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REACH

2,2,2-trichloro-1-phenylethyl acetate

EC number: 201-972-0 | CAS number: 90-17-5

Life Cycle description
Uses advised against

Ecotoxicological information

Long-term toxicity to fish

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Administrative data

Link to relevant study record(s)

Reference

Endpoint:: fish early-life stage toxicity
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 2 (reliable with restrictions)
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:: no
Analytical monitoring:: yes
Remarks:: UV-Vis spectrophotometer
Details on sampling:: - Concentrations: Test chemical conc. used for the study were 0 (Control), 0.625, 1.25, 2.5, 5 and 10 mg/l, respectively.
- Sampling method: The stock solution of test chemical was prepared by dissolving 2000 mg in 2000 mL of test media to get the final concentration of 1000 mg/L followed by analytical determination. The final solubility obtained after analytical determination was 13.25 mg/L which was later on used to prepare the remaining test solutions by diluting the above stock solution.
- 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 test water and the selected test chemical concentrations were prepared by dilution of a stock solution. The solution was prepared by simply mixing.
- Controls: Reconstituted 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: In-house ESSEM breed, India
- Age: Fertilized eggs (before cleavage of the blastodisc commences)
- Length: 11 mm

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 and eggs were rinsed with reconstituted water after collection from spawning traps.

POST-HATCH FEEDING
Commercial dry food was provided for 30 days. From day 15 onwards live food (artemia) was provided along with dry food.
- Start date: 4 days post hatch
- Type of feed: Dry flake food and Brine shrimp Artemia
- Source of feed: Brine shrimp (Artemia sp.) nauplii of appropriate size obtained from an uncontaminated source were given as feed.
- Amount given: ad libitum
- Frequency of feeding: Dry flake food was given thrice 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:: 100 to 125 mg/L CaCO3
Test temperature:: 26.5°C
pH:: 6.8 to 7.4
Dissolved oxygen:: 84.72 to 99.25% air saturation value
Nominal and measured concentrations:: Test chemical conc. used for the study were 0 (Control), 0.625, 1.25, 2.5, 5 and 10 mg/l, respectively which were spaced by a constant factor 2.
Details on test conditions:: TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): Glass aquaria
- Test vessel: 2L Glass aquaria
- Type (delete if not applicable): Test vessel were covered with glass lids in order to minimize evaporation and disturbance.
- Material, size, headspace, fill volume: 2L Glass aquaria
- 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: Reconstituted water
- Total organic carbon: 2 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: 3.58 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: 16 light: dark conditions
- Light intensity: Fluorescent light with a light intensity of 500 to 1000 lux

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

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY: No

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.:: 1.25 mg/L
Nominal / measured:: nominal
Conc. based on:: test mat.
Basis for effect:: other: mortality and survival larvae
Duration:: 30 d
Dose descriptor:: LOEC
Effect conc.:: 2.5 mg/L
Nominal / measured:: nominal
Conc. based on:: test mat.
Basis for effect:: other: mortality and survival larvae
Duration:: 30 d
Dose descriptor:: EC50
Effect conc.:: 6.93 mg/L
Nominal / measured:: nominal
Conc. based on:: test mat.
Basis for effect:: other: survival larvae
Remarks on result:: other: EC50 = 6.93± 0.51 mg/l
Duration:: 30 d
Dose descriptor:: LC50
Effect conc.:: 5.39 mg/L
Nominal / measured:: nominal
Conc. based on:: test mat.
Basis for effect:: mortality
Remarks on result:: other: LC50 = 5.39±0.499 mg/l (95% C. I. = 4.34 to 6.70 mg/l)
Duration:: 30 d
Dose descriptor:: NOEC
Effect conc.:: 2.5 mg/L
Nominal / measured:: nominal
Conc. based on:: test mat.
Basis for effect:: fertility
Duration:: 30 d
Dose descriptor:: LOEC
Effect conc.:: 5 mg/L
Nominal / measured:: nominal
Conc. based on:: test mat.
Basis for effect:: fertility
Duration:: 30 d
Dose descriptor:: EC10
Effect conc.:: 4.42 mg/L
Nominal / measured:: nominal
Conc. based on:: test mat.
Basis for effect:: fertility
Remarks on result:: other: EC10 = 4.42±0.55 mg/l
Details on results:: - Mortality/survival at embryo, larval, juvenile, and adult stages: 100 percent survival rate was observed at embryos stage in control, 20% mortality was observed in control after 30 days.
After at the end of exposure to test chemical at concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/L cumulative mortality of embryos, larvae and juvenile fish was 7.81, 14.06, 37.5, 53.13 and 59.38%, respectively.
- 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: 73 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 11.28 mm and 25.48 g
- Number of healthy fish at end of test: all fishes were healthy in 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.
Validity criteria fulfilled:: yes
Remarks:: Water temp was maintained at 26 ± 1.5 °C throughout the exposure. The DO in all groups was between 68 to 78% of saturation. Hatching rate in control was 92.5% and larvae survival in control was evaluated to be 87.83%.
Conclusions:: Based on the effect on mortality of test fishes, the 30 d NOEC, LOEC and LC50 value was determined to be 1.25, 2.5 and 5.39±0.499 mg/l (95% C. I. = 4.34 to 6.70 mg/l)l, respectively. On the basis of the effect on survival larvae of test fishes, the 30 d NOEC, LOEC and EC50 value was determined to be 1.25, 2.5 and 6.93± 0.51 mg/l mg/l, respectively. Where, based on hatching success, the 30 d NOEC, LOEC and EC10 value was determined to be 2.5, 5 and 4.42±0.55 mg/l, respectively.
Executive summary:: Chronic toxicity study to aquatic fishes was carried for 30 days post hatch for assessing the effect of test chemical on survival rate and hatching success. Study was performed following the principles of the OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) under semi-static conditions. The stock solution of test chemical was prepared by dissolving 2000 mg in 2000 mL of test media to get the final concentration of 1000 mg/L followed by analytical determination. The final solubility obtained after analytical determination was 13.25 mg/L which was later on used to prepare the remaining test solutions by diluting the above stock solution. The received samples were stirred and done filtrations for the preparation of stock solution. This was later analyzed under UV-Vis spectrophotometer. Internal calibration was done and LOD, LOQ was determined to be 0.654164 and 1.982315, respectively. In spent samples of the test substance concentrations, the determined concentrations of test item were between 72 and 129.6% of the concentrations, respectively. Therefore, the concentrations of test item were stable during 96 h under test conditions. 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. Which were collected from breeding groups, mixed, and randomly selected for 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. The fertilised eggs were collected and eggs were rinsed with reconstituted water after collection from spawning traps. Post hatch feeding was given to test fishes. Which was as follows, Commercial dry food was provided for 30 days. From day 15 onwards live food (artemia) was provided along with dry food. Dry flake food was given thrice a day and Brine shrimp Artemia was given to test fishes once daily. Surplus food and feces were removed where required, to avoid accumulation of waste. Reconstituted water was used as a test medium. Test fishes (total 20embryos/vessel) were exposed to different test chemical conc. (i.e., 0 (Control), 0 (Control), 0.625, 1.25, 2.5, 5 and 10 mg/l) in a 2 lit glass aquaria. 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: 16 light: dark conditions, Light intensity ranges from 500 to 1000 lux, temperature at the beginning of the test: 26.5°C, pH of 6.8 to 7.4 and dissolved oxygen of 84.72 to 99.25% air saturation value, respectively. Data were analyzed using 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 7.2 & 7.1 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. 20% mortality was observed in control after 30 days whereas mortality at the end of exposure to test chemical at concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/l was 7.81, 14.06, 37.5, 53.13 and 59.38%, respectively. At 96 hr, all the embryos were hatched in control. Hatching success in the control and in the tested concentration of 0.625, 1.25, 2.5, 5 and 10 mg/ L was 91.25%, 90%, 87.5%, 86.25%, 81.25% and 75%, respectively. Larval survival until day 30 post-hatch in the control group was 87.67% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%) whereas in the group treated with test chemical at concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/l was 81.94%, 78.57%, 57.97%, 46.15% and 43.33%,respectively.All surviving juveniles were healthy in the control. For fish total lengths, determined on Day 30 post-hatch were 11.28, 9.33, 8.78, 8.41, 7.87 and 7.72 mm for test item at concentrations control, 0.625, 1.25, 2.5, 5 and 10mg/L, respectively.In fresh samples of the test substance concentrations, the determined concentrations of test chemical were 89.92 to 120% for 0.0625 mg/L, 89.8 to 104.8% for 1.25 mg/L, 100 to 115.6% for 2.5 mg/l, 95.4% to 111.8% for 5 mg/l and 88.9 to 111.8% for 10 mg/L, respectively whereas in old samples of the test substance concentrations, the determined concentrations of test chemical were 72 to 129.6% for 0.0625 mg/L, 89.6 to 126.4% for 1.25 mg/L, 94 to 170% for 2.5 mg/l, 83.6% to 136.2% for 5 mg/l and 88.2 to 129.8% for 10 mg/L, respectively. The results confirm that the test substance concentrations were within the acceptability range. All validity criteria were satisfied during the test, therefore the test was considered to be valid. Based on the effect on mortality of test fishes, the 30 d NOEC, LOEC and LC50 value was determined to be 1.25, 2.5 and 5.39±0.499 mg/l (95% C. I. = 4.34 to 6.70 mg/l), respectively. On the basis of the effect on survival larvae of test fishes, the 30 d NOEC, LOEC and EC50 value was determined to be 1.25, 2.5 and 6.93± 0.51 mg/l mg/l, respectively. Where, based on hatching success, the 30 d NOEC, LOEC and EC10 value was determined to be 2.5, 5 and 4.42±0.55 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.

Description of key information

Chronic toxicity study to aquatic fishes was carried for 30 days post hatch for assessing the effect of test chemical on survival rate and hatching success. Study was performed following the principles of the OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) under semi-static conditions. The stock solution of test chemical was prepared by dissolving 2000 mg in 2000 mL of test media to get the final concentration of 1000 mg/L followed by analytical determination. The final solubility obtained after analytical determination was 13.25 mg/L which was later on used to prepare the remaining test solutions by diluting the above stock solution. The received samples were stirred and done filtrations for the preparation of stock solution. This was later analyzed under UV-Vis spectrophotometer. Internal calibration was done and LOD, LOQ was determined to be 0.654164 and 1.982315, respectively. In spent samples of the test substance concentrations, the determined concentrations of test item were between 72 and 129.6% of the concentrations, respectively. Therefore, the concentrations of test item were stable during 96 h under test conditions. 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. Which were collected from breeding groups, mixed, and randomly selected for 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. The fertilised eggs were collected and eggs were rinsed with reconstituted water after collection from spawning traps. Post hatch feeding was given to test fishes. Which was as follows, Commercial dry food was provided for 30 days. From day 15 onwards live food (artemia) was provided along with dry food. Dry flake food was given thrice a day and Brine shrimp Artemia was given to test fishes once daily. Surplus food and feces were removed where required, to avoid accumulation of waste. Reconstituted water was used as a test medium. Test fishes (total 20embryos/vessel) were exposed to different test chemical conc. (i.e., 0 (Control), 0 (Control), 0.625, 1.25, 2.5, 5 and 10 mg/l) in a 2 lit glass aquaria. 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: 16 light: dark conditions, Light intensity ranges from 500 to 1000 lux, temperature at the beginning of the test: 26.5°C, pH of 6.8 to 7.4 and dissolved oxygen of 84.72 to 99.25% air saturation value, respectively. Data were analyzed using 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 7.2 & 7.1 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. 20% mortality was observed in control after 30 days whereas mortality at the end of exposure to test chemical at concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/l was 7.81, 14.06, 37.5, 53.13 and 59.38%, respectively. At 96 hr, all the embryos were hatched in control. Hatching success in the control and in the tested concentration of 0.625, 1.25, 2.5, 5 and 10 mg/ L was 91.25%, 90%, 87.5%, 86.25%, 81.25% and 75%, respectively. Larval survival until day 30 post-hatch in the control group was 87.67% thereby exceeding and satisfying the validity criteria for post-hatch survival (>75%) whereas in the group treated with test chemical at concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/l was 81.94%, 78.57%, 57.97%, 46.15% and 43.33%,respectively.All surviving juveniles were healthy in the control. For fish total lengths, determined on Day 30 post-hatch were 11.28, 9.33, 8.78, 8.41, 7.87 and 7.72 mm for test item at concentrations control, 0.625, 1.25, 2.5, 5 and 10mg/L, respectively.In fresh samples of the test substance concentrations, the determined concentrations of test chemical were 89.92 to 120% for 0.0625 mg/L, 89.8 to 104.8% for 1.25 mg/L, 100 to 115.6% for 2.5 mg/l, 95.4% to 111.8% for 5 mg/l and 88.9 to 111.8% for 10 mg/L, respectively whereas in old samples of the test substance concentrations, the determined concentrations of test chemical were 72 to 129.6% for 0.0625 mg/L, 89.6 to 126.4% for 1.25 mg/L, 94 to 170% for 2.5 mg/l, 83.6% to 136.2% for 5 mg/l and 88.2 to 129.8% for 10 mg/L, respectively. The results confirm that the test substance concentrations were within the acceptability range. All validity criteria were satisfied during the test, therefore the test was considered to be valid. Based on the effect on mortality of test fishes, the 30 d NOEC, LOEC and LC50 value was determined to be 1.25, 2.5 and 5.39±0.499 mg/l (95% C. I. = 4.34 to 6.70 mg/l), respectively. On the basis of the effect on survival larvae of test fishes, the 30 d NOEC, LOEC and EC50 value was determined to be 1.25, 2.5 and 6.93± 0.51 mg/l mg/l, respectively. Where, based on hatching success, the 30 d NOEC, LOEC and EC10 value was determined to be 2.5, 5 and 4.42±0.55 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.

Key value for chemical safety assessment

Fresh water fish

Effect concentration:: 1.25 mg/L

Additional information

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Stock:2000mgoftestsubstancewasdissolvedin2000mLoftestmedium
Sample/concentration (mg/L)		Volumeof Stock(ml)	Volumeofreconstitutedwater
T5	0.625mg/L	47.17	filledupto1L
T4	1.25mg/L	94.34	filledupto1L
T3	2.5mg/L	188.68	filledupto1L
T2	5mg/L	377.36	filledupto1L
T1	10mg/L	754.72	filledupto1L
Control	0.0	-	1L

S.No.	5mg/LConcentration
	R1	R2	R3	R4	R1	R2	R3	R4
	Length (mm)	Length (mm)	Length (mm)	Length (mm)	Dry Weight (mg)	Dry Weight (mg)	Dry Weight (mg)	Dry Weight (mg)
1	10	8	7	8	24	20	16	17
2	9	7	8	7	23	12	17	12
3	9	7	7	7	24	14	13	14
4	8	8	8	8	20	20	20	16
5	8	8	8	7	20	20	19	14
6	9	-	7	7	19	-	16	13
7	9	-	8	6	18	-	18	11
8	-	-	8	8	-	-	18	18
9	-	-	9	7	-	-	19	16
10	-	-	-	-	-	-	-	-
11	-	-	-	-	-	-	-	-
12	-	-	-	-	-	-	-	-
13	-	-	-	-	-	-	-	-
14	-	-	-	-	-	-	-	-
15	-	-	-	-	-	-	-	-
16	-	-	-	-	-	-	-	-
17	-	-	-	-	-	-	-	-
18	-	-	-	-	-	-	-	-
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Individual mean	8.86	7.6	7.78	7.22	21.14	17.2	17.33	14.56
Totalmean	7.87				17.56
Minimum	6				11
Maximum	10				24

SampleName	Week	Days	DoseFormulation (mg/L)	Measured Conc.(mg/L)	80-120(%)
90-17-5	2nd	11th	Control	-	-
			0.625	0.81	129.6
			1.25	1.60	120.8
			2.5	4.25	170
			5	6.81	136.2
			10	12.98	129.8
	3rd	18th	Control	-	-
			0.625	0.64	102.4
			1.25	1.12	89.6
			2.5	2.40	96
			5	4.18	83.6
			10	9.5	95
	4th	25th	Control	-	-
			0.625	0.51	81.6
			1.25	1.2	96
			2.5	2.35	94
			5	4.73	94.6
			10	8.82	88.2
	5th	2nd	Control	-	-
			0.625	0.78	124.8
			1.25	1.51	120.8
			2.5	2.53	101.2
			5	5.09	101.8
			10	9.87	98.7
	6th	9th	Control	-	-
			0.625	0.45	72
			1.25	1.58	126.4
			2.5	2.5	100
			5	4.35	87
			10	8.95	89.5

TestConcentration	Replicates	No.ofembryoshatched	Totalhatch	HatchingPercent	Totalsurvived Afterhatching	Total Survive	Percentsurvival Afterhatching
Control	R1	18	73	91.25	16	64	87.67
	R2	19			14
	R3	17			16
	R4	19			18
0.625mg/L	R1	18	72	90	15	59	81.94
	R2	19			17
	R3	17			15
	R4	18			12
1.25mg/L	R1	19	70	87.5	16	55	78.57
	R2	18			12
	R3	16			13
	R4	17			14
2.5mg/L	R1	18	69	86.25	10	40	57.97
	R2	16			9
	R3	16			11
	R4	19			10
5mg/L	R1	15	65	81.25	7	30	46.15
	R2	16			5
	R3	18			9
	R4	16			9
10mg/L	R1	17	60	75	8	26	43.33
	R2	16			7
	R3	13			5
	R4	14			6

S.No.	ControlConcentration
	R1	R2	R3	R4	R1	R2	R3	R4
	Length (mm)	Length (mm)	Length (mm)	Length (mm)	Dry Weight(mg)	Dry Weight(mg)	Dry Weight(mg)	Dry Weight(mg)
1	10	13	9	11	25	27	21	26
2	13	11	11	12	33	25	25	28
3	11	12	10	10	28	27	29	22
4	10	11	11	14	26	22	25	30
5	11	10	10	11	29	21	20	26
6	12	11	10	11	24	28	28	25
7	11	12	9	12	21	27	21	28
8	11	11	11	13	29	25	25	29
9	10	13	11	12	23	21	26	26
10	12	11	11	12	27	24	21	26
11	11	11	12	11	24	29	29	23
12	13	11	13	10	25	28	22	21
13	10	11	14	9	27	25	34	21
14	12	11	15	12	28	22	36	26
15	11	-	9	11	25	-	20	25
16	11	-	11	11	27	-	24	21
17	-	-	-	13	-	-	-	27
18	-	-	-	12	-	-	-	24
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Individualmean	11.19	11.36	11.06	11.5	26.25	25.07	25.38	25.22
Totalmean	11.28				25.48
Minimum	9				20
Maximum	15				36

S.No.	0.625Concentration
	R1	R2	R3	R4	R1	R2	R3	R4
	Length (mm)	Length (mm)	Length (mm)	Length (mm)	Dry Weight(mg)	Dry Weight(mg)	Dry Weight(mg)	Dry Weight(mg)
1	10	9	8	10	24	19	20	26
2	8	10	10	9	20	25	25	23
3	9	9	8	10	24	22	24	25
4	11	10	9	11	26	27	25	27
5	9	9	8	12	24	20	22	28
6	9	8	8	9	21	21	21	22
7	11	10	7	9	25	27	17	21
8	9	10	7	8	21	23	17	20
9	11	10	8	9	26	24	19	23
10	9	10	9	9	19	27	23	23
11	9	9	10	10	21	23	26	26
12	9	8	11	11	20	24	27	25
13	10	8	12	-	25	17	28	-
14	8	9	9	-	20	20	22	-
15	7	10	10	-	22	21	26	-
16	-	11	-	-	-	27	-	-
17	-	9	-	-	-	20	-	-
18	-	-	-	-	-	-	-	-
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Individualmean	9.27	9.35	8.93	9.75	22.53	22.76	22.8	24.08
Total mean	9.33				23.03
Minimum	7				17
Maximum	12				28

S.No.	1.25mg/l Concentration
	R1	R2	R3	R4	R1	R2	R3	R4
	Length (mm)	Length (mm)	Length (mm)	Length (mm)	Dry Weight (mg)	Dry Weight (mg)	Dry Weight (mg)	Dry Weight (mg)
1	10	10	9	9	24	22	20	21
2	11	9	11	9	29	20	25	22
3	10	9	9	10	27	20	24	25
4	10	8	9	10	22	19	25	24
5	7	10	8	11	17	23	22	28
6	9	8	8	9	20	18	21	22
7	9	10	7	8	19	24	17	20
8	7	9	8	8	16	23	17	20
9	7	8	7	9	16	22	19	21
10	8	9	9	9	19	20	23	21
11	7	10	10	7	17	23	26	16
12	8	10	10	9	19	24	27	20
13	11	-	11	8	21	-	28	19
14	8	-	-	9	17	-	-	21
15	9	-	-	-	16	-	-	-
16	10	-	-	-	20	-	-	-
17	-	-	-	-	-	-	-	-
18	-	-	-	-	-	-	-	-
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Individualmean	8.81	8.46	8.92	8.93	19.94	21.5	22.6	21.43
Totalmean	8.78				21.37
Minimum	7				16
Maximum	11				29

S.No.	2.5mg/LConcentration
	R1	R2	R3	R4	R1	R2	R3	R4
	Length (mm)	Length (mm)	Length (mm)	Length (mm)	Dry Weight (mg)	Dry Weight (mg)	Dry Weight (mg)	Dry Weight (mg)
1	7	7	7	6	18	14	16	13
2	9	7	8	8	24	14	18	17
3	11	8	9	10	28	17	20	26
4	7	9	8	8	18	22	19	17
5	9	8	9	8	22	20	21	18
6	10	8	10	6	25	19	22	14
7	8	9	10	8	20	21	22	17
8	8	9	10	8	16	20	23	18
9	10	8	10	8	25	18	24	15
10	9	-	8	8	21	-	19	16
11	-	-	9	-	-	-	20	-
12	-	-	-	-	-	-	-	-
13	-	-	-	-	-	-	-	-
14	-	-	-	-	-	-	-	-
15	-	-	-	-	-	-	-	-
16	-	-	-	-	-	-	-	-
17	-	-	-	-	-	-	-	-
18	-	-	-	-	-	-	-	-
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Individual mean	8.8	8.11	8.91	7.8	21.7	18.33	20.36	17.1
Totalmean	8.41				19.37
Minimum	6				13
Maximum	11				28

S.No.	10mg/LConcentration
	R1	R2	R3	R4	R1	R2	R3	R4
	Length (mm)	Length (mm)	Length (mm)	Length (mm)	Dry Weight (mg)	Dry Weight (mg)	Dry Weight (mg)	Dry Weight (mg)
1	7	7	9	8	13	18	20	15
2	8	9	8	8	14	19	13	14
3	7	8	8	6	16	17	16	15
4	8	8	9	8	15	17	18	17
5	9	9	8	6	16	19	14	10
6	7	7	-	6	14	16	-	11
7	7	7	-	-	15	15	-	-
8	8	-	-	-	17	-	-	-
9	-	-	-	-	-	-	-	-
10	-	-	-	-	-	-	-	-
11	-	-	-	-	-	-	-	-
12	-	-	-	-	-	-	-	-
13	-	-	-	-	-	-	-	-
14	-	-	-	-	-	-	-	-
15	-	-	-	-	-	-	-	-
16	-	-	-	-	-	-	-	-
17	-	-	-	-	-	-	-	-
18	-	-	-	-	-	-	-	-
19	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
Individual mean	7.63	7.86	8.4	7	15	17.29	16.2	13.67
Totalmean	7.72				15.54
Minimum	6				10
Maximum	9				20

WEEK	Parameter	Concentrations(mg/L)
WEEK	Parameter	Control	0.625	1.25	2.5	5	10
1st	pH	7.2	7.2	7.2	7.2	7.2	7.2
	Temperature(°C)	26.5	26.5	26.5	26.5	26.5	26.5
	Dissolvedoxygen(%)	8.0	8.0	8.0	8.0	8.0	8.0
	Hardness(CaCO3)	125	125	125	125	125	125
2nd	pH	7.1	7.1	7.1	7.1	7.1	7.1
	Temperature(°C)	26.5	26.5	26.5	26.5	26.5	26.5
	Dissolvedoxygen(%)	7.0	7.0	7.0	7.0	7.0	7.0
	Hardness(CaCO3)	125	125	125	125	125	125
3rd	pH	7.1	7.3	7.2	7.4	7.3	7.2
	Temperature(°C)	26.5	26.5	26.5	26.5	26.5	26.5
	Dissolvedoxygen(%)	8.2	8.1	8.1	8.1	8.1	8.1
	Hardness(CaCO3)	100	100	100	100	100	100
4th	pH	7.1	7.0	7.1	7.2	7.2	7.2
	Temperature(°C)	26.5	26.5	26.5	26.5	26.5	26.5
	Dissolvedoxygen(%)	7.9	7.7	7.5	7.7	7.8	7.7
	Hardness(CaCO3)	125	125	125	125	125	125
5th	pH	7.0	6.9	6.9	6.8	6.9	6.8
	Temperature(°C)	26.5	26.5	26.5	26.5	26.5	26.5
	Dissolvedoxygen(%)	7.7	7.8	7.7	7.5	7.7	7.9
	Hardness(CaCO3)	100	100	100	100	100	100
6th	pH	7.0	7.0	7.1	7.1	7.0	7.0
	Temperature(°C)	26.5	26.5	26.5	26.5	26.5	26.5
	Dissolvedoxygen(%)	8.2	8.1	8.2	8.1	8.1	8.1
	Hardness(CaCO3)	125	125	125	125	125	125

Concentration (mg/L)	R	No.of eggsaddedatthestartoftest	Mortality
			Pre-hatch					Post-hatch
			Embryostage					Larvaand Juvenilesstage
			Days
			1	2	3	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
Control	1	20	0	0	1	1	0	0	0	0	0	0	1	0	1	0	0	0	0	0	0
	2	20	0	1	0	0	0	0	0	1	0	0	0	0	0	0	0	0	1	0	0
	3	20	0	1	1	1	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0
	4	20	1	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0
0.625	1	20	2	0	0	0	0	1	1	0	0	0	1	0	0	0	0	0	0	0	0
	2	20	1	0	0	0	0	0	1	0	0	1	0	0	0	0	0	0	0	0	0
	3	20	0	1	2	0	0	0	0	0	0	0	0	0	0	0	0	0	2	0	0
	4	20	0	1	1	0	0	0	0	2	0	0	0	2	0	0	1	0	0	1	0
1.25	1	20	1	0	0	0	0	0	3	0	0	0	0	0	0	0	0	0	0	0	0
	2	20	2	0	0	0	1	0	2	0	0	0	1	1	0	0	0	0	0	0	1
	3	20	0	3	1	0	0	0	0	0	0	0	0	0	0	0	1	0	1	0	0
	4	20	1	0	0	2	0	0	0	0	0	1	0	0	2	0	0	0	0	0	0
2.5	1	20	2	0	0	0	0	1	1	0	0	1	0	0	0	1	0	2	0	0	2
	2	20	3	0	1	0	0	0	2	1	0	0	0	0	2	1	0	1	0	0	0
	3	20	0	1	1	2	0	0	0	0	0	0	0	0	0	0	2	0	2	0	1
	4	20	1	0	0	0	0	0	0	0	3	0	0	0	0	0	0	3	0	0	0
5	1	20	5	0	0	0	0	0	1	0	0	1	2	0	0	0	0	0	1	0	0
	2	20	3	1	0	0	0	0	0	0	1	0	0	2	0	0	3	1	0	0	0
	3	20	0	2	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0	2
	4	20	2	0	1	1	0	0	0	0	0	0	0	2	0	0	0	5	0	0	0
10	1	20	0	1	2	0	0	2	1	0	0	0	1	2	2	0	0	0	1	0	0
	2	20	2	1	0	1	1	0	2	0	0	0	0	1	2	0	0	1	0	0	2
	3	20	1	2	1	3	0	0	0	0	2	0	0	0	0	3	0	0	1	0	0
	4	20	2	1	1	2	0	2	0	1	0	1	0	0	0	1	0	0	0	0	0