Prometryn

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:

10 Dec 1987 to 14 Dec 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA /OTS guidelines for testing the effects of chemicals on fish (No. 72 - 3)

Version / remarks:

1985,1987

Qualifier:

according to guideline

Guideline:

other: ASTM 1980: Standard Practice for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

A sample of each test solution and the controls was obtained at test initiation (0-hour) and termination (96-hour) from the approximate midpoint of each aquaria and submitted for analyses of the test substance.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

For use during the definitive test, a cloudy nominal stock solution of 100 mg a.i./mL was prepared by diluting 5.0968 grams of the test substance with acetone to volume in a 50 mL volumetric flask. The stock solution was sonicated for approximately ten minutes to improve solubility. The appropriate volume of stock solution was then added to 15 L of dilution water in each test aquaria. Each solution was mixed for approximately 30 seconds with a T-Line 105 laboratory stirrer. For this test two control aquaria were established containing the same dilution water and maintained under the same conditions as the test aquaria: one control aquaria contained no test substance; the other control aquaria contained the maximum quantity of solvent present in any test vessel (0.1 mL/L) and was designated the solvent control.

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Sheepshead minnow
- Bionomics lot: 87A59
- Total length at study initiation: Mean = 29 mm; range = 22 - 36 mm; N = 30
- Wet weight at study initiation: Mean = 0.42 g; range = 0.14 - 0.69 g; N = 30

ACCLIMATION
- Acclimation period: minimum of 14 days
- Acclimation conditions: In 500 L fiberglass tank with a closed loop recirculating filtration system provided natural seawater with a salinity range of 32 - 35 ‰
- pH 7.3 - 7.4
- Temperature: 20 - 21 °C
- Dissolved oxygen concentration: 88 - 92 % of saturation
- Photoperiod: 16 hours/ 8 hours (Light/ dark)
- Type and amount of food during acclimation: Dry commercial pelleted food, ad libitum
- Feeding frequency during acclimation: Daily
- Health during acclimation (any mortality observed): There was no mortality of the test fish population during the 48 hours prior to test initiation (Daily Record of Fish Holding Conditions).

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

22 ± 1°C

pH:

7.7

Dissolved oxygen:

- At 48 hour: control 58% of saturation; the other test solutions ranged of 65 - 69% of saturation
- 48 - 96 hour: 93 - 107 % of saturation in all test solutions (aeration started from 48 hour)

Salinity:

33‰

Nominal and measured concentrations:

Nominal concentration: 1.3, 2.2, 3.6, 6.0 and 10 mg/L
Measured concentration: 0.88, 1.9, 2.8, 4.6 and 9.1 mg/L, respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: 18.9 L glass aquaria with 15 L of test solution
- Test solution: 18.4 cm depth x 816 cm2
- Aeration: Since dissolved oxygen levels fell below 60% of saturation, test solutions were aerated after 48 hours with oil-free air using a air pump.
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 2
- Biomass loading rate: 0.28 g /L
- Photoperiod: 16 hours/8 hours (light/dark)
- Light intensity and location: 25 footcandles; at the solution surface
- Fish were not fed during exposure

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used during the definitive exposure was natural seawater collected from the Cape Cod Canal, Bourne, Massachusetts. The seawater was filtered through a 5-µm porosity polypropylene core filter and an activated carbon canister before use. This water had a salinity of 33‰, pH of 7.7 and specific conductivity of 21,000 µmhos/cm.
- Intervals: At the test initiation and daily thereafter, temperature, pH, salinity and dissolved oxygen concentration were measured in each test vessel.

BIOLOGICAL OBSERVATION: At the start of the test and at 24- hour intervals thereafter, observations of stress, abnormal behavior (including immobilisation and loss of equilibrium ) and mortality were made. Dead fish were removed from the test vessels at these intervals. In addition, characteristics of the test solutions (such as precipitated materials, cloudiness, etc. ) were observed and recorded.

TEST CONCENTRATIONS
- Range finding study : A preliminary range-finding test was performed exposing sheepshead minnow to the test substance at concentrations of 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: All fish exposed to the test substance concentrations > 10 mg/L died. Sheepshead minnow exposed for 96 hours to 1.0 mg/L the test substance were unaffected. Based on these preliminary results, a definitive
toxicity test was conducted exposing sheepshead minnow to the following nominal concentrations of the test substance: 1.3, 2.2, 3.6, 6.0 and 10 mg/L.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

5.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

95% C.L.: 4.0 - 7.0 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.88 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

Overview of the results are provided in the "Any other information on results incl. tables".
After 96 hour of exposure, all test fish survived in the controls (both seawater control and the solvent control) and in the 0.88 mg/L treatment. No mortality was observed in the 1.9 mg/L treatment neither, but 20 - 50% of the surviving fish were lethargic. Same amount of lethargic surviving fish were shown in the 2.8 mg/L treatment as well, which also had a 10% mortality rate after 96 hour exposure. The mortality raised to 40% in 4.6 mg/L treatment and 90% in the 9.1 mg/L treatment. In addition, all survived fish in 4.6 mg/L were lethargic and all survived fish in the highest exposure (9.1mg/L) exhibited a complete loss of equilibrium. Based on the findings, the 96-hour NOEC for the acute definitive toxicity test was calculated to be 0.88 mg/L and the LC50 was determined to be 5.1 mg/L with 95% confidence limits of 4.0 to 7.0 mg/L.

Reported statistics and error estimates:

The mean measured concentrations (0- and 96-hour analyses) tested and the corresponding mortality data derived from the toxicity test were used to estimate the median lethal concentrations (LC50) and 95% confidence intervals for each observation interval of the exposure period. The LC50 is defined as the concentration of the test material in dilution water lethal to 50% of the test organism population at the stated time interval. If at least one test concentration caused mortality of greater than or equal to 50% of the test population, then a computer program modified from the program of C. Stephan (Peltier, 1985) was used to calculate the LC50 values and 95% confidence intervals.
Three statistical methods were available in the computer program: moving average angle analysis, probit analysis, and nonlinear interpolation with 95% confidence intervals calculated by binomial probability. Moving average angle and probit analyses yield statistically sound results only if at least two concentrations produce a mortality of between 0 and 100% of the test organism population. The selection of reported LC50 values and 95% confidence intervals was based upon an examination of the data base and the results of the computer analysis. Selection criteria included the establishment of a concentration-effect relationship (mortality), the number of concentrations causing partial responses, and the span of responses bracketing the LC50 value. If two or more statistical methods produced acceptable results, then the method which yielded the smallest 95% confidence interval was selected. The No Observed Effect Concentration (NOEC), defined as the highest concentration tested at and below which there were no toxicant-related mortalities or physical and behavioral abnormalities (e.g. lethargy, loss of equilibrium, darkened pigmentation) observed during the 96-hour exposure period was also determined.

Sublethal observations / clinical signs:

Table 2.Concentrations tested, corresponding cumulative mortalities and observations made during the 96 hour static exposure of sheepshead minnow (Cyprinodon variegatus) to the test substance

Mean measured concentration (mg/L)	Cumulative mortality (%)
	24-hour	48-hour	72-hour	96-hour
Control	0	0	0	0
Solvent control	0	0	0	0
0.88	0	0	0	0
1.9	0	0	0^a	0^b
2.8	0^cd	0^c	10^a	10^b
4.6	0^be	0^f	10^fj	40^a
9.1	0^gh	0^i	60^i	90^i

a All of the surviving fish were lethargic.
b 20 - 50% of the surviving fish were lethargic.
c One of the surviving fish was lethargic.
d One of the surviving fish exhibited a complete loss of equilibrium.
e One of the surviving fish exhibited a partial loss of equilibrium.
f 20 - 50% of the surviving fish exhibited a partial loss of equilibrium and a darkened pigmentation.
g > 50% of the surviving fish exhibited a complete loss of equilibrium.
h One of the surviving fish was at the surface of the test solution.
i All of the surviving fish exhibited a complete loss of equilibrium.
j > 50% of the surviving fish were lethargic.

Validity criteria fulfilled:

yes

Remarks:

See Validation criteria in "Any other information on materials and method incl. tables"

Conclusions:

Based on the findings, the 96-hour NOEC was determined to be 0.88 mg/L and the LC50 value was determined to be 5.1 mg/L (corresponding 95% confidence intervals of 4.0 to 7.0 mg/L).

Executive summary:

The acute toxicity of the test substance to sheepshead minnow (Cyprinodon variegatus) was studied in a 96-hour static test. The test was conducted according to the EPA test guideline No. 72 - 3 and the “Standard Practice for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians” (ASTM 1980). The study was in compliance with GLP. The test organisms were acclimated in 500 L fiberglass tanks at 20 – 21 °C with a closed loop recirculating filtration system (5 µm porosity polypropylene filter plus activated carbon filter) providing natural seawater with a salinity range of 32 - 35‰ for 14 days. There was no mortality of the test fish population during the 48 hours prior to test initiation. Twenty fish per concentration (ten per vessel containing 15 L test medium) were exposed to the acetone dissolved compound at the mean measured concentrations of 0.88, 1.9, 2.8, 4.6 and 9.1 mg/L (measured by HPLC; the nominal concentrations were 1.3, 2.2, 3.6, 6.0 and 10 mg/L, respectively). There were seawater control and carrier control (0.1 mL acetone/L) groups in the test as well. The test was carried out under the following conditions: salinity 32-33‰, pH 7.5-7.9 and temperature 22 ± 1 °C. At 48 hours, the dissolved oxygen concentration in the control solution was 58% of saturation and ranged from 65 – 69% in the test solutions. Therefore, aeration was initiated at 48 hours to both control solutions and all test solutions. Throughout the remainder of the 96-hour test, the dissolved oxygen concentration in all solutions ranged from 93 to 107% of saturation. The photoperiod regime was 16 hours light (25 footcandles) and 8 hours darkness.

After 96 hours of exposure, all test fish survived in the controls (both blank seawater and solvent control) and in the 0.88 mg/L treatment group. No mortality was observed also in the 1.9 mg/L treatment group, but 20 - 50% of the surviving fish were lethargic. The same fraction of lethargic surviving fish was observed in the 2.8 mg/L treatment group, which also had a 10% mortality rate after 96 hours of exposure. Percentage of mortality raised to 40% in the 4.6 mg/L treatment group and 90% in the 9.1 mg/L treatment group at the end of the test period. In addition, all surviving fish at 4.6 mg/L were lethargic and all surviving fish in the highest exposure group (9.1 mg/L) exhibited a complete loss of equilibrium. Based on the findings, the 96 hour LC50 was determined to be 5.1 mg/L with 95% confidence limits of 4.0 to 7.0 mg/L.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 Apr 1985 to 14 Apr 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

not GLP compliant

Qualifier:

according to guideline

Guideline:

other: ASTM 1980: Standard Practice for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

Water samples from each treatment, carrier control, and control, taken on April 10, 1985 (0 hour) and were analyzed by gas chromatograph.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

After the test concentrations were delineated via the range-finding test, a 80,000 mg/L stock solution is prepared. The stock solution was prepared by adding 2 g of the test substance into 25ml acetone.
Considering the purity of the test substance (97%), the correction factor for preparing the test concentrations was calculated as 1.03. Thus, to obtain 20L of each test concentration, different amount of 80,000 mg/L solution was used.
- 10 mg/L test concentration: 2.58 mL 80,000 mg/L solution
- 5 mg/L test concentration: 1.29 mL 80,000 mg/L solution
- 2.5 mg/L test concentration: 0.64 mL 80,000 mg/L solution
- 1.25 mg/L test concentration: 0.32 mL 80,000 mg/L solution
- 0.625 mg/L test concentration: 0.16 mL 80,000 mg/L solution
Each of the five test solutions was then divided into the duplicate containers. A 500 mg/L acetone solution was used as carrier control in the test.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Obtained from a trout farm, at 11 °C, on 29 March 1985
- Age at study initiation: Approximately 30 days post-hatch
- Length at study initiation: Approximately 3 cm
- Weight at study initiation: Approximately 1.5 g
- Holding and acclimation: After transportation to the laboratory, fish are quarantined for 10 days for observation. They are then transferred to acclimation tanks. Temperature is raised or lowered 1 °C a day till the desired test temperature is reached. This also applies to fish that have been held in the lab. They are acclimated to the test temperature and dilution water for 2 days before the test is initiated.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

20 - 36 mg/L as CaCO3

Test temperature:

11.8 - 13.2 °C

pH:

7.5

Dissolved oxygen:

- 6.1 - 9.0 mg O2/L
- > 60% of saturation

Nominal and measured concentrations:

Nominal concentration: control, carrier control, 0.625, 1.25, 2.5, 5 and 10 mg/L
Measured concentration: 0.0, 0.0, 0.68, 1.54, 2.55, 4.77 and 8.16 mg/L, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: 36 L glass aquaria (25 cm width x 50 cm length x 30 cm height)
- Vessel fill volume: 20 L test solution
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 2
- Photoperiod: 16 hours/8 hours (light/dark)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Domestic water was used for dilutions and controls in the test. The Fort Collins domestic water supply of dechlorinated water (approximately 28 ppm hardness as CaCO3) was filtered through an in - house battery of five automatic backflushing dechlorination filters. No additional treatment was required.
- Intervals of water quality measurement: Temperature and DO are measured once daily. Alkalinity, pH, hardness , and conductivity were measured at 0 and 96 hours in one replicate from the control groups, low, medium, and high concentration test vessels.

EFFECT PARAMETERS MEASURED: Mortality and toxicity effect criteria were recorded at 1, 4, 24, 48, 72, 96 hours of exposure. Criteria of effect are immobilization, lack of gill movements, and response to gentle prodding.

TEST CONCENTRATIONS
- Range finding study : Nominal test concentrations for the static range-finding test were 20.0, 2.0, and 0.2 mg/L the test substance. Five rainbow trout were introduced into the three glass test aquaria, one carrier control , and one control aquarium containing 20L of test solution and dilution water. Test containers were held in a water bath maintained at 12 °± 1°C.
- Results used to determine the conditions for the definitive study: Forty-eight hours after introduction into the test chambers, all fish died in the 20 mg/L treatment. No other mortality or effect was noted in any other test group or control. The 48-h LC50 for this range - finding test was calculated to be 6.325 mg/L with 95% confidence limits of 5.536 and 7.225 mg/L.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

5.46 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

95% C.L.: 5.183 - 5.771 mg/L

Details on results:

Overview of the results are provided in the "Any other information on results incl. tables".
After 1 hour of exposure to 8.16 mg/L the test substance all test fish were observed as very darkly pigmented, however, no mortality was observed. At 24 hours of exposure to 8.16 mg/L the test substance, 13 fish were dead and 2 were moribund. At 24 hours, all fish in 4.77 mg/L the test substance were darkly pigmented with 1 fish moribund. By 48 hours of exposure to 8.16 mg/L the test substance, 18 fish were dead. At 48 hours 1 fish was dead in 4.77 mg/L the test substance with the remaining fish being observed as very black (dark). At 72 hours of exposure to 8.16 mg/L the test substance all test fish were dead. At 72 and 96 hours of exposure to 4.77 mg/L 2 and 3 fish were dead, respectively (cumulative total).
The 96 hour LC50 for the acute definitive toxicity test was calculated to be 5.460 mg/L with 95 percent confidence limits of 5.183 and 5.771 mg/L by the moving average method. The binomial test provides an estimate of the LC50 (at 95% confidence) to be between 4.77 and 8.16 mg/L the test substance.

Sublethal observations / clinical signs:

Table 1 Mortality, dissolved oxygen and temperature during the definitive static acute toxicity test

Nominal concentration (mg/L)	Measured concentration (mg/L)	Number of fish dead at 96 hours	Mean dissolved oxygen (mg/L)	Mean temperature (°C)
0.0 (Control)	0.0	0	7.9	12.1
0.0 (Carrier control)	0.0	0	7.7	12.5
0.625	0.68	0	7.8	12.4
1.25	1.54	0	7.9	12.3
2.50	2.55	0	7.6	12.5
5.0	4.77	3	7.1	12.6
10	8.19	20	7.3	12.4

Validity criteria fulfilled:

yes

Remarks:

See validation criteria in "Any other information on material and method incl. tables"

Conclusions:

The 96-hour LC50 value was determined to be 5.460 mg/L (corresponding 95% confidence intervals of 5.183 to 5.771 mg/L).

Executive summary:

The acute toxicity of the test substance to rainbow trout (Oncorhynchus mykiss) was studied in a 96-hour static test. The test was conducted according to “Standard Practice for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians” (ASTM 1980) and without compliance to GLP. The study followed the principles of the guideline strictly and the test procedures and results are well documented, so that the study is considered as reliable (with restrictions) and fully valid for the hazard and risk characterisation. The test organisms were acclimated at the test temperature (12 ± 1 °C) in dilution water (dechlorinated tap water, 28 ppm hardness as CaCO3) for 2 days before the exposure started. The fish were exposed to the acetone-dissolved compound at the mean measured concentrations of 0.68, 1.54, 2.55, 4.77 and 8.16 mg/L (measured by gas chromatography; the nominal concentrations were 0.625, 1.25, 2.50, 5.0 and 10 mg/L, respectively). There were negative control and carrier control groups in the test as well.Two replicate test chambers each comprising 10 fish in 20 L test medium were set up for each test group. The test was carried out under the following conditions: pH 7.5 (control and 8.16 mg/L treatment), dissolved oxygen concentration 6.1 – 9.0 mg/L (>60% of saturation) and, 16-hour light and 8-hour dark photoperiod. 

 

After 1 hour of exposure to 8.16 mg/L of the test substance all test fish appeared very darkly pigmented, however, no mortality was observed. At 24 hours of exposure to 8.16 mg/L, 13 fish were dead and 2 were moribund. The number of dead fish in this dose group increased to 18 after 48 hours, and all fish in this group were dead after 72 hours of exposure. All fish exposed to 4.77 mg/L of the test substance were darkly pigmented after 24 hours and one fish was moribund. After 48 hours of exposure to 4.77 mg/L, one fish was dead and the remaining fish appeared very black (dark). The number of dead fish in this dose group increased to two and three after 72 and 96 hours of exposure, respectively (cumulative total). No adverse effects on test fish were observed in the other dose groups. Based on these findings, the 96-hour LC50 was calculated to be 5.460 mg/L with 95 percent confidence limits of 5.183 and 5.771 mg/L by the moving average method.

Description of key information

All available data were assessed and the studies representing the worst-case effects are included here as key. The results can be considered worst-case and are selected for the CSA.

Freshwater, 96-h LC50 = 5.460 mg/L (corresponding 95% confidence intervals of 5.183 to 5.771 mg/L), Salmo gairdneri, mortality, No guideline followed, Hamaker 1985

Saltwater, 96-h LC50 = 5.1 mg/L (corresponding 95% confidence intervals of 4.0 to 7.0 mg/L), Cyprinodon variegatus, mortality, EPA FIFRA TG 72 - 3, Surprenant 1988

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

5.46 mg/L

Marine water fish

Marine water fish

Dose descriptor:

LC50

Effect concentration:

5.1 mg/L

Additional information

Fresh water

The acute toxicity of the test substance to rainbow trout (Oncorhynchus mykiss) was studied in a 96-hour static test. The test was conducted according to “Standard Practice for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians” (ASTM 1980) and without compliance to GLP. The study followed the principles of the guideline strictly and the test procedures and results are well documented, so that the study is considered as reliable (with restrictions) and fully valid for the hazard and risk characterisation. The test organisms were acclimated at the test temperature (12 ± 1 °C) in dilution water (dechlorinated tap water, 28 ppm hardness as CaCO3) for 2 days before the exposure started. The fish were exposed to the acetone-dissolved compound at the mean measured concentrations of 0.68, 1.54, 2.55, 4.77 and 8.16 mg/L (measured by gas chromatography; the nominal concentrations were 0.625, 1.25, 2.50, 5.0 and 10 mg/L, respectively). There were negative control and carrier control groups in the test as well. Two replicate test chambers each comprising 10 fish in 20 L test medium were set up for each test group. The test was carried out under the following conditions: pH 7.5 (control and 8.16 mg/L treatment), dissolved oxygen concentration 6.1 – 9.0 mg/L (>60% of saturation) and, 16-hour light and 8-hour dark photoperiod. After 1 hour of exposure to 8.16 mg/L of the test substance all test fish appeared very darkly pigmented, however, no mortality was observed. At 24 hours of exposure to 8.16 mg/L, 13 fish were dead and 2 were moribund. The number of dead fish in this dose group increased to 18 after 48 hours, and all fish in this group were dead after 72 hours of exposure. All fish exposed to 4.77 mg/L of the test substance were darkly pigmented after 24 hours and one fish was moribund. After 48 hours of exposure to 4.77 mg/L, one fish was dead and the remaining fish appeared very black (dark). The number of dead fish in this dose group increased to two and three after 72 and 96 hours of exposure, respectively (cumulative total). No adverse effects on test fish were observed in the other dose groups. Based on these findings, the 96-hour LC50 was calculated to be 5.460 mg/L with 95 percent confidence limits of 5.183 and 5.771 mg/L by the moving average method.

Marine water

The acute toxicity of the test substance to sheepshead minnow (Cyprinodon variegatus) was studied in a 96-hour static test. The test was conducted according to the EPA test guideline No. 72 - 3 and the “Standard Practice for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians” (ASTM 1980). The study was in compliance with GLP. The test organisms were acclimated in 500 L fiberglass tanks at 20 – 21 °C with a closed loop recirculating filtration system (5 µm porosity polypropylene filter plus activated carbon filter) providing natural seawater with a salinity range of 32 - 35‰ for 14 days. There was no mortality of the test fish population during the 48 hours prior to test initiation. Twenty fish per concentration (ten per vessel containing 15 L test medium) were exposed to the acetone dissolved compound at the mean measured concentrations of 0.88, 1.9, 2.8, 4.6 and 9.1 mg/L (measured by HPLC; the nominal concentrations were 1.3, 2.2, 3.6, 6.0 and 10 mg/L, respectively). There were seawater control and carrier control (0.1 mL acetone/L) groups in the test as well. The test was carried out under the following conditions: salinity 32-33‰, pH 7.5-7.9 and temperature 22 ± 1 °C. At 48 hours, the dissolved oxygen concentration in the control solution was 58% of saturation and ranged from 65 – 69% in the test solutions. Therefore, aeration was initiated at 48 hours to both control solutions and all test solutions. Throughout the remainder of the 96-hour test, the dissolved oxygen concentration in all solutions ranged from 93 to 107% of saturation. The photoperiod regime was 16 hours light (25 footcandles) and 8 hours darkness. After 96 hours of exposure, all test fish survived in the controls (both blank seawater and solvent control) and in the 0.88 mg/L treatment group. No mortality was observed also in the 1.9 mg/L treatment group, but 20 - 50% of the surviving fish were lethargic. The same fraction of lethargic surviving fish was observed in the 2.8 mg/L treatment group, which also had a 10% mortality rate after 96 hours of exposure. Percentage of mortality raised to 40% in the 4.6 mg/L treatment group and 90% in the 9.1 mg/L treatment group at the end of the test period. In addition, all surviving fish at 4.6 mg/L were lethargic and all surviving fish in the highest exposure group (9.1 mg/L) exhibited a complete loss of equilibrium. Based on the findings, the 96 hour LC50 was determined to be 5.1 mg/L with 95% confidence limits of 4.0 to 7.0 mg/L.