3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol

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:

Study Initiation Date: 9th November, 2021 to Study Completion Date: 7th July, 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control (0 mg/L), Solvent control (0 mg/L), test conc. 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441, and 6.104 mg/L, respectively
- Sampling method: Preparation of test solution from the stock solution and stock solutions were prepared by direct weighing method.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Solubility of the test item was performed by weighing 25.7 mg of test item in a glass vial and dissolved in 25 µL of acetone and transferred into the 250 ml volumetric flask, and made up to the mark using natural water for solubility check. Test solutions of the selected concentrations were prepared by dilution of a stock solution. The solution was prepared by simply mixing. The solution preparation details are presented in appendix 5-5H.
- Controls: 0 mg/L
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone is one of the recommended vehicle by OECD Test Guideline 210 and same was used as solvent control (acetone+ natural water).
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): solvent control 0 mg/L
- Test concentration separation factor: not exceeding 3.2
- Other relevant information: Natural water (Dilution water) is one of the recommended water by OECD Test Guideline 210 and same was used as dilution water (natural water). Water quality parameters were analysed externally (bi-annual basis).

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Zebrafish (Wild type , India)
- Source: The fishes were supplied by Tarun Fish Aquarium, manimangalam, chennai, Tamilnadu, India.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): Parental fishes of 8 male and 4 female (at ratio of 2 male and 1 female
- Method of collection of fertilised eggs: Handling of Fertilised Eggs, Embryos and Larvae Zebra fish, (Parental fishes of 8 male and 4 female (at ratio of 2 male and 1 female) were place in a breeding container for eggs production one day before evening of the exposure. To prevent predation of eggs by adult zebra fish, the spawn traps were covered with inert wire mesh of appropriate mesh size (approx. 2.5±0.5 mm). Mating, spawning and fertilisation were take place within 30 min after the onset of light.
- Subsequent handling of eggs: Adult zebra fish were removed carefully with fish collection net and placed in a maintenance tank and the spawn traps were carefully removed. The fertilised eggs were collected by using Pasteur pipette on next day morning (on the day of exposure) and ferilized eggs were rinsed with natural water after collection from spawning traps. Fertilized eggs were placed into respective test vessels by using transparent plastic pipette for hatching.

POST-HATCH FEEDING
- Start date: Commercial dry food was provided for 14 days. From day 15 onwards live food (artemia) was provided along with dry food. Start date of feeding: 2 days post hatch.
- Type of feed: Dry powder and Brine shrimp, Artemia
- Source of feed: Feed (Peqon Blue Ring Brine Shrimp Eggs/ Artemia Cyst 1 Strip (10 Capsules) was commercially procured.
- Amount given: a very small amount food was given
- Frequency of feeding: Dry feed (twice a day) and Brine shrimp Artemia(once daily). Surplus food and faeces were removed where required, to avoid accumulation of waste.

- Other: Total mean weight in the control and solvent control groups were 0.034 and 0.032 g whereas, 0.028 g, 0.027 g, 0.025 g, 0.023 g, 0.022 g, 0.021 g, 0.018 g and 0.015 g in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Total mean lengths in the control and solvent control groups were 11 mm whereas, 10.93 mm, 10.90 mm, 10.64 mm, 10.57 mm, 10.52 mm, 10.03 mm, 9.80 mm and 9.42 mm in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 d

Hardness:

170 mg/L as CaCO3

Test temperature:

26.0°C to 26.5°C

pH:

7.3 to 7.9 at the beginning of the test and 7.3 to 7.6 at test termination.

Dissolved oxygen:

74.5 to 91.5% ASV

Nominal and measured concentrations:

Control (0 mg/L), Solvent control (0 mg/L), test conc. 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441, and 6.104 mg/L (spacing factor = 2.5), respectively.

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): Petriplate size 90 mm x 15 mm
- Test vessel: 2 L Glass beaker
- Material, size, headspace, fill volume: 50 ml/ pertriplate (during hatiching), 150 --250 ml/ test vessel (post hatching)
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): 96 hours
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates):4
- No. of vessels per vehicle control (replicates):4
- Biomass loading rate: 80 eggs per concentration
- Other: Brood Fish Maintenance: A breeding stock of unexposed, Zebrafish with good fertilization rate of eggs were procured from an external source. Loading capacity of fish during this period were in the ratio of 1 gram wet weight fish per litre. Water was changed once in a week. The temperature in the holding tanks were maintained at 26 ± l .5°C and the photoperiod at 12 hours light. The fish were provided with commercial dry food and live newly hatched Artemia, Daphnia (3-5 times daily). The excess food and feces were removed one hour after feedings.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural Water
- Metals:
Copper as Cu (IS 3025(Part-42)): BLQ(LOQ : 1.0) g/L
Silver as Ag (IS 13428): BLQ(LOQ: 100) ng/L
Aluminum as Al (IS 3025(Part-55)): BLQ(LOQ: 1.0) g/L
Total Arsenic as As (IS 3025(Par1-37)): BLQ(LOO: 1.0) g/L
Sodium as Na (IS 3025(Part-45)): 20.8 mg/L
Potassium as K (IS 3025(Part-45)): BLQ (LO01.0) mg/L
Chlorine as Cl (IS 3025(Part-32)): 63.6 mg/L
Sulphate as SO4 (IS 3025(Part-24)): 4.03 mg/L
Iron as Fe (IS 3025(Part-53)): BLQ(LOQ : 1.0) 9/L
- Chlorine: BLQ(LOQ : 0.02) mg/L
- Ca/mg ratio: 47.3 mg/L: 12.5 mg/L
- Intervals of water quality measurement: Conductivity IS 3025(Part-14): 2013 329 Ls/cm
- Other:
Particulate Matter (IS 3025 (Part-15)): 1.87 mg/L
Residual Free Chlorine (IS 3025(Part-26)): BLQ(LOQ : 0.02) mg/L
Nitrate as NO: 8.7 mg/L
Chemical Oxygen Demand (IS 3025(Part-58)): BLQ (LOQ:4.0) mg/L
Total Suspended Solids (IS 3025(Part-17)): BLQ (LOQ:1.0) mg/L


OTHER TEST CONDITIONS
- Photoperiod: The photoperiod 12 hours light and 12 hours dark
- Light intensity: The mean intensity of light ranged from 694 to 768 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : During the definitive test, no clinical sign exhibited in control group, solvent control group and in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391 and 0.977 mg/ L, whereas loss of buoyancy control (2+2 larvae) behaviour was observed in the tested concentration of 2.441 and 6.104 mg/ L. Abnormal behaviour like hypoactivity (2 larvae) observed in in the tested concentration of 2.441 mg/ L for a period of 30 after post hatch.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY

- Test concentrations: Range finding test was conducted with test concentrations at 0 (Control), 0 (Solvent Control), 5, 10, 25, 50 and 100 mg/ L. Each concentration contained two replicate and each replicates were exposed 20 fertilized eggs. Test item was formulated in acetone + natural water (not exceeding 100 µL /L). Hatching (percent) observed in control and solvent control groups were 92.5% and 85.0% whereas, 82.5% and 2.5% in the tested concentration of 5 and 10 mg/L for a period of 96 hours. Non-detachment of the tail observed in the control (3), solvent control (6) and in the
tested concentration of 5 mg/ L (7) for a period of 96 hours. Coagulation observed was 39 in the tested concentration of 10 mg/ L, wheras Coagulation observed (40) in the tested concentration of 25, 50 and 100 mg/ L for a period of 96 hours .
- Results used to determine the conditions for the definitive study: Based on the results of range finding test, the definitive was conducted with 8 test
concentrations at 0 (Control), 0 (Solvent Control), 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/L, in a geometric series with a spacing factor of 2.5 by static renewal method. Test item was formulated in acetone+ natural water (not exceeding lO0µL /L). At the start of the test, each test concentration contained four replicate and each replicates were randomly distributed 20 fertilized eggs.

POST-HATCH DETAILS

- No. of hatched eggs (alevins)/treatment released to the test chamber: Hatching success in the control and solvent control groups were 98.75% and 95.00% whereas, 93.75%, 90.00%, 88.75%, 87.50%, 71.25%, 63.75%, 57.50% and 47.50%, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L
- Other: Post-hatch larval survival in the control and solvent control groups were 91.139% and 92.105% whereas, 89.333%, 88.889%, 85.915%, 84.286%, 78.947%, 76.471 %, 69.565% and 60.526%, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Larval mortality (Corrected mortality) was 4.29%, 8.57%, 2.86%, 15.71 %, 35.71 %, 44.29%, 54.29% and 67.14% in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L.

Key result

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

0.063 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: larvae mortality

Remarks on result:

other: LOEC: 0.156 mg/L

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

0.156 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: larvae mortality

Remarks on result:

other: Other deetails not known

Key result

Duration:

30 d

Dose descriptor:

LC50

Effect conc.:

1.64 mg/L

95% CI:

> 1.062 - < 2.218

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

larvae mortality

Remarks on result:

other: details not given

Key result

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

0.156 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: hatching success and post-hatching survival

Remarks on result:

other: LOEC: 0.391 mg/L

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

0.391 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: larvae survival and ferility reduction

Remarks on result:

other: Other details not known

Key result

Duration:

30 d

Dose descriptor:

EC50

Effect conc.:

5.037 mg/L

95% CI:

> 2.544 - < 7.531

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: hatching reduction

Remarks on result:

other: details not given

Key result

Duration:

30 d

Dose descriptor:

EC50

Effect conc.:

> 6.104

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: surviving larval reduction

Remarks on result:

other: details not given

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: Larval mortality (Corrected mortality) was 4.29%, 8.57%, 12.86%, 15.71%, 35.71%,
44.29%, 54.29% and 67.14% in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L.
- Days to hatch or time to release of young: Hatching success in the control and solvent control groups were 98.75% and 95.00%
- Numbers hatched: Total 80 embryos hatched in both control and solvent control. It is 98.75% and 95% in control and solvent control, respectively.
- Observations on body length and weight of young and/or exposed parents at one or more time periods: Total mean length of of zebrafish larvae in the control and solvent control was minimum about 11mm .

Reported statistics and error estimates:

Based on the mortality data, LC,o /EC, value (Based on the larvae mortality, Fertility reduction (%) and Surviving larval reduction) was determined statistically by probit analysis using NCSS software 2022, version V22.0.2. the 95% confidence limits were calculated by using the formula: C,o/EC5«±1.96xstandard error. Based on the mortality data, NOEC and LOEC value were determined statistically by Fisher's Exact test (p value > 0.05) using NCSS software 2022, version V22.0.2. Based on the hatching success data, NOEC and LOEC value were determined statistically by Kruskal-Wallis Multiple-Comparison Z-Value Test (z value> 1.96) using NCSS software 2022, version V22.0.2. Based on the hatching success data, NOEC and LOEC value were determined statistically by Dunnett's Two-Sided Multiple-Comparison Test (Alpha value > 0.05) using NCSS software 2022, version V22.0.2.

Range Finding Test:

Hatching (percent) observed in control and solvent control groups were 92.5% and 85.0% whereas, 82.5% and 2.5% in the tested concentration of 5 and 10 mg/L for a period of 96 hours. Non-detachment of the tail observed in the control (3), solvent control (6) and in the tested concentration of 5 mg/ L (7) for a period of 96 hours. Coagulation observed was 39 in the tested concentration of 10 mg/ L, wheras Coagulation observed (40) in the tested concentration of 25, 50 and 100 mg/ L for a period of 96 hours.

 

Definitive Test

1. Hatching and Survival:

Hatching success in the control and solvent control groups were 98.75% and 95.00% whereas, 93.75%, 90.00%, 88.75%, 87.50%, 71.25%, 63.75%, 57.50% and 47.50 %, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/L. Summary of the results are presented in Table 1 and distribution of fertilised eggs are presented in Table 2. The results of hatching success are presented in Table 3.

 

Table 1: Larvae mortality, Hatching success and fish larvae survival -Definitive Test

Concentration mg/L	Mortality		Hatching success and fish larvae surviva
	Total (%)	Corrected (%)	Mean% of hatched larvae	Fertility reduction (%)	Mean% of surviving larvae	Surviving larval reduction
Control	10.00	NA	98.75	NA	91.139	NA
Solvent control	12.50	NA	95.00	NA	92.105	NA
Test chemical substance
0.010	16.25	4.29	93.75	1.32	89.333	3.01
0.025	20.00	8.57	90.00	5.26	88.889	3.49
0.063	23.75	12.86	88.75	6.58	85.915	6.72
0.156	26.25	15.71	87.50	7.89	84.286	8.49
0.391	43.75	35.71	71.25	25.00	78.947	14.29
0.977	51.25	44.29	63.75	32.89	76.471	16.97
2.441	60.00	54.29	57.50	39.47	69.565	24.47
6.104	71.25	67.14	47.50	50.00	60.526	34.29
LC50 mortality	1.640 mg/ L		EC50 rer 1 unity	5.037 mg/L	ECa survival	Greater than 6.104 mg/L
NOEC mortalitv	0.063 mg/ L		NOEC	0.156	NOEC survival	0.156
LOEC mortalitv	0.156 mg/ L		LOEC	0.391	LOEC survival	0.391

Key : mg = milligram, L=Litre, % = Percentage

 

Table 2: Distribution of Fertilised Eggs - Definitive Test

Concentrations (mg/L)	Replication				Number o Eggs Released
	Rl	R2	R3	R4
Control	20	20	20	20	80
Solvent control	20	20	20	20	80
0.010	20	20	20	20	80
0.025	20	20	20	20	80
0.063	20	20	20	20	80
0.156	20	20	20	20	80
0.391	20	20	20	20	80
0.977	20	20	20	20	80
2.441	20	20	20	20	80
6.104	20	20	20	20	80

Key : mg = milligram, L=Litre

 

Table 3: Number of Eggs Hatched (Definitive test)

 

Cone. (mg/L)	24 hrs				No. of eggs hatched	48 hrs				No. of eggs hatched	72 hrs				No. of eggs hatched
	Rl	R2	R3	R4		Rl	R2	R3	R4		Rl	R2	R3	R4
Control	0	0	0	0	0	0	0	0	0	0	10	8	6	10	34
Solvent control	0	0	0	0	0	0	0	0	0	0	9	6	8	7	30
0.010	0	0	0	0	0	0	0	0	0	0	8	6	8	9	31
0.025	0	0	0	0	0	0	0	0	0	0	8	6	7	10	31
0.063	0	0	0	0	0	0	0	0	0	0	10	5	7	8	30
0.156	0	0	0	0	0	0	0	0	0	0	8	7	7	8	30
0.391	0	0	0	0	0	0	0	0	0	0	6	6	7	8	27
0.977	0	0	0	0	0	0	0	0	0	0	5	4	5	7	27
2.441	0	0	0	0	0	0	0	0	0	0	4	3	5	5	17
6.104	0	0	0	0	0	0	0	0	0	0	2	2	4	3	11

 

Cone. (mg/L)	96 hrs				No. of eggs hatched	Cummulative numbers				No. of eggs hatched (cummulative)	Eggs hatched (%)	Fertility reduction (%)
	Rl	R2	R3	R4		Rl	R2	R3	R4
Control	10	12	14	9	45	20	20	20	19	79	98.75	NA
Solvent control	10	12	11	13	46	19	19	19	20	76	95.00	NA
0.010	12	11	10	11	44	20	20	18	20	75	93.75	1.32
0.025	10	12	9	10	41	18	18	16	20	72	90.00	5.26
0.063	8	10	11	12	41	18	18	18	20	71	88.75	6.58
0.156	10	8	13	9	40	18	18	20	17	70	87.50	7.89
0.391	8	7	8	7	30	14	14	15	15	57	71.25	25.00
0.977	6	8	7	9	30	11	11	12	16	51	63.75	32.89
2.441	5	8	9	7	29	9	9	14	12	46	57.50	39.47
6.104	6	6	7	8	27	8	8	11	11	35	47.50	50.00

Key : mg = milligram, L=Litre, R=Replicate, NA=Not applicable
A Eggs hatched(%) = Number of eggs hatched/ Total Number of eggs exposed x 100
B Fertility reduction (%) = Eggs hatched (%) in Solvent control - Eggs hatched (%) in Treatment I Eggs hatched (%) in Solvent control x 100

 

Embryo dead (pre hatch) in the control and solvent control groups were 1 and 4 whereas, 5, 8, 9, 10, 23, 29, 34 and 42 in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. The results are presented in Table 4.

 

Table 4: Mortality in prehatch on each day

 

	Mortality in Pre hatch at each day							Cumulative Mortality	Total mortality
Conc.	R	Eggs	Day 0	Day 1	Day 2	Day 3	Day 4
Control	R1	20	0	0	0	0	0	0	1
	R2	20	0	0	0	0	0	0
	R3	20	0	0	0	0	0	0
	R4	20	0	0	0	0	1	1
Solvent control	R1	20	0	0	0	0	1	1	4
	R2	20	0	0	0	0	2	2
	R3	20	0	0	0	0	1	1
	R4	20	0	0	0	0	0	0
0.010	R1	20	0	0	0	0	0	0	5
	R2	20	0	0	0	0	3	3
	R3	20	0	0	0	0	2	2
	R4	20	0	0	0	0	0	0
0.025	R1	20	0	0	0	0	2	2	8
	R2	20	0	0	0	0	2	2
	R3	20	0	0	0	0	4	4
	R4	20	0	0	0	0	0	0
0.063	R1	20	0	0	0	0	2	2	9
	R2	20	0	0	0	0	5	5
	R3	20	0	0	0	0	2	2
	R4	20	0	0	0	0	0	0
0.156	R1	20	0	0	0	0	2	2	10
	R2	20	0	0	0	0	5	5
	R3	20	0	0	0	0	0	0
	R4	20	0	0	0	0	3	3
0.391	R1	20	0	0	0	2	4	6	23
	R2	20	0	0	0	5	2	7
	R3	20	0	0	0	1	4	5
	R4	20	0	0	0	2	3	5
0.977	R1	20	0	0	0	4	5	9	29
	R2	20	0	0	0	2	6	8
	R3	20	0	0	0	3	5	8
	R4	20	0	0	0	1	3	4
2.441	R1	20	0	0	0	5	6	11	34
	R2	20	0	0	0	3	6	9
	R3	20	0	0	0	2	4	6
	R4	20	0	0	0	2	6	8
6.104	R1	20	0	0	0	8	4	12	42
	R2	20	0	0	0	5	7	12
	R3	20	0	0	0	3	6	9
	R4	20	0	0	0	4	5	9

 Key: Cone.= Concentration (mg/ L), R = Replication, Eggs= Number of eggs added at the start

Larval mortality (Corrected mortality) was 4.29%, 8.57%, 12.86%, 15.71 %, 35.71 %, 44.29%, 54.29% and 67.14% in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. The results of larvae mortality are presented in Table 5.

 

Table 5: Larvae Mortality

Concentration (mg/L)	Replication				Larval mortality (sum of 4 replicates)	Dead embryos
	R1	R2	R3	R4
Control	3	1	2	1	7	1
Solvent control	2	1	2	1	6	4
0.010	1	2	3	2	8	5
0.025	2	3	2	1	8	8
0.063	4	2	1	3	10	9
0.156	2	2	3	4	11	10
0.391	3	4	3	2	12	23
0.977	3	4	2	3	12	29
2.441	4	3	3	4	14	34
6.104	5	4	3	3	15	42

Key : mg = milligram, L=Litre, % = Percentage, NA=Not applicable

Total Larvae Mortality (%) = Total Larvae Mortality/ Total Number of Larvae exposed x 100
** Corrected Mortality(%) = Larvae Mortality in Treatment (%) - Larvae Mortality in Solvent control (%) I 100 - Larvae Mortalityin Solvent
control (%) x 100

Post-hatch larval survival in the control and solvent control groups were 91.139% and 92.105% whereas, 89.333%, 88.889%, 85.915%, 84.286%, 78.947%, 76.471 %, 69.565% and 60.526 %, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. The detailed results are presented in Table 6.

 

Table 6: Larvae survival

Concentration (mg/L)	Number of eggs hatched	Replication				Total larvae survival	Survival larvae	Survival larvae reduction
		R1	R2	R3	R4		%	%
Control	79	17	19	18	18	72	91.139	NA
Solvent control	76	17	17	17	19	70	92.105	NA
0.010	75	19	15	15	18	67	89.333	3.01
0.025	72	16	15	14	19	64	88.889	3.49
0.063	71	14	13	17	17	61	85.915	6.72
0.156	70	16	13	17	13	59	84.286	8.49
0.391	57	11	9	12	13	45	78.947	14.29
0.977	51	8	8	10	13	39	76.471	16.97
2.441	46	5	8	11	8	32	69.565	24.47
6.104	38	3	4	8	8	23	60.526	34.29

Survival larvae % = Total Larvae Survived/ Number of larvae hatched * 100

Survival larvae reduction% = Number of larvae survived in solvent control (%) - Number of larvae survived in treatment/ Number of larvae survived in solvent control * 100

 

Abnormal Appearance and Behaviour:

During the definitive test, no clinical sign exhibited in control group, solvent control group and in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391 and 0.977 mg/L, whereas loss of buoyancy control (2+2 larvae) behaviour was observed in the tested concentration of 2.441 and 6.104 mg/ L. Abnormal behaviour like hypoactivity (2 larvae) observed in in the tested concentration of 2.441 mg/ L for a period of 30 after post hatch. The detailed results are presented in Table 7.

 

3. Total Weight and Length (Total mean):

Total mean weight in the control and solvent control groups were 0.034 and 0.032 g whereas, 0.028 g, 0.027 g, 0.026 g, 0.023 g, 0.022 g, 0.020 g, 0.018 g and 0.017 gin the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Total mean lengths in the control and solvent control groups were about 11 mm whereas, 10.92 mm, 10.87 mm, 10.64 mm, 10.55 mm, 10.48 mm, 10.05 mm, 9.83 mm and 9.57 mm in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. The detailed results are presented in Table 8 to 17.

Validity criteria fulfilled:

yes

Remarks:

Water temp was maintained at 26 ± 1.5 °C throughout the exposure. The DO in all groups was between 74.5 to 91.5% of saturation. Hatching rate in control and solvent control was 98.75% & 95%. Larvae survival in control &solvent control was91.139% & 92.1%.

Conclusions:

The LC50 of the test chemical in Dania rerio larvae mortality (30 days post hatch) was found to be 1.640 mg/L with 95% confidence limits between 1.062 mg/L and 2.218 mg/L. EC50 value for hatching reduction was found to be 5.037 mg/L with 95% confidence limits between 2.544 mg/L and 7.531 mg/L. EC50 value for surviving larval reduction was found to be > 6.104 mg/L. The NOEC and LOEC for larvae mortality was found to be 0.063 mg/L and 0.156 mg/L, respectively. On the basis of survival effect, the NOEC and LOEC were found to be 0.156 mg/L and 0.391 mg/L, respectively.

Executive summary:

The Early-Life Stage Fish Toxicity Test was performed using Danio rerio (zebrafish). The study was performed in compliance with OECD 210 Guideline for the testing of chemicals; Fish, Early-life Stage Toxicity Test, (adopted 26 July, 2013). Solubility of the test item was performed by weighing 25.7 mg of test item in a glass vial and dissolved in 25 µL of acetone and transferring into the 250 ml volumetric flask, and made up to the mark using natural water for solubility check. The stability of the test item in natural water determined by analyzing the test concentrations of 0.01 and 100 mg/L at 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (100.55% to 101.51 % for 0.01 mg/L and 98.66% to 99.94% for 100 mg/L).Range finding test was conducted with test concentrations at 0 (Control), 0 (Solvent Control), 5, 10, 25, 50 and 100 mg/ L. Each concentration contained two replicate and each replicates were exposed 20 fertilized eggs. Test item was formulated in acetone + natural water (not exceeding 100 µL /L). Hatching (percent) observed in control and solvent control groups were 92.5% and 85.0% whereas, 82.5% and 2.5% in the tested concentration of 5 and 10 mg/L for a period of 96 hours. Non-detachment of the tail observed in the control (3), solvent control (6) and in the tested concentration of 5 mg/ L (7) for a period of 96 hours. Coagulation observed was 39 in the tested concentration of 10 mg/ L, wheras Coagulation observed (40) in the tested concentration of 25, 50 and 100 mg/ L for a period of 96 hours. Based on the results of range finding test, the definitive was conducted with 8 test concentrations at 0 (Control), 0 (Solvent Control), 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/L, in a geometric series with a spacing factor of 2.5 by static renewal method. Test item was formulated in acetone+ natural water (not exceeding lO0µL /L). At the start of the test, each test concentration contained four replicate and each replicates were randomly distributed 20 fertilized eggs. Hatching success in the control and solvent control groups were 98.75% and 95.00% whereas, 93.75%, 90.00%, 88.75%, 87.50%, 71.25%, 63.75%, 57.50% and 47.50%, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Post-hatch larval survival in the control and solvent control groups were 91.139% and 92.105% whereas,  89.333%, 88.889%, 85.915%, 84.286%, 78.947%, 76.471 %, 69.565% and 60.526%, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Larval mortality (Corrected mortality) was 4.29%, 8.57%, 12.86%, 15.71 %, 35.71 %, 44.29%,54.29% and  67.14% in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. During the definitive test, no clinical sign exhibited in control group, solvent control group and in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391 and 0.977 mg/ L, whereas loss of buoyancy control (2+2 larvae) behaviour was observed in the tested concentration of 2.441 and 6.104 mg/ L. Abnormal behaviour like hypoactivity (2 larvae) observed in in the tested concentration of 2.441 mg/ L for a period of 30 after post hatch. Total mean weight in the control and solvent control groups were 0.034 and 0.032 g whereas, 0.028 g, 0.027 g, 0.025 g, 0.023 g, 0.022 g, 0.021 g, 0.018 g and 0.015 g in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Total mean lengths in the control and solvent control groups were 11 mm whereas, 10.93 mm, 10.90 mm, 10.64 mm, 10.57 mm, 10.52 mm, 10.03 mm, 9.80 mm and 9.42 mm in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. During the definitive test period, all the beakers were incubated in the room under test conditions. The pH of the control and solvent control at the test start 7.3 and 7.2 and end of the test was 7.1 and 7.2 and therefore did not vary more than 1.5 units during the study. The pH of  all the tested concentrations was 7.3 to 7.9 at the beginning of the test and 7.3 to 7.6 at test termination. The temperature at the beginning was 26.0°C to 26.5°C and at test termination was 26.1 °C to 26.5°C. The Dissolved oxygen of the concentrations ranged from 74.5 to 91.5% ASV. The mean intensity of light ranged from 698 to 735 Lux. The test item content obtained in during the dose concentration analysis New sample solution range was 99.94 % to 101.22 % for 0.01 mg/L, 99.27 % to 100.84 % for 0.391 mg/Land 99.98 % to 101.22 % for 6.104 mg/L. For old sample solution range was 99.76 % to 101.56 % for 0.01 mg/L, 99.52 % to 101.01 % for 0.391 mg/Land 99.05 % to 101.04 % for 6.104 mg/L. Based on the mortality data, LC5o /EC,o value (Based on the larvae mortality, Fertility reduction (%) and Surviving larval reduction) was determined statistically by probit analysis using NCSS software 2022, v22.0.2. the 95% confidence limits were calculated by using the formula: LC,oIEC,o±1.96xstandard error. Based on the mortality data, NOEC and LOEC value were determined statistically by Fisher's Exact test (p value > 0.05) using NCSS software 2022, v22.0.2. Based on the hatching success data, NOEC and LOEC value were determined statistically by Kruskal-Wallis Multiple. Comparison Z-Value Test (z value > 1.96) using NCSS software 2022, v22.0.2. Based on the hatching success data, NOEC and LOEC value were determined statistically by Dunnett's Two Sided Multiple-Comparison Test (Alpha value > 0.05) using NCSS software 2022, v22.0.2. The LC50 of the test chemical in Dania rerio larvae mortality (30 days post hatch) was found to be 1.640 mg/L with 95% confidence limits between 1.062 mg/L and 2.218 mg/L. EC50 value for hatching reduction was found to be 5.037 mg/L with 95% confidence limits between 2.544 mg/L and 7.531 mg/L. EC50 value for surviving larval reduction was found to be > 6.104 mg/L. The NOEC and LOEC for larvae mortality was found to be 0.063 mg/L and 0.156 mg/L, respectively. On the basis of survival effect, the NOEC and LOEC were found to be 0.156 mg/L and 0.391 mg/L, respectively. Based on the NOEC value (i.e., 0.156 mg/l) (based on larave survival effect), the test chemical was considered to be classified in ''aquatic chronic category 2'' as per CLP classification criteria.

Description of key information

The Early-Life Stage Fish Toxicity Test was performed using Danio rerio (zebrafish). The study was performed in compliance with OECD 210 Guideline for the testing of chemicals; Fish, Early-life Stage Toxicity Test, (adopted 26 July, 2013). Solubility of the test item was performed by weighing 25.7 mg of test item in a glass vial and dissolved in 25 µL of acetone and transferring into the 250 ml volumetric flask, and made up to the mark using natural water for solubility check. The stability of the test item in natural water determined by analyzing the test concentrations of 0.01 and 100 mg/L at 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (100.55% to 101.51 % for 0.01 mg/L and 98.66% to 99.94% for 100 mg/L).Range finding test was conducted with test concentrations at 0 (Control), 0 (Solvent Control), 5, 10, 25, 50 and 100 mg/ L. Each concentration contained two replicate and each replicates were exposed 20 fertilized eggs. Test item was formulated in acetone + natural water (not exceeding 100 µL /L). Hatching (percent) observed in control and solvent control groups were 92.5% and 85.0% whereas, 82.5% and 2.5% in the tested concentration of 5 and 10 mg/L for a period of 96 hours. Non-detachment of the tail observed in the control (3), solvent control (6) and in the tested concentration of 5 mg/ L (7) for a period of 96 hours. Coagulation observed was 39 in the tested concentration of 10 mg/ L, wheras Coagulation observed (40) in the tested concentration of 25, 50 and 100 mg/ L for a period of 96 hours. Based on the results of range finding test, the definitive was conducted with 8 test concentrations at 0 (Control), 0 (Solvent Control), 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/L, in a geometric series with a spacing factor of 2.5 by static renewal method. Test item was formulated in acetone+ natural water (not exceeding lO0µL /L). At the start of the test, each test concentration contained four replicate and each replicates were randomly distributed 20 fertilized eggs. Hatching success in the control and solvent control groups were 98.75% and 95.00% whereas, 93.75%, 90.00%, 88.75%, 87.50%, 71.25%, 63.75%, 57.50% and 47.50%, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Post-hatch larval survival in the control and solvent control groups were 91.139% and 92.105% whereas,  89.333%, 88.889%, 85.915%, 84.286%, 78.947%, 76.471 %, 69.565% and 60.526%, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Larval mortality (Corrected mortality) was 4.29%, 8.57%, 12.86%, 15.71 %, 35.71 %, 44.29%,54.29% and  67.14% in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. During the definitive test, no clinical sign exhibited in control group, solvent control group and in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391 and 0.977 mg/ L, whereas loss of buoyancy control (2+2 larvae) behaviour was observed in the tested concentration of 2.441 and 6.104 mg/ L. Abnormal behaviour like hypoactivity (2 larvae) observed in in the tested concentration of 2.441 mg/ L for a period of 30 after post hatch. Total mean weight in the control and solvent control groups were 0.034 and 0.032 g whereas, 0.028 g, 0.027 g, 0.025 g, 0.023 g, 0.022 g, 0.021 g, 0.018 g and 0.015 g in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Total mean lengths in the control and solvent control groups were 11 mm whereas, 10.93 mm, 10.90 mm, 10.64 mm, 10.57 mm, 10.52 mm, 10.03 mm, 9.80 mm and 9.42 mm in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. During the definitive test period, all the beakers were incubated in the room under test conditions. The pH of the control and solvent control at the test start 7.3 and 7.2 and end of the test was 7.1 and 7.2 and therefore did not vary more than 1.5 units during the study. The pH of  all the tested concentrations was 7.3 to 7.9 at the beginning of the test and 7.3 to 7.6 at test termination. The temperature at the beginning was 26.0°C to 26.5°C and at test termination was 26.1 °C to 26.5°C. The Dissolved oxygen of the concentrations ranged from 74.5 to 91.5% ASV. The mean intensity of light ranged from 698 to 735 Lux. The test item content obtained in during the dose concentration analysis New sample solution range was 99.94 % to 101.22 % for 0.01 mg/L, 99.27 % to 100.84 % for 0.391 mg/Land 99.98 % to 101.22 % for 6.104 mg/L. For old sample solution range was 99.76 % to 101.56 % for 0.01 mg/L, 99.52 % to 101.01 % for 0.391 mg/Land 99.05 % to 101.04 % for 6.104 mg/L. Based on the mortality data, LC5o /EC,o value (Based on the larvae mortality, Fertility reduction (%) and Surviving larval reduction) was determined statistically by probit analysis using NCSS software 2022, v22.0.2. the 95% confidence limits were calculated by using the formula: LC,oIEC,o±1.96xstandard error. Based on the mortality data, NOEC and LOEC value were determined statistically by Fisher's Exact test (p value > 0.05) using NCSS software 2022, v22.0.2. Based on the hatching success data, NOEC and LOEC value were determined statistically by Kruskal-Wallis Multiple. Comparison Z-Value Test (z value > 1.96) using NCSS software 2022, v22.0.2. Based on the hatching success data, NOEC and LOEC value were determined statistically by Dunnett's Two Sided Multiple-Comparison Test (Alpha value > 0.05) using NCSS software 2022, v22.0.2. The LC50 of the test chemical in Dania rerio larvae mortality (30 days post hatch) was found to be 1.640 mg/L with 95% confidence limits between 1.062 mg/L and 2.218 mg/L. EC50 value for hatching reduction was found to be 5.037 mg/L with 95% confidence limits between 2.544 mg/L and 7.531 mg/L. EC50 value for surviving larval reduction was found to be > 6.104 mg/L. The NOEC and LOEC for larvae mortality was found to be 0.063 mg/L and 0.156 mg/L, respectively. On the basis of survival effect, the NOEC and LOEC were found to be 0.156 mg/L and 0.391 mg/L, respectively. Based on the NOEC value (i.e., 0.156 mg/l) (based on larave survival effect), the test chemical was considered to be classified in ''aquatic chronic category 2'' as per CLP classification criteria.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.156 mg/L

Additional information

The Early-Life Stage Fish Toxicity Test was performed using Danio rerio (zebrafish). The study was performed in compliance with OECD 210 Guideline for the testing of chemicals; Fish, Early-life Stage Toxicity Test, (adopted 26 July, 2013). Solubility of the test item was performed by weighing 25.7 mg of test item in a glass vial and dissolved in 25 µL of acetone and transferring into the 250 ml volumetric flask, and made up to the mark using natural water for solubility check. The stability of the test item in natural water determined by analyzing the test concentrations of 0.01 and 100 mg/L at 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (100.55% to 101.51 % for 0.01 mg/L and 98.66% to 99.94% for 100 mg/L).Range finding test was conducted with test concentrations at 0 (Control), 0 (Solvent Control), 5, 10, 25, 50 and 100 mg/ L. Each concentration contained two replicate and each replicates were exposed 20 fertilized eggs. Test item was formulated in acetone + natural water (not exceeding 100 µL /L). Hatching (percent) observed in control and solvent control groups were 92.5% and 85.0% whereas, 82.5% and 2.5% in the tested concentration of 5 and 10 mg/L for a period of 96 hours. Non-detachment of the tail observed in the control (3), solvent control (6) and in the tested concentration of 5 mg/ L (7) for a period of 96 hours. Coagulation observed was 39 in the tested concentration of 10 mg/ L, wheras Coagulation observed (40) in the tested concentration of 25, 50 and 100 mg/ L for a period of 96 hours. Based on the results of range finding test, the definitive was conducted with 8 test concentrations at 0 (Control), 0 (Solvent Control), 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/L, in a geometric series with a spacing factor of 2.5 by static renewal method. Test item was formulated in acetone+ natural water (not exceeding lO0µL /L). At the start of the test, each test concentration contained four replicate and each replicates were randomly distributed 20 fertilized eggs. Hatching success in the control and solvent control groups were 98.75% and 95.00% whereas, 93.75%, 90.00%, 88.75%, 87.50%, 71.25%, 63.75%, 57.50% and 47.50%, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Post-hatch larval survival in the control and solvent control groups were 91.139% and 92.105% whereas,  89.333%, 88.889%, 85.915%, 84.286%, 78.947%, 76.471 %, 69.565% and 60.526%, in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Larval mortality (Corrected mortality) was 4.29%, 8.57%, 12.86%, 15.71 %, 35.71 %, 44.29%,54.29% and  67.14% in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. During the definitive test, no clinical sign exhibited in control group, solvent control group and in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391 and 0.977 mg/ L, whereas loss of buoyancy control (2+2 larvae) behaviour was observed in the tested concentration of 2.441 and 6.104 mg/ L. Abnormal behaviour like hypoactivity (2 larvae) observed in in the tested concentration of 2.441 mg/ L for a period of 30 after post hatch. Total mean weight in the control and solvent control groups were 0.034 and 0.032 g whereas, 0.028 g, 0.027 g, 0.025 g, 0.023 g, 0.022 g, 0.021 g, 0.018 g and 0.015 g in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. Total mean lengths in the control and solvent control groups were 11 mm whereas, 10.93 mm, 10.90 mm, 10.64 mm, 10.57 mm, 10.52 mm, 10.03 mm, 9.80 mm and 9.42 mm in the tested concentration of 0.010, 0.025, 0.063, 0.156, 0.391, 0.977, 2.441 and 6.104 mg/ L. During the definitive test period, all the beakers were incubated in the room under test conditions. The pH of the control and solvent control at the test start 7.3 and 7.2 and end of the test was 7.1 and 7.2 and therefore did not vary more than 1.5 units during the study. The pH of  all the tested concentrations was 7.3 to 7.9 at the beginning of the test and 7.3 to 7.6 at test termination. The temperature at the beginning was 26.0°C to 26.5°C and at test termination was 26.1 °C to 26.5°C. The Dissolved oxygen of the concentrations ranged from 74.5 to 91.5% ASV. The mean intensity of light ranged from 698 to 735 Lux. The test item content obtained in during the dose concentration analysis New sample solution range was 99.94 % to 101.22 % for 0.01 mg/L, 99.27 % to 100.84 % for 0.391 mg/Land 99.98 % to 101.22 % for 6.104 mg/L. For old sample solution range was 99.76 % to 101.56 % for 0.01 mg/L, 99.52 % to 101.01 % for 0.391 mg/Land 99.05 % to 101.04 % for 6.104 mg/L. Based on the mortality data, LC5o /EC,o value (Based on the larvae mortality, Fertility reduction (%) and Surviving larval reduction) was determined statistically by probit analysis using NCSS software 2022, v22.0.2. the 95% confidence limits were calculated by using the formula: LC,oIEC,o±1.96xstandard error. Based on the mortality data, NOEC and LOEC value were determined statistically by Fisher's Exact test (p value > 0.05) using NCSS software 2022, v22.0.2. Based on the hatching success data, NOEC and LOEC value were determined statistically by Kruskal-Wallis Multiple. Comparison Z-Value Test (z value > 1.96) using NCSS software 2022, v22.0.2. Based on the hatching success data, NOEC and LOEC value were determined statistically by Dunnett's Two Sided Multiple-Comparison Test (Alpha value > 0.05) using NCSS software 2022, v22.0.2. The LC50 of the test chemical in Dania rerio larvae mortality (30 days post hatch) was found to be 1.640 mg/L with 95% confidence limits between 1.062 mg/L and 2.218 mg/L. EC50 value for hatching reduction was found to be 5.037 mg/L with 95% confidence limits between 2.544 mg/L and 7.531 mg/L. EC50 value for surviving larval reduction was found to be > 6.104 mg/L. The NOEC and LOEC for larvae mortality was found to be 0.063 mg/L and 0.156 mg/L, respectively. On the basis of survival effect, the NOEC and LOEC were found to be 0.156 mg/L and 0.391 mg/L, respectively. Based on the NOEC value (i.e., 0.156 mg/l) (based on larave survival effect), the test chemical was considered to be classified in ''aquatic chronic category 2'' as per CLP classification criteria.