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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 March - 16 May 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Qualifier:
according to
Guideline:
EPA OPP 72-2 (Aquatic Invertebrate Acute Toxicity Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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 fiom 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 48 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 20 + 1°C with five concentrations of test substance and a dilution water control. Stock solutions with a nominal concentration of 2,600 mg/L were prepared daily during the test by bringing 2.7571 to 2.7573 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 2.6 mg/L ). Nominal concentrations of 2-methyl-4-isothiazolin-3-one, Technical were: 0 mg/L (control), 0.34, 0.57, 0.94, 1.6, and 2.6 mg/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
Juvenile daphnids, Daphnia magna, less than 24-hours old, employed as test organisms were from a single source and were identified using an appropriate taxonomic key. They were produced from an in-house culture by adult daphnids that were maintained under test conditions at T.R. Wilbury Laboratories. The original culture was obtained from Aquatic Biosystems Inc., Fort Collins, Colorado, on April 10, 2001. At the conclusion of the test the control daphnids had an average wet weight of 0.15 mg.

Prior to testing, the daphnid culture was maintained in 100% dilution water under static, renewal conditions, and the test organisms were not treated for disease. No mortality was observed during the 48 hours prior to the test and at the beginning of the test organisms were apparently free of disease, injuries, and abnormalities. The culture produced young by day 9.

During the 21 days prior to the beginning of the test, the culture was maintained under static, renewal conditions in a 3.8-L glass vessel containing dilution water. The temperature of the culture ranged from 19.2 to 21.0°C, the pH ranged from 7.1 to 8.1, and the dissolved oxygen concentration was always at least 7.9 mg/L. Test organisms were fed the freshwater algae, Selenastrum capricornutum, and a mixture of yeast and trout chow (Lot number DC03) once each day before the test. Daphnids were not fed during the test.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 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 160 to 180 mg/L as CaC03. Water was stored in polyethylene tanks where it was aerated and recirculated through particle filters, activated carbon, and an ultraviolet sterilizer. A sample of dilution water collected at the start of the definitive toxicity test had an hardness of 168 mg/L as CaC03.
Test temperature:
temperature ranged from 19.0 to 21.0°C (mean = 20. 1°C)
pH:
the pH ranged from 7.2 to 7.7, the , and the
Dissolved oxygen:
dissolved oxygen concentration ranged from 8.9 to 9.2 mg/L (mean = 9.1 mg/L).
Salinity:
No data.
Nominal and measured concentrations:
Nominal concentrations of 2-methyl-4-isothiazolin-3-one, Technical were: 0 mg/L (control), 0.34, 0.57, 0.94, 1.6, and 2.6 mg/L.
Details on test conditions:
A range-finding test was conducted under static conditions from March 28 to 30, 2001. The test was performed with a dilution water control and five nominal concentrations of 2-methyl-4-isothiazolin-3 -one, Technical (0.10, 1.0, 5.0, 10, and 100 mg/L) . After 48 hours, there was 100% survival in the control and at 0.10 mg/L, 80% survival at 1.0 mg/L, and 0% survival at 5.0, 10, and 100 mg/L. No sublethal effects were observed during the test.
No insoluble material was observed during the test.

The definitive toxicity test was performed from May 14 to 16, 2001. The test was conducted at a temperature of 20 +/- 1°C with five concentrations of test substance and a dilution water control. Stock solutions with a nominal concentration of 2,600 mg/L were prepared daily during the test by bringing 2.7571 to 2.7573 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 2.6 mg/L). Nominal concentrations of 2-methyl-4-isothiazolin-3-one, Technical were: 0 mg/L (control), 0.34, 0.57, 0.94, 1.6, and 2.6 mg/L.

Ten daphnids 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 19 cm). The loading rate was <1 daphnid per liter. Daphnids were exposed in glass cylinders (typically 8 cm in height x 8 cm in diameter) with Nitex screen (typically 350 um mesh) attached to the bottom with silicone adhesive. These exposure chambers were suspended within each test vessel. Test vessels were arranged in a water bath during the 48-hour test (random numbers tables were used to select the location of each vessel, as well as 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 Teflon su rfaces. The diluter was calibrated before and after the test and monitored for normal operation twice per day. The diluter was in operation for approximately 73 hours prior to the start of the definitive toxicity test. During the test the diluter was activated 320 times, resulting in an average of 5.3 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 41 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 and 48 hours. Dissolved oxygen (YSI Model 57 meter; instrument number l), 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 and 48 hours. The temperature in a control vessel of water was recorded continuously.
Reference substance (positive control):
not specified
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
0.934 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 0.765 to 1.16 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), 0.34, 0.57, 0.94, 1.6, and 2.6 mg/L. Mean measured concentrations of 2-methyl-4-isothiazolin-3-one were: ND (not detected at or above the limit of quantitation of 0.00134 mg/L, control), 0.275, 0.524, 0.865, 1.44, and 2.39 mg/L. Mean measured concentrations of 2-methyl-4-isothiazolin-3-one ranged from 81 to 92% of nominal concentrations.

Ninety-five percent survival occurred in the control with no immobilization or other sublethal effects observed. During the definitive toxicity test, the conductivity ranged from 450 to 560 umhos/cm (mean = 510 umhos/cm), the pH ranged from 7.2 to 7.7, the temperature ranged from 19.0 to 21.0°C (mean = 20. 1°C), and the dissolved oxygen concentration ranged from 8.9 to 9.2 mg/L (mean = 9.1 mg/L).
Results with reference substance (positive control):
No data.
Reported statistics and error estimates:
Exposure of the daphnid, Daphnia magna, to 2-methyl-4-isothiazolin-3-one, Technical resulted in a 48-hour LC50 of 0.934 mg/L, with a 95% confidence interval of 0.765 to 1.16 mg/L, and a 48-hour EC50 of 0.850 mg/L with a 95% confidence interval of 0.681 to 1.06 mg/L (Table 1). The 48-hour NOEC is <0.275 mg/L 2-methyl-4-isothiazolin-3-one, the lowest tested concentration.

Table 1. Median effective concentrations (EC50s) and median lethal concentrations (LC50s) from the toxicity test with 2-methyl-4-isothiazolin-3-one, Technical and the daphnid, Daphnia magna.

 Exposure Period  Endpoint1  95 percent confidence limit  LC50/EC50
 EC50         
 24 hours  >2.39 mg/L  --  --
 48 hours  0.850 mg/L  0.681 - 1.06  moving average
 LC50         
 24 hours  >2.39 mg/L  --  --
 48 hours  0.934 mg/L  0.765 - 1.16  moving average

1Based on mean measured concentrations of 2-methyl-4-isothiazolin-3-one.

Validity criteria fulfilled:
yes
Conclusions:
Exposure of the daphnid, Daphnia magna, to 2-methyl-4-isothiazolin-3-one, Technical resulted in a 48-hour median lethal concentration (LC50) of 0.934 mg/L, with a 95% confidence interval of 0.765 to 1.16 mg/L, and a 48-hour median effective concentration (EC50) of 0.850 mg/L, with a 95% confidence interval of 0.681 to 1.06 mg/L. The 48-hour no-observed effect concentration (NOEC) was <0.275 mg/L 2-methyl-4-isothiazolin-3-one, the lowest tested concentration.
Executive summary:

The acute toxicity of 2-methyl-4-isothiazolin-3-one, Technical to the daphnid, Daphnia magna is described in this report. The test was conducted for Rohm and Haas Company for 48 hours from May 14 to 16, 2001, at T.R. Wilbury Laboratories, Inc., in Marblehead, Massachusetts. 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 reported % a.i. of 94.3%. Nominal test concentrations were corrected for the percent purity.

The test was performed under flow-through conditions with five concentrations of test substance and a dilution water control at a mean temperature of 20.1°C. The dilution water was filtered deionized water collected at Marblehead, Massachusetts and adjusted to a hardness of 160 to 180 mg/L as CaC03 and a pH <8.0. Water quality measurements were taken throughout the definitive toxicity test and were within acceptable limits. During the definitive toxicity test, the conductivity ranged from 450 to 560 p μmhos/cm (mean = 510 μmhos/cm), the pH ranged from 7.2 to 7.7, the temperature ranged from 19.0 to 21.0°C (mean = 20.1°C), and the dissolved oxygen concentration ranged from 8.9 to 9.2 mg/L (mean = 9.1 mg/L).

Nominal concentrations of 2-methyl-4-isothiazolin-3-one, Technical were: 0 mg/L (control), 0.34, 0.57, 0.94, 1.6, and 2.6 mg/L. Mean measured concentrations of 2-methyl-4-isothiazolin-3-one were: ND (not detected at or above the limit of quantitation of 0.00134 mg/L, control), 0.275, 0.524, 0.865, 1.44, and 2.39 mg/L. Mean measured concentrations of 2-methyl-4-isothiazolin-3-one ranged from 81 to 92% of nominal concentrations.

Exposure of the daphnid, Daphnia magna, to 2-methyl-4-isothiazolin-3-one, Technical resulted in a 48-hour median lethal concentration (LC50) of 0.934 mg/L, with a 95% confidence interval of 0.765 to 1.16 mg/L, and a 48-hour median effective concentration (EC50) of 0.850 mg/L, with a 95% confidence interval of 0.681 to 1.06 mg/L. The 48-hour no-observed effect concentration (NOEC) was <0.275 mg/L 2-methyl-4-isothiazolin-3-one, the lowest tested concentration.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 April - 18 May 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 850.1035 (Mysid 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:
Test solution samples were collected from all control and treatment levels and diluter stock during system equilibration (day-N), test initiation (0 hour), and test termination (96 hours). At each sample point, 10 ml composite samples (5.0 ml from each replicate) were collected from each treatment. Quality control fortifications of dilution water were prepared at this time. Any dilutions were made with ABC reagent water. The samples were then analyzed by HPLC.
Vehicle:
no
Details on test solutions:
Test material was used as is and diluted with sea water with a syringe injector.
Test organisms (species):
Americamysis bahia (previous name: Mysidopsis bahia)
Details on test organisms:
Mysid shrimp (Americamysis bahia) used for testing were obtained from an in-house culture. Post-larvae (<24 hours old) collected from the culture on 14 May 2004, were used for test initiation. The culture was maintained in laboratory saltwater (~20%) at approximately 25C. During culture, the mysids were fed live brine shrimp (Artemia salina) nauplii and shrimp food once a day.
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
No data
Hardness:
ranged between 130 and 160 mg CaCO3/l.
Test temperature:
Test solution temperature ranged from 23.4 to 24.7C. The continuous temperature recording indicated a periodic average temperature of 25.2 +/- 0.0C. ranging from 25.0 to 25.4C.
pH:
8.1 to 8.2 in all solutions during the test.
Dissolved oxygen:
ranged from 7.3 to 7.6 mg/l (100 to 104% saturation) at test initiation and remained >/=7.0 mg/l (99% saturation) throughout the test.
Salinity:
ranged from 21.2 to 21.3%.
Nominal and measured concentrations:
Nominal concentrations of 0 (control), 0.10, 1.0, 10, 100, and 1000 mg a.i./l. Analytical concentrations at 0 hr were
Details on test conditions:
A 96-hour static range-finding test was conducted from April 26 to 30, 2004 at nominal concentrations of 0 (Control), 0.10, 1.0, 10, 100 and 1000 mg a.i./l. After 96 hours, mortality was 0, 0, 10, 100, 100, and 100% in the 0 (Control), 0.10, 1.0, 10, 100, and 1000 mg a.i./l treatments. Based on the results fo this range-finding test, nominal concentrations selected for the definitive exposure were of 0 (control), 0.63, 1.3, 2.5, 5.0 and 10 mg a.i./l.

The definitive test was conducted from May 14 to 18, 2004. An intermittent-flow proportional diluter system similar to that described by Mount and Brungs (2), with a Hamilton Model 420 syringe injector, was used for the intermittent introduction of an aqueous solution of 2-methyl-4-isothiazolin-3-one and control dilution water to exposure chambers. The diluter system provided five test substance treatment levels with a 50% dilution factor between treatments and delivered approximately 250 ml to each test chamber with each cycle. Diluter stock solutions were prepared in deionized water at a target concentration of 1940 mg a.i./l by mixing approximately 1.917 g (0.983 g active ingredient) of test substance into 0.50 l of deionized water. Stock solution usage was monitored on a daily basis. The diluter system was volumetrically calibrated before test initiation. Proper operation of the diluter and all mechanical systems was verified at least twice each day during the definitive test. The diluter system was labeled with study number and treatments.

Test chambers consisted of two 1-l glass beakers in each treatment with a screened overflow notch. Each beaker measured approximately 15 cm high and 10 cm in diameter. Test solution ddepth was maintained at approximately 12 cm. This provided a test solution volume of approximately 1000 ml. The two beakers in each treatment were grouped together to facilitate the delivery of the test solutions into each replicate. The beakers were arranged in a temperature controlled waterbath. Illumination was provided by a wide-spectrum fluorescent bulbs controlled by an electronic timer. A 16-hour light:8-hour dark photoperiod with a 30-minute simulated dawn and dusk transition period was provided.

The test exposure was initiated on May 14, 2004, with the impartial addition of 10 mysid shrimp to each beaker. Observations of mortality and sublethal responses were made at approximately 24, 48, 72, and 96 hours. Over the course of the study, an average of 6.0 l of control diluton water and test solution was delivered to each chamber each day. This rate was sufficient to provide an average of 6.0 volume additions in a 24-hour period.

Temperature, pH, dissolved oxygen concentration, and salinity were measured at 0, 24, 48, 72 and 96 hours in each test chamber, unless 100 percent mortality was observed in a test chamber. If a test chamber had 100 percent mortality, water quality data was recorded at that time and further measurements were discontinued. In addition, a continuous record of the temperature in the waterbath was collected with an electronic data logger with thermistor probe. Temperature was measured with either a WTW OXi 330i dissolved oxygen meter or with a Denver Instruments pH meter. The pH was measured with a Denver Instruments pH meter. Dissolved oxygen concentration was measured with a WTW OXi 330i dissolved oxygen meter. Salinity was measured with an Orion Model 140 conductivity/salinity meter. Light intensity was measured with a Li-Cor Model LI-189 light meter equipped with a photometric sensor.
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.81 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 1.30 - 2.52 mg a.i./l
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
After 96 hours of exposure, mortality in the 0 (control), 0.632, 1.30, 2.52, 4.92, and 10.1 mg a.i./l treatments was 0, 0, 0, 100, 100, and 100%, respectively. There were no sublethal effects observed in any treatment at test termination.

.
Results with reference substance (positive control):
No data.
Reported statistics and error estimates:
The 24-hour LC50 for mysid shrimp exposed to 2-methyl-4-isothiazolin-3-one was estimated to be >10.1 mg a.i./l and the 95% confidence limits could not be estimated. The 48-hour LC50 was calculated to be 2.98 mg a.i./l and the 95% confidence limits were 2.62 to 3.39 mg a.i./l. The 72-hour LC50 was calculated to be 1.94 mg a.i./l and the 95% confidence limits were 1.77 to 2.12 mg a.i./l. The 96-hour LC50 was calculated to be 1.81 mg a.i./l and the best estimate of the 95% confidence limits was 1.30 to 2.52 mg a.i./l. The 96 hour no-observed-effect concentration (NOEC) was 1.30 mg a.i./l based on the lack of significantly mortality at this and the lower concentration. The slope of the 96-hour concentration-response line could not be calculated

No additional information available.

Validity criteria fulfilled:
yes
Conclusions:
The 96 -hour LC50 was calculated to be 1.81 mg a.i./L and the best estimate of the 95% confidence limits was 1.30 to 2.52 mg a.i./l. The 96 -hour no-observed-effect concentration (NOEC) was 1.30 mg a.i./l based on the lack of significant mortality at ths and lower concentration.
Executive summary:

A test was conducted to estimate the potential acute toxicity of 2 -methyl-4 -isothiazoline-3 -one (supplied as a 50% solution in water known as Kordek 573F Industrial Microbiocide) to the mysid shrimp, Americamysis bahia. Mysid shrimp were exposed for 96 hours under flow-through conditions to nominal concentrations of 0 (control), 0.63, 1.3, 2.5, 5.0 and 10 mg a.i./l 2 -methyl-4 -isothiazolin-3 -one (Lot No. 0000454102, purity of 51.252%). The guideline followed was the US Environmental Protection Agency method OPPTS 850.1035.

Mean measured concentrations of 2 -methyl-4 -isothiazolin-3 -one were <MQL (control), 0.632, 1.30, 2.52, 4.92, and 10.1 mg a.i./l, which represented recoveries of 100, 100, 101, 98, and 101% of the nominal treatment concentrations. Water quality characteristics of temperature, dissolved oxygen, pH, and salinity, measured daily, remained within acceptable limits throughout the exposure. All test solutions appeared clear and colorless with no visible particulates, surface film, undissolved test substance or precipitate throughout the duration of the exposure. The 4.92 and 10.1 mg a.i./l treatments were discontinued after 100% mortality was observed after 48 hours.

After 96 hours, mortality was 0, 0, 0, 100, 100, and 100% in the 0 (control), 0.632, 1.30, 2.52, 4.92, and 10.1 mg a.i./l treatments, respectively. No sublethal observations were noted at test termination. Based on mean measured concentrations, the 96 -hour LC50 for mysid shrimp, Americamysis bahia, exposed to 2 -methyl-4 -isothiazolin-3 -one was calculated to be 1.81 mg a.i./l with best estimates of 1.30 to 2.52 mg a.i./l. The 96 -hour no-observed-effect concentration was the mean measured concentration of 1.30 mg a.i./l, based on the absence of significant mortality at this concentration and the lower concentration.

Description of key information

There were two identified key studies conducted on freshwater and marine water species.

Freshwater species - Exposure of the daphnid, Daphnia magna, to 2-methyl-4-isothiazolin-3-one, Technical resulted in a 48 -hour median lethal concentration (LC50) of 0.934 mg/L, with a 95% confidence interval of 0.765 to 1.16 mg/L, and a 48-hour median effective concentration (EC50) of 0.850 mg/L, with a 95% confidence interval of 0.681 to 1.06 mg/L. The 48-hour no-observed effect concentration (NOEC) was <0.275 mg/L 2-methyl-4-isothiazolin-3-one, the lowest tested concentration

Marine water species - A test was conducted to estimate the potential acute toxicity of 2 -methyl-4 -isothiazoline-3 -one (supplied as a 50% solution in water known as Kordek 573F Industrial Microbiocide) to the mysid shrimp, Americamysis bahia. The 48 -hour LC50 was calculated to be 2.98 mg a.i./l and the 95% confidence limits were 2.62 to 3.39 mg a.i./l. The 96 -hour LC50 was calculated to be 1.81 mg a.i./L and the best estimate of the 95% confidence limits was 1.30 to 2.52 mg a.i./l. The 96 -hour no-observed-effect concentration (NOEC) was 1.30 mg a.i./l based on the lack of significant mortality at tested concentrations

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.934 mg/L
EC50/LC50 for marine water invertebrates:
2.98 mg/L

Additional information