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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 April - 9 June 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction 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 the control, each test substance treatment, and diluter stock solution during system equilibration (day -N; May 17,2004) and on days 0 (test initiation), 7, 14, and 2 1 (test termination). At each sample point, 10-mL composite samples (2.5 mL from each replicate) were collected from each treatment. Quality control fortifications were prepared at each sample point. If necessary, samples were diluted with ABC reagent water prior to analysis. The samples were then analyzed by HPLC.
Vehicle:
no
Details on test solutions:
A 0.5-L proportional diluter system similar to that described by Mount and Brungs (3) was used for the intermittent introduction of a 0.40 mg a.i./L test solution, dilutions of the 0.40-mg a.i./L test solution, and dilution water to quadruplicate test chambers. A Hamilton Model 420 syringe dispenser was used for the intermittent introduction of the 392 mg a.i./L 2-Methyl-4-isothiazolin-3-one diluter stock solution to the diluter system. Mixing cells delivered the dilution water control and each of the six test solutions to flow-splitting cells. Each flow-splitting cell divided each 500-mL volume four ways. This split resulted in a volume of approximately 125 mL being delivered to each test chamber. The accuracy of the diluter was verified by volumetric measurement before test initiation. Proper operation of the proportional diluter and all mechanical systems was verified at least twice each day up to test termination. The diluter system maintained a cycle rate of approximately 2.0 cycles/hour during the test.

Dilution Water
The dilution water was a moderately hard freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis (RO). The well water and RO water was blended to yield a total hardness of 130 to 160 mg/L as CaC03 and the blended water was biologically aged (stored in a tank containing aquatic organisms) prior to use. Prior to delivery to the test chambers, the dilution water was passed through a sediment filter and UV sterilizer.
Test organisms (species):
Daphnia magna
Details on test organisms:
First-instar (<24 hours old) Daphnia magna neonates were obtained from an in-house culture. The adult daphnids were cultured in a temperature-controlled waterbath at approximately 20°C. The culture was maintained on a 16-hour daylight photoperiod with 30-minute dawn/dusk transitions periods. During the holding period, the adults in the culture were fed a suspension of Selenastrum capricornutum at least once a day, supplemented by an artificial diet prepared according to ABC SOP'S. Neonates for the test were collected from one culture that contained adults that were approximately 18-days old and exhibited no signs of stress (i.e., no ephippia) or physical damage. One day prior to test initiation, the adults in each culture were transferred to a fresh culture/food suspension and/or observed for the presence of young.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
No data
Hardness:
Hardness, measured in a composite sample collected from the control and highest test substance treatment replicates ranged from 138 to 144 mg/L as CaC03 in the control samples and ranged from 138 to 142 in the highest test substance treatment samples.
Test temperature:
Temperature of the treatment solutions remained between 20 +/- 1°C throughout the test and ranged from 19.9 to 20.7°C.
pH:
Test solution pH ranged from 8.27 to 8.62 and the deviation of pH from the initial values was 4.5 units
Dissolved oxygen:
Dissolved oxygen concentrations in treatment solutions ranged from 7.4 to 8.5 mg/L and the percent saturation ranged from 85 to 98%.
Salinity:
No data.
Nominal and measured concentrations:
Nominal concentrations of 0 (control), .013, 0.025, 0.10, 0.20, and 0.40 mg a.i./L

Mean measured concentrations of 2-Methyl-4-isothiazolin-3-one in the test substance treatments were 0.01 17, 0.0209, 0.0442, 0.0889, 0.183, and 0.359 mg a.i./L, which represented recoveries of 90, 84, 88, 89, 92, and 90% of the nominal treatment concentrations.
Details on test conditions:
Range-Finding Test
A 10-day flow-through range-finding test was conducted April 30 to May 10,2004, to evaluate the potential chronic toxicity of 2-Methyl-4-isothiazolin-3-one. The test was conducted at nominal concentrations of 0 (control), 0.25, 0.50, 1.0, 2.0, 4.0, and 8.0 mg a.i./L. After 10 days, 100% immobility was observed at concentrations 22.0 mg a.i./L. Immobility in the 0 (control), 0.25, 0.50, and 1.0 mg a.i./L treatments was 0, 15, 15, and 50%. First brood release occurred on day 7 in the control, day 9 in the 0.25 and 0.50 mg a.i./L treatments, and day 10 in the 1.0 mg a.i./L treatment. No young were produced in treatments 22.0 mg a.i.L. The total number of young produced by adult daphnids in the test substance treatment ranged from 137 in the 1.0 mg a.i./L treatment to 221 in the 0.50 mg a.i./L treatment. The total number of young produced by adult daphnids in the control was 410. Based on these results, nominal concentrations of 0 (control), 0.013, 0.025, 0.050, 0.10, 0.20, and 0.40 mg a.i./L were selected for the definitive test.

Definitive Test
The in-life phase of the flow-through definitive test was conducted from May 19 to June 9, 2004. A 0.5-L proportional diluter system similar to that described by Mount and Brungs (3) was used for the intermittent introduction of a 0.40 mg a.i./L test solution, dilutions of the 0.40-mg a.i./L test solution, and dilution water to quadruplicate test chambers. A Hamilton Model 420 syringe dispenser was used for the intermittent introduction of the 392 mg a.i./L 2-Methyl-4-isothiazolin-3-one diluter stock solution to the diluter system. Mixing cells delivered the dilution water control and each of the six test solutions to flow-splitting cells. Each flow-splitting cell divided each 500-mL volume four ways. This split resulted in a volume of approximately 125 mL being delivered to each test chamber. The accuracy of the diluter was verified by volumetric measurement before test initiation. Proper operation of the proportional diluter and all mechanical systems was verified at least twice each day up to test termination. The diluter system maintained a cycle rate of approximately 2.0 cycles/hour during the test.

The diluter stock solution was prepared May 14, May 20, and June 3, 2004, at a target concentration of 392 mg a.i./L by diluting approximately 0.3874 g (0.196 g corrected for purity) of 2-Methyl-4-isothiazolin-3-one with deionized water to a final volume of 500 mL. The syringe dispenser introduced 1.0 mL volumes of the diluter stock solution to the diluter system where the solution was diluted with approximately 980 mL of dilution water. Operation of the diluter system and delivery of the test substance was initiated on May 14, 2004.

Test chambers consisted of four 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 depth was maintained at approximately 12 cm. This provided a test solution volume of approximately 1,000 mL that was replaced at least five times per 24-hour period. The four test chambers in each treatment were grouped together to facilitate the delivery of the test solutions into each replicate. The test chambers were arranged in a temperature-controlled waterbath. Illumination was provided by 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. Light intensity, measured with a LI-COR Model LI-189 light meter equipped with a photometric sensor, ranged between 634 and 668 lux.

The test was initiated on May 19,2004, when 10 neonates were distributed to each of four test chambers for the dilution water control and each test substance treatment. Daphnids were assigned to a particular test chamber and treatment using a random number table. Observations were made daily for immobility, occurrence of sublethal effects (e.g., surfacing, trailing extraneous material), and production of first young. The criterion for immobility was the lack of response to disturbance. Immobile daphnids were discarded, therefore, immobility was synonymous with mortality.

Beginning on day 9 of the definitive test, neonates were collected and test chambers were cleaned on a Monday-Wednesday-Friday schedule by gently removing the adult daphnids from each beaker with a large-bore pipet and pouring the contents of the beaker through a fine mesh screen into a clean beaker. Following cleaning, the test chambers were refilled with test solution, the adults were returned, and the test chambers returned to the waterbath. The neonates collected on the screen were rinsed into small jars and set aside for counting. The neonates produced between each collection period were enumerated and discarded.

The daphnids were fed a diet consisting of a concentrated algal suspension (Selenastrum capricornutum) with each diluter cycle and supplemented twice daily except for test initiation and termination with 0.5 mL or 1.0 mL of a 5-mg/mL suspension of an artificial invertebrate diet. A total volume of approximately 21 mL of concentrated algal suspension at a minimum cell density of 1.0 x 10(6) cells/mL was delivered to each control and test substance treatment solution with each diluter cycle to maintain a minimum algal density of 2.52 x10(4) cells/mL. Representative samples of the food were screened for contaminants and the data are on file at ABC Laboratories.

At test termination, the length (head to base of spine) of each surviving adult was measured by use of a calibrated ocular micrometer of an Bausch & Lomb Stereo Zoom 4 microscope. After length measurements, the daphnids from each replicate were pooled and placed in preweighed foil pans and dried for approximately 44 hours at approximately 73C. After drying, the foil pans were placed in a desiccator to cool and then weighed.

Water quality characteristics of temperature, dissolved oxygen concentration, and pH were measured in each replicate at initiation and once each week following initiation. Temperature and dissolved oxygen concentration were measured with a WTW Oxi 330 dissolved oxygen meter. The pH was measured with a Denver Instruments pH meter. No aeration was provided to any control or test substance treatment during the test. A continuous recording of the waterbath temperature was made during the test using a datalogger and thermistor probe. Hardness of pooled replicate samples collected from the control and highest test substance treatment was measured at test initiation once each week following initiation. Hardness was measured using standard titrimetric techniques.
Reference substance (positive control):
not specified
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.044 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: survival, reproduction, and growth at this concentration
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.089 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth
Remarks:
Statistically significant weight effects at this and higher treatments.
Details on results:
After 21 days of exposure, survival of parent Daphnia magna exposed to 2-Methyl-4-isothiazolin-3-one ranged from 87% in the 0.359 mg a.i./L treatment to 97% in the 0.0209 mg a.i./L treatment (Table 4, Appendix D). Survival in the control treatment was 93%. Replicate A of the 0.0209 mg a.i./L treatment and replicate B of the 0.359 mg a.i./L treatment were determined to be statistical outliers based on survival; therefore, these replicates were excluded from all other statistical analyses. The anomalous mortality observed in replicate A of the 0.0209 mg a.i./L treatment (80% mortality) and replicate B of the 0.359 mg a.i./L treatment (90% mortality) was not attributed to the test substance or water quality since all other replicates in these treatments were exposed to the same conditions. No statistically significant decrease of survival in all test substance treatments, as compared to the control, was detected. Based on survival, the no-observed-effect concentration (NOEC) was 0.359 mg a.i./L and the lowest-observed- effect concentration (LOEC) was >0.359 mg a.i./L. The 21-day EC50 was estimated to be >0.359 mg a.i./L, the highest treatment tested, since immobility in all test substance treatments was <50%.

Young were first observed in the control on day 7 and the parent daphnids had produced young in all replicates of the controls by day 9. In the test substance treatments, young were first observed on day 8 and the parent daphnids had produced young in all replicates by day 10. The total number of young produced per parent present at test initiation in test substance treatments ranged from 105 at 0.183 mg a.i./L to 140 at 0.0117 mg a.i./L. The total number of young produced per parent present at test initiation in the control was 135. No immobility or morphological abnormalities were observed in the young in any treatment. There was no statistically significant increase in the day young were first observed in all test substance treatments, as compared to the control. No statistically significant decrease of total number of young produced per parent for all test substance treatments, as compared to the control, was detected. Based on total number of young produced per parent, the NOEC was 0.359 mg a.i./L and the LOEC was >0.359 mg a.i./L. The 21-day EC50 was estimated to be >0.359 mg a.i./L, the highest treatment tested, since the percent difference of total number of young produced per parent in all test substance treatments was <50% as compared to the control.

Mean length of the control daphnids was 4.9 mm. Mean length of daphnids in the test substance treatments ranged from 4.8 to 4.9 mm. No statistically significant decrease in length, as compared to the control, was detected. Based on length, the NOEC was 0.359 mg a.i./L and the LOEC was >0.359 mg a.i./L. The 21-day EC50 was estimated to be >0.359 mg a.i./L, the highest treatment tested, since the percent difference of length in all test substance treatments was 40 % as compared to the control.

Mean dry weight of parent daphnids in the control was 1.01 mg. Mean dry weight of parent daphnids in the test substance treatments ranged from 0.60 mg at 0.359 mg a.i./L to 0.98 mg at 0.0442 mg a.i./L. The mean dry weight of daphnids in the 0.0889 and 0.359 mg a.i./L treatments were statistically significantly less than the control. Based on dry weight, the NOEC was 0.0442 mg a.i./L and the LOEC was 0.0889 mg a.i./L. The 21-day ECS0 was estimated to be >0.359 mg a.i./L, the highest treatment tested, since the percent difference of dry weight in all test substance treatments was <50% as compared to the control.

The acceptance criteria for the control (i.e., <20% mortality of parent daphnids, no ephippia production, and >/=60 young produced per parent control daphnid surviving a the end of the test.) were all met. The overall NOEC was 0.0442 mg a.i./L based upon the lack of statistically significant effects on survival, reproduction, and growth at this concentration. The overall LOEC was 0.0889 mg a.i./L based upon statistically significant weight effects at this and higher treatments.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
Based on survival, the no-observed-effect concentration (NOEC) was 0.359 mg a.i./L and the lowest-observed-effect concentration (LOEC) was >0.359 mg a.i./L. The 21-day EC50 was estimated to be >0.359 mg a.i./L, the highest treatment tested, since immobility in all test substance treatments was <50%.

Based on total number of young produced per parent, the NOEC was 0.359 mg a.i./L and the LOEC was >0.359 mg a.i./L. The 21-day EC50 was estimated to be >0.359 mg a.i./L, the highest treatment tested, since the percent difference of total number of young produced per parent in all test substance treatments was <50% as compared to the control.

Based on length, the NOEC was 0.359 mg a.i./L and the LOEC was >0.359 mg a.i./L. The 21-day EC50 was estimated to be >0.359 mg a.i./L, the highest treatment tested, since the percent difference of length in all test substance treatments was 40 % as compared to the control.

Based on dry weight, the NOEC was 0.0442 mg a.i./L and the LOEC was 0.0889 mg a.i./L. The 21-day ECS0 was estimated to be >0.359 mg a.i./L, the highest treatment tested, since the percent difference of dry weight in all test substance treatments was <50% as compared to the control.

The overall NOEC was 0.0442 mg a.i./L based upon the lack of statistically significant effects on survival, reproduction, and growth at this concentration. The overall LOEC was 0.0889 mg a.i./L based upon statistically significant weight effects at this and higher treatments.

Method Validation Results

Recoveries of 2-Methyl-4-isothiazolin-3-one ranged from 95 to 103% of the nominal concentrations. No residues of 2-Methyl-4-isothiazolin-3-one were detected in the control samples above the MQL of 0.0106 mg a.i./L. The validation results indicated that the method was suitable for recovering 2-Methyl-4-isothiazolin-3-one from blended freshwater.

Analytical Chemistry Results

Measured concentrations of 2-Methyl-4-isothiazolin-3-one in the test substance treatments ranged from 0.0102 to 0.384 mg a.i./L and ranged from 78 to 98% ofthe nominal concentrations during the exposure. The mean measured concentrations for the test substance treatments ranged from 0.01 17 to 0.359 mg a.i./L and ranged from 84 to 92 of the nominal concentrations. The mean measured test concentrations were used to present all biological results. The recoveries of 2-Methyl-4-isothiazolin-3-one from the test substance treatments indicate that 2-Methyl-4-isothiazolin-3-one exposure concentrations were maintained during the exposure. Measured concentrations of 2-Methyl-4-isothiazolin-3-one in the 392 mg a.i. /L diluter stock solutions ranged from 97 to 105% of the nominal concentration. No residues of 2-Methyl-4-isothiazolin-3-one were detected in the control samples above the MQL of 0.00530 mg a.i./L. Recoveries of 2-Methyl-4-isothiazolin-3-one from the QC samples ranged from 91 to 113% of the nominal concentrations.

There was no notable presence of undissolved test substance or surface film in test solutions a throughout the test.

Water Quality

Dissolved oxygen concentrations in treatment solutions ranged from 7.4 to 8.5 mg/L and the percent saturation ranged from 85 to 98%. Temperature of the treatment solutions remained between 20 + l°C throughout the test and ranged from 19.9 to 20.7°

C. Continuous temperature measurement in the water bath, monitored by an electronic datalogger, indicated consistency with the test solution measurements. Test solution pH ranged from 8.27 to 8.62 and the deviation of pH from the initial values was 4.5 units. Hardness, measured in a composite sample collected from the control and highest test substance treatment replicates ranged from 138 to 144 mg/L as CaC03 in the control samples and ranged from 138 to 142 in the highest test substance treatment samples.

Table 1 Summary of Results

 Mean Measured Concentration  Cumulative Number Surviving  Young per  Helmet-Spline Length  Mean Dry Weight per Organismc
 (mg a.i./L)  (Percent Survival)  Parenta  Weighted Grand Meanb (mm) (mg) 
 Control  37 (93)a  135  4.9  1.01
 0.0117  38 (95)  140  4.8  0.94
 0.0209  29 (97)a  130  4.8  0.97
 0.0442  38 (95)  130  4.9  0.98
 0.0889  36 (90)  121  4.8  0.83**
 0.183  32 (80)  105  4.8  0.85
 0.359  26 (87)a  134  4.8  0.60**

a Values were rounded to the nearest whole number

b Mean of all individual daphnid lengths for the treatment.

c The weighted mean is calculated by dividing the sum of all treatment replicates by the total number of organisms weighed for the treatment.

d Value was rounded to the nearest whole number

** Statistically significant reduction (P = 0.0000 to 0.0339) when compared to the control.

Validity criteria fulfilled:
yes
Conclusions:
After 21 days, survival in the control, 0.0117, 0.0209, 0.0442, 0.0889, 0.183, and 0.359 mg a.i./L treatments was 93, 95, 97, 95, 90, 80, and 87%, respectively. The number of offspring produced per parent present at test initiation was 135 in the control. The 21-day LOEC and NOEC for Daphnia magna exposed to 2-Methyl-4-isothiazolin-3-one was 0.0889 and 0.0442 mg a.i./L, respectively, based on dry weight, the most sensitive parameter. The 21 -Day NOEC for the parameters of survival, reproduction, and length was 0.359 mg a.i/L, the highest treatment tested. The 21-day EC50 values for survival, reproduction, and growth were estimated to be >0.359 mg a.i./L, the highest treatment tested.
Executive summary:

A test was conducted to estimate the potential chronic toxicity of 2-Methyl-4-isothiazolin-3-one (supplied as a 50% solution in water known as Kordek™ 573F Industrial Microbiocide) to the water flea, Daphnia magna, under flow-through conditions (ABC Study No. 48836, Rohm and Haas Protocol No. 04P-024). Daphnids were exposed for 21 days under flow-through conditions to nominal concentrations of 0 (control), 0.013, 0.025, 0.10, 0.20, and 0.40 mg a.i./L 2-Methyl-4- isothiazolin-3-one (Lot No. 0000454102, TD No. 04-005, purity of 51.252%, expiration date of July 3, 2008).

Mean measured concentrations of 2-Methyl-4-isothiazolin-3-one in the test substance treatments were 0.0117, 0.0209, 0.0442, 0.0889, 0.183, and 0.359 mg a.i./L, which represented recoveries of 90, 84, 88, 89, 92, and 90% of the nominal treatment concentrations. All biological endpoints were based on these mean measured concentrations. No residues of 2-Methyl-4-isothiazolin-3- one were detected in the control at or above the MQL of 0.00530 mg a.i./L. Water quality characteristics of temperature, dissolved oxygen concentration, and pH, measured on days 0, 7, 14, and 21, remained within acceptable limits throughout the exposure. All test solutions appeared clear and colorless with no visible particulates, surface film, and undissolved test substance or precipitate throughout the duration of the exposure.

After 21 days, survival in the control, 0.0117, 0.0209, 0.0442, 0.0889, 0.183, and 0.359 mg a.i./L treatments was 93, 95, 97, 95, 90, 80, and 87%, respectively. The number of offspring produced per parent present at test initiation was 135 in the control. The 21-day LOEC and NOEC for Daphnia magna exposed to 2-Methyl-4-isothiazolin-3-one was 0.0889 and 0.0442 mg a.i./L, respectively, based on dry weight, the most sensitive parameter. The 21 -Day NOEC for the parameters of survival, reproduction, and length was 0.359 mg a.i/L, the highest treatment tested. The 21-day EC50 values for survival, reproduction, and growth were estimated to be >0.359 mg a.i./L, the highest treatment tested.

Description of key information

A test was conducted to estimate the potential chronic toxicity of 2-Methyl-4-isothiazolin-3-one (supplied as a 50% solution in water known as Kordek™ 573F Industrial Microbiocide) to the water flea, Daphnia magna, under flow-through conditions. The 21 -day LOEC and NOEC for Daphnia magna exposed to 2-Methyl-4-isothiazolin-3-one was 0.0889 and 0.0442 mg a.i./L, respectively, based on dry weight, the most sensitive parameter. The 21-Day NOEC for the parameters of survival, reproduction, and length was 0.359 mg a.i/L, the highest treatment tested. The 21-day EC50 values for survival, reproduction, and growth were estimated to be >0.359 mg a.i./L, the highest treatment tested

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.044 mg/L

Additional information