4-methylstyrene

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 16, 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA - 660/3-75-009

Version / remarks:

April 1975

Deviations:

not specified

GLP compliance:

yes

Remarks:

FDA 21 CRF Part 58

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 12.0, 6.1, 3.0,1.5 and 0.76 mg/L
- Sampling method: water samples
- Sample storage conditions before analysis: refrigerated storage prior to analysis.
- sampling frequency: days 0, 1, 4, 7.

Vehicle:

yes

Details on test solutions:

On received at the laboratory the test substance was diluted in nanograde acetone. Test solutions: 12.0, 6.1, 3.0, 1.5 and 0.76 mg/L.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: Bluegill sunfish
- Source: Fattig fish hatchery, Brady, Nebraska.
- Feeding during test
- Food type: commercial fish food
- Amount:3% of body weight
- Frequency: daily
- Acclimation period:fourteen days
- Photoperiod: 16 h daylight
- Health during acclimation: observed daily no mortality recorded

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Hardness:

CaCO3 - 255 ppm

Test temperature:

21-22 °C

pH:

7.5 to 8.2 over the test period

Dissolved oxygen:

9.3 ppm

Conductivity:

50 umhos/cm

Nominal and measured concentrations:

Nominal: 0.76 mg/L, 1.5 mg/L, 3.0 mg/L, 6.1 mg/L, 12 mg/L
Measured: 0.41 mg/L, 0.66 mg/L, 1.2 mg/L, 2.3 mg/L and 5.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquaria 30 L
- Type of flow-through: proportional dilutor system described by Mount and Brungs with the modifications of McAllister et. al.
- Renewal rate of test solution: 250 ml/min (replacement rate 10 x in 24 hours)
- The test aquaria were immersed in a circulating water bath held at 22°C by submerged heating elements
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Source/preparation of dilution water: well water
- Metals: <0.004 ppmd
- Alkalinity: 368 ppm
- Dissolved oxygen: 9.3 ppm
- Intervals of water quality measurement: 0, 1, 4, 7 days

Reference substance (positive control):

yes

Remarks:

Antimysin A

Key result

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.66 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

The LC50’s and 95% confidence limits were calculated by a computerized LC50 program developed by Stephan et al. A lethal threshold for the test substance to bluegill sunfish was reached within 7 days of the study since mortality on the last 3 days had not exceeded 5 /5 of the total population and 10 % in any test concentration. The weight and analysis indicated no significant effects on growth of the test fish at any test concentrations.
Adverse behavior observations noted during the study were loss of equilibrium and surfacing. Mortality was preceded by surfacing behavior in most cases.
Ananalytical results: analytical results from the study showed the mean measured test substance concentrations at: 0.41 mg/L, 0.66 mg/L, 1.2 mg/L, 2.3 mg/L and 5.5 mg/L. The nominal concentrations were approximately 45 % of the measured values. The volatile nature of the test substance is thought to be responsible for the reduced concentrations in test solutions.

Reported statistics and error estimates:

ANOVA - F test.

Sublethal observations / clinical signs:

Analytical data.

Aquaria Number	Nominal conc mg/L	Measured Concentrations of the test substance mg/L
		Day 0	Day 1	Day 4	Day 7	Mean	SD
1	Control	0.2	0.8	0.7	0.6
2	0.76	0.545	0.297	0.442	0.37	0.41	0.11
3	1.5	0.649	0.572	0.763	0.649	0.66	10.079
4	3.0	1.11	1.13	1.4	1.07	1.2	0.15
5	6.1	2.25	2.37	2.54	2.18	2.3	0.16
6	12	4.51	5.46	6.58	5.09	5.4	0.87
Spiking	1.0mg/L	0.804	0.847	0.855	0.989	0.87	0.08
	5.0 mg/L	4.04	4.18	4.1	4.34	4.2	0.13
	10.0 mg/L	8.94	8.98	10.2	9.09	9.3	0.6

test substance mortality data.

Nominal Conc mg/L	Day of Exposure
	1	2	3	4	5	6	7
Control	0	0	0	0	0	0	0
0.76	0	0	0	0	0	0	0
1.5	0	0	0	0	0	0	0
3.0	0	0	0	0	0	0	0
6.1	0	0	15	30	35	40	40
12.0	95	95	100	100	100	100	100

test substance LC₅₀ data based on measured concentrations.

	test substance concentration ppm
Day of exposure	LC50	95% confidence limits
1 & 2	3.7	2.3 - 5.4
3	3.1	2.3 - 5.4
4	2.8	1.2 - 5.4
5	2.7	1.2 - 5.4
6 & 7	2.6	1.2 - 5.4

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 7 d mean measured LC50 was determined to be 2.6 mg/L and 7 d NOEC was established at 0.66 mg/L.

Executive summary:

A study was conducted to determine the toxicity of the test substance to Bluegill sunfish (Lepomis macrochirus) according to a method equivalent to EPA - 660/3-75-009 (April 1975), in compliance with GLP. Groups of 20 fish were exposed to the test substance for 7 d at nominal concentrations of 0.76, 1.5, 3.0, 6.1 and 12 mg/L in a dynamic flow-through system. Analytical dose verification was conducted using liquid chromatography. The mean measured concentrations (0.41, 0.66, 1.2, 2.3 and 5.5 mg/L) were equivalent to approximately 45 % of the measured values due to test substance volatility. Under the study conditions, the 7 d mean measured LC50 was determined to be 2.6 mg/L and 7 d NOEC was established at 0.66 mg/L (Johnson, 1981).

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 01, 2001 to March 05, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

tests conducted in sealed vessels

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

Although the test substance is stable in water, it is volatile, therefore both the range finding and definitive study were conducted in sealed vessels. The study design used was static renewal with a proposed water change at 48 hours. Test solutions were sampled at the start and end of each water change.

Vehicle:

no

Details on test solutions:

Suitable stock solutions were prepared undiluted directly into testing water.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Aquatic Biosystems, Colorado
- Age at study initiation: juvenile fish weighing 3.6 mg (10 fish therefore give a biological loading of 0.018 g/L in study aquaria)
- Feeding during test: no

ACCLIMATION
- Acclimation period: fourteen days
- Acclimation conditions: virtually the same as in the test (temperature 22°C)
- Type and amount of food: Artemia salina
- Feeding frequency: once per day
- Health during acclimation: 0% mortality and good health

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

40 - 48 mg/L calcium carbonate. In the definitive study the hardness of water reduced by 4.0 mg/Lof calcium carbonate between changes

Test temperature:

22 +/- 1°C

pH:

6.7 - 7.5 (measured)

Dissolved oxygen:

6.8-9.0 mg/L (minimum saturation level 78%)

Nominal and measured concentrations:

Nominal concentrations
Range finding study as listed above.
Definitive study: 0 (untreated control), 1.6, 2.5, 4.0, 6.4 & 10 mg/L. Mean measured concentrations: 0 (untreated control), 0.82, 1.3, 2.6, 4.2 & 6.8 mg/L.

Details on test conditions:

TEST SYSTEM
The test was performed under static, renewal conditions in sealed ½ gallon vessels that contained approximately 2 L of the test solution in order to minimum head space and the losses of the test substance.

- Test vessel: 3.8 L
- Renewal rate of test solution: 48-hours
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: average fish weight: 3.6 mg/L
- Source/preparation of dilution water: local supply, carbon filtered and deionized. Water was adjusted to hardness 40 to 48 mg/L as CaCO3 and stored in polyethylene tanks were it was aerated and recirculated through particle filters, activated carbon and an ultraviolet sterilizer.
- Photoperiod: 16 hours light
- Light intensity: 83 foot candles
- Dissolved oxygen: minimum noted 78 % saturation
- Effects monitored: mortality and toxic effects

TEST CONCENTRATIONS
- Spacing factor for test concentrations: as the protocol
- Range finding study
- Test concentrations: untreated control, 0.1, 1.0, 5.0, 10.0 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

5.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

- Behavioral abnormalities: at concentrations above 4.2 mg/L affected fish were either intermittently or completely mobilised/lost equilibrium or were lethargic.
- Observations on body length and weight: none recorded.
- Other biological observations: none other than those recorded above.
- Mortality of control: no mortality.
- Effect concentrations exceeding solubility of substance in test medium: not applicable test concentrations were within the solubility range of test material.
- Nominal concentrations: the results are recorded as nominal concentrations however due to volatility of the test substance losses were experienced during the preparation of test solutions and transfer of test solutions to test vessels. Results of analysis showed that during preparation nominal concentrations of 5.0 mg/L reduced to 3.7-4.3 mg/L but there was no loss between water changes (analysed concentrations range from 103 – 125 % of the initial measured concentration).

Reported statistics and error estimates:

Standard statistical techniques used as recommended in the guideline. LC50 calculated using the probit method (Stephan et al 1978).

Sublethal observations / clinical signs:

Information on the nominal and measured concentration in the definitive study is shown in table below.

Nominal conc mg/L	Measured concentration mg/L					% of nominal
	0 hrs	48 hrs(a)	48 hrs(b)	96 hrs	Mean
0	ND	ND	ND	ND	ND	-
1.6	0.73	0.91	0.76	0.89	0.82	51
2.5	1.2	1.2	1.2	1.5	1.3	52
4.0	2.5	2.5	2.7	2.6	2.6	65
6.4	4.1	4.3	3.8	4.6	4.2	66
10.0	7.1	6.2	6.5	7.3	6.8	68

The results of assessments of toxicity in the definitive study are shown in the table below.

Measured conc mg/L	% mortality
	0 hrs		24 hrs		48 hrs		72 hrs		96 hrs
Replicate	1	2	1	2	1	2	0	0	0	0
0	0	0	0	0	0	0	0	0	0	0
0.82	0	0	0	0	0	0	0	0	0	0
1.3	0	0	0	0	0	0	0	0	0	0
2.6	0	0	0	0	0	0	0	0	0	0
4.2	0	0	0	0	20	0	20	0	30	10
6.8	0	0	10	0	10	0	70	40	80	90

The results of effects assessments are shown in the table below.

Measured conc mg/L	% showing effects
	0 hrs		24 hrs		48 hrs		72 hrs		96 hrs
Replicate	1	2	1	2	1	2	0	0	0	0
0	0	0	0	0	0	0	0	0	0	0
0.82	0	0	0	0	0	0	0	0	0	0
1.3	0	0	0	0	0	0	0	0	0	0
2.6	0	0	0	0	0	0	0	0	0	0
4.2	0	0	0	30	50	100	80	90	100	100
6.8	0	50	80	80	100	100	30	60	100	100

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 96 h mean measured LC50 was 5.2 mg/L (confidence intervals: 4.6 to 5.9 mg/L) and the 96 h NOEC was 2.6 mg/L.

Executive summary:

A study was conducted to determine the toxicity of the test substance to Fathead minnow (Pimephales promelas) according to OECD Guideline 203, in compliance with GLP. The study was carried out in sealed test vessels due to the volatility of the substance. Groups of 10 fish were exposed to nominal concentrations of 0, 1.6, 2.5, 4.0, 6.4 and 10 mg/L for 96 h under semi-static conditions (48 h renewal). Larger than recommended aquaria were used to ensure that oxygen concentrations in the test media were adequate for fish survival between renewal dates. Analytical dose verification showed that measured concentrations were equivalent to 0, 0.82, 1.3, 2.6, 4.2 and 6.8 mg/L. Losses of the test substance occurred during mixing and loading of the aquaria. However, the test substance was stable in water between changes. Final measured concentrations were 103 and 125% of initial levels, indicating that once the aqueous solutions of the test substance were sealed into the test vessels, concentrations remained constant. Oxygen concentrations were adequate for fish survival (no mortalities in the untreated control). Under the study conditions, the 96 h mean measured LC50 was 5.2 mg/L (confidence intervals: 4.6 to 5.9 mg/L) and the 96 h NOEC was 2.6 mg/L (Wyskiel, 2000).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.6 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance to Bluegill sunfish (Lepomis macrochirus) according to a method equivalent to EPA - 660/3-75-009 (April 1975), in compliance with GLP. Groups of 20 fish were exposed to the test substance for 7 d at nominal concentrations of 0.76, 1.5, 3.0, 6.1 and 12 mg/L in a dynamic flow-through system. Analytical dose verification was conducted using liquid chromatography. The mean measured concentrations (0.41, 0.66, 1.2, 2.3 and 5.5 mg/L) were equivalent to approximately 45 % of the measured values due to test substance volatility. Under the study conditions, the 7 d mean measured LC50 was determined to be 2.6 mg/L and 7 d NOEC was established at 0.66 mg/L (Johnson, 1981).

A study was conducted to determine the toxicity of the test substance to Fathead minnow (Pimephales promelas) according to OECD Guideline 203, in compliance with GLP. The study was carried out in sealed test vessels due to the volatility of the substance. Groups of 10 fish were exposed to nominal concentrations of 0, 1.6, 2.5, 4.0, 6.4 and 10 mg/L for 96 h under semi-static conditions (48 h renewal). Larger than recommended aquaria were used to ensure that oxygen concentrations in the test media were adequate for fish survival between renewal dates. Analytical dose verification showed that measured concentrations were equivalent to 0, 0.82, 1.3, 2.6, 4.2 and 6.8 mg/L. Losses of the test substance occurred during mixing and loading of the aquaria. However, the test substance was stable in water between changes. Final measured concentrations were 103 and 125% of initial levels, indicating that once the aqueous solutions of the test substance were sealed into the test vessels, concentrations remained constant. Oxygen concentrations were adequate for fish survival (no mortalities in the untreated control). Under the study conditions, the 96 h mean measured LC50 was 5.2 mg/L (confidence intervals: 4.6 to 5.9 mg/L) and the 96 h NOEC was 2.6 mg/L (Wyskiel, 2000).