Phenethyl benzoate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Experimental test result performed according to the OECD test guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 6.25, 12.5, 25, 50 and 100 mg/l,
- Sampling method: Samples for analytical determination was collected from four corners and center of the aquarium for each test concentrations send for chemical analysis
- Sample storage conditions before analysis: Samples were immediately analysed after sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test chemical was prepared by dissolving 5 g of test chemical in 5 l of potable water (passed through reverse osmosis system). After stirring the stock solution was filtered and analytically detected and the concentration found to be 236.34 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. Test chemical concentrations used for the study were 0, 6.25, 12.5, 25, 50 and 100 mg/l, respectively.
- Controls: RO water 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 applicable

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra Fish
- Length at study initiation (length definition, mean, range and SD): average 1.5 ± 0.2 cm
- Weight at study initiation (mean and range, SD): average 0.09 ± 0.01 g
- Housing:The fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration.
- Duration of housing: 12 days
- Photoperiod: 16 hour light and 8 hour darkness
- Diet : Standard brand feed, once daily
- Acclimatization: seven days
- Feeding frequency: Once in a day


TEST ORGANISM
- Common name: Zebra fish
- Strain: Tubingen
- Source: Fishes obatined Center for cellular and molecular biology, India were reared and culture in test facility
- Age at study initiation (mean and range, SD): all fishes were of same age less than 3 months
- Length at study initiation (length definition, mean, range and SD): 1.5 ± 0.2 cm
- Weight at study initiation (mean and range, SD): 0.09 ± 0.01 g
-

ACCLIMATION
- Acclimation period: Total 9 days ( 2 days of settling period plus 7 days of acclimitisation)
- Acclimation conditions (same as test or not): Similar as test conditions
- Type and amount of food during acclimation: Live feed ( artemia salina) and Standard brand feed, once daily
- Feeding frequency during acclimation: once daily
- Health during acclimation (any mortality observed): All fishes were healthy during acclimitisation no mortality was observed


FEEDING DURING TEST
- Food type: Feeding was not provided during test.
- Amount:
- Frequency:

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

40 mg of CaCO3

Test temperature:

21-22°C

pH:

7.3 to 7.9 in control groups during all the exposure duration

Dissolved oxygen:

6.8 to 4.8 mg/l of DO at 0 hour and 96 hour

Nominal and measured concentrations:

Test chemical concentrations used for the study were 0, 6.25, 12.5, 25, 50 and 100 mg/l, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: Polypropylene (PP) fish tank
- Material, size, headspace, fill volume: 5 liters of test vessel filled with 4 liter of water
- Aeration: Aeration in test vessels was provided 1 day before the start of experiment
- No. of organisms per vessel: 7 fishes

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8-hour darkness
TEST SYSTEM
- Test vessel: Poly propylene (PP) fish tank
- Material, size, headspace, fill volume: FIll volume of 4 Liters and test medium used in study is 2 liters
- Aeration: Not provided during study
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms per vessel: 7 test organisms per vessel
- No. of vessels per concentration (replicates): one test tank per concentrations
- No. of vessels per control (replicates): 1 vessel per control
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 0.14g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: RO water obatined from the RO water filter systems
- Total organic carbon: TOC 1.48 mg/L
- Particulate matter: 187 µg/l
- Metals: Aluminium (Al), Arsenic (As), Chromium (Cr), Cobalt (Co), Copper (Cu), Iron (Fe), Lead (Pb), Nickel (Ni), Zinc (Zn), were less than < 0.01 Cadmium (Cd), mercury and silver are <100ng/L,
- POly chlorinated Bis phenol <10 ng/L
- Free residual Chlorine: 2 ng/L
- Culture medium different from test medium: same as test medium
- Intervals of water quality measurement: twice in year

OTHER TEST CONDITIONS
- Adjustment of pH: Not adjusted
- Photoperiod: 16 hours light and 8 hours dark
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study Main stusy was conducted based on the details on main study

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 25 - < 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

37.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: In 25 mg/L at 3 and 6 hours duration all fishes were showing slow moment. At same concentration out of 7 fishes 3 have shown normal behaviour and 4 fishes have shown surface water swimming. At 48 hours, 72 hours and 96 hours all test organisms were normal.
-At higher conenrations i.e, 50 and 100 mg/l at 3 hours and 6 hours moments were slow when compared with contorl and inverted swimming was observed in all fishes
- Other biological observations: None
- Mortality of control: No mortality was observed in control
- Other adverse effects control: None
- Abnormal responses: None in control and test concentrations

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Based on the mortality LC50 was recorded by taking average of test concentrations of those where 0 and 100% mortality was observed.

Sublethal observations / clinical signs:

OBSERVATIONS

Table: Assessment of test concentrations

Sr. no.	Concentrations (mg/l)	Wavelength (nm)	Absorbance	Temperature (°C)
1	blank	199	0.0001	22
2	0.25	199	0.0683	22
3	0.5	199	0.1407	22
4	1.0	199	0.2410	22
5	2.0	199	0.4178	22
6	3.0	199	0.6592	22
7	4.0	199	0.8454	22
8	5.0	199	1.0198	22
9	6.0	199	1.1887	22
10	7.0	199	1.3409	22
11	7.86	199	1.4886	22

 

The absorbance and concentrations were recorded at 199 nm.

Table: Concentration after analytical Determination

Sr. No	Concentrations (mg/L)	Analytical Concentrations (0 hour)	Analytical Concentrations (96 hour)
1	blank	0.00	0.00
2	6.25	5.9	7.29
3	12.5	13	11.3
4	25.5	20.8	19.3
5	50	72.59	71.6
6	100	87.2	89.9

Mortality and symptoms

Nominal Concentration (mg/l)	Number of fish	Mortality (cumulated) after
		3h	6h	24h		48h	72h	96h
Control	7	-		-	-	-	-	1
6.25	7	-	-	-	-	-	-	-
12.5	7	-	-	-	-	-	-	-
25	7	-	-	-	-	-	-	-
50	7	4	-	3	-	-	-	-
100	7	7	-	-	-	-	-	-

Visible Symptoms: 

Nominal Concentration (mg/l)	Number of fish	Symptoms after
		3h	6h	24h	48h	72h	96h
Control	7	-	-	-	-	SM1	-
6.25	7	-	-	-	-	-	-
12.5	7	-	-	-	-	-	-
25	7	M7	M7	NS3SC7	-	-	-
50	7	B7M7IS7	M7	NS3	-	-	-
100	7	B7M7IS7	-	-	-	-	-

Explanation of symptoms:

A:apathy

N:Narcotic-like state

C:Swimming near water surface

M: Slow movements compared to control

NS:No abnormal symptoms 

S:Surfacing behaviour

L:Loss of equilibrium

SM:Shimming movement

H1:Hyperactivity

H2:Hypo activity

B:Bending behavior

F:Fin rot

IS:Invert Swimming

SB:Swimming behaviour

RF:Respiratory Function

P:Pigmentation

 

pH and oxygen content:

Nominal Concentration (mg/l)	Number of fish	pH readings after
		24h	48h	72h	96h
Control	7	7.3	7.5	7.5	7.9
6.25	7	6.9	7.3	7.1	7.1
12.5	7	6.6	7.2	7.1	7.2
25	7	6.9	7.2	7.3	7.2
50	7	-	-	-	-
100	7	-	-	-	-

 

Nominal Concentration (mg/l)	Number of fish	Oxygen content (mg/L) after
		24h	48h	72h	96h
Control	7	6.6	5.3	4.9	4.8
6.25	7	5.6	4.0	4.3	4.6
12.5	7	4.4	4.6	4.3	4.9
25	7	5.5	5.2	5.5	5.5
50	7	-	-	-	-
100	7	-	-	-	-

 

Test temperature

Nominal Concentration (mg/l)	Number of fish	Temperature (°C) after
		24h	48h	72h	96h
Control	7	22	22	22	21
6.25	7	22	22	22	21
12.5	7	22	22	22	21
25	7	22	22	22	21
50	7	-	-	-	-
100	7	-	-	-	-

Standard graph:

SAMPLE NAME	ABSORBANCE	CONCENTRATION (mg/L)
Blank	0.0001	0
Standard 1	0.1407	0.5
Standard 2	0.241	1
Standard 3	0.4178	2
Standard 4	0.6592	3
Standard 5	0.8454	4
Standard 6	1.0198	5
Standard 7	1.1887	6
Standard 8	1.3409	7
Standard 9	0.0683	0.25
Standard 10	1.4886	7.86

Analytical at Day 0:

SAMPLE NAME	ABSORBANCE	CONCENTRATION (mg/L)	after dilution(mg/L)
Blank	0.0006	0.003
Conc_6.25mgL_Day0_R1	1.1832	5.9871	5.99
Conc_6.25mgL_Day0_R2	1.1785	5.9636	5.96
Conc_6.25mgL_Day0_R3	1.1821	5.9819	5.98
Conc_12.5mgL_5dil_Day0_R1	0.5123	2.5922	12.96
Conc_12.5mgL_5dil_Day0_R2	0.5149	2.6054	13
Conc_12.5mgL_5dil_Day0_R3	0.5174	2.6181	13.1
Conc_25mgL_5Dil_Day0_R1	0.8222	4.1604	20.8
Conc_25mgL_5Dil_Day0_R2	0.8229	4.1639	20.8
Conc_25mgL_5Dil_Day0_R3	0.8228	4.1634	20.8
Conc_50mgL_10Dil_Day0_R1	1.0281	5.2052	52.05
Conc_50mgL_10Dil_Day0_R2	1.0347	5.2386	52.38
Conc_50mgL_10Dil_Day0_R3	1.0408	5.2695	52.69
Conc_100mgL_20Dil_Day0_R1	0.8612	4.3581	87.2
Conc_100mgL_20Dil_Day0_R2	0.8624	4.3642	87.2
Conc_100mgL_20Dil_Day0_R3	0.8626	4.3649	87.2

Analytical at Day 4:

SAMPLE NAME	ABSORBANCE	CONCENTRATION (mg/L)	after dilution(mg/L)
Blank	0.0001	0.0004	0.0004
Conc_6.25mgL_Day4_R1	1.3765	6.9653	6.97
Conc_6.25mgL_Day4_R2	1.3651	6.9115	6.91
Conc_6.25mgL_Day4_R3	1.3722	6.9727	6.97
Conc_12.5mgL_5dil_Day4_R1	0.453	2.2922	11.45
Conc_12.5mgL_5dil_Day4_R2	0.4603	2.3294	11.65
Conc_12.5mgL_5dil_Day4_R3	0.4582	2.3186	11.6
Conc_25mgL_5Dil_Day4_R1	0.8427	4.2665	21.33
Conc_25mgL_5Dil_Day4_R2	0.8506	4.3065	21.53
Conc_25mgL_5Dil_Day4_R3	0.8303	4.2016	21
Conc_50mgL_10Dil_Day4_R1	1.0154	5.1409	51.4
Conc_50mgL_10Dil_Day4_R2	1.0362	5.2462	52.46
Conc_50mgL_10Dil_Day4_R3	1.0316	5.2229	52.22
Conc_100mgL_20Dil_Day4_R1	0.88	4.4554	89.108
Conc_100mgL_20Dil_Day4_R2	0.8967	4.5399	90.67
Conc_100mgL_20Dil_Day4_R3	0.898	4.5465	90.93

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal concentrations, the median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be > 25 to < 50 mg/L.

Executive summary:

An acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 1.5 ± 0.2 cm and average weight of 0.09 ± 0.01 g was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. Aeration in test vessels was provided till 1 day before the start of the experiment. The test conditions during the housing of the test organisms were oxygen content of 8.8 mg/l, pH 7.5, water temperature 22°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. The test chemical was prepared by dissolving 5 gm of test chemical in 5 lit of potable water (passed through reverse osmosis system). After stirring, the stock solution was filtered and analytically detected & the concentration was found to be 236.34 mg/l. The remaining test solutions were prepared by dilution from the stock solution. Test chemical concentrations were analytically determined by UV-VIS spectrophotometer. Test chemical concentrations used for the study were 0, 6.25, 12.5, 50 and 100 mg/l, respectively. Total 7 fishes with biomass of 0.14g/L were exposed to test chemical in a 4 lit Polypropylene (PP) fish tank containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 21 -22°C, pH of control at 0 and 96 hr was 7.3 & 7.9 and DO of control at 0 and 96 hr was 6.8 & 4.8 mg/l and under a photoperiod of 16:8 hr light: dark conditions, respectively. Mortality in the control was 0%. The dissolved oxygen concentration remained above 60% of the air saturation value throughout the exposure period. Thus, fulfilling the validity criterion. All the test concentrations were analytical determined at 0 our and 96 hours of the exposure durations which were maintained with in range of 95.92 - 111.12%, 104.16 - 92.48%, 83.2 - 85.12%, 104.74 - 104.04%, 87.2 - 90.23% at 6.25, 12.5, 25, 50 and 100 mg/L concentrations. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mortality of the test organism, the 96 hr median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was reported to be in the range of > 25 to < 50 mg/L, 37.5 mg/L (based on average of LC0 and LC100) . Thus, test chemical was considered as toxic to aquatic fishes and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Description of key information

An acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 1.5 ± 0.2 cm and average weight of 0.09 ± 0.01 g was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. Aeration in test vessels was provided till 1 day before the start of the experiment. The test conditions during the housing of the test organisms were oxygen content of 8.8 mg/l, pH 7.5, water temperature 22°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. The test chemical was prepared by dissolving 5 gm of test chemical in 5 lit of potable water (passed through reverse osmosis system). After stirring, the stock solution was filtered and analytically detected & the concentration was found to be 236.34 mg/l. The remaining test solutions were prepared by dilution from the stock solution. Test chemical concentrations were analytically determined by UV-VIS spectrophotometer. Test chemical concentrations used for the study were 0, 6.25, 12.5, 50 and 100 mg/l, respectively. Total 7 fishes with biomass of 0.14g/L were exposed to test chemical in a 4 lit Polypropylene (PP) fish tank containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 21 -22°C, pH of control at 0 and 96 hr was 7.3 & 7.9 and DO of control at 0 and 96 hr was 6.8 & 4.8 mg/l and under a photoperiod of 16:8 hr light: dark conditions, respectively. Mortality in the control was 0%. The dissolved oxygen concentration remained above 60% of the air saturation value throughout the exposure period. Thus, fulfilling the validity criterion. All the test concentrations were analytical determined at 0 our and 96 hours of the exposure durations which were maintained with in range of 95.92 - 111.12%, 104.16 - 92.48%, 83.2 - 85.12%, 104.74 - 104.04%, 87.2 - 90.23% at 6.25, 12.5, 25, 50 and 100 mg/L concentrations. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mortality of the test organism, the 96 hr median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was reported to be in the range of > 25 to < 50 mg/L, 37.5 mg/L (based on average of LC0 and LC100) . Thus, test chemical was considered as toxic to aquatic fishes and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

37.5 mg/L

Additional information

Various experimental studies of the test chemical and supporting weight of evidence studies for its structurally and functionally similar read across chemical were reviewed for short term toxicity to aquatic fish end point which are summarized as below:

 

An acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 1.5 ± 0.2 cm and average weight of 0.09 ± 0.01 g was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. Aeration in test vessels was provided till 1 day before the start of the experiment. The test conditions during the housing of the test organisms were oxygen content of 8.8 mg/l, pH 7.5, water temperature 22°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. The test chemical was prepared by dissolving 5 gm of test chemical in 5 lit of potable water (passed through reverse osmosis system). After stirring, the stock solution was filtered and analytically detected & the concentration was found to be 236.34 mg/l. The remaining test solutions were prepared by dilution from the stock solution. Test chemical concentrations were analytically determined by UV-VIS spectrophotometer. Test chemical concentrations used for the study were 0, 6.25, 12.5, 50 and 100 mg/l, respectively. Total 7 fishes with biomass of 0.14g/L were exposed to test chemical in a 4 lit Polypropylene (PP) fish tank containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 21 -22°C, pH of control at 0 and 96 hr was 7.3 & 7.9 and DO of control at 0 and 96 hr was 6.8 & 4.8 mg/l and under a photoperiod of 16:8 hr light: dark conditions, respectively. Mortality in the control was 0%. The dissolved oxygen concentration remained above 60% of the air saturation value throughout the exposure period. Thus, fulfilling the validity criterion. All the test concentrations were analytical determined at 0 our and 96 hours of the exposure durations which were maintained with in range of 95.92 - 111.12%, 104.16 - 92.48%, 83.2 - 85.12%, 104.74 - 104.04%, 87.2 - 90.23% at 6.25, 12.5, 25, 50 and 100 mg/L concentrations. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mortality of the test organism, the 96 hr median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was reported to be in the range of > 25 to < 50 mg/L, 37.5 mg/L (based on average of LC0 and LC100) . Thus, test chemical was considered as toxic to aquatic fishes and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

 

In a prediction done using EPI Suite ECOSAR version 1.11, the short-term toxicity of the test chemical to fish was predicted. On the basis of effect of chemical observed on the mortality of the test organism, the 96 hr lethal effect concentration (LC50) was estimated to be 2.81 mg/l. Thus, based on the LC50 value, chemical can be considered as toxic to fish and hence, considered to be classified in ‘aquatic chronic category 2’ as per CLP classification criteria.

 

Another short term fish toxicity study was conducted for 120 hrs for assessing the effect of test chemical. Cyprinus carpio (Common carp) of weight 1 -10 pounds (average weight of around 3 pounds) captured with an alternating-current electric boat shocker in the New York State Barge Canal was used as a test organism for the study. After transportation of test organism to the laboratory, oxygen was supplied. Test fishes were acclimatized for weeks at spring temperature of 47˜F. Test chemical concentrations were not verified analytically. Test chemical concentrations used for the study were 66, 132 and 155 mg/Kg (nominal concentrations), respectively. Study was performed using fishes in a flow through system at 18.33°C (65°F) temperature, 6.7 pH and alkalinity of 10 mg/l CaCO3. Test fishes were exposed to test chemical in 350 and 550 gallons glass-fronted, fiber glass tank for 120 hr. After exposure period of 120 hr, mortality and other visual symptoms of the test organism was noted. No abnormal responses of the test fish were observed. On the basis of the effect on mortality of the test organism Cyprinus carpio, the 120 hr NOEC was determined to be 66-155 mg/Kg (nominal concentration).

 

In a supporting weight of evidence study from peer reviewed journals, authoritative database and secondary source, short term fish toxicity was conducted for 96 hrs for assessing the effect of test chemical. The study was performed following the OECD Guideline 203 (Fish, Acute Toxicity Test) under flow through conditions. Oryzias latipes (Japanese Medaka) of 28 -43 days old and 18 -71 mg weight obtained from the Environmental Research Laboratory-Duluth (ERL-D) culture unit was used as a test organism. Test fishes were nurtured in tank at 25°C and fed with live Biomaine brand brine shrimp. Before 24 hr or during the study, test organism was not fed. 5 different concentrations of test chemical along with the control were taken for the study. Stock solutions of test chemical was prepared by dissolving the test chemical in Lake Superior water, using a high speed stirrer. Stock solutions were then transferred to a glass stock bottle inside the vented diluter enclosure using Teflon tubing and air pressure. During each test, a predetermined volume (ml/min) of stock solution was continuously pumped from the stock bottle into the mixing cell of the diluter system. Test chemical concentrations were verified analytically and analysis was carried out bya Hewlett Packard 5730A gas chromatograph (GC) equipped with a flame ionization detector (FID) linked to an HP 3350 lab automation system. All test analyses were accomplished using direct aqueous injection. GC column consists of a wall-coated open tubular silica column, 0.53 mm I.D. x 15 cm, with a 2.5 µ phase of bonded polyethlene glycol at isothermal oven temperature of 85, 120 and 110°C, respectively. Total 20 fishes/conc (10 organisms/replicate) were exposed to test chemical in a2.0 lit glass aquaria tank.2.0 l glass aquaria tank has a dimension of 18.5 X 14.0 X 13.0 cm deep. It has a 8.6 cm standpipe which resulted in a total volume of 2.0 lit. Continuous-flow mini diluter exposure system with vented enclosures was used for the study. Flow rate during the study was 25 ml/min with 90% replacement times of 2.8 hr. Lake superior water was used. It was filtered before use through sand, a 50-micron filter; a 5-micron filter; and then exposed to ultraviolet light before heating to the test temperature of 25±1°C.The test vessels were placed in a room at a temperature of 25±1°C, pH 7.88 ± 0.18 (7.31 to 8.85), dissolve oxygen (D. O) 6.8 ± 0.7 (5.0 to 8.5), hardness of water 45.8 (38.0 to 52.0) mg/l as CaCO3, alkalinity 45.9 (35.0 to 58.5 mg/l) as CaCO3 and under a 16 hr photoperiod with a light intensity of 12 to 25 lumens provided by fluorescent lamps for 96 hrs. All experiments were performed in replicate.95% confidence intervals were calculated using the binomial tests. Dissolved oxygen (D.O.) was measured by a dissolved oxygen meter. pH was determined on one set of replicate tanks atleast once and often twice during the test. On the other hand, hardness and alkalinity determinations were done at a minimum on a control, one intermediate and one at high test concentration tank; it was carried out once or twice during the study. Mortality was noted after an exposure period of 96 hrs. No mortalities were observed in the control vessel, the dissolved oxygen concentration was evaluated to be ≥ 60% (i.e, reported as 82.3%) of the air saturation value in test vessels throughout the study period and analytical monitoring of test concentrations has been carried out, thus fulfilling the validity criteria of the study. As the test concentrations were maintained within ±20% of the initial measured concentrations throughout the study, all results will be reported in nominal concentrations. On the basis of the effect on mortality of the test organismOryzias latipes(Japanese Medaka), the 96 hr LC50 was determined to be 4.0 mg/l (95% C. I. = 3.48 to 4.60 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to fish and hence, considered to be classified in ‘aquatic chronic category 2’ as per the CLP classification criteria.

 

For the test chemical, an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average weight 0.473 g and average length of 1.76 cm was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. The test conditions during the housing of the test organisms were oxygen content of 7.8 mg/l, pH 7.65, water temperature 24.5°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. Test chemical conentrations were not verified analytically. Nominal test chemical concentrations selected for the study were 0, 0.625,1.25, 2.5, 5 and 10 mg/L. respectively. Total 8 fishes were exposed to test chemical in a 5 lit bowl aquaria containing 4 liters of potable water. The test vessels were placed in a room at a temperature of 24.4°C, pH 7.03, hardness of water 152.5 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions, respectively. Aeration in test vessels was provided 1 day before the start of the experiment. The fishes were moving slowly in the test chemical conc. as compared to the control. No mortalities were observed in the control vessel and the dissolved oxygen concentration was evaluated to be ≥ 60% (i.e, reported as 90.96%) of the air saturation value in test vessels throughout the study period. On the basis of effect of test chemical on mortality of the test organism, the median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was determined to be > 2.5 to < 5.0 mg/L. Thus, test chemical was considered as toxic to aquatic fishes and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

 

On the basis of the above results, it can be concluded that the test chemicalwas considered as toxic to aquatic fishes and hence, considered to be classified in ‘aquatic chronic category 2’ as per the CLP classification criteria.