): demineralised water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): stock solution 1000 mg/L, test solutions 10 mg/L and 1 mg/L

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Danio rerio
- Strain: Danio rerio HAMILTON-BUCHANAN
- Source: University Heidelberg

POST-HATCH FEEDING
- Type/source of feed: powdered flake food
- Frequency of feeding: 1 - 3 times per working day

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 d

Remarks on exposure duration:

approx. 30 days post hatch

Test temperature:

26.0 +/- 1.5 °C

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

> 9.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Key result

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 9.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: 99 % (surviving rate)
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: 75 % (hatching rate)
- Observations on body length and weight of young and/or exposed parents at one or more time periods: 11.84 mm (mean length)
- Number of healthy fish at end of test: 99 %
- Type of and number with morphological abnormalities: 0
- Type of and number with behavioural abnormalities: 0
- Type and number of developmental effects: 0
- Other biological observations: 0

Table 1: Range of measured pH, O2 and Temperature values

Nominal concentration [mg/L]	Replicate	pH range	O2 range [mg/L & % ASV]	Temp. range [°C]
Blank control	1	7.3 – 7.5	7.1 – 7.8 89.5 – 97.1	26.2 – 26.6
	2	7.3 – 7.6	7.2 – 7.8 90.3 – 97.5	26.2 – 26.5
	3	7.2 – 7.5	7.3 – 7.8 91.4 – 96.5	25.9 – 26.4
	4	7.3 – 7.5	7.1 – 7.8 89.8 – 96.5	26.0 – 26.5
1 mg/L	1	7.3 – 7.5	7.0 – 7.8 87.9 – 96.5	26.0 – 26.5
	2	7.3 – 7.5	6.9 – 7.8 86.4 – 96.9	25.9 – 26.4
	3	7.3 – 7.5	6.9 – 7.8 86.7 – 96.5	25.9 – 26.5
	4	7.3 – 7.5	7.0 – 7.8 88.0 – 96.2	25.9 – 26.4
10 mg/L	1	7.2 – 7.5	6.5 – 7.8 81.6 – 96.6	25.9 – 26.5
	2	7.2 – 7.5	6.3 – 7.8 78.5 – 96.7	25.9 – 26.4
	3	7.3 – 7.5	6.1 – 7.8 76.2 – 96.7	25.8 – 26.4
	4	7.3 – 7.5	6.8 – 7.8 85.1 – 96.8	26.0 – 26.5

 

Table 2: Hatching data

Nominal concentration [mg/L]	Replicate	Number of fish at the start	Number of fish hatched	Total hatched	Hatching rate [%]
Blank control	1	30	19	89	74
	2	30	17
	3	30	26
	4	30	27
1 mg/L	1	30	17	86	72
	2	30	18
	3	30	22
	4	30	29
10 mg/L	1	30	24	90	75
	2	30	22
	3	30	20
	4	30	24

 

Table 3: Mortality data

Nominal concentration [mg/L]	Replicate	Number of fish hatched	Dead fish	Number of survived fish at the end	Total survived	Surviving rate [%]
Blank control	1	19	3	16	84	94
	2	17	2	15
	3	26	0	26
	4	27	0	27
1 mg/L	1	17	0	17	86	100
	2	18	0	18
	3	22	0	22
	4	29	0	29
10 mg/L	1	24	0	24	88	98
	2	22	2	20
	3	20	0	20
	4	24	0	24

 

Table 4: Weight of surviving fish

Nominal concentration [mg/L]	Replicate	Mean weight fish per replicate [mg]	Total mean
Blank control	1	10.72	12.85
	2	15.24
	3	10.20
	4	15.23
1 mg/L	1	13.91	14.68
	2	18.72
	3	13.83
	4	12.26
10 mg/L	1	17.55	16.19
	2	18.32
	3	15.03
	4	13.88

 

Table 5: Length of surviving fish

Nominal concentration [mg/L]	Replicate	Length per fish [mm]	Mean
Blank control	1	11.25	11.62
	2	12.13
	3	11.00
	4	12.11
1 mg/L	1	12.41	11.92
	2	12.78
	3	11.09
	4	11.41
10 mg/L	1	12.25	11.76
	2	12.45
	3	10.50
	4	11.13

Validity criteria fulfilled:

yes

Conclusions:

The tested concentrations showed no toxicity for all endpoints. Hatchability, post hatch survival, wet weight and length in the highest tested concentration was slightly higher than in the control. Therefore, a two-sided hypothesis test was used for the NOEC determination.
The statistical evaluation did not show a significant difference to the control for any endpoint. The slight stimulation at the tested concentrations was within the normal range of a biological system.
The NOEC was determined to be >= 9.90 mg/L.

Executive summary:

One valid experiment was performed.

The toxicity of the test item was tested in the Early-Life Stage Toxicity Test agains Danio rerio. The study was performed using the two concentrations 1 mg/L and 10 mg/L (nominal). For each treatment, 30 fish per replicate were exposed to the test item for 35 days in a flow-through system. Dilution water was added with flow meters. Test item solutions were added with tubing pumps. Test item solutions and dilution water were mixed in the aquarium inlet. Fresh test item solutions were prepared daily.

The concentration of test item was measured at eight time points in each treatment group during the study period of 35 days using HPLC. The measured concentrations were between 60 % and 122 % of the nominal concentration.

Adjusting a stable test concentration under flow-through conditions is technically challenging. Of the total of 16 measured concentrations, 3 values were not in the range of the +/- 20 % nominal concentration. The variation within the measured concentrations was within a normal range for a flow-through test. However, because the measured concentrations were not within the range of 80 % - 120 % of the nominal concentration, the determination of the results was based on the time-weighted mean of the measured concentrations namely 0.80 mg/L (80 % of the nominal concentration of 1 mg/L) and 9.90 mg/L (99 % of the nominal concentration of 10 mg/L).

The tested concentrations showed no toxicity for all endpoints. Hatchability, post hatch survival, wet weight and length in the highest tested concentration was slightly higher than in the control. Therefore, a two-sided hypothesis test was sued for the NOEC determination. The statistical evaluation did not show a significant difference to the control for any endpoint. The slight stimulation at the tested concentrations was within the normal range of a biological system.

The following results for the test item were determined:

Endpoint	NOEC	LOEC
Hatchability	≥ 9.90 mg/L	> 9.90 mg/L
Post hatch survival	≥ 9.90 mg/L	> 9.90 mg/L
Wet weight	≥ 9.90 mg/L	> 9.90 mg/L
Length	≥ 9.90 mg/L	> 9.90 mg/L

Description of key information

Endpoint	NOEC	LOEC
Hatchability	≥ 9.90 mg/L	> 9.90 mg/L
Post hatch survival	≥ 9.90 mg/L	> 9.90 mg/L
Wet weight	≥ 9.90 mg/L	> 9.90 mg/L
Length	≥ 9.90 mg/L	> 9.90 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

>= 9.9 mg/L