Tetrabutylammonium bromide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

- Concentrations: Test chemical conc. used for the study were 0 (Control), 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/l, respectively.
- Sampling method: test sample taken for the analysis, is collected from four corners and centre of the test vessels, The received test sample concentrations (0.0164 mg/L, 0.64 mg/L and 10 mg/L on 0-hour fresh samples, 96-Hour spent and fresh samples and 48-hour spent samples) were mixed well with uniformity. An aliquot of required volume of sample solutions was transfer to a clean round bottom flask (RB) and dry on a 60°C heating condition. After dry eluate of sample solutions made up to the final volume using acetonitrile and analyzed under High performance liquid chromatography.
- Sample storage conditions before analysis: Samples were analysed immediately after preparation.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: The test chemical was dissolved in the dilution water and the selected test chemical concentrations were prepared by dilution of a stock solution. The solution was prepared by simply mixing.
- Controls: Diliution water was used as control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not aplicable

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: The fishes were supplied by Tarun Fish Aquarium, manimangalam, India.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): Zebra fishes of both sex of ratio (2 male : 1female) total 12 fishes were used for the spawning
- Method of collection of fertilised eggs: Eggs were collected from breeding groups, mixed and randomly selected. Spawning traps were placed in the breeding tank for the collection of eggs. To prevent predation of eggs by adult zebra fish, the spawn traps were covered with inert wire mesh of size (2.5±0.5 mm).
- Subsequent handling of eggs: The spawn traps with the collected eggs were carefully removed. The fertilised eggs were collected by using pasteur pipette on next day morning (on the day of exposure) and 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
Commercial dry food was provided for 14 days. From day 15 onwards live food (artemia) was provided along with dry food.
- Start date: 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: ad libitum
- Frequency of feeding: Dry feed was given twice a day and Brine shrimp Artemia was given to test fishes once daily.
- Other: Surplus food and faeces were removed where required, to avoid accumulation of waste.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

34 d

Remarks on exposure duration:

Total exposure duration 34 days (30 days post hatch duration)

Hardness:

170 mg/L CaCO3

Test temperature:

At the beginning of the test: 26.5°C to 27.0°C
At the end of the test: 26.2°C to 26.5°C

pH:

At the beginning of the test: 7.5 to 8.0
At the end of the test: 7.5 to 8.2

Dissolved oxygen:

69.8 to 94.3% air saturation value

Nominal and measured concentrations:

Test chemical conc. used for the study were 0 (Control), 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/l, respectively which were spaced by a constant factor 2.5.

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): Glass vessel of size 90 mm × 15 mm fill volume 50 ml used before post hatching period
- Test vessel: 2 L Glass beaker
- Type (delete if not applicable): loosely closed to avoid the crossover and contamination
- Material, size, headspace, fill volume: 2L Glass 250 mL
- Aeration: No aeration was provided during study
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Semi static condition with 96 hours of renewal rate
- 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): Not applicable
- Biomass loading rate: 80 eggs/conc.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon: 1.48 mg/L
- Particulate matter: <1.0 mg/L
- Metals: Aluminium < 1µg/L, Arsenic- < 1µg/L, Chromium < 1µg/L, Cobalt< 1µg/L, Copper< 1µg/L, Iron< 1µg/L, Lead< 1µg/L, Nickel< 1µg/L,Zinc< 1µg/L Cadmium <100 ng/L, Mercury <100ng/L, Silver<100ng/L.
- Pesticides: Organo phosphates were below Detection limit of <0.01µg/L
- Chlorine:63.6 mg/L
- Ca/mg ratio: 3.78 mg/L
- Culture medium different from test medium: Same as test medium
- Intervals of water quality measurement: every 6 months

OTHER TEST CONDITIONS
- Adjustment of pH: Not adusted
- Photoperiod: 12: 12 light: dark conditions
- Light intensity: Light intensity ranges from 694 to 768 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, hatcing sucess, survival rate,

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 0 (Control), 5, 10, 25, 50 and 100 mg/l, respectively.
- Results used to determine the conditions for the definitive study: Mortlity and hatcing sucess along with behavioural and morphological abnormalities were uesd to determine the conditions for the test

POST-HATCH DETAILS
- Begin of post-hatch period: 4 days after embryos spawning
- No. of hatched eggs (alevins)/treatment released to the test chamber: 20 embryos released in chamber
- Release of alevins from incubation cups to test chamber on day no.: 4

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 80 eggs
- Removal of eggs to check the embryonic development on day no.: every day from day1

Reference substance (positive control):

not required

Key result

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: survival larvae

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: survival larvae

Duration:

30 d

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: survival larvae

Duration:

30 d

Dose descriptor:

LC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: 100 percent survival rate was observed at embryos stage in control, 7.5% mortality was observed in control after 30 days
- Days to hatch or time to release of young: 96 hours all the embryos were hatched in control
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: 80 embryos hatched in control,
- Observations on body length and weight of young and/or exposed parents at one or more time periods: length and weight was measured after 30 days post hatch, in control reported to be 11.20 mm and 0.032 g
- Number of healthy fish at end of test: all fishes were healthy in control
- Type of and number with morphological/behavioural/developmental abnormalities: At concentration 1.6 mg/L in two hatchling out of which one has tail deformities and other was hypoactive.
At concentraion 4 mg/L one hatchling was hypoactive.
At concentraion 10 mg/L one hatchling had oedema and other has Loss of buoyancy control,

Reported statistics and error estimates:

Data were analysed using appropriate statistical methods to calculate the EC50, LOEC and NOEC value if applicable. The 95% confidence Limits were calculated.

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% (98.37% to 99.09% for 0.01 mg/L and 98.29% to 99.03% for 100 mg/L) with respect to initial measured concentration and hence the dose verification for definitive test was performed at the beginning and at the termination of the test.

Range Finding Test

Hatching (percent) observed in control group 100% whereas, 97.5%, 92.5%, 90.0%, 87.5% and 85.0% in the tested concentration of 5, 10, 25, 50 and 100 mg/L for a period of 96 hours. All fertilised eggs hatched in the control group whereas non-detachment of the tail observed in the tested concentration of 5, 10, 25, 50 and 100 mg/ L for a period of 96 hours.

Tabl: Larvae mortality, Hatching success and fish larvae survival – Definitive Test

Concentration mg/L	Mortality		Hatching success and fish larvae survival
	Total (%)	Corrected	Mean % of hatched larvae	Fertility reduction (%)	Mean % of surviving larvae	Surviving larval reduction (%)
		(%)
Control	7.5	NA	100	NA	92.5	NA
Tetrabutylammonium bromide
0.0164	11.25	4.05	100	0	88.8	4.05
0.041	10.00	2.70	100	0	90.0	2.70
0.102	10.00	2.70	100	0	90.0	2.70
0.256	11.25	4.05	100	0	88.8	4.05
0.64	12.5	5.41	100	0	87.5	5.41
1.600	13.75	6.76	100	0	86.3	6.76
4.000	17.5	10.81	96	4	81.8	11.55
10.000	17.5	10.81	90	10	80.6	12.91
LC50mortality	Greater than 10.000 mg/ L		EC50fer1tility	Greater than 10.000 mg/ L	EC50survival	Greater than 10.000 mg/ L
NOECmortality	Greater than or equal to 10.000 mg/ L		NOECfertility	1.600 mg/ L	NOECsurvival	1.600 mg/ L
LOECmortality			LOECfertility	4.000 mg/ L	LOECsurvival	4.000 mg/ L

 

Table : FishTotal Length (mm) andWeight (g) : Control[E1] 

Number of larvae	Control
	Length (mm)				Total weight (g)
	R1	R2	R3	R4	R1	R2	R3	R4
1	11.02	11.35	11.18	10.98	0.049	0.032	0.027	0.032
2	11.05	11.42	11.05	10.96	0.035	0.038	0.034	0.027
3	11.21	11.12	11.2	10.99	0.031	0.031	0.032	0.031
4	11.41	11.33	11.41	11.32	0.042	0.029	0.024	0.035
5	11.29	11.02	11.29	11.29	0.022	0.032	0.041	0.042
6	11.13	11.31	11.27	11.27	0.038	0.035	0.038	0.039
7	11.41	11.21	11.21	11.23	0.025	0.028	0.025	0.032
8	11.08	11.25	11.08	10.99	0.019	0.031	0.029	0.025
9	11.23	11.34	11.17	11.42	0.035	0.034	0.031	0.045
10	11.3	11.05	11.21	11.03	0.027	0.035	0.022	0.029
11	11.42	11.23	10.59	11.35	0.041	0.032	0.028	0.035
12	11.35	11.38	11.23	11.41	0.032	0.029	0.035	0.038
13	11.6	11.34	11.6	11.22	0.029	0.036	0.026	0.031
14	11.12	11.42	11.17	11.05	0.028	0.035	0.031	0.041
15	11.17	11.12	10.98	10.97	0.029	0.032	0.023	0.023
16	11.24	11.18	10.92	11.32	0.035	0.028	0.021	0.029
17	11.05	10.81	11.23	11.26	0.032	0.031	0.032	0.035
18	11.22	11.02	11.28	11.28	0.029	0.032	0.035	0.031
19	-	-	11.05	11.35	-	-	0.034	0.035
20	-	-	-	-	-	-	-	-
Individual mean	11.24	11.22	11.16	11.19	0.032	0.032	0.030	0.033
Total mean	11.20				0.032
Individual SD	0.16	0.17	0.21	0.16	0.007	0.003	0.006	0.006
Total SD	0.17				0.005
Minimum	10.59				0.019
Maximum	11.6				0.049

Key: - Not applicable due to larvae dead, SD = Standard deviation, mm = millimeter,

g = gram,mg = milligram, L=Litre, R=Replicate

Table : FishTotal Length (mm) andWeight (g) : 0.0164 mg/ L

Number of larvae	Concentration: 0.0164 mg/ L
	Length (mm)				Total weight (g)
	R1	R2	R3	R4	R1	R2	R3	R4
1	10.54	11.28	10.95	10.98	0.033	0.029	0.018	0.019
2	10.92	11.05	11	10.61	0.031	0.022	0.025	0.022
3	11.25	10.98	11.22	11.25	0.045	0.019	0.029	0.035
4	11.24	10.85	11.32	10.88	0.041	0.025	0.035	0.0298
5	10.68	10.71	11.02	10.23	0.028	0.021	0.027	0.045
6	11.21	11.35	10.58	10.87	0.029	0.038	0.025	0.029
7	11.32	11.21	10.98	11.18	0.035	0.032	0.03	0.034
8	10.91	11.22	10.85	11.05	0.018	0.035	0.022	0.024
9	10.34	11.05	10.99	10.68	0.025	0.025	0.027	0.018
10	11.34	11.24	11.32	11.35	0.035	0.028	0.034	0.041
11	11.25	11.35	11.05	11.25	0.032	0.031	0.032	0.039
12	11.02	11.41	10.88	11.04	0.029	0.042	0.027	0.042
13	11.12	11.02	11.25	11.32	0.027	0.031	0.032	0.045
14	10.65	11.35	11.32	11.36	0.025	0.035	0.039	0.052
15	11.02	11.05	11.41	11.08	0.029	0.029	0.034	0.028
16	10.55	11.12	11.24	11.06	0.028	0.034	0.027	0.024
17	10.96	11.05	11.13	11.08	0.024	0.028	0.028	0.021
18	-	10.97	11.18	11.15	-	0.019	0.031	0.025
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Individual mean	10.96	11.13	11.09	11.02	0.030	0.029	0.029	0.032
Total mean	11.05				0.030
Individual SD	0.31	0.19	0.21	0.29	0.006	0.006	0.005	0.010
Total SD	11.05				0.007
Minimum	10.23				0.018
Maximum	11.41				0.052

Key: - Not applicable due to larvae dead, SD = Standard deviation, mm = millimeter,

g = gram,mg = milligram, L=Litre, R=Replicate

Table : Fish Total Length (mm) and Weight (g) : 0.041 mg/ L

Number of larvae	Concentration: 0.041 mg/ L
	Length (mm)				Total weight (g)
	R1	R2	R3	R4	R1	R2	R3	R4
1	10.52	11.02	11.02	11.35	0.022	0.028	0.035	0.039
2	11.02	10.75	11.35	11.21	0.025	0.024	0.041	0.028
3	11.13	11.35	11.41	10.23	0.037	0.035	0.052	0.021
4	11.27	11.28	11.25	11.28	0.047	0.018	0.031	0.032
5	10.54	11.15	10.65	11.14	0.023	0.021	0.028	0.035
6	11.02	11.25	10.89	10.63	0.029	0.023	0.032	0.016
7	11.39	11.05	11.05	11.25	0.036	0.028	0.025	0.018
8	10.59	10.58	10.65	10.95	0.022	0.027	0.021	0.022
9	10.57	11.05	11.25	11.02	0.024	0.031	0.014	0.032
10	10.58	11.32	10.85	10.52	0.023	0.035	0.025	0.034
11	11.27	10.88	11.03	10.85	0.037	0.017	0.036	0.024
12	10.65	11.29	10.88	10.85	0.018	0.035	0.028	0.025
13	10.88	11.14	11.29	11.32	0.022	0.012	0.035	0.032
14	11.21	10.85	11.14	11.25	0.028	0.021	0.032	0.035
15	10.25	11.25	11.17	11.04	0.025	0.037	0.029	0.028
16	11.00	11.31	10.92	11.03	0.032	0.035	0.025	0.031
17	11.31	10.35	11.35	11.42	0.039	0.026	0.032	0.025
18	11.05	-	-	11.05	0.025	-	-	0.018
19	-	-	-	11.25	-	-	-	0.025
20	-	-	-	10.78	-	-	-	0.032
Individual mean	10.90	11.05	11.07	11.02	0.029	0.027	0.031	0.028
Total mean	11.01				0.028
Individual SD	0.34	0.29	0.24	0.31	0.008	0.007	0.008	0.006
Total SD	0.29				0.007
Minimum	10.23				0.012
Maximum	11.42				0.052

Key: - Not applicable due to larvae dead, SD = Standard deviation, mm = millimeter,

g = gram,mg = milligram, L=Litre, R=Replicate

 

Table : Fish Total Length (mm) and Weight (g) : 0.102 mg/ L

Number of larvae	Concentration: 0.102 mg/ L
	Length (mm)				Total weight (g)
	R1	R2	R3	R4	R1	R2	R3	R4
1	10.89	11.14	10.65	11.05	0.028	0.025	0.018	0.028
2	10.54	11.05	11.25	11.06	0.027	0.022	0.025	0.027
3	11.25	10.98	10.65	10.58	0.031	0.028	0.014	0.018
4	10.97	11.15	10.49	10.35	0.029	0.026	0.019	0.012
5	11.14	10.89	11.25	11.24	0.026	0.028	0.032	0.035
6	11.25	11.35	11.32	11.15	0.025	0.024	0.035	0.028
7	11.23	11.05	11.05	11.25	0.029	0.031	0.031	0.032
8	11.26	11.21	11.25	10.58	0.023	0.022	0.035	0.024
9	10.97	10.78	10.85	11.35	0.029	0.024	0.024	0.035
10	11.05	10.95	10.78	11.41	0.022	0.022	0.022	0.041
11	11.32	11.31	10.89	11.15	0.029	0.015	0.018	0.030
12	11.32	10.85	10.56	11.14	0.027	0.021	0.023	0.025
13	11.14	11.36	11.32	10.95	0.026	0.036	0.032	0.031
14	11.05	10.25	11.27	10.68	0.027	0.025	0.037	0.022
15	11.26	10.95	10.75	11.14	0.023	0.028	0.024	0.019
16	11.25	11.63	10.35	11.32	0.034	0.046	0.025	0.025
17	11.14	11.35	11.41	11.25	0.021	0.028	0.038	0.029
18	11.21	-	10.95	11.21	0.022	-	0.025	0.032
19	-	-	10.45	-	-	-	0.037	-
20	-	-	-	-	-	-	-	-
Individual mean	11.12	11.07	10.92	11.05	0.027	0.027	0.027	0.027
Total mean	11.04				0.027
Individual SD	0.19	0.31	0.34	0.30	0.003	0.007	0.007	0.007
Total SD	0.29				0.006
Minimum	10.25				0.012
Maximum	11.63				0.046

Key: - Not applicable due to larvae dead, SD = Standard deviation, mm = millimeter,

g = gram,mg = milligram, L=Litre, R=Replicate

Table : Fish Total Length (mm) and Weight (g) : 0.256 mg/ L

Number of larvae	Concentration: 0.256 mg/ L
	Length (mm)				Total weight (g)
	R1	R2	R3	R4	R1	R2	R3	R4
1	10.25	10.14	9.09	10.61	0.022	0.018	0.014	0.023
2	9.12	10.35	10.66	9.58	0.014	0.016	0.026	0.013
3	10.09	10.65	9.75	10.92	0.012	0.018	0.012	0.024
4	11.05	10.84	9.52	11.05	0.028	0.021	0.016	0.031
5	8.17	11.32	10.21	11.14	0.012	0.035	0.028	0.032
6	10.32	10.54	9.86	10.66	0.024	0.029	0.011	0.029
7	11.05	10.68	9.72	10.38	0.031	0.024	0.014	0.022
8	11.23	9.87	11.35	9.88	0.027	0.012	0.032	0.013
9	10.56	11.06	8.54	10.21	0.024	0.032	0.012	0.022
10	10.14	10.65	10.68	10.75	0.022	0.028	0.024	0.025
11	10.35	10.82	10.35	10.58	0.028	0.025	0.026	0.024
12	11.05	9.89	11.25	10.67	0.032	0.015	0.035	0.026
13	11.35	9.65	10.96	10.55	0.035	0.012	0.029	0.019
14	10.47	10.32	10.25	10.15	0.021	0.019	0.022	0.028
15	10.65	10.85	9.88	10.96	0.015	0.022	0.014	0.012
16	11.24	10.68	10.57	9.85	0.018	0.025	0.012	0.022
17	10.46	10.67	11.25	10.36	0.023	0.028	0.018	0.028
18	-	-	11.31	10.65	-	-	0.025	0.019
19	-	-	-	10.55	-	-	-	0.021
20	-	-	-	-	-	-	-	-
Individual mean	10.44	10.53	10.29	10.50	0.023	0.022	0.021	0.023
Total mean	10.44				0.022
Individual SD	0.81	0.44	0.80	0.42	0.007	0.007	0.008	0.006
Total SD	0.62				0.007
Minimum	8.17				0.011
Maximum	11.35				0.035

Key: - Not applicable due to larvae dead, SD = Standard deviation, mm = millimeter,

g = gram,mg = milligram, L=Litre, R=Replicate

 

Table : Fish Total Length (mm) and Weight (g) : 0.640 mg/ L

Number of larvae	Concentration: 0.640 mg/ L
	Length (mm)				Total weight (g)
	R1	R2	R3	R4	R1	R2	R3	R4
1	9.89	9.65	9.85	9.96	0.017	0.014	0.015	0.013
2	9.15	10.78	10.25	9.56	0.015	0.022	0.021	0.012
3	10.32	8.98	9.99	10.23	0.023	0.012	0.014	0.026
4	11.15	10.47	9.32	10.27	0.032	0.025	0.012	0.027
5	11.02	10.69	10.25	8.98	0.031	0.022	0.018	0.013
6	10.35	11.06	10.48	9.78	0.024	0.035	0.028	0.015
7	10.48	11.14	10.78	10.35	0.022	0.031	0.025	0.031
8	10.88	9.25	10.36	11.01	0.025	0.014	0.022	0.032
9	10.69	10.27	10.55	9.65	0.028	0.016	0.021	0.015
10	10.22	9.69	11.25	10.63	0.022	0.012	0.032	0.023
11	11.35	10.58	9.65	11.03	0.035	0.019	0.014	0.031
12	9.15	11.05	10.52	10.55	0.012	0.031	0.023	0.032
13	9.48	10.18	10.21	7.99	0.014	0.024	0.022	0.014
14	9.74	10.22	10.32	8.59	0.015	0.022	0.018	0.012
15	9.99	9.85	9.87	10.23	0.023	0.016	0.012	0.021
16	9.58	10.55	10.25	9.99	0.013	0.029	0.015	0.018
17	10.07	9.11	10.26	10.66	0.014	0.013	0.022	0.019
18	-	9.88	-	10.21	-	0.014	-	0.017
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Individual mean	10.21	10.19	10.24	9.98	0.021	0.021	0.020	0.021
Total mean	10.16				0.021
Individual SD	0.67	0.67	0.44	0.80	0.007	0.007	0.006	0.007
Total SD	0.65				0.007
Minimum	7.99				0.012
Maximum	11.35				0.035

Key: - Not applicable due to larvae dead, SD = Standard deviation, mm = millimeter,

g = gram,mg = milligram, L=Litre, R=Replicate

Table : Fish Total Length (mm) and Weight (g) : 1.600 mg/ L

Number of larvae	Concentration: 1.600 mg/ L
	Length (mm)				Total weight (g)
	R1	R2	R3	R4	R1	R2	R3	R4
1	9.02	8.88	9.25	9.78	0.014	0.012	0.021	0.028
2	9.09	9.75	8.88	9.45	0.019	0.015	0.015	0.031
3	9.15	7.78	8.97	7.19	0.012	0.011	0.019	0.012
4	8.65	10.89	9.56	8.96	0.017	0.023	0.022	0.014
5	9.23	9.29	9.15	8.56	0.012	0.014	0.025	0.018
6	10.34	9.86	10.12	9.17	0.021	0.016	0.031	0.023
7	11.42	9.72	8.82	9.25	0.045	0.019	0.016	0.028
8	11.02	7.22	8.55	8.63	0.032	0.012	0.012	0.014
9	10.48	9.17	9.78	8.77	0.014	0.015	0.025	0.019
10	9.12	9.78	9.56	8.96	0.012	0.022	0.031	0.016
11	8.32	8.52	9.98	10.65	0.022	0.025	0.035	0.028
12	10.25	9.55	8.96	8.99	0.013	0.021	0.012	0.016
13	9.22	9.38	7.69	9.64	0.015	0.023	0.025	0.022
14	8.54	9.27	8.17	9.89	0.014	0.013	0.031	0.023
15	9.65	8.17	10.23	10.36	0.018	0.015	0.028	0.031
16	7.58	7.12	11.29	9.73	0.012	0.012	0.023	0.038
17	8.34	-	9.66	9.45	0.021	-	0.029	0.012
18	7.65	-	-	-	0.014	-	-	-
19	9.12	-	-	-	0.022	-	-	-
20	-	-	-	-	-	-	-	-
Individual mean	9.27	9.02	9.33	9.26	0.018	0.017	0.024	0.022
Total mean	9.22				0.020
Individual SD	1.05	1.02	0.85	0.79	0.008	0.005	0.007	0.008
Total SD	0.93				0.007
Minimum	7.12				0.011
Maximum	11.42				0.045

Key: - Not applicable due to larvae dead, SD = Standard deviation, mm = millimeter,

g = gram,mg = milligram, L=Litre, R=Replicate

Table : Fish Total Length (mm) and Weight (g) : 4.000 mg/ L

Number of larvae	Concentration: 4.000 mg/ L
	Length (mm)				Total weight (g)
	R1	R2	R3	R4	R1	R2	R3	R4
1	8.12	9.65	7.61	7.96	0.025	0.029	0.013	0.011
2	6.79	6.89	7.89	8.78	0.012	0.012	0.016	0.013
3	8.23	7.88	8.25	8.23	0.029	0.015	0.025	0.019
4	7.14	9.82	8.64	10.18	0.022	0.014	0.026	0.016
5	7.65	7.77	10.41	10.64	0.011	0.013	0.034	0.022
6	7.02	6.59	8.97	9.48	0.012	0.012	0.022	0.024
7	8.82	10.62	9.14	9.12	0.011	0.012	0.025	0.023
8	7.69	10.49	10.36	7.56	0.014	0.032	0.015	0.015
9	8.52	9.87	11.06	7.19	0.016	0.029	0.016	0.011
10	8.74	7.19	9.17	7.89	0.021	0.024	0.018	0.013
11	9.53	7.45	7.13	9.25	0.025	0.012	0.011	0.017
12	10.13	7.69	7.08	7.15	0.022	0.023	0.014	0.018
13	8.85	8.89	7.87	7.69	0.032	0.013	0.021	0.013
14	8.97	8.14	8.99	8.22	0.016	0.025	0.023	0.019
15	7.65	8.19	8.76	8.23	0.015	0.013	0.029	0.013
16	8.25	7.63	7.45	7.54	0.021	0.011	0.013	0.014
17	-	7.15	8.65	-	-	0.013	0.019	-
18	-	-	-	-	-	-	-	-
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Individual mean	8.26	8.35	8.67	8.44	0.019	0.018	0.020	0.016
Total mean	8.43				0.018
Individual SD	0.92	1.29	1.15	1.04	0.007	0.007	0.006	0.004
Total SD	1.10				0.006
Minimum	6.59				0.011
Maximum	11.06				0.034

Key: - Not applicable due to larvae dead, SD = Standard deviation, mm = millimeter,

g = gram,mg = milligram, L=Litre, R=Replicate

 

Table : Fish Total Length (mm) and Weight (g) : 10.000 mg/ L

Number of larvae	Concentration: 10.000 mg/ L
	Length (mm)				Total weight (g)
	R1	R2	R3	R4	R1	R2	R3	R4
1	7.18	6.98	6.54	6.18	0.015	0.011	0.015	0.012
2	6.54	7.17	7.14	7.52	0.012	0.014	0.018	0.018
3	7.09	6.54	7.65	7.18	0.011	0.023	0.017	0.013
4	8.16	7.12	7.83	8.19	0.013	0.019	0.017	0.025
5	7.12	7.09	6.32	7.65	0.021	0.019	0.014	0.013
6	7.27	8.12	8.65	7.81	0.019	0.016	0.012	0.012
7	6.62	8.16	6.15	7.09	0.016	0.021	0.014	0.017
8	8.16	7.14	8.16	7.45	0.013	0.014	0.017	0.016
9	7.02	6.68	8.13	7.14	0.011	0.011	0.014	0.014
10	6.54	6.98	7.15	6.75	0.016	0.013	0.016	0.012
11	7.15	7.16	7.12	6.98	0.012	0.015	0.018	0.011
12	6.78	7.74	6.65	7.63	0.015	0.017	0.021	0.016
13	8.12	7.18	7.12	6.74	0.014	0.012	0.014	0.014
14	8.16	7.21	6.55	6.79	0.012	0.014	0.012	0.017
15	8.23	6.98	7.83	6.92	0.019	0.015	0.016	0.022
16	6.79	-	7.59	6.85	0.014	-	0.013	0.012
17	7.35	-	6.52	-	0.018	-	0.018	-
18	-	-	7.62	-	-	-	0.022	-
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Individual mean	7.31	7.22	7.26	7.18	0.015	0.016	0.016	0.015
Total mean	7.24				0.015
Individual SD	0.62	0.46	0.71	0.50	0.003	0.004	0.003	0.004
Total SD	0.57				0.003
Minimum	6.15				0.011
Maximum	8.65				0.025

Key: - Not applicable due to larvae dead, SD = Standard deviation, mm = millimeter,

g = gram,mg = milligram, L=Litre, R=Replicate

Table 3: Number of Eggs Hatched

Concentration (mg/L)	24 hours				No. of eggs hatched	48 hours				No. of eggs hatched
	R1	R2	R3	R4		R1	R2	R3	R4
Control	0	0	0	0	0	0	0	0	0	0
0.0164	0	0	0	0	0	0	0	0	0	0
0.041	0	0	0	0	0	0	0	0	0	0
0.102	0	0	0	0	0	0	0	0	0	0
0.256	0	0	0	0	0	0	0	0	0	0
0.640	0	0	0	0	0	0	0	0	0	0
1.600	0	0	0	0	0	0	0	0	0	0
4.000	0	0	0	0	0	0	0	0	0	0
10.000	0	0	0	0	0	0	0	0	0	0

 

Concentration (mg/L)	72 hours				No. of eggs hatched	96 hours				No. of eggs hatched	Cumulative nos.	Eggs hatched (%)
	R1	R2	R3	R4		R1	R2	R3	R4
Control	10	11	14	10	45	10	9	6	10	35	80	100.0
0.0164	11	12	8	11	42	9	8	12	9	38	80	100.0
0.041	10	8	9	13	40	10	12	11	7	40	80	100.0
0.102	8	9	9	10	36	12	11	11	10	44	80	100.0
0.256	7	8	9	7	31	13	12	11	13	49	80	100.0
0.640	13	10	9	12	44	7	10	11	8	36	80	100.0
1.600	12	11	13	9	45	8	9	7	11	35	80	100.0
4.000	8	7	10	12	37	11	12	10	7	40	77	96.3
10.000	10	8	7	11	36	8	11	10	7	36	72	90.0

 

 

 

 

 

 

 

Table 4: Mortaliy in prehatch at each day

Mortaliy 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	0
	R2	20	0	0	0	0	0	0
	R3	20	0	0	0	0	0	0
	R4	20	0	0	0	0	0	0
0.0164	R1	20	0	0	0	0	0	0	0
	R2	20	0	0	0	0	0	0
	R3	20	0	0	0	0	0	0
	R4	20	0	0	0	0	0	0
0.041	R1	20	0	0	0	0	0	0	0
	R2	20	0	0	0	0	0	0
	R3	20	0	0	0	0	0	0
	R4	20	0	0	0	0	0	0
0.041	R1	20	0	0	0	0	0	0	0
	R2	20	0	0	0	0	0	0
	R3	20	0	0	0	0	0	0
	R4	20	0	0	0	0	0	0
0.102	R1	20	0	0	0	0	0	0	0
	R2	20	0	0	0	0	0	0
	R3	20	0	0	0	0	0	0
	R4	20	0	0	0	0	0	0
0.256	R1	20	0	0	0	0	0	0	0
	R2	20	0	0	0	0	0	0
	R3	20	0	0	0	0	0	0
	R4	20	0	0	0	0	0	0
0.640	R1	20	0	0	0	0	0	0	0
	R2	20	0	0	0	0	0	0
	R3	20	0	0	0	0	0	0
	R4	20	0	0	0	0	0	0
1.600	R1	20	0	0	0	0	0	0	0
	R2	20	0	0	0	0	0	0
	R3	20	0	0	0	0	0	0
	R4	20	0	0	0	0	0	0
4.000	R1	20	0	0	0	1	0	1	3
	R2	20	0	0	0	1	0	1
	R3	20	0	0	0	0	0	0
	R4	20	0	0	0	1	0	1
10.000	R1	20	0	0	0	2	0	2	8
	R2	20	0	0	0	1	0	1
	R3	20	0	0	0	3	0	3
	R4	20	0	0	0	2	0	2

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

Table:Larvae Mortality

Conc. mg/ L	R	Number of eggs added at the start of the test	Mortality (Larvae and juveniles stage)																															LM	TLM	LM
																																				T	C
			Days																																	%	%
			0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	26	27	28	29	30
Control	R1	20	0	0	0	0	0	0	0	1	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2	6	7.5	NA
	R2	20	0	0	0	0	0	0	0	0	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2
	R3	20	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1
	R4	20	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1
0.0164	R1	20	0	0	0	0	0	0	0	1	0	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3	9	11.25	4.05
	R2	20	0	0	0	0	0	0	0	1	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2
	R3	20	0	0	0	0	0	0	0	0	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2
	R4	20	0	0	0	0	0	0	0	0	1	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2
0.041	R1	20	0	0	0	0	0	0	0	0	1	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2	8	10.00	2.70
	R2	20	0	0	0	0	0	0	0	1	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3
	R3	20	0	0	0	0	0	0	0	1	0	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3
	R4	20	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
0.102	R1	20	0	0	0	0	0	0	0	1	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2	8	10.00	2.70
	R2	20	0	0	0	0	0	0	0	2	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3
	R3	20	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1
	R4	20	0	0	0	0	0	0	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2
0.256	R1	20	0	0	0	0	0	0	0	0	2	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3	9	11.25	4.05
	R2	20	0	0	0	0	0	0	0	0	0	1	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3
	R3	20	0	0	0	0	0	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2
	R4	20	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1
0.640	R1	20	0	0	0	0	0	0	1	0	0	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3	10	12.5	5.41
	R2	20	0	0	0	0	0	0	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2
	R3	20	0	0	0	0	0	0	0	1	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3
	R4	20	0	0	0	0	0	0	1	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2
1.600	R1	20	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	11	13.75	6.76
	R2	20	0	0	0	0	0	0	1	0	1	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	4
	R3	20	0	0	0	0	0	0	2	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3
	R4	20	0	0	0	0	0	0	0	0	2	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3
4.000	R1	20	0	0	0	0	0	0	1	0	1	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	4	14	17.5	10.81
	R2	20	0	0	0	0	0	0	1	1	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3
	R3	20	0	0	0	0	0	0	0	2	0	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3
	R4	20	0	0	0	0	0	0	0	1	2	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	4
10.000	R1	20	0	0	0	0	0	0	0	0	0	2	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	3	14	17.5	10.81
	R2	20	0	0	0	0	0	0	0	2	0	3	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	5
	R3	20	0	0	0	0	0	0	0	0	0	0	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2
	R4	20	0	0	0	0	0	0	0	2	0	2	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	4

Key :Conc.-Concentration, mg - milligram, L-Litre, R-Replication, LM-Larvae mortality, TLM- Total larvae mortality, T –Total, C- Corrected mortality

Table 6:Fish Clinical Sign 

Conc. mg/ L	R	Number of eggs added at the start of the test	Fish Clinical Sign
			Days
			0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	26	27	28	29	30
Control	R1	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R2	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R3	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R4	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
0.0164	R1	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R2	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R3	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R4	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
0.041	R1	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R2	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R3	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R4	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
0.102	R1	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R2	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R3	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R4	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
0.256	R1	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R2	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R3	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R4	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
0.640	R1	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R2	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R3	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R4	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
1.600	R1	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R2	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1, 32(1)	1
	R3	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1, 5(1)	1	1	1	1	1
	R4	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
4.000	R1	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1, 5(1)	1	1	1	1	1
	R2	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R3	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
	R4	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
10.000	R1	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1, 27(1)	1	1	1
	R2	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1, 4(1)	1	1
	R3	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1, 5	1	1	1	1	1
	R4	20	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1

Key :Key : mg = milligram, L=Litre, R=Replicate1 – Normal, 4 – Loss of buoyancy control, 5- Hypoactivity, 27 – Oedema, 32 – Deformities found in tail

 

Validity criteria fulfilled:

yes

Remarks:

Water temp was maintained at 26 ± 1.5 °C throughout the exposure. The DO in all groups was between 69.8 to 94.3% of saturation. Hatching rate in control was 100% and larvae survival in control was evaluated to be 92.5%.

Conclusions:

Based on the effect on mortality of test fishes, the 30 d NOEC and LC50 value was determined to be ≥ 10 mg/l and >10 mg/l, respectively. On the basis of the effect on hatching success and survival larvae of test fishes, the 30 d NOEC, LOEC and EC50 value was determined to be 1.6, 4 and >10 mg/l, respectively.

Executive summary:

Chronic toxicity study to aquatic fishes was carried for 30 dayspost hatchfor assessing the effect of test chemicalon survival rate and hatchingsuccess. Study was performed following the principles of the OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) under semi-static conditions. The test chemical was dissolved in the dilution water and the selected test chemical concentrations were prepared by dilution of a stock solution. Solubility of the test item was performed by weighed 25.5 mg of the test item in a 250 ml volumetric flask dissolved and made up to the mark.  The stability test was conducted toanalysethe chemical stability for the duration of 96 hours(at regular intervals24 hours).At 0.01 mg/L and 100 mg/Lat 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (98.37% to 99.09% for 0.01 mg/L and 98.29% to 99.03% for 100 mg/L) with respect to initial measured concentrationthus semi static conditions were opted for main study. Danio rerio (Zebra fish) of both sex of ratio (2 male: 1female)were used for spawning.Spawning traps were placed in the breeding tank for the collection of eggs.  Whichwere collected from breeding groups,mixed,and randomly selectedfor the exposure. To prevent predation of eggs by adult zebra fish, the spawn traps were covered with inert wire mesh of size (2.5±0.5 mm). The spawn traps with the collected eggs were carefully removed. Thefertilizedeggs were collected by usingpasteurpipette on next day morning (on the day of exposure) and 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 wasgivento test fishes.Which was as follows,Commercial dry food was provided for 14 days. From day 15 onwards live food (artemia) was provided along with dry food. Dry feed was given twice a day and Brine shrimp Artemia was given to test fishes once daily. Surplus food andfeceswere removed where required, to avoid accumulation of waste.dilutionwater was used as atest medium.Range finding test was conducted with test concentrations at 0 (Control), 5, 10, 25, 50 and 100 mg/. Each concentration contained two replicate and eachreplicatewere exposed 20 fertilized eggs. Test item was formulated inDilutionwater. Hatching (percent) observed in control group was 100% whereas, 97.5%, 92.5%, 90.0%, 87.5% and 85.0% in the tested concentration of 5, 10, 25, 50 and 100 mg/L for a period of 96 hours. Allfertilisedeggs hatched in the control group whereas non-detachment of the tail observed in the tested concentration of 5, 10, 25, 50 and  100 mg/ L for a period of 96 hours. Based on the mortality of embryos and hatching success concentrations for the main study was determined.The concentrations ranging from0 (Control), 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/lwith geometric factor 2.5 was selected formianstudy.Test fishes (total 20embryos/vessel) were exposed to different test chemical conc. (i.e., 0 (Control), 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/l) in a 2 lit glass beaker. No aeration was provided during the study. Renewal rate of test solution was 96 hrs. Biomass loading rate contains 80 eggs/conc. All control and test experiments were performed in 4 replicates. Test conditions involve a photoperiod of 12: 12 light: dark conditions, Light intensity ranges from 694 to 768 lux, temperature at the beginning of the test: 26.5°C to 27.0°C and at the end of the test: 26.2°C to 26.5°C, pH at the beginning of the test: 7.5 to 8.0 and at the end of the test: 7.5 to 8.2 and dissolved oxygen of 69.8 to 94.3% air saturation value, respectively. Data wereanalyzedusing appropriate statistical methods to calculate the EC50, LOEC and NOEC. The 95% confidence Limits were calculated. The pH of the control at the test start and end was 7.5 and therefore did not vary more than 1.5 units during the study. No mortality/100% survival was observed at embryo stage in the control; 7.5% mortality was observed in control after 30 days whereas mortality at the end of exposure to test chemical at concentrations of 0.0164, 0.041, 0.102, 0.256, 0.64,1.6, 4 and 10 mg/l was 4.05%, 2.70%, 2.70%, 4.05%, 5.41%, 6.76%, 10.81% and 10.81%, respectively. At 96hr, all the embryos were hatched in control. Hatching success in the control and in the tested concentration of 0.0164, 0.041, 0.102, 0.256, 0.64 and 1.6 mg/ L was 100%, whereas 96.3% and 90.0 %, in the tested concentration of 4 and 10 mg/l. Larval survival until day 30 post-hatch in the control group was 92.5% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%).whereas in the group treated with test chemical at concentrations of 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/l was 88.8, 90, 90, 88.8, 87.5, 86.3, 81.8 and 80.6%, respectively. All fishes were healthy in the control. For fish total lengths, the NOEC and LOEC determined on Day 30 post-hatch were 0.102 and 0.256 mg/L, respectively. At concentration of 1.6 mg/L in two hatching out of which one has tail deformities and other was hypoactive, at concentration 4 mg/L one hatchling was hypoactive and at concentration of 10 mg/L one hatchling had oedema and other has loss of buoyancy control. Length and weightweremeasured after 30 days post hatch, in control reported to be 11.20 mm and 0.032 g. Samples wereanalytically determined at pre and post renewal. The received test sample concentrations (0.0164 mg/L, 0.64 mg/L and 10 mg/L on 0-hour fresh samples, 96-Hour spent and fresh samples and 48-hour spent samples) were mixed well with uniformity. An aliquot of required volume of sample solutions was transfer to a clean round bottom flask (RB) and dry on a 60°C heating condition. After dry eluate of sample solutions made up to the final volume using acetonitrile and analyzed under high performance liquid chromatography (HPLC).In fresh samples of the test substance concentrations, the determined concentrations of test chemicalwere93.19 % to 95.79 % for 0.0164 mg/L, 91.37 % to 94.48 % for 0.640 mg/L and 93.60 % to 95.34 % for 10 mg/L, respectively. The results confirm that the test substance concentrations were within the acceptability range. In spent samples of the test substance concentrations, the determined concentrations of test chemical was 93.94 % to 96.03 % for 0.0164 mg/L, 92.67 % to 94.55 % for 0.640 mg/L and 94.05 % to 95.67 % for 10 mg/L, respectively. All validity criteria were satisfied during the test, therefore the test was considered to be valid. Based on the results and effect on mortality of test fishes, the 30 d NOEC and LC50 value was determined to be ≥ 10 mg/l and >10 mg/l, respectively. On the basis of the effect on hatching success and survival larvae of test fishes,the 30 d NOEC, LOEC and EC50 value was determined to be 1.6, 4 and >10 mg/l, respectively. Thus, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.Thus based on the above results chemical cannot be classified as per CLP classification criteria. 

Description of key information

Chronic toxicity study to aquatic fishes was carried for 30 dayspost hatchfor assessing the effect of test chemicalon survival rate and hatchingsuccess. Study was performed following the principles of the OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) under semi-static conditions. The test chemical was dissolved in the dilution water and the selected test chemical concentrations were prepared by dilution of a stock solution. Solubility of the test item was performed by weighed 25.5 mg of the test item in a 250 ml volumetric flask dissolved and made up to the mark.  The stability test was conducted toanalysethe chemical stability for the duration of 96 hours(at regular intervals24 hours).At 0.01 mg/L and 100 mg/Lat 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (98.37% to 99.09% for 0.01 mg/L and 98.29% to 99.03% for 100 mg/L) with respect to initial measured concentrationthus semi static conditions were opted for main study. Danio rerio (Zebra fish) of both sex of ratio (2 male: 1female)were used for spawning.Spawning traps were placed in the breeding tank for the collection of eggs.  Whichwere collected from breeding groups,mixed,and randomly selectedfor the exposure. To prevent predation of eggs by adult zebra fish, the spawn traps were covered with inert wire mesh of size (2.5±0.5 mm). The spawn traps with the collected eggs were carefully removed. Thefertilizedeggs were collected by usingpasteurpipette on next day morning (on the day of exposure) and 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 wasgivento test fishes.Which was as follows,Commercial dry food was provided for 14 days. From day 15 onwards live food (artemia) was provided along with dry food. Dry feed was given twice a day and Brine shrimp Artemia was given to test fishes once daily. Surplus food andfeceswere removed where required, to avoid accumulation of waste.dilutionwater was used as atest medium.Range finding test was conducted with test concentrations at 0 (Control), 5, 10, 25, 50 and 100 mg/. Each concentration contained two replicate and eachreplicatewere exposed 20 fertilized eggs. Test item was formulated inDilutionwater. Hatching (percent) observed in control group was 100% whereas, 97.5%, 92.5%, 90.0%, 87.5% and 85.0% in the tested concentration of 5, 10, 25, 50 and 100 mg/L for a period of 96 hours. Allfertilisedeggs hatched in the control group whereas non-detachment of the tail observed in the tested concentration of 5, 10, 25, 50 and  100 mg/ L for a period of 96 hours. Based on the mortality of embryos and hatching success concentrations for the main study was determined.The concentrations ranging from0 (Control), 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/lwith geometric factor 2.5 was selected formianstudy.Test fishes (total 20embryos/vessel) were exposed to different test chemical conc. (i.e., 0 (Control), 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/l) in a 2 lit glass beaker. No aeration was provided during the study. Renewal rate of test solution was 96 hrs. Biomass loading rate contains 80 eggs/conc. All control and test experiments were performed in 4 replicates. Test conditions involve a photoperiod of 12: 12 light: dark conditions, Light intensity ranges from 694 to 768 lux, temperature at the beginning of the test: 26.5°C to 27.0°C and at the end of the test: 26.2°C to 26.5°C, pH at the beginning of the test: 7.5 to 8.0 and at the end of the test: 7.5 to 8.2 and dissolved oxygen of 69.8 to 94.3% air saturation value, respectively. Data wereanalyzedusing appropriate statistical methods to calculate the EC50, LOEC and NOEC. The 95% confidence Limits were calculated. The pH of the control at the test start and end was 7.5 and therefore did not vary more than 1.5 units during the study. No mortality/100% survival was observed at embryo stage in the control; 7.5% mortality was observed in control after 30 days whereas mortality at the end of exposure to test chemical at concentrations of 0.0164, 0.041, 0.102, 0.256, 0.64,1.6, 4 and 10 mg/l was 4.05%, 2.70%, 2.70%, 4.05%, 5.41%, 6.76%, 10.81% and 10.81%, respectively. At 96hr, all the embryos were hatched in control. Hatching success in the control and in the tested concentration of 0.0164, 0.041, 0.102, 0.256, 0.64 and 1.6 mg/ L was 100%, whereas 96.3% and 90.0 %, in the tested concentration of 4 and 10 mg/l. Larval survival until day 30 post-hatch in the control group was 92.5% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%).whereas in the group treated with test chemical at concentrations of 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/l was 88.8, 90, 90, 88.8, 87.5, 86.3, 81.8 and 80.6%, respectively. All fishes were healthy in the control. For fish total lengths, the NOEC and LOEC determined on Day 30 post-hatch were 0.102 and 0.256 mg/L, respectively. At concentration of 1.6 mg/L in two hatching out of which one has tail deformities and other was hypoactive, at concentration 4 mg/L one hatchling was hypoactive and at concentration of 10 mg/L one hatchling had oedema and other has loss of buoyancy control. Length and weightweremeasured after 30 days post hatch, in control reported to be 11.20 mm and 0.032 g. Samples wereanalytically determined at pre and post renewal. The received test sample concentrations (0.0164 mg/L, 0.64 mg/L and 10 mg/L on 0-hour fresh samples, 96-Hour spent and fresh samples and 48-hour spent samples) were mixed well with uniformity. An aliquot of required volume of sample solutions was transfer to a clean round bottom flask (RB) and dry on a 60°C heating condition. After dry eluate of sample solutions made up to the final volume using acetonitrile and analyzed under high performance liquid chromatography (HPLC).In fresh samples of the test substance concentrations, the determined concentrations of test chemicalwere93.19 % to 95.79 % for 0.0164 mg/L, 91.37 % to 94.48 % for 0.640 mg/L and 93.60 % to 95.34 % for 10 mg/L, respectively. The results confirm that the test substance concentrations were within the acceptability range. In spent samples of the test substance concentrations, the determined concentrations of test chemical was 93.94 % to 96.03 % for 0.0164 mg/L, 92.67 % to 94.55 % for 0.640 mg/L and 94.05 % to 95.67 % for 10 mg/L, respectively. All validity criteria were satisfied during the test, therefore the test was considered to be valid. Based on the results and effect on mortality of test fishes, the 30 d NOEC and LC50 value was determined to be ≥ 10 mg/l and >10 mg/l, respectively. On the basis of the effect on hatching success and survival larvae of test fishes,the 30 d NOEC, LOEC and EC50 value was determined to be 1.6, 4 and >10 mg/l, respectively. Thus, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.Thus based on the above results chemical cannot be classified as per CLP classification criteria. 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.6 mg/L

Additional information

Chronic toxicity study to aquatic fishes was carried for 30 dayspost hatchfor assessing the effect of test chemicalon survival rate and hatchingsuccess. Study was performed following the principles of the OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) under semi-static conditions. The test chemical was dissolved in the dilution water and the selected test chemical concentrations were prepared by dilution of a stock solution. Solubility of the test item was performed by weighed 25.5 mg of the test item in a 250 ml volumetric flask dissolved and made up to the mark.  The stability test was conducted toanalysethe chemical stability for the duration of 96 hours(at regular intervals24 hours).At 0.01 mg/L and 100 mg/Lat 0 hour, 24 hour, 48 hour, 72 hour and 96 hour showed that the test item concentration remained 80% to 120% (98.37% to 99.09% for 0.01 mg/L and 98.29% to 99.03% for 100 mg/L) with respect to initial measured concentrationthus semi static conditions were opted for main study. Danio rerio (Zebra fish) of both sex of ratio (2 male: 1female)were used for spawning.Spawning traps were placed in the breeding tank for the collection of eggs.  Whichwere collected from breeding groups,mixed,and randomly selectedfor the exposure. To prevent predation of eggs by adult zebra fish, the spawn traps were covered with inert wire mesh of size (2.5±0.5 mm). The spawn traps with the collected eggs were carefully removed. Thefertilizedeggs were collected by usingpasteurpipette on next day morning (on the day of exposure) and 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 wasgivento test fishes.Which was as follows,Commercial dry food was provided for 14 days. From day 15 onwards live food (artemia) was provided along with dry food. Dry feed was given twice a day and Brine shrimp Artemia was given to test fishes once daily. Surplus food andfeceswere removed where required, to avoid accumulation of waste.dilutionwater was used as atest medium.Range finding test was conducted with test concentrations at 0 (Control), 5, 10, 25, 50 and 100 mg/. Each concentration contained two replicate and eachreplicatewere exposed 20 fertilized eggs. Test item was formulated inDilutionwater. Hatching (percent) observed in control group was 100% whereas, 97.5%, 92.5%, 90.0%, 87.5% and 85.0% in the tested concentration of 5, 10, 25, 50 and 100 mg/L for a period of 96 hours. Allfertilisedeggs hatched in the control group whereas non-detachment of the tail observed in the tested concentration of 5, 10, 25, 50 and  100 mg/ L for a period of 96 hours. Based on the mortality of embryos and hatching success concentrations for the main study was determined.The concentrations ranging from0 (Control), 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/lwith geometric factor 2.5 was selected formianstudy.Test fishes (total 20embryos/vessel) were exposed to different test chemical conc. (i.e., 0 (Control), 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/l) in a 2 lit glass beaker. No aeration was provided during the study. Renewal rate of test solution was 96 hrs. Biomass loading rate contains 80 eggs/conc. All control and test experiments were performed in 4 replicates. Test conditions involve a photoperiod of 12: 12 light: dark conditions, Light intensity ranges from 694 to 768 lux, temperature at the beginning of the test: 26.5°C to 27.0°C and at the end of the test: 26.2°C to 26.5°C, pH at the beginning of the test: 7.5 to 8.0 and at the end of the test: 7.5 to 8.2 and dissolved oxygen of 69.8 to 94.3% air saturation value, respectively. Data wereanalyzedusing appropriate statistical methods to calculate the EC50, LOEC and NOEC. The 95% confidence Limits were calculated. The pH of the control at the test start and end was 7.5 and therefore did not vary more than 1.5 units during the study. No mortality/100% survival was observed at embryo stage in the control; 7.5% mortality was observed in control after 30 days whereas mortality at the end of exposure to test chemical at concentrations of 0.0164, 0.041, 0.102, 0.256, 0.64,1.6, 4 and 10 mg/l was 4.05%, 2.70%, 2.70%, 4.05%, 5.41%, 6.76%, 10.81% and 10.81%, respectively. At 96hr, all the embryos were hatched in control. Hatching success in the control and in the tested concentration of 0.0164, 0.041, 0.102, 0.256, 0.64 and 1.6 mg/ L was 100%, whereas 96.3% and 90.0 %, in the tested concentration of 4 and 10 mg/l. Larval survival until day 30 post-hatch in the control group was 92.5% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%).whereas in the group treated with test chemical at concentrations of 0.0164, 0.041, 0.102, 0.256, 0.64, 1.6, 4 and 10 mg/l was 88.8, 90, 90, 88.8, 87.5, 86.3, 81.8 and 80.6%, respectively. All fishes were healthy in the control. For fish total lengths, the NOEC and LOEC determined on Day 30 post-hatch were 0.102 and 0.256 mg/L, respectively. At concentration of 1.6 mg/L in two hatching out of which one has tail deformities and other was hypoactive, at concentration 4 mg/L one hatchling was hypoactive and at concentration of 10 mg/L one hatchling had oedema and other has loss of buoyancy control. Length and weightweremeasured after 30 days post hatch, in control reported to be 11.20 mm and 0.032 g. Samples wereanalytically determined at pre and post renewal. The received test sample concentrations (0.0164 mg/L, 0.64 mg/L and 10 mg/L on 0-hour fresh samples, 96-Hour spent and fresh samples and 48-hour spent samples) were mixed well with uniformity. An aliquot of required volume of sample solutions was transfer to a clean round bottom flask (RB) and dry on a 60°C heating condition. After dry eluate of sample solutions made up to the final volume using acetonitrile and analyzed under high performance liquid chromatography (HPLC).In fresh samples of the test substance concentrations, the determined concentrations of test chemicalwere93.19 % to 95.79 % for 0.0164 mg/L, 91.37 % to 94.48 % for 0.640 mg/L and 93.60 % to 95.34 % for 10 mg/L, respectively. The results confirm that the test substance concentrations were within the acceptability range. In spent samples of the test substance concentrations, the determined concentrations of test chemical was 93.94 % to 96.03 % for 0.0164 mg/L, 92.67 % to 94.55 % for 0.640 mg/L and 94.05 % to 95.67 % for 10 mg/L, respectively. All validity criteria were satisfied during the test, therefore the test was considered to be valid. Based on the results and effect on mortality of test fishes, the 30 d NOEC and LC50 value was determined to be ≥ 10 mg/l and >10 mg/l, respectively. On the basis of the effect on hatching success and survival larvae of test fishes,the 30 d NOEC, LOEC and EC50 value was determined to be 1.6, 4 and >10 mg/l, respectively. Thus, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.Thus based on the above results chemical cannot be classified as per CLP classification criteria.