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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 March - 8 June 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline
Qualifier:
according to
Guideline:
EPA OPP 72-1 (Fish Acute Toxicity Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
not specified
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
yes
Details on sampling:
Analytical determination of the test substance active ingredient (2-methyl-4-isothiazolin-3-one) concentrations were performed with 40 mL samples collected from each replicate test vessel of each concentration at the beginning and end of the test. Secondary stock solution samples (prepared by the diluter at the highest tested concentration of 2-methyl-4-isothiazolin-3-one) were also collected at these times. Samples were collected into 40-mL amber glass vials. The 0 and 96 hour samples were accompanied by 2 matrix spike samples prepared in water from a control vessel, a laboratory control spike sample prepared in dilution water, and a dilution water blank.
Vehicle:
no
Details on test solutions:
The test was conducted at a temperature of 12 +/- 1C with five concentrations of test substance and a dilution water control. Stock solutions with a nominal concentration of 10,000 mg a.i./L were prepared daily during the test by bringing 10.6028 to 10.6060 g of test substance to a total volume of 1,000 mL with deionized water. Appropriate amounts of the stock solution were added directly to dilution water by a proportional diluter (3.0 mL added to 3,000 mL of dilution water to yield a secondary stock solution with a nominal concentration of 10 mg a.i./L, based on the initial diluter calibration). Nominal concentrations of 2-methyl-4-isothiazolin-3-one, Technical were: 0 mg/L (control), 1.3, 2.2, 3.6, 6.0, and 10 mg a.i./L, based on the initial diluter calibration.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Juvenile rainbow trout, Oncorhynchus mykiss, used as test organisms were from a single source and identified using an appropriate taxonomic key. They were procured from a commercial supplier (Thomas Fish Company, Anderson, California) on March 8, 2001. Prior to testing, the fish were maintained under flow-through conditions in a 280 liter fiberglass tank.

During acclimation, fish were not treated for disease and they were free of apparent disease, injuries, and abnormalities at the beginning of the test. Fish were fed dry commercial fish food (TetraMin Staple Food, Lot # TM09 and trout chow, Lot # TC03) once per day except that they were not fed during the 48-hour period immediately preceding the toxicity test or during the toxicity test. During the 14 day period before the start of the test, the acclimation temperature range was 11.6 to 12.8C, the pH ranged from 7.2 to 8.0, and the dissolved oxygen concentration was always at least 9.0 mg/L. Mortality during the last 48 hours of acclimation was 0%. At the conclusion of the test, the control fish had an average total length of 52.0 mm and an average wet weight of 1.3 g (loading rate was approximately 0.87 g/L at any one time and 0.12 g/L/24 hours).
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
No data
Hardness:
Water used for the acclimation of test organisms and for all toxicity testing was carbon filtered, deionized water collected at T.R. Wilbury Laboratories in Marblehead, Massachusetts. Water was adjusted to a hardness of 40 to 48 mg/L as CaCO3 . A sample of dilution water collected at the start of the definitive toxicity test had an hardness of 44 mg/L as CaCO,.
Test temperature:
Temperature ranged from 11.1 to 12.7C (mean = 11.9C)
pH:
pH ranged from 7.5 to 7.9,, and the
Dissolved oxygen:
dissolved oxygen concentration ranged from 9.8 to 10.6 mg/L (mean = 10.1 mg/L).
Salinity:
No data.
Nominal and measured concentrations:
Nominal concentrations of 2-methyl-4-isothiazolin-3-one, Technical were: 0 mg/L (control), 1.3, 2.2, 3.6, 6.0, and 10 mg a.i./L.

Mean measured concentrations of 2-methyl-4-isothiazolin-3-one were: ND (not detected at or above the limit of quantitation of 0.00561 mg a.i./L, control), 1.06, 2.01, 3.46, 5.67, and 9.70 mg a.i./L. Mean measured concentrations of 2-methyl-4-isothiazolin-3-one ranged from 82 to 97% of nominal concentrations
Details on test conditions:
A range-finding test was conducted under static conditions from March 27 to 31, 2001. The test was performed with a dilution water control and five nominal concentrations of 2 methyl-4-isothiazolin-3-one, Technical (0.094, 0.94, 4.7, 9.4, and 94 mg a.i./L). After 96 hours, there was 100% survival in the control and at 0.094, 0.94, and 4.7 mg a.i./L, and 0% survival at 9.4 and 94 mg a.i./L. No sublethal effects were observed at the end of the test and no insoluble material was observed during the test.

The definitive toxicity test was performed from June 4 to 8, 2001. The test was conducted at a temperature of 12 +/- 1 C with five concentrations of test substance and a dilution water control. Stock solutions with a nominal concentration of 10,000 mg a.i./L were prepared daily during the test by bringing 10.6028 to 10.6060 g of test substance to a total volume of 1,000 mL with deionized water. Appropriate amounts of the stock solution were added directly to dilution water by a proportional diluter (3.0 mL added to 3,000 mL of dilution water to yield a secondary stock solution with a nominal concentration of 10 mg a.i./L, based on the initial diluter calibration). Nominal concentrations of 2-methyl-4-isothiazolin-3-one, Technical were: 0 mg/L (control), 1.3, 2.2, 3.6, 6.0, and 10 mg a.i./L, based on the initial diluter calibration.

Ten rainbow trout were randomly distributed among two randomly positioned replicates of each treatment. The test was performed in 20 liter glass aquaria that contained 15 liters of test solution (water depth was approximately 18 cm). Test vessels were arranged in a water bath during the test (random numbers tables were used to assign concentrations to test vessels and to select the location of each vessel and the assignment of test organisms).

The test substance was supplied to the test vessels under flow-through conditions by an intermittent flow proportional diluter. The diluter, which was constructed at T.R. Wilbury Laboratories, allowed test substance to contact only glass or ~ef lon@sur faces. The diluter was calibrated before and after the test and monitored for normal operation twice per day. The diluter was in operation for approximately 67 hours prior to the start of the definitive toxicity test. During the test the diluter was activated 838 times, resulting in an average of 7.0 volume additions per 24 hours in each test vessel.

A 16 hour light and 8 hour dark photoperiod was automatically maintained with cool-white fluorescent lights that provided a light intensity of approximately 39 foot-candles. A 15 minute transition period was provided between dark and light. Aeration was not required to maintain dissolved oxygen concentrations above acceptable levels. The number of surviving organisms and the occurrence of sublethal effects were determined visually and recorded initially and after 24, 48, 72, and 96 hours. Dissolved oxygen (YSI Model 57 meter; instrument number I), pH (Beckman model 320 meter; instrument numbers 202), conductivity (Cole-Parmer conductivity meter; instrument number 94), and temperature (Beckman model 320 meter; instrument number 202 probe pHT-15) were measured and recorded daily in each test chamber. Observations of test substance insolubility were recorded initially and after 24, 48, 72, and 96 hours. The temperature in a control vessel was recorded continuously.
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
4.77 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 4.21 to 5.42 mg/L
Details on results:
No cloudiness or insoluble material was observed in any test vessel during the definitive test. Nominal concentrations of 2-methyl-4-isothiazolin-3-one, Technical were: 0 mg/L (control), 1.3, 2.2, 3.6, 6.0, and 10 mg a.i./L. Mean measured concentrations of 2-methyl-4-isothiazolin-3-one were: ND (not detected at or above the limit of quantitation of 0.00561 mg a.i./L, control), 1.06, 2.01, 3.46, 5.67, and 9.70 mg a.i./L. Mean measured concentrations of 2-methyl-4-isothiazolin-3-one ranged from 82 to 97% of nominal concentrations
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
Exposure of rainbow trout, Oncorhynchus mykiss, to 2-methyl-4-isothiazolin-3-one, Technical resulted in a 96-hour LC50 of 4.77 mg a.i./L, with a 95% confidence interval of 4.21 to 5.42 mg a.i./L (Table 1). The 96-hour NOEC for 2-methyl-4-isothiazolin-3-one is 2.01 mg a.i./L and the slope of the concentration response curve is 9.4.

Table 1. Median lethal concentrations (LC5Os) from the toxicity test with 2-methyl-4- isothiazolin-3-one, Technical and the rainbow trout, Oncorhynchus mykiss.

 Exposure Period  LC501 (mg a.i./L)  95 percent confidence limit  LC50 calculation method
 24 hrs  6.54  5.71 - 7.54  probit
 48 hrs  5.51  4.85 - 6.30  probit
 72 hrs  5.00  4.39 - 5.73  probit
 96 hrs  4.77  4.21 - 5.42  probit
Validity criteria fulfilled:
yes
Conclusions:
Exposure of the rainbow trout, Oncorhynchus mykiss, to 2-methyl-4-isothiazolin-3-one.Technical resulted in a 96-hour median lethal concentration (LC50) of 4.77 mg a.i./L, with a 95% confidence interval of 4.21 to 5.42 mg a.i./L. The 96-hour no-observed effect concentration (NOEC) for 2-methyl-4-isothiazolin-3-one was 2.01 mg a.i./L and the slope of the concentration response curve is 9.4.
Executive summary:

The acute toxicity of 2-methyl-4-isothiazolin-3-one, Technical to the rainbow trout, Oncorhynchus mykiss, is described in this report). The test substance, 2-methyl-4-isothiazolin-3-one, Technical, was supplied by the sponsor and is synonymous with RH-573 and RH-57,399 Technical. The sample was identified as Lot No. B- 1001, TD No. 01-043, with a % a.i. of 94.3%.

The test was performed under flow-through conditions with five concentrations of test substance and a dilution water control at a mean temperature of 11.9°C. The dilution water was filtered deionized water collected at Marblehead, Massachusetts and adjusted to a hardness of 40 to 48 mg/L as CaCO3- No cloudiness or insoluble material was observed in any test vessel during the definitive test. All water quality parameters (dissolved oxygen concentration, temperature, pH, and conductivity) were within acceptable limits.

Based on the initial diluter calibration, nominal concentrations of 2-methyl-4-isothiazolin-3-one, Technical were: 0 mg/L (control), 1.3, 2.2, 3.6, 6.0, and 10 mg a.i./L. Mean measured concentrations of 2-methyl-4-isothiazolin-3- one were: ND (not detected at or above the limit of quantitation of 0.00561 mg a.i./L, control), 1.06, 2.01, 3.46, 5.67, 9.70 mg a.i./L. Mean measured concentrations of 2-methyl-4-isothiazolin-3-one ranged from 82 to 97% of nominal concentrations.

Exposure of the rainbow trout, Oncorhynchus mykiss, to 2-methyl-4-isothiazolin-3-one.Technical resulted in a 96-hour median lethal concentration (LC50) of 4.77 mg a.i./L, with a 95% confidence interval of 4.21 to 5.42 mg a.i./L. The 96-hour no-observed effect concentration (NOEC) for 2-methyl-4-isothiazolin-3-one was 2.01 mg a.i. /L and the slope of the concentration response curve is 9.4.

Description of key information

The results of the key study indicated that exposure of the rainbow trout, Oncorhynchus mykiss, to 2-methyl-4-isothiazolin-3-one.Technical resulted in a 96-hour median lethal concentration (LC50) of 4.77 mg a.i./L, with a 95% confidence interval of 4.21 to 5.42 mg a.i./L.

Key value for chemical safety assessment

LC50 for freshwater fish:
4.77 mg/L

Additional information